Isolation of chlorophylls and carotenoids from freshwater algae using different extraction methods

Usually marine algae are an excellent source of pigments for different commercial sectors. Freshwater macroalgae can be exploited as a good source of biologically active compounds provided an appropriate extraction method is developed. The efficiency of four methods, like microwave‐assisted (MAE), ultrasound‐assisted extraction (UAE), supercritical fluid extraction (SFE) with ethanol as a co‐solvent, as well as conventional Soxhlet extraction were studied in the same conditions (time, solvent and temperature) for the recovery of chlorophylls and carotenoids from three freshwater green algae species: Cladophora glomerata, Cladophora rivularis and Ulva flexuosa. UV‐Vis spectrophotometry was used to determine chlorophyll a, chlorophyll b and total carotenoid content in obtained extracts. The results of this study showed that the advantages of novel extraction techniques (MAE and UAE) include higher yield and, in consequence, lower costs compared to traditional solvent extraction techniques. These methods were much more efficient in freshwater green algae pigment recovery than the classic Soxhlet extraction as well as SFE.     

ANALYSIS OF ANCIENT DNA FROM FOSSIL CORALLINES (CORALLINALES,RHODOPHYTA)1

The field of molecular paleontology has recently made significant contributions to anthropology and biology. Hundreds of ancient DNA studies have been published, but none has targeted fossil coralline algae. Using regions of the SSU gene, we analyzed rDNA from fossil coralline algae of varying ages and states of preservation from Spain, Papua New Guinea (PNG), and the Great Barrier Reef (GBR). Specimens from PNG, GBR, and some localities from Spain did not contain endogenous ancient DNA. Reproducible sequence data were obtained from specimens ～550 years old from near Cadiz, Spain, and from rocky‐shore deposits in Carboneras, Almeria Province of Spain (～78,000 years before present [YBP]). Based on BLAST searches and a phylogenetic analysis of sequences, an undescribed coralline alga belonging to the Melobesioideae was discovered in the Carboneras material as well as the following coralline genera: Jania, Lithophyllum, Lithothamnion, Mesophyllum, and Phymatolithon. DNA from fleshy brown and red macroalgae was also discovered in the specimens from Carboneras. The coralline algae identified using molecular techniques were in agreement with those based on morphological methods. The identified taxa are common in the present‐day southeastern Spain littoral zone. Amino acid racemization, concentration ratios, and specific concentrations failed to show a correlation between biomolecular preservation and PCR amplification success. Results suggest that molecular investigations on fossil algae, although limited by technical difficulties, are feasible. Validity of our results was established using authentication criteria and a self‐critical approach to compliance.     

A simple method for DNA extraction from sporophyte in the brown alga <Emphasis Type="Italic">Laminaria japonica</Emphasis>

A simple method was developed for extracting DNA from brown algae Laminaria japonica, which possess large amounts of acidic polysaccharides. Firstly, the sporophyte were washed by eliminating polysaccaride buffer to remove the polysaccharides and then ground in liquid nitrogen. Secondly, the powders were treated with lysing buffer. Thirdly, KAc was used to eliminate the remaining acidic polysaccharides. The extracted DNA was purified using a chloroform-isoamyl alcohol (24:1 v/v), and precipitated in cold isopropanol. The yield was from 18.7 to 37.5 μg g⁻¹ (wet weight) and the purity of total DNA was determined spectrophotometrically as the ratio of A₂₆₀/A₂₈₀, which was about 1.7–1.9. The extracted DNA was of high quality and suitable for molecular analyses, such as PCR, restriction enzyme digestion. This method is a reproducible, simple, and rapid technique for routine DNA extraction from sporophyte in Laminaria japonica. Furthermore, the low cost of this method makes it attractive for large-scale studies.     

OPTIMIZATION OF DNA EXTRACTION FROM BROWN ALGAE (PHAEOPHYCEAE) BASED ON A COMMERCIAL KIT1

Large‐scale DNA molecular studies require reliable and efficient tools for DNA extractions. However, for some plant species and brown algae, isolation of high‐quality DNA is difficult. We developed a novel method for isolating high‐quality DNA from the polysaccharide‐rich and polyphenol‐rich brown algae based on a commercial kit and protocol (Qiagen) by optimizing the lysis step and including a chloroform/isoamyl alcohol supplementary purification step. DNAs from 24 brown algal species extracted using the original and the modified Qiagen protocol were compared for yield, quality, and effectiveness in PCR amplification. There was no significant difference in the yields between protocols. However, a statistically significant increase in DNA purity was obtained with the modified protocol, for which the A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀ absorbance ratios averaged 1.66 ± 0.05 and 1.31 ± 0.01, respectively, compared to 1.37 ± 0.04 and 0.52 ± 0.04 with the original protocol. DNAs extracted by the modified procedure were more successfully amplified by PCR (nuclear, mitochondrial, and chloroplastic regions) than DNAs extracted using the original commercial kit and protocol. Importantly, the modified protocol can be applied in a high‐throughput (e.g., 96‐well plate) format, allowing a higher efficiency for downstream molecular analysis. In addition, improved DNA quality could increase its stability for long‐term storage.     

Comparative evaluation and selection of a method for lipid and fatty acid extraction from macroalgae

A comparative evaluation of Bligh and Dyer, Folch, and Cequier-Sánchez methods for quantitative determination of total lipids (TLs) and fatty acids (FAs) was accomplished in selective green (Ulva fasciata), red (Gracilaria corticata), and brown algae (Sargassum tenerrimum) using a full factorial categorical design. Applications of sonication and buffer individually on lipid extraction solvent systems were also evaluated. The FA recoveries obtained from the aforementioned methods were compared with those of direct transesterification (DT) methods to identify the best extraction methods. The experimental design showed that macroalgal matrix, extraction method, and buffer were key determinants for TL and FA recoveries (P ? 0.05), exhibiting significant interactions. But sonication gave erratic results with no interaction with any of the factors investigated. The buffered solvent system of Folch rendered the highest TL yield in U. fasciata and G. corticata while the buffered system of Bligh and Dyer gave the highest yield in S. tenerrimum. DT methods were more convenient and accurate for FA quantification and rendered 1.5–2 times higher yields when compared with the best conventional method, minimizing the use of chlorinated solvents, their cost of analysis, and disposal. The buffered solvent system was found to be the most appropriate for lipid research in macroalgae.     

Induction and genetic identification of a callus‐like growth developed in the brown alga Fucus vesiculosus

Induction of an axenic filamentous‐like callus growth from the brown algae Fucus vesiculosus is described. Different treatments were investigated in various combinations to develop axenic cultures based on identification of surface symbionts via 18S ribosomal RNA. Moreover, viability was confirmed after such processes by 2,3,5‐triphenyl tetrazolium chloride assay that demonstrated an average viability of 29%, relative to nonsterilized explants. After six weeks of a phototrophic cultivation on artificial sea water‐12‐nitrilotriacetic acid (0.5% w/v agar), a filamentous‐like callus growth was observed, which was identified genetically through its mitochondrial DNA after subculturing. Achievement of confirmed marine callus cultures might enrich old previously established blue biotechnology techniques and open new chances for cultivation of brown algae for production of good manufacturing practice‐compliant bioproducts.     

Antitumor activity of marine algae

Powdered tissue from 46 species of air-dried marine algae (four green, 21 brown and 21 red algae) were screened for antitumor activity. Significant activity against Ehrlich carcinoma was found in the brown algae Scytosiphon lomentaria (69.8% inhibition), Lessonia nigrescens (60.0%), Laminaria japonica (57.6%), Sargassum ringgoldianum (46.5%), the red algae Porphyra yezoensis (53.2%) and Eucheuma gelatinae (52.1%) and the green alga Enteromorpha prolifera (51.7%). Five brown and four red algae showed appreciable antitumor activity against Meth-A fibrosarcoma. To identify specific molecules with antitumor activity, 15 kinds of polysaccharide preparations of seaweed origin and 24 kinds of lipid fractions extracted from various seaweeds were tested. Appreciable inhibition of Ehrlich carcinoma was found for fucoidan preparations from Undaria pinnatifida and Sargassum ringgoldianum, for carrageenans and for porphyran. Several glycolipid and phospholipid fractions from brown and red algae were effective against Meth-A fibrosarcoma.     

GENOMIC INSIGHTS INTO EVOLUTIONARY RELATIONSHIPS AMONG HETEROKONT LINEAGES EMPHASIZING THE PHAEOPHYCEAE1

Heterokonts comprise a large and diverse group of organisms unified by the heterokont biflagellate condition. Monophyly of many of these lineages is well established, but evolutionary relationships among the various lineages remain elusive. Among these lineages, the brown algae (Phaeophyceae) are a monophyletic, taxonomically diverse, and ecologically critical group common to marine environments. Despite their biological and scientific importance, consensus regarding brown algal phylogeny and taxonomic relationships is missing. Our long‐term research goal is to produce a well‐resolved taxon‐rich phylogeny of the class to assess evolutionary patterns and taxonomic relationships among brown algal lineages and their relationship to other closely related heterokont groups. To accomplish this goal and augment existing loci for phaeophycean‐wide systematic studies, we generated expressed sequence tags (ESTs) from several major brown algal lineages and from the heterokont lineage representing the closest sister group to brown algae. To date, we have successfully constructed cDNA libraries for two lineages (Choristocarpus tenellus Zanardini and Schizocladia ischiensis E. C. Henry, Okuda et H. Kawai) and in the library test phase obtained up to 1,600 ESTs per organism. Annotation results showed a gene discovery rate of 45%–50% for each library revealing 500–700 unique genes from each organism. We have identified several potential genes for phylogenetic inference and used these loci for preliminary molecular clock analyses. Our molecular clock analysis suggests that the basal divergence in brown algae occurred around the time of the pennate‐centric diatom divergence. Here we report this analysis and other uses of ESTs in brown algal phylogenomics and the utility of these data for resolving the phylogeny of this group.     

Noncryogenic Preservation of Mammalian Tissues for DNA Extraction: An Assessment of Storage Methods

Reliable field methods for the storage of tissues to be used for DNA extraction and amplification are critical to many studies employing molecular techniques. Protection from DNA degradation was compared among three commonly used methods of noncryogenic storage of tissues over a time scale of 2 years. All three methods prevented DNA degradation during storage for at least 6 months. DMSO (dimethyl sulfoxide)-salt solution provided the best protection from DNA degradation of tissues stored for up to 2 years. High molecular weight DNA was recovered from lysis buffer in which tissue was stored for 2 years, however, moderate amounts of degraded DNA was also present. High molecular weight DNA was recovered from tissues stored in ethanol for 2 years, however, the yield was relatively small compared to the other two noncryogenic storage techniques. Much of the DNA degradation in ethanol preserved tissues appeared to occur during the extraction procedure and can be reduced by soaking the tissue in lysis buffer for a few hours prior to beginning the extraction. The yield of PCR products was greatest from DNA extracted from DMSO-salt solution preserved tissues, whereas DNA from tissues stored in either lysis buffer or ethanol produced lower yields.     

Dye affinity chromatography for fast and simple purification of fucoidan from marine brown algae

Fucoidan is a sulfated polysaccharide with promising pharmacological applications. Due to its medicinal properties, there is a demand for a separation technique that yields a high purification grade. Here, we present a novel purification tool for recovering fucoidan from the marine brown macroalgae Fucus vesiculosus. The developed method is based on amino‐derivatized Sepabeads^® EC‐EA. The beads were modified with toluidine blue (TB), a thiazine derivative, to exploit the strong donor acceptor interactions between the cationic dye and the anionic polysaccharide. The adsorption kinetics and the binding capacity of the resin were analyzed. A Sips model was used to approximate the adsorption isotherm, resulting in a maximum capacity of 127.7 mg fucoidan per g adsorbent. Investigation of the effect of adsorption step's pH on purity and chemical structure was performed by TB and Fourier transform infra‐red spectroscopy assays. Results showed that adsorption at pH 1 and 6 had negligible effects on fucoidan's chemical structure. However, purity was actually improved by 1.55‐ and 1.69‐fold at pH 1 and 6, respectively, with an average yield of 5 g/100 g dried algae powder. In contrast, only a 1.46‐fold increment was observed in fucoidan purified by the traditional method at pH 2, with a yield of 7.5 g/100 g dried algae powder. Furthermore, fucoidan purified by this method at pH 6 complies with, or even exceeds the quality of the commercially available (≥95% pure) fucoidan (Sigma‐Aldrich^®) with respect to molecular weight and sulfur content. Therefore, dye affinity chromatography provides more advantages than the classically used techniques for fucoidan purification.     

Utility of rbcS gene as a novel target DNA region for brown algal molecular systematics

The usefulness of molecular phylogenetic studies has increased remarkably as the quantity and quality of available DNA sequences has increased. When compared with the progress that has occurred in angiosperms and animals, there have been relatively few target DNA regions identified for use in taxonomic studies of brown algae. Therefore, in this study, we developed a new set of primers to amplify Rubisco small subunit (rbcS) gene sequences and determined the rbcS gene sequences of various species of brown algae including those belonging to Dictyotales, Ectocarpales, Fucales and Sphacelariales. The level of sequence variations in the rbcS gene varied according to the brown algal lineages. When focusing on the relationship of species within the genus Sargassum, the rbcS gene sequences provided useful information regarding the phylogenetic relationship among sections of the subgenus Bactrophycus. Based on the broad applicability and phylogenetic utility of the rbcS gene, we suggest that the sequence be used as a new target region for the molecular systematics of brown algae.     

The biogeography of polyphenolic compounds in marine macroalgae: temperate brown algal defenses deter feeding by tropical herbivorous fishes

Summary Many tropical brown algae have low levels of polyphenolic compounds and are readily consumed by herbivorous fish. In contrast, temperate brown algae often produce large quantities of phenolic compounds causing them to be distasteful to herbivorous gastropods and sea urchins. We hypothesized that tropical brown algae do not use phenolic compounds as antiherbivore defenses because these compounds are not effective deterrents against tropical fish. To test our hypothesis, we assessed the ability of extracts from 8 tropical and 13 temperate algae with a broad range of phenolic levels to deter feeding by herbivorous fishes on Guam. Extracts of the high-phenolic (>2% d.w.) temperate brown algae consistently deterred feeding by herbivorous fishes, whereas extracts from low phenolic (<2% d.w.) temperate and 6 of 8 low-phenolic tropical brown algae did not. Thus, phenolic compounds could be effective feeding deterrents towards herbivorous fishes on Guam, but for unknown reasons they are not used by Guamanian brown algae.     

Proboscidean DNA from Museum and Fossil Specimens: An Assessment of Ancient DNA Extraction and Amplification Techniques

Applications of reliable DNA extraction and amplification techniques to postmortem samples are critical to ancient DNA research. Commonly used methods for isolating DNA from ancient material were tested and compared using both soft tissue and bones from fossil and contemporary museum proboscideans. DNAs isolated using three principal methods served as templates in subsequent PCR amplifications, and the PCR products were directly sequenced. Authentication of the ancient origin of obtained nucleotide sequences was established by demonstrating reproducibility under a blind testing system and by phylogenetic analysis. Our results indicate that ancient samples may respond differently to extraction buffers or purification procedures, and no single method was universally successful. A CTAB buffer method, modified from plant DNA extraction protocols, was found to have the highest success rate. Nested PCR was shown to be a reliable approach to amplify ancient DNA templates that failed in primary amplification.     

Amino acid composition, protein content and calculation of nitrogen-to-protein conversion factors for 19 tropical seaweeds

The use of nitrogen‐to‐protein conversion factors (N‐Prot factors) is the most practical way of determining protein content. The accuracy of protein determination by this method depends on the establishment of N‐Prot factors specific to individual species. Experimental data are needed to allow the use of this methodology with seaweeds. The present study was designed to characterize the amino acid composition and to establish specific N‐Prot factors for six green, four brown and nine red marine algae. Mean values for individual amino acids tended to be similar among the three groups, but some differences were found. Green algae tended to show lower percentages of both aspartic acid and glutamic acid than the other two groups of algae. The percentages of both lysine and arginine were higher in red algae, while brown algae tended to show more methionine than green and red algae. The actual protein content of the species, based on the sum of amino acid residues, varied from 10.8% (Chnoospora minima, brown algae) to 23.1% (Aglaothamnion uru‐guayense, red algae) of the dry weight. Nitrogen‐to‐protein conversion factors were established for the species studied, based on the ratio of amino acid residues to total nitrogen, with values ranging from 3.75 (Cryptonemia seminervis, red algae) to 5.72 (Padina gymnospora, brown algae). The relative importance of non‐protein nitrogen is greater in red algae, and consequently lower N‐Prot factors were calculated for these species (average value 4.59). Conversely, protein nitrogen content in both green and brown algae tends to be higher, and average N‐Prot factors were 5.13 and 5.38, respectively. An overall average N‐Prot factor for all species studied of 4.92 ± 0.59 (n = 57) was established. This study confirms that the use of the traditional factor 6.25 is unsuitable for seaweeds, and the use of the N‐Prot factors proposed here is recommended.     

On the utility of DNA barcoding for species differentiation among brown macroalgae (Phaeophyceae) including a novel extraction protocol

The generation of a species-rich DNA barcode database in combination with rapid and affordable sequencing techniques will dramatically change specimen identification in ecological, biogeographical and taxonomic applications. Though cytochrome c oxidase 1 has been shown to be a useful tool for differentiating some groups of marine algae, its wide application in the Phaeophyceae has yet to be studied. The presence of polymerase chain reaction (PCR) inhibiting compounds in members of the Fucales, Laminariales and Tilopteridales, that are often co-extracted with DNA, has hampered the rapid processing associated with barcode projects. Polyphenolics and polysaccharides are present in concentrations such that DNA extraction methods typically include extensive series of washes, organelle extractions and/or cesium columns. In this paper we examine the utility of cytochrome c oxidase 1 for barcoding the Phaeophyceae and present a method for extracting PCR friendly DNA from brown macroalgae in about 2 h, dramatically reducing the time required from previous methods, some of which take days. This method is easily adapted to a 96 well, high-throughput format and may have applications in other organisms where the presence of similar PCR inhibiting compounds hinders molecular analyses. We extracted DNA from 106 isolates representing 29 species from 20 genera in nine families from five orders of Phaeophyceae. We were able to amplify the barcode marker (cytochrome c oxidase 1) from all samples and a nuclear marker (internal transcribed spacer region) from 54 selected samples. Cytochrome c oxidase 1 was able to differentiate clearly among species, showing within species divergence of 0.00–0.46%, with the exception of one previously studied genus, and between species divergences of greater than 3%.     

DNA EXTRACTION METHODS FOR KELP (LAMINARIALES) TISSUE1

Newly adapted extraction methods for harvesting total cellular DNA from kelp (Laminariales; Phaeophyta) exploit the life-history stages of these heteromorphic algae. Earlier techniques, developed primarily for chloroplast DNA extraction, were time consuming and labor intensive and required large quantities of fresh sporophyte tissue. In contrast, the new methods expedite DNA extraction by employing dried sporophyte laminae or fresh gametophyte filaments and meiospores. The current methods require less tissue and procedural manipulation, reducing the time and labor involved in DNA extraction. Additionally, the current methods were successfully employed to extract DNA from herbarium specimens up to 22 years old. Total cellular DNA yields were 12.8 and 26.4 μg:g^?1 from dried laminae,: 1.9 and 11 μg from small volumes of concentrated gametophyte filaments (25 μL), and 43 to 54.5 μg from pellets of isolated meiospores (50 μL). Following purification on Sepharose columns, DNA from all three sources was sufficiently pure for molecular applications, including restriction endonuclease digestion, Southern blot-hybridizations, and amplification via the polymerase chain reaction.     

GENOMIC DNA ISOLATION FROM GREEN AND BROWN ALGAE (CAULERPALES AND FUCALES) FOR MICROSATELLITE LIBRARY CONSTRUCTION1

A method for isolating high‐quality DNA is presented for the green algae Caulerpa sp. (C. racemosa, C. prolifera, and C. taxifolia) and the brown alga Sargassum muticum. These are introduced, and invasive species in Europe, except for the native C. prolifera. Previous methods of extraction, using cetyl trimethyl ammonium bromide or various commercial kits, were used to isolate genomic DNA but either no DNA or DNA of very low quality was obtained. Genomic libraries were attempted with Caulerpa sp. on three occasions and either the restriction enzyme, the Taq polymerase, or the T4 ligase was inhibited, probably by the large amount of polysaccharides in these algae. The method presented here consists of the rapid isolation of stable nuclei, followed by DNA extraction. Yields of 6–10 μ g genomic DNA from 1 g fresh blades were obtained. After genomic DNA was isolated from fresh material, the quality was checked by agarose gel. Quantification of DNA concentration was performed using UV spectrophotometric measurement of the A ₂₆₀/ A ₂₈₀ ratio. The DNA was suitable for PCR, cloning, and hybridization. The DNA isolated using this method allowed successful construction of microsatellite libraries for Caulerpa species and S. muticum . The technique is inexpensive and appropriate for the isolation of multiple samples of DNA from a small amount of fresh material.     

A technique for the rapid extraction of microalgal DNA from single live and preserved cells

Molecular methods are increasingly being used in the study of harmful microalgae; however, DNA extraction techniques have imposed limitations on the species and questions studied, with research primarily restricted to cultured specimens. Here we describe a simple method that merges two existing techniques for DNA extraction from live and preserved single dinoflagellate cells. DNA was successfully isolated from live single cells of Gambierdiscus toxicus Adachi et Fukuyo, 1979 and cells preserved using formalin/methanol fixation. This method supplements existing techniques and expands the scope of genetics studies conducted on dinoflagellates to include routine molecular analysis of single cells isolated from field samples.     

海藻多糖化学及生物活性的研究进展

海藻多糖具有多种生物活性,在生物体内起着重要作用,现已成为海藻研究的热点。本文综述了海藻多糖的种类,海藻多糖的提取、分离与提纯技术,海藻多糖性质的测定方法及海藻多糖的生物活性。