Maeve Kelly

Biogas from Macroalgae: is it time to revisit the idea?

The economic and environmental viability of dedicated terrestrial energy crops is in doubt. The production of large scale biomass (macroalgae) for biofuels in the marine environment was first tested in the late 1960’s. The culture attempts failed due to the engineering challenges of farming offshore. However the energy conversion via anaerobic digestion was successful as the biochemical composition of macroalgae makes it an ideal feedstock. The technology for the mass production of macroalgae has developed principally in China and Asia over the last 50 years to such a degree that it is now the single largest product of aquaculture. There has also been significant technology transfer and macroalgal cultivation is now well tried and tested in Europe and America. The inherent advantage of production of biofuel feedstock in the marine environment is that it does not compete with food production for land or fresh water. Here we revisit the idea of the large scale cultivation of macroalgae at sea for subsequent anaerobic digestion to produce biogas as a source of renewable energy, using a European case study as an example.

Fucans, but Not Fucomannoglucuronans, Determine the Biological Activities of Sulfated Polysaccharides from Laminaria saccharina Brown Seaweed

Sulfated polysaccharides from Laminaria saccharina (new name: Saccharina latissima) brown seaweed show promising activity for the treatment of inflammation, thrombosis, and cancer; yet the molecular mechanisms underlying these properties remain poorly understood. The aim of this work was to characterize, using in vitro and in vivo strategies, the anti-inflammatory, anti-coagulant, anti-angiogenic, and anti-tumor activities of two main sulfated polysaccharide fractions obtained from L. saccharina: a) L.s.-1.0 fraction mainly consisting of O-sulfated mannoglucuronofucans and b) L.s.-1.25 fraction mainly composed of sulfated fucans. Both fractions inhibited leukocyte recruitment in a model of inflammation in rats, although L.s.-1.25 appeared to be more active than L.s.-1.0. Also, these fractions inhibited neutrophil adhesion to platelets under flow. Only fraction L.s.-1.25, but not L.s.-1.0, displayed anticoagulant activity as measured by the activated partial thromboplastin time. Investigation of these fractions in angiogenesis settings revealed that only L.s.-1.25 strongly inhibited fetal bovine serum (FBS) induced in vitro tubulogenesis. This effect correlated with a reduction in plasminogen activator inhibitor-1 (PAI-1) levels in L.s.-1.25-treated endothelial cells. Furthermore, only parent sulfated polysaccharides from L. saccharina (L.s.-P) and its fraction L.s.-1.25 were powerful inhibitors of basic fibroblast growth factor (bFGF) induced pathways. Consistently, the L.s.-1.25 fraction as well as L.s.-P successfully interfered with fibroblast binding to human bFGF. The incorporation of L.s.-P or L.s.-1.25, but not L.s.-1.0 into Matrigel plugs containing melanoma cells induced a significant reduction in hemoglobin content as well in the frequency of tumor-associated blood vessels. Moreover, i.p. administrations of L.s.-1.25, as well as L.s.-P, but not L.s.-1.0, resulted in a significant reduction of tumor growth when inoculated into syngeneic mice. Finally, L.s.-1.25 markedly inhibited breast cancer cell adhesion to human platelet-coated surfaces. Thus, sulfated fucans are mainly responsible for the anti-inflammatory, anticoagulant, antiangiogenic, and antitumor activities of sulfated polysaccharides from L. saccharina brown seaweed.

Fatty acid compositions of gonadal material and diets of the sea urchin, Psammechinus miliaris: trophic and nutritional implications

The fatty acid compositions of gonadal material was examined for the sea urchin Psammechinus miliaris (Gmelin) held in aquaria and fed either salmon feed pellets or the macroalga, Laminaria saccharina for 18 months. Gonadal material was also examined from P. miliaris collected from four field sites, including commercial scallop lines encrusted with the mussel, Mytilus edulis, sea cages stocked with Atlantic salmon Salmo salar and two intertidal sea-loch sites, characterised by either a fine mud or a macroalgal substratum. The fatty acid compositions of known and potential dietary material was examined. The proportions of certain fatty acids in the gonads of P. miliaris were significantly affected by diet type and location. Docosahexaenoic acid (DHA) 22:6 n-3 was significantly higher in the gonads of the sea urchins fed salmon feed in aquaria and collected from the salmon cages and scallop lines than in the gonads of the sea urchins fed L. saccharina in aquaria and collected from the intertidal sea loch sites. The salmon feed and the mussel tissue also contained a high proportion of this fatty acid. Stearidonic acid 18:4 n-3 and arachidonic acid 20:4 n-6, however, were found in significantly higher proportions than DHA in the gonads of the sea urchins fed L. saccharina and collected from the two intertidal sea-loch sites. L. saccharina was also found to contain high proportions of stearidonic and arachidonic acid. The gonads of the sea urchins collected from the intertidal site, characterised by a mud substratum, and from the scallop lines were found to contain a lower 18:1 n-9/18:1 n-7 ratio and a higher proportion of branched and odd-chained fatty acids, signifying a high dietary bacterial input, than the sea urchins held in the aquaria and collected from the salmon cage. 20:2 and 22:2 non-methylene-interrupted dienoic fatty acids (NMIDs) were found in P. miliaris fed diets lacking these fatty acids suggesting de novo biosynthesis. These results, therefore, suggest that the proportions/ratios of certain fatty acids in the gonads of P. miliaris could be used to give an indication of the predominant diet type of this species in the wild.

Further studies on the composition and structure of a fucoidan preparation from the brown alga Saccharina latissima

The polysaccharide composition of a fucoidan preparation isolated from the brown alga Saccharina latissima (formerly Laminaria saccharina) was reinvestigated. The preparation was fractionated by anion-exchange chromatography, and the fractions obtained were analyzed by chemical methods combined with NMR spectroscopy. Several 2D procedures, including HSQC, HMQC-TOCSY, and HMQC-NOESY, were used to obtain reliable structural information from the complex spectra, and the signal assignments were additionally confirmed by comparison with the literature spectra of the related polysaccharides and synthetic oligosaccharides. In accordance with the previous data, the main polysaccharide component was shown to be a fucan sulfate containing a backbone of 3-linked alpha-l-fucopyranose residues sulfated at C-4 and/or at C-2 and branched at C-2 by single sulfated alpha-l-fucopyranose residues. In addition, three other types of sulfated polysaccharide molecules were detected in the total fucoidan preparation: (i) a fucogalactan having a backbone of 6-linked beta-d-galactopyranose residues branched mainly at C-4 and containing both terminal galactose and fucose residues; (ii) a fucoglucuronomannan having a backbone of alternating 4-linked beta-d-glucopyranosyluronic acid and 2-linked alpha-d-mannopyranose residues with alpha-l-fucopyranose residues as single branches at C-3 of alpha-d-Manp; and (iii) a fucoglucuronan having a backbone of 3-linked beta-d-glucopyranosyluronic acid residues with alpha-l-fucopyranose residues as single branches at C-4. Hence, even a single algal species may contain, at least in minor amounts, several sulfated polysaccharides differing in molecular structure. Partial resolution of these polysaccharides has been accomplished, but unambiguous evidence on their presence as separate entities was not obtained.

Morphology and survivorship of larval Psammechinus miliaris (Gmelin) (Echinodermata: Echinoidea) in response to varying food quantity and quality

Abstract Experiments were conducted with the aim of defining the optimal culture conditions for Psammechinus miliaris larvae. Larval response to varying food rations of the microalgae, Pleurocrysis elongata, was assessed by recording the morphological parameters of developing larvae. Larvae on a high ration (4000 cells ml−1) showed an extreme reduction in postoral arm length and were unable to maintain their position in the water column. Larvae fed an optimal ration (1500–4000 cells ml−1 according to developmental stage) displayed a more typical morphology, whereas larvae fed a low ration (500 cells ml−1) failed to develop to metamorphosis. Survivorship of the larvae to metamorphosis was at best 61%. Larval response to various diet types was measured both in terms of the larval morphology, survivorship during metamorphosis and growth over the post-larval period. The microalgae, Dunaliella tertiolecta, produced more morphologically typical larvae and gave better results in terms of survivorship at metamorphosis (65.8%) than Pl. carterae (48.2%). The resulting juveniles, measured at 10 days post-settlement, were also significantly larger when the larvae had been fed D. tertiolecta. Survivorship over the post-larval period was more consistent when larvae were provided with a substrate coated with a natural biofilm compared with a substrate coated with the microalgae, Tetraselmis suecica. The data suggest that it is possible to produce large numbers of juvenile P. miliaris using these methods.

Fucosylated chondroitin sulfates from the body wall of the sea cucumber Holothuria forskali. Conformation, selectin binding and biological activity

Background: An acidic polysaccharide, fCS, from the sea cucumber Holothuria forskali has a range of biological activities. Results: The conformation of fCS was determined, and resulting oligosaccharides were shown to retain desirable biological properties. Conclusion: The conformation of the fCS repeating unit underpins binding to L- and P-selectins. Significance: Exploitation of the fCS-selectin interaction may open new avenues for therapeutic intervention using fCS fragments or their mimetics. Fucosylated chondroitin sulfate (fCS) extracted from the sea cucumber Holothuria forskali is composed of the following repeating trisaccharide unit: →3)GalNAcβ4,6S(1→4) [FucαX(1→3)]GlcAβ(1→, where X stands for different sulfation patterns of fucose (X = 3,4S (46%), 2,4S (39%), and 4S (15%)). As revealed by NMR and molecular dynamics simulations, the fCS repeating unit adopts a conformation similar to that of the Lex blood group determinant, bringing several sulfate groups into close proximity and creating large negative patches distributed along the helical skeleton of the CS backbone. This may explain the high affinity of fCS oligosaccharides for L- and P-selectins as determined by microarray binding of fCS oligosaccharides prepared by Cu2+-catalyzed Fenton-type and photochemical depolymerization. No binding to E-selectin was observed. fCS poly- and oligosaccharides display low cytotoxicity in vitro, inhibit human neutrophil elastase activity, and inhibit the migration of neutrophils through an endothelial cell layer in vitro. Although the polysaccharide showed some anti-coagulant activity, small oligosaccharide fCS fragments had much reduced anticoagulant properties, with activity mainly via heparin cofactor II. The fCS polysaccharides showed prekallikrein activation comparable with dextran sulfate, whereas the fCS oligosaccharides caused almost no effect. The H. forskali fCS oligosaccharides were also tested in a mouse peritoneal inflammation model, where they caused a reduction in neutrophil infiltration. Overall, the data presented support the action of fCS as an inhibitor of selectin interactions, which play vital roles in inflammation and metastasis progression. Future studies of fCS-selectin interaction using fCS fragments or their mimetics may open new avenues for therapeutic intervention.

Environmental parameters controlling gametogenesis in the echinoid Psammechinus miliaris

Abstract The role of photoperiod and temperature as stimuli for the onset of gametogenesis in the echinoid Psammechinus miliaris was examined, and the resulting effect on energy allocation between gonadal and somatic growth was evaluated. The hypothesis tested was that P. miliaris is a ‘lengthening day’ species and that photoperiod and not temperature is the primary stimulus for gametogenesis. Six-month-old, hatchery reared P. miliaris were maintained under different photoperiod and temperature regimes from December 1988 to June 1999. The photoperiod treatments were either ambient, i.e. days lengthening, or fixed at 7 h of light, equivalent to the shortest day, to remove the stimulus of increasing spring day length. The temperature treatments were either ambient (6–14 °C) or heated so that the seawater temperature never fell below 9 °C to remove the cue of increasing spring water temperatures. Gonad indices, alimentary indices, test diameter and test organic content were measured monthly. Once gonad development commenced, samples were examined using phase-contrast microscopy on wet-squash preparations and also preserved for histological examination of the reproductive state. There was no significant difference in the gonad index (GI) between urchins from the various treatments either during or at the end of the trial. By the end of the trial, the urchins in the fixed photoperiod regime were of significantly smaller test diameter than those in the temperature treatments. In early spring (May), the urchins in all treatments were of a similar reproductive status with developing gametes. However, subsequent samples (June) showed a significant proportion of the urchins of both sexes in the fixed photoperiod regime, and female urchins in the temperature-controlled treatment had not completed gametogenesis, remaining premature. Failure to complete gametogenesis did not result in a measurable increase in energy allocation to somatic growth. The data suggest that lengthening days are an important cue for the completion of gametogenesis in P. miliaris in both males and females and that experiencing low temperature is also an important cue for the completion of vitellogenesis.

Biosynthesis of eicosapentaenoic acid in the sea urchin Psammechinus miliaris.

The sea urchin Psammechinus miliaris (Gmelin) (Echinodermata: Echinoidea) was shown by using a deuterated tracer (D5−18∶3n−3) and quantitation by negative chemical ionization gas chromatography-mass spectrometry to convert 18∶3n−3 to 20∶5n−3. The rate of conversion was very slow, corresponding to 0.09 μg/g tissue/mg 18∶3n−3 eaten over 14 d. Deuterated arachidonic acid (D8−20∶4n−6) was also included in the diet to give a measure of the relative amounts of diet eaten by the different animals. The recovery of this fatty acid in tissue lipids was 33.7% compared with only 0.95% recovery of D5−18∶3n−3 and its anabolites, indicating that the majority of the D5-tracer was catabolized. Considerable elongation of D5−18∶3n−3 into 20∶3n−3 and a trace of 22∶3n−3 was found, and these were accompanied by minor amounts of the intermediates 18∶4n−3 and 20∶4n−3. No deuterated 22∶6n−3 was found.

The reproductive cycle of the sea urchin Psammechinus miliaris (Echinodermata: Echinoidea) in a Scottish sea loch

The sea urchin Psammechinus miliaris was sampled at monthly intervals from two replicate sites at contrasting locations (littoral and subtidal habitats) on the west coast of Scotland. Samples were collected from November 1995 to October 1997. A study of gonad development showed a clearly defined annual cycle of gametogenesis with a single spawning period. Gonad indices (GIs) varied between locations, between sites at the same location and between study years. Gonad indices were maximal prior to the onset of the spawning period in June and July. Ripe gametes were shed by dissected urchins from June to September in 1996 and June to August in 1997 coinciding with the time of year when the gonad colour was best in terms of what is desired in the market place. Over winter the GIs were very low and gonads became very dark in colour. Higher summer GIs in the urchins from one littoral site suggested that the urchins there had a seasonal influx of a more nutritious or more abundant food resource. Histological determination of the reproductive state showed that for these food-limited populations high GIs do equate with the spawning period. The gametogenic cycle fits the six stage pattern described for other echinoid species. In females oocytes of various developmental stages were present throughout the annual cycle. Recruitment to the littoral location was monitored over two seasons by quantifying the number of urchins with a test diameter < 5 mm appearing in the population. Recruitment appeared variable between sites and between seasons suggesting that the collection of juveniles resulting from natural settlement may not be a reliable alternative to hatchery produced spat. Although P miliaris has commercial potential when in cultivation, this study provided no evidence of an opportunity for a commercial fishery.

The effect of diet type and quantity on the development of common sea urchin larvae Echinus esculentus

Abstract Several species of sea urchins are now being cultivated for commercial purposes and with the continued increased demand for sea urchin gonads as a food product, new species are being assessed for their aquaculture and market potential. This study focussed on establishing protocols for the production of common sea urchin Echinus esculentus larvae and juveniles to assess its potential as an echinoculture species. Two trials were carried out, the first trial evaluated the influence of three microalgal diets (D= Dunaliella tertiolecta only, mixed D/P= D. tertiolecta plus Phaeodactylum tricornutum and P= P. tricornutum only) on larval morphology. Larval length, width, post-oral arm length and rudiment length were significantly effected by diet. Diets D and D/P prompted more rapid metamorphosis. In the second trial, the effects of different rations of D. tertiolecta were tested. The food ration, standard ration (SR; 1000, 3000, and 5000 cells ml −1 ) and high ration (HR; 3000, 9000, and 15,000 cells ml −1 ) were increased as the larvae acquired the 3rd and 4th pair of larval arms. Larvae fed the SR were significantly larger (longer and wider) and had significantly longer rudiments than those in the HR treatment. The number of larvae metamorphosing and settling onto substrates was significantly higher in treatment SR compared to HR. Optimising the larval diet shortened the larval stage from 21–23 days in the first trial to 16 days in the second trial. The maximum percentage of metamorphosing individuals which survived to post-larvae or juveniles (10 days after they were first judged competent to settle) was 46.6%, suggesting E. esculentus is a viable aquaculture candidate.