Study of increasing lipid production from fresh water microalgae Chlorella vulgaris was conducted by investigating several important factors such as the effect of CO 2 concentration, nitrogen depletion and harvesting time as well as the method of extraction. The drying temperature during lipid extraction from algal biomass was found to affect not only the lipid composition but also lipid content. Drying at very low temperature under vacuum gave the best result but drying at 60 °C still retained the composition of lipid while total lipid content decreased only slightly. Drying at higher temperature decreased the content of triacylglyceride (TG). As long as enough pulverization was applied to dried algal sample, ultrasonication gave no effect whether on lipid content or on extraction time. In addition to the increase of total lipid content in microalgal cells as a result of cultivating in nitrogen depletion media, it was found that changing from normal nutrient to nitrogen depletion media will gradually change the lipid composition from free fatty acid-rich lipid to lipid mostly contained TG. Since higher lipid content was obtained when the growth was very slow due to nitrogen starvation, compromising between lipid content and harvesting time should be taken in order to obtain higher values of both the lipid content and lipid productivity. As the growth was much enhanced by increasing CO 2 concentration, CO 2 concentration played an important role in the increase of lipid productivity. At low until moderate CO 2 concentration, the highest lipid productivity could be obtained during N depletion which could surpassed the productivity during normal nutrition. At high-CO 2 concentration, harvesting at the end of linear phase during normal nutrition gave the highest lipid productivity. However, by reducing the incubation time of N depletion, higher lipid content as well as higher lipid productivity may still be achieved under this condition.

Study of increasing lipid production from fresh water microalgae Chlorella vulgaris

We have analyzed the genetic profiles of dinoflagellate populations obtained from the Pacific coast sea anemone Anthopleura elegantissima (Brandt) at collection sites from Washington to California. Genetic differences within the symbiont populations of California anemones have been uncovered by restriction length polymorphism (RFLP) analysis of the small subunit (SSU) and large subunit (LSU) ribosomal RNA genes, and by denaturing gradient gel electrophoresis (DGGE) of the internal transcribed spacer region 2 (ITS 2). The existence of two Symbiodinium species is substantiated by sequence analysis of the variable regions V1, V2, and V3 of the SSUrDNA, which also establishes their phylogenetic relatedness to other members of the genus Symbiodinium. Anemones from Washington and Oregon harbor a single dinoflagellate species, for which we propose the name S. muscatinei sp. nov. At these northern locations, S. muscatinei either exists alone or co-occurs with the Chlorella-like green algal symbiont. Our results i...

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Biogeography of two species of Symbiodinium (Freudenthal) inhabiting the intertidal sea anemone Anthopleura elegantissima (Brandt).

We present a simple protocol for DNA isolation from climbing cacti, genera Hylocereus and Selenicereus. The abundant polysaccharides present in Hylocereus and Selenicereus species interfere with DNA isolation, and DNA extracts, rich in polysaccharides, are poor templates for amplification using polymerase chain reaction (PCR). We used roots as the source tissue due to the lower viscosity of the extracts relative to that of other tissues. The extraction and isolation procedure we devised consists of the following steps: (1) three washes of ground tissue with the extraction buffer to remove the polysaccharides; (2) extraction with high-salt (4 M NaCl) cetyltrimethylammonium bromide (CTAB) buffer to remove the remaining polysaccharides; (3) removal of RNA by RNase; (4) phenol:chloroform extraction to remove proteins; (5) chloroform extraction to remove remaining phenols. The yields ranged from 10 to 20 μg DNA/g fresh roots. DNA samples prepared by our method were consistently amplifiable in the RAPD reaction and gave reproducible profiles.

Modified CTAB Procedure for DNA Isolation from Epiphytic Cacti of the Genera Hylocereus and Selenicereus (Cactaceae)

One of the most fundamental and precarious stages in the life history of a marine benthic alga is the colonization of a new substratum. For the majority of algae this is achieved by the formation and detachment of different types of highly specialized reproductive propagules which are then dispersed via the pelagic zone. This paper reviews the process of “settlement”, “attachment” and “establishment” of these propagules. Particular attention is given to the influence of substratum surface properties, both physical and chemical, on these processes, along with aspects of the derivation, chemical composition, mechanisms of release and mode of action of the various adhesives secreted by the attaching spores and germlings.

https://www.tandfonline.com/doi/pdf/10.1080/00071619200650281

The settlement, attachment and establishment of marine algal spores

Despite half a century of research, the biology of dinoflagellates remains enigmatic: they defy many functional and genetic traits attributed to typical eukaryotic cells. Genomic approaches to study dinoflagellates are often stymied due to their large, multi-gigabase genomes. Members of the genus Symbiodinium are photosynthetic endosymbionts of stony corals that provide the foundation of coral reef ecosystems. Their smaller genome sizes provide an opportunity to interrogate evolution and functionality of dinoflagellate genomes and endosymbiosis. We sequenced the genome of the ancestral Symbiodinium microadriaticum and compared it to the genomes of the more derived Symbiodinium minutum and Symbiodinium kawagutii and eukaryote model systems as well as transcriptomes from other dinoflagellates. Comparative analyses of genome and transcriptome protein sets show that all dinoflagellates, not only Symbiodinium, possess significantly more transmembrane transporters involved in the exchange of amino acids, lipids, and glycerol than other eukaryotes. Importantly, we find that only Symbiodinium harbor an extensive transporter repertoire associated with the provisioning of carbon and nitrogen. Analyses of these transporters show species-specific expansions, which provides a genomic basis to explain differential compatibilities to an array of hosts and environments, and highlights the putative importance of gene duplications as an evolutionary mechanism in dinoflagellates and Symbiodinium.

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Genomes of coral dinoflagellate symbionts highlight evolutionary adaptations conducive to a symbiotic lifestyle

Four areas containing different cell morphologies were mapped on Porphyra blades and five different cell types (i.e. tapered with long extensions, large and vacuolated, vegetative and dividing, and reproductive: males and females) were identified in them. Tissues from these areas were dissociated, and protoplasts and single cells were isolated from the dissociated tissue of each distinct region. Regeneration rates of the isolated cells and protoplasts (isolates) varied depending on their morphological type. Regeneration rates were lowest in cultured isolates from the area just above the holdfast (ca. 1 %) and increased gradually to over 80% in isolates from areas of vegetative and reproductive regions away from the holdfast. Four distinct morphological patterns were observed among the regenerating plants. Cells isolated from vegetative areas developed into leafy plants while in liquid culture, and into calli when grown on solid medium. Isolates from reproductive areas developed into either a long thin or short thick filamentous plant. Those from ripe patches of carposporangia developed into thin conchocelis filaments, while isolates from non-differentiated cells bordering the ripe reproductive patches developed into thick filaments resembling the morphology of conchosporangial branches. The blade of Porphyra appears simple as it consists of a single cell layer; however, it is complex both morphologically and physiologically.

Developmental studies in porphyra. i. blade differentiation in porphyra perforata as expressed by morphology, enzymatic digestion, and protoplast regeneration1

A rapid and economical method of DNA extraction from a red seaweedPorphyra perforata J. Agardh has been developed by the use of lithium chloride. This paper describes the optimization of extraction conditions. Heat treatment of tissues in a solution (0.8 M LiCl, 0.6% Sarkosyl, 10 mM EDTA, 0.2% PVPP, 5% s-mercaptoethanol, pH 9.0) at 55 °C for 10 min extracts DNA that is of sufficient quality to be used as a template for PCR amplification. Total DNA yield was approximately 30 to 50μg g−1 t of partially dried tissue. Total RNA yield was approximately 400μg g−1 of partially dried tissue. Carbohydrate was contained as approximately 40 to 90 mM (expressed as glucose equivalents) from 1 g tissue, and protein contamination calculated as the O.D. 260/280 ratio was in the range of 1.4 to 1.7. The DNA was characterized by high molecular weight larger than 50 kb.

DNA extraction conditions from Porphyra perforata using LiCl

Lithium chloride facilitates the softening of cell walls resulting in a simple, quick (2 h) method for DNA extraction from the red seaweed Porphyra perforata J. Agardh. A 5-min treatment of tissues in Lid at 55°C extracts DNA that is relatively free of the viscous polysaccharides and proteins that are usually coextracted in large amounts from cell walls and cytoplasm. This protocol does not require grinding of tissues, hydroxyapatite binding, cetyl trimethyl ammonium bromide treatments, enzymatic treatments, phenol extraction, or CsCl-gradient centrifugation. The resulting DNA is of sufficient quality to be used as a template for polymerase chain reaction amplification.

Lithium chloride extraction of dna from the seaweed porphyra perforata (rhodophyta)1

Protoplasts isolated from thalli of four Porphyra species regenerated successfully into differentiated plantlets. The efficiency of protoplast isolation and the developmental patterns of the regenerating protoplasts depended on the type of tissues from which they were isolated. However, culture conditions greatly influenced the patterns of development at the cellular and organismal levels. Sorbitol, nitrogen, and agar concentration in the medium controlled rates of cell division, thickening of cell walls, development of rhizoids, and formation of calluses or differentiated blades. Agitation disturbed the attachment of the protoplasts to a substrate. Cells in agitated cultures produced suspensions of single cells and non-polarized small calluses. Calluses which developed from protoplasts survived in storage for over two years. The stored calluses, and cells and protoplasts that were isolated from them, were subcultured successfully. We forsee extensive use of Porphyra cell suspensions for strain selection and vegetative propagation of cultivars. This technology, which makes vegetative cloning of selected Porphyra plants possible, may eliminate the need for cultivation and storage of the conchocelis phase. Protoplasts are also being used as tools for studies in genetic engineering of these commercial species.

Developmental studies in porphyra (rhodophyceae). iii. effect of culture conditions on wall regeneration and differentiation of protoplasts1

The marine amoeba Trichosphaerium Am-I-7 was used as a tool for preparing unialgal axenic cultures of nondigestible Symbiodinium and Porphyridium species. The resistance of these unicellular algae to the amoebal digestive enzymes, and the differential digestion of bacteria, protozoans, and other algae, resulted in cleansed cells of Symbiodinium and Porphyridium that remained in the amoebal food vacuoles. During multiple fission, the amoeba evacuated its food vacuoles and released the trapped and intact algae, which were then successfully cultured. This method of cleaning was especially useful with algal species that were sensitive to antibiotics or other germicidal agents.

Miriam Polne-Fuller

Papers

Developmental studies in porphyra. i. blade differentiation in porphyra perforata as expressed by morphology, enzymatic digestion, and protoplast regeneration1

DNA extraction conditions from Porphyra perforata using LiCl

Lithium chloride extraction of dna from the seaweed porphyra perforata (rhodophyta)1

Developmental studies in porphyra (rhodophyceae). iii. effect of culture conditions on wall regeneration and differentiation of protoplasts1

A novel technique for preparation of axenic cultures of symbiodinium (pyrrophyta) through selective digestion by amoebae1