Patricia I. Leonardi

Lipid analysis in Haematococcus pluvialis to assess its potential use as a biodiesel feedstock

The lipid content and composition of Haematococcuspluvialis exposed to stress conditions were analyzed to assess the potential of this microalga as a biodiesel feedstock. The total lipid content of control cells was 15.61% dw, whereas that of cells exposed to continuous high light intensity with nitrogen-sufficient medium (A-stress condition) or under continuous high light intensity with nitrogen-deprivation medium (B-stress condition) was 34.85% dw and 32.99% dw, respectively. The fatty acid profile was similar under all conditions and indicated that the main components were palmitic, stearic, oleic, linoleic, linolenic and linolelaidic acids. The neutral lipid fraction increased about 2-fold under both stress conditions. The percentage of saturated fatty acids in the neutral lipid fraction was 30.36% and 29.62% in cultures grown under A-stress and B-stress, respectively, and 27.81% under control conditions. The monounsaturated fatty acid content was not significantly different in control and A-stress cultures (20.07% and 19.91%, respectively), but was 18.96% under B-stress. The content of polyunsaturated fatty acids was 47.23% under B-stress and 43.15% under A-stress. Growth-rate was higher under A-stress compared to B-stress. This is the first study of H.pluvialis that provides a detailed characterization of its lipid content in relation to bioenergy. The results indicate the potential of this microalga as a biodiesel feedstock; however, culture conditions still have to be improved in order to achieve an adequate energy balance in mass culture.

Diversity, phenomenology and epidemiology of epiphytism in farmed Gracilaria chilensis (Rhodophyta) in northern Chile

This study identified the most common epiphytes infecting the algal host Gracilaria chilensis on a farm in northern Chile. Simultaneously, the types of host–epiphyte interfaces were characterized and their relative abundance and temporal variability were monitored. Five types of anatomical relationships were detected. Infection type I included the epiphytes weakly attached to the surface of the host and not associated with damage of host tissues (i.e. Hincksia mitchelliae, H. granulosa and Ectocarpus acutus). Infection type II included those epiphytes strongly attached to the surface of the host but not associated with any host tissue damage (i.e. Acrochaetium sp., Antithamnionella sp. and Colpomenia sinuosa). Infection type III included all the epiphytes that penetrated the outer layer of the host wall without damaging its cortical cells (i.e. Xenococcus sp. and Sahlingia subintegra). Infection type IV included epiphytes penetrating deep into the host cell wall, disorganizing the cortical tissue (i.e. Ulva lactuca and Acrosorium corallinarum). Infection type V included epiphytes that penetrated deeply into the cortex, reached the medullary tissue and caused destruction of the hosts cells in the area around the infection (i.e. Ceramium rubrum and Polysiphonia harveyi). Prevalence varied with time and with infection type, with types II and III reaching up to 80% and 90% of the thalli respectively. Severity of epiphyte infection was similar to the distribution of infection prevalence, with crustose epiphytes colonizing up to 80% of the host surface.

Neochloris oleoabundans grown in enriched natural seawater for biodiesel feedstock: evaluation of its growth and biochemical composition.

The freshwater microalga Neochloris oleoabundans was used to study algal lipid production in enriched natural seawater, in order to assess its suitability as biodiesel feedstock. Optimal and nitrogen-stress (N-stress) conditions were analyzed. Under optimal conditions, the strains growth rate was 0.73 div day(-1) and the biomass concentration was 1.5 g L(-1), while it had a maximum lipid yield under N-stress conditions (lipid content: 26% of dry weigh and lipid productivity: 56 mg L(-1) day(-1)). Lipid accumulation was mainly due to a significant increase of triacylglycerol content. Neutral lipids were characterized by a dominance of monounsaturated fatty acids and displayed a fatty acid profile that is suitable for biodiesel. This work offers an interesting alternative for sustainable microalgal oil synthesis for biodiesel production without using freshwater resources. However, further studies are necessary in order to optimize the lipid productivities required for commercial biodiesel production.

Ultrastructure of the cyst wall of Haematococcus pluvialis (Chlorophyceae): wall development and behaviour during cyst germination

M.C. Damiani, P.I. Leonardi, O.I. Pieroni and E.J. Cáceres. 2006. Ultrastructure of the cyst wall of Haematococcus pluvialis (Chlorophyceae): wall development and behaviour during cyst germination. Phycologia 45: 616–623. DOI: 10.2216/05-27.1 The cell wall structure and behaviour of cysts of Argentinian strains of Haematococcus pluvialis were studied during cyst germination and zooid release. The cyst wall is composed of an outer primary wall, a trilaminar sheath, a secondary wall, and a tertiary wall. The primary wall disintegrates as the cyst ages. During germination the trilaminar sheath breaks down, carrying the adjacent secondary wall with it and the extensible tertiary wall accompanies the cyst in its size increase and shape changes. Zooid release subsequently follows as a result of the apical breakage of this third wall. Although the secondary and tertiary walls exhibit the same conformation in mature cysts, the arrangement of their components during their formation is different. This could be the reason why the behaviour of the two walls is different during germination. The chemical characterization carried out in the present research showed that the external trilaminar sheath of the wall is formed by algaenan, while fluorescence labelling suggested the presence of mannose and cellulose in the secondary and tertiary walls. Because of the physical difficulty of breaking cysts during astaxanthin extraction, cyst germination may provide a natural, alternative wall-breaking mechanism.

Agar from Gracilaria gracilis (Gracilariales, Rhodophyta) of the Patagonic coast of Argentina--content, structure and physical properties.

Milled summer thalli of Gracilaria gracilis from Argentina were sequentially extracted with water at room temperature (RTW1-3), 70 degrees C (W701-3) and 90 degrees C (W901-2). Both W701 and W901 consisted of high molecular weight polysaccharides (ca. 540,000Da), but polydispersity was higher for the major product W701 (yield, 72% of the recovered). Structural analyzes by methylation and (13)C NMR spectroscopy revealed that W701 was mainly agarose. Alkaline treatment, together with structural analyzes, indicated a negligible proportion of precursor l-galactose 6-sulfate residues in this product, while they were clearly detected in the (13)C NMR spectra of RTW2-3. The presence of floridean starch in W901 had an antagonistic effect on its gel strength, which resulted nearly three times lower than that of fraction W701. Ultrastructural observation by transmission electron microscopy showed that, after extraction with hot water, a partial loss of cell wall stratification and disorganization of the cuticle had occurred. Final cellular debris exhibited swelling in the microfibrillar component. After this first thorough study of the chemical composition and physical properties of the products of G. gracilis from Bahía Bustamante we conclude that a good quality agarose is obtained in high yield after extraction with water at 70 degrees C without the requirement of alkaline pretreatment, which usually produces degradation of the polysaccharide.

Dissection of two distinct defense-related responses to agar oligosaccharides in Gracilaria chilensis (Rhodophyta) and Gracilaria conferta (Rhodophyta)

The two agar‐producing red algae, Gracilaria chilensis C. J. Bird, McLachlan & E. C. Oliveira and Gracilaria conferta (Schousboe ex Montagne) Montagne, responded with hydrogen peroxide (H2O2) release when agar oligosaccharides were added to the medium. In G. conferta, a transient release was observed, followed by a refractory state of 6 h. This response was sensitive to chemical inhibitors of NADPH oxidase, protein kinases, protein phosphatases, and calcium translocation in the cell, whereas it was insensitive to inhibitors of metalloenzymes. Transmission electron microscopic observations of the H2O2‐dependent formation of cerium peroxide from cerium chloride indicated oxygen activation at the plasma membrane of G. conferta. A putative system, consisting of a receptor specific to agar oligosaccharides and a plasma membrane‐located NADPH oxidase, appears to be responsible for the release of H2O2 in G. conferta. Subcellular examination of G. chilensis showed that the H2O2 release was located in the cell wall. It was sensitive to inhibitors of metalloenzymes and flavoenzymes, and no refractory state was observed. The release was correlated with accumulation of an aldehyde in the algal medium, suggesting that an agar oligosaccharide oxidase is present in the apoplast of G. chilensis. The presence of this enzyme could also be demonstrated by polyacrylamide electrophoresis under nondenaturating conditions and proven to be variable. Cultivation of G. chilensis at 16 to 17°C resulted in significantly stronger expression of agar oligosaccharide oxidase than cultivation at 12°C, which indicates that the enzyme is used under conditions that generally favor microbial agar macerating activity.

Ultrastructure and X-ray microanalysis of Euglena gracilis (Euglenophyta) under chromium stress

I. Rocchetta, P.I. Leonardi, G.M. Amado Filho, M. del Carmen Ríos de Molina and V. Conforti. 2007. Ultrastructure and X-ray microanalysis of Euglena gracilis (Euglenophyta) under chromium stress. Phycologia 46: 300–306. DOI: 10.2216/06-49.1 The effect of chromium on the biology of Euglena gracilis was studied. The ultrastructural modifications caused by this metal and its location within the cell were analyzed by TEM and EDXA, respectively. The effects of chromium on protein, pigment, and lipid contents were also studied in order to evaluate the metabolic responses to metal exposure. Two strains of Euglena gracilis, UTEX 753 (from the Culture Collection of Texas University) and MAT (isolated from the Matanza River), were used in this research. Both were grown in photoauxotrophic and photoheterotrophic conditions and exposed to different metal concentrations. In all treated cells, increases in total protein and lipid contents, changes in chlorophyll amount, and alterations in fine structure were observed, especially with the higher concentration tested. In photosynthetic treated cells, assays showed chloroplast thylakoid disorganization, the presence of cytoplasm lipid globules, and several vacuoles with electron-dense inclusions and remnants of membranes inside. Nuclei presented lobulations, and eventually total fragmentation in some cells treated with the highest chromium concentration was seen, suggesting that chromium cytotoxicity leads to cellular death. The EDXA spectrum showed well-defined Cr and S peaks in the vacuoles containing electron-dense inclusions and remnants of membranes from autotrophic MAT samples. These results indicate that the different defense mechanisms against chromium depend on strain type and culture conditions. The S peak detected in MAT would suggest that sulfur-rich proteins groups play an important role in the detoxification system inducing metal-complex accumulation into vacuoles.

Feedstocks for Second-Generation Biodiesel: Microalgae’s Biology and Oil Composition

The solar energy is an inexhaustible source, while other energy reserves, like fossil and nuclear fuels, are limited in quantity and are depleted as years go by. Renewable energy is necessary to replace petroleum-derived fuels. The first generation biofuels, which are produced from oil seeds and crops, are a possible alternative, but they are limited in their capacity to provide all the energy demanded in the world. Therefore, new sources for the sustainable production of renewable energy are being looked for. This concern has promoted the keen interest in developing second generation biofuels, which are produced from other feedstocks, such as microalgal oils (Schenk et al., 2008; Mata et al., 2010). Some microalgal species are capable of producing biomass yields containing high percentages of oils (Aaronson et. al., 1980). In addition, microalgal systems can use low value natural resources, such as arid lands and saline water, thus offering the potential for large biomass energy contributions without competing for prime agricultural or forest land. Most microalgae grow photoautotrophically by using solar energy and mainly carbon dioxide as carbon source. Alternatively, some species can grow heterotrophically or mixotrophically using organic compounds as energy and carbon sources (Kitano et al., 1997; Hu & Gao, 2003; Xu et al., 2006; Liang et al., 2009). Some microalgae are called oleaginous because they synthesize and accumulate substantial amounts of neutral lipids, mainly as triacylglycerol (TAG), under diverse stress conditions (Bigogno et al., 2002; Hu et al., 2008; Gardner et al., 2010; Damiani et al., 2010). TAGs as storage lipids are the best substrate to produce biodiesel (Xu et al., 2006; Schenk et al., 2008). This biofuel is obtained by transesterification of oil or fat with a monohydric alcohol, yielding the corresponding mono-alkyl esters (Knothe, 2005). Since transesterification maintains the relative ratio of fatty acids present in the feedstock (Costa Neto et al., 2000), the profile of the fatty acid methyl esters is a reflection of the feedstock fatty-acid composition (Lang et al. 2001; Ferrari et al. 2005). Biodiesel production from microalgae is technically feasible (Xu et al., 2006; Patil et al., 2008; Francisco et al., 2010), but for an effective use of this renewable resource as biofuel, it is necessary to be able to modify microalgal growth conditions in order to obtain high biomass productivity and the desired lipid quantity and quality. Those interested in

CELL WALL CARBOHYDRATE EPITOPES IN THE GREEN ALGA OEDOGONIUM BHARUCHAE F. MINOR (OEDOGONIALES, CHLOROPHYTA)1

Cell wall changes in vegetative and suffultory cells (SCs) and in oogonial structures from Oedogonium bharuchae N. D. Kamat f. minor Vélez were characterized using monoclonal antibodies against several carbohydrate epitopes. Vegetative cells and SCs develop only a primary cell wall (PCW), whereas mature oogonial cells secrete a second wall, the oogonium cell wall (OCW). Based on histochemical and immunolabeling results, (1→4)‐β‐glucans in the form of crystalline cellulose together with a variable degree of Me‐esterified homogalacturonans (HGs) and hydroxyproline‐rich glycoprotein (HRGP) epitopes were detected in the PCW. The OCW showed arabinosides of the extensin type and low levels of arabinogalactan‐protein (AGP) glycans but lacked cellulose, at least in its crystalline form. Surprisingly, strong colabeling in the cytoplasm of mature oogonia cells with three different antibodies (LM‐5, LM‐6, and CCRC‐M2) was found, suggesting the presence of rhamnogalacturonan I (RG‐I)–like structures. Our results are discussed relating the possible functions of these cell wall epitopes with polysaccharides and O‐glycoproteins during oogonium differentiation. This study represents the first attempt to characterize these two types of cell walls in O. bharuchae, comparing their similarities and differences with those from other green algae and land plants. This work represents a contribution to the understanding of how cell walls have evolved from simple few‐celled to complex multicelled organisms.

Pseudulvella Americana belongs to the order Chaetopeltidales (class chlorophyceae), evidence from ultrastructure and SSU rDNA sequence data

The genus Pseudulvella Wille 1909 includes epiphytic, freshwater, or marine disk‐shaped green microalgae that form quadriflagellate zoospores. No ultrastructural or molecular studies have been conducted on the genus, and its evolutionary relationships remain unclear. The purpose of the present study is to describe the life history, ultrastructural features, and phylogenetic affiliations of Pseudulvella americana (Snow) Wille, the type species of the genus. Thalli of this microalga were prostrate and composed of radiating branched filaments that coalesced to form a disk. Vegetative cells had a pyrenoid encircled by starch plates and traversed by one or two convoluted cytoplasmic channels. They had well‐defined cell walls without plasmodesmata. Asexual reproduction was by means of tetraflagellate zoospores formed in numbers of two to eight from central cells of the thallus. The flagellar apparatus of zoospores was cruciate, with four basal bodies and four microtubular roots. The paired basal bodies lay directly opposite (DO) one another. The microtubular root system had a 5‐2‐5‐2 alternation pattern, where the “s” roots contained five microtubules in a four‐over‐one configuration. A tetralobate nonstriated distal fiber connected all four basal bodies. A wedge‐shaped proximal sheath subtended each of the basal bodies. The ultrastructural features of the zoospores were those of members of the order Chaetopeltidales. Phylogenetic analyses based on SSU rDNA placed P. americana sister to Chaetopeltis orbicularis in a well‐supported Chaetopeltidales clade. Such a combination of features confirmed that this alga is a member of the order Chaetopeltidales.