Development of koji by culturing Aspergillus oryzae on nori (Pyropia yezoensis).

Koji is a traditional fermentation culture medium, based on Aspergillus oryzae, which is commonly used in the manufacture process of Japanese fermented products such as soy sauce, miso, and sake, and promote enzymatic degradation. Koji is usually prepared by culturing a mold on cereals such as wheat flour, soybean, or rice, but that cultured on seaweeds has not been developed yet. This study prepared the koji by culturing A. oryzae on seaweed nori (dried piece of Pyropia yezoensis), and, then, characterized on this nori koji. The nori koji contained 0.85 µg N-acetylglucosamine, estimated as 6.1 µg mold cells, per gram dry matter and showed various kind of enzymatic activities in glycosidase, protease, and phosphatase as well as traditional soy sauce koji and rice koji. The suitability of these characteristics for degradation of nori was tested on nori sauce culture with and without the addition of the nori koji. After 167 days of culture, the fermentation tank with the nori koji showed over 74% recovery of supernatant while that without the nori koji had less than 57% recovery. The supernatant of culture mashes contained more than two times larger quantity of total nitrogen compounds in nori koji test group against control group. The present study prepared koji on seaweed nori for the first time and demonstrated its advantages to shorten the culture period and increase taste value in nori sauce manufacture. Development of seaweed koji enables a method to prepare cereal allergen free fermented sauces from seaweeds.

Characteristics of hydrogen-producing enrichment cultures from marine sediment using macroalgae Laminaria japonica as a feedstock.

This study aimed to investigate the characteristics of hydrogen production by mixed cultures using Laminaria japonica hydrolysates. The hydrolysates of L. japonica were prepared by pretreatment methods, including heat (100°C or 121°C) and acid (HCl or H2SO4) pretreatments. The mixed cultures could produce hydrogen using L. japonica as a substrate, with the highest cumulative hydrogen production of 825 ± 14 mL/L from HCl-pretreated L. japonica. High-throughput sequencing of the 16S rRNA gene revealed that the microbial community in the hydrolysate of HCl-pretreated L. japonica was the most diverse among all the samples, with a Shannon diversity index of 5.253. The mixed culture from HCl-pretreated L. japonica and those from heat-pretreated (100°C and 121°C) L. japonica occupied different regions in a principal component analysis (PCA) plot. The dominant population in the hydrolysate of HCl-pretreated L. japonica was represented by hydrogen-producing bacteria, Clostridium spp. and Bacillus spp. The results suggested that L. japonica was an optimal feedstock for hydrogen production. The acid (HCl) pretreatment method could effectively enhance the hydrogen production from L. japonica.

Bending strategies of convergently evolved, articulated coralline algae.

The evolution of uncalcified genicula in upright calcified corallines has occurred at least three times independently, resulting in articulated corallines within Corallinoideae, Lithophylloideae, and Metagoniolithoideae. Genicula confer flexibility to otherwise rigid thalli, and the localization of bending at discrete intervals amplifies bending stress in genicular tissue. Genicular morphology must, therefore, be balanced between maintaining flexibility while mitigating or resisting stress. Genicula in the three articulated lineages differ in both cellular construction and development, which may result in different constraints on morphology. By studying the interaction between flexibility and morphological variation in multiple species, we investigate whether representatives of convergently evolving clades follow similar strategies to generate mechanically successful articulated fronds. By using computational models to explore different bending strategies, we show that there are multiple ways to generate flexibility in upright corallines but not all morphological strategies are mechanically equivalent. Corallinoids have many joints, lithophylloids have pliant joints, and metagoniolithoids have longer joints-while these strategies can lead to comparable thallus flexibility, they also lead to different levels of stress amplification in bending. Moreover, genicula at greatest risk of stress amplification are typically the strongest, universally mitigating the trade-off between flexibility and stress reduction.

The brown seaweed Sargassum cymosum: changes in metabolism and cellular organization after long-term exposure to cadmium.

Sargassum cymosum was exposed to cadmium (Cd) to determine any physiological and ultrastructural effects. To accomplish this, S. cymosum samples were cultivated under photosynthetic active radiation (PAR) and Cd (0, 0.1, 0.2, 0.4 and 0.8 mg L-1) during 7 and 14 days in laboratory-controlled conditions (0 mg L-1 Cd at both exposure times as control). Seaweeds had high retention capacity (over 90 %) for both exposure times. Growth rates showed significant increases by 14 days, especially for 0.1 and 0.4 mg L-1 Cd. Photosynthetic parameters were unaffected by Cd treatments. Chlorophyll contents were present in higher concentrations for all Cd treatments compared to respective control. Carotenoid profile showed significant differences in total composition and proportion of fucoxanthin and ß-carotene, and no lutein was detected at 14 days. Phenolic and flavonoid compounds showed major accumulation at 14 days. Transmission electron microscopy (TEM) analyses presented major alterations in Cd-treated samples, when compared with respective control, in particular disorganization of cell wall fibrils. When compared to respective control samples, multivariate analyses showed disparate and complex interactions among metabolites in Cd-exposed seaweeds, giving evidence of physiological defence response. Thus, it can be concluded that Cd is a stressor for S. cymosum, resulting in physiological and structural alterations related to defence mechanisms against oxidative stress and toxicological effects resulting from long-term metal exposure. However, in the present paper, some observed changes also appear to result from acclimation mechanisms under lower concentration of Cd relative to the tolerance of S. cymosum to experimental conditions.

A microfluidic chip for studying the reproduction of Enteromorpha prolifera.

In recent years, green tides caused by water eutrophication, has brought serious environmental problems. Enteromorpha prolifera (E. prolifera), an opportunistic macroalgae, is one of the main source contributing to the formation of green tides. It has been estimated that the excessive growth of E. prolifera is closely related to various reproductive ways of germ cells which are at the micrometer scale. Here we report a microfluidic device named Germ Cell Capture Chip (GCChip) to investigate the E. prolifera reproductive mechanism. GCChip integrates the functions of algal growing, and the release, capture and selective culture of germ cells. Automatic separation and capture of germ cells on the chip allows to study germ cells' response to different stimuli. The novel device greatly facilitates long-term live-cell imaging at cellular resolution and implements the rapid and accurate exchange of growth medium without manual intervention. Results showed that the starting time of germ cell releases were earlier on the chip than that of traditional experiments with more concentrated breakout. Moreover, GCChip can be widely applied on the study of other algae. The study of algae growth process, including the elongation of somatic cell, the generation, and the release of reproductive cells, can all be improved by using this microfluidic platform.

Delimitating cryptic species in the Gracilaria domingensis complex (Gracilariaceae, Rhodophyta) using molecular and morphological data.

Species in the genus Gracilaria that display conspicuously flattened vegetative morphologies are a taxonomically challenging group of marine benthic red algae. This is a result of their species richness, morphological similarity, and broad phenotypic plasticity. Within this group, the Gracilaria domingensis complex is one of the most common, conspicuous, and morphologically variable species along the tropical western Atlantic Ocean. Previous research has identified that members of this complex belong to two distantly related clades. However, despite this increased phylogentic resolution, species delimitations within each of these clades remain unclear. Our study assessed the species diversity within this difficult complex using morphological and molecular data from three genetic markers (cox1, UPA, and rbcL). We additionally applied six single-marker species delimitation methods (SDM: ABGD, GMYCs, GMYCm, SPN, bPTP, and PTP) to rbcL, which were largely in agreement regarding species delimitation. These results, combined with our analysis of morphology, indicate that the G. domingensis complex includes seven distinct species, each of which are not all most closely related: G. cervicornis; a ressurected G. ferox; G. apiculata subsp. apiculata; a new species, Gracilaria baiana sp. nov.; G. intermedia subsp. intermedia; G. venezuelensis; and G. domingensis sensu stricto, which includes the later heterotypic synonym, G. yoneshigueana. Our study demonstrates the value of multipronged strategies, including the use of both molecular and morphological approaches, to decipher cryptic species of red algae.

Ploidy Distribution of the Harmful Bloom Forming Macroalgae Ulva spp. in Narragansett Bay, Rhode Island, USA, Using Flow Cytometry Methods.

Macroalgal blooms occur worldwide and have the potential to cause severe ecological and economic damage. Narragansett Bay, RI is a eutrophic system that experiences summer macroalgal blooms composed mostly of Ulva compressa and Ulva rigida, which have biphasic life cycles with separate haploid and diploid phases. In this study, we used flow cytometry to assess ploidy levels of U. compressa and U. rigida populations from five sites in Narragansett Bay, RI, USA, to assess the relative contribution of both phases to bloom formation. Both haploid gametophytes and diploid sporophytes were present for both species. Sites ranged from a relative overabundance of gametophytes to a relative overabundance of sporophytes, compared to the null model prediction of √2 gametophytes: 1 sporophyte. We found significant differences in cell area between ploidy levels for each species, with sporophyte cells significantly larger than gametophyte cells in U. compressa and U. rigida. We found no differences in relative growth rate between ploidy levels for each species. Our results indicate the presence of both phases of each of the two dominant bloom forming species throughout the bloom season, and represent one of the first studies of in situ Ulva life cycle dynamics.

Extraction and Enrichment of Protein from Red and Green Macroalgae.

Macroalgae, in particular red and green species, are gaining interest as protein-rich foods for human consumption and sources of proteinaceous biofunctional peptide ingredients. During protein extraction the starting raw material, the cell disruption method utilized and the reagents employed have a major effect on the yield of protein recovered. A method is described herein for extraction and semi-purification of food-grade aqueous and alkaline soluble proteins from red and green macroalgae. Dried milled macroalgae are disrupted by osmotic shock with subsequent removal of aqueous soluble proteins by centrifugation. Alkaline soluble proteins are removed following consecutive treatment of the resultant pellet with an alkaline solution. Aqueous and alkaline soluble proteins are then enriched from the crude extracts by isoelectric precipitation.

Comparación de las propiedades antioxidantes y contenido de polifenoles de extractos acuosos de las algas marinas Bryothamnion triquetrum y Halimeda opuntia / Comparation of antioxidants properties and polyphenols content of aqueous extract from seaweeds Bryothamnion triquetrum and Halimeda opuntia

Objetivos. Evaluar y comparar las propiedades antioxidantes mediante ensayos in vitro de extractos acuosos de las algas roja Bryothamnion triquetrum y verde Halimeda opuntia y su relación con el contenido de polifenoles. Material y Métodos. Se utilizaron las técnicas in vitro: DPPH, Capacidad reductora, Inhibición de la peroxidación lipídica e inhibición de la hemólisis inducida por AAPH. Resultados. B. triquetrum: DPPH; CI50=1,15 ± 0,06, capacidad reductora a concentración; 128 mg/mL, DO=2,798, inhibición de la peroxidación lipídica; CI50=5,09± 0,25 e inhibición de la hemólisis con 12 mg/ mL; 35 %. H. opuntia: DPPH; CI50=12,34 ± 0,30 mg/mL, capacidad reductora; DO=0,800, inhibición de la peroxidación lipídica; CI50=1,25± 0,31 mg/mL e inhibición de la hemólisis; 82%. Conclusiones. Los resultados evidencian que B triquetrum resulto mucho más eficiente en los ensayos de DPPH y Capacidad reductora mientras H opuntia resulta más eficiente en Inhibición de la peroxidación lipídica e inhibición de la hemólisis. Se discuten algunos aspectos acerca de sus posibles mecanismos de acción

Objective. To evaluate and compare the antioxidant activity displayed by seaweed H. opuntia and B. triquetrum using different experimental in vitro antioxidant assessment models. Material and Methods. The following techniques are utilized: DPPH, Reducing power, inhibition of lipid peroxidation and inhibition of haemolysis. Results. B. triquetrum: DPPH; IC =1.15 ± 0.06, Reducing power (concentration 128 mg/mL), OD=2.798, inhibition of lipid peroxidation; IC 50 =5.09± 0.25 and inhibition of haemolysis; with 12 mg/mL; 35 %. H. opuntia: DPPH; IC 50 50 =12.34 ± 0.30 mg/mL, reducing power; OD=0.800, inhibition of lipid peroxidation; IC 50 =1.25± 0.31 mg/mL, inhibition of lipid peroxidation; C =1.25± 0.31 mg/mL and inhibition of haemolysis; 82%. 50 Conclusion. It was demonstrated that B.triquetrum extract was more effective than H. opuntia in reducing power and DPPH assays while H.opuntia was more effective in inhibition of lipid peroxidation in rat brain homogenates and the inhibition of red blood cell (RBC) haemolysis induced by AAPH. We discuss some aspects about their possible mechanisms of action

Polysaccharides from the green seaweed Codium decorticatum. Structure and cell wall distribution.

The cell wall polysaccharides from Codium decorticatum and their assembly were studied and these results were compared with those obtained previously for this genus. The water soluble polysaccharides are: (i) Pyruvylated and sulfated 3- and 6-linked ß-D-galactans with sulfate mainly on C-4 and also on C-6. Pyruvate ketals are linked to O-3 and O-4 of terminal ß-D-galactose or O-4 and O-6 of 3-linked ß-D-galactose. (ii) Sulfated 3-linked ß-L-arabinans substituted on C-2 or C-2 and C-4 predominantly with sulfate, but also with single stubs of arabinose, and (iii) 4-linked ß-D-mannans with a low degree of sulfation on C-2. The whole polysaccharide system comprises 6.9% of sulfated polysaccharides and 32.9% of fibrillar polysaccharides, mostly insoluble mannans. By in situ localization it was possible to detect two similar fibrillar layers separated by a zone rich in charged polymers. Besides, arabinogalactan proteins co-localized with the fibrillar components.

Simple growth patterns can create complex trajectories for the ontogeny of constitutive chemical defences in seaweeds.

All of the theory and most of the data on the ecology and evolution of chemical defences derive from terrestrial plants, which have considerable capacity for internal movement of resources. In contrast, most macroalgae--seaweeds--have no or very limited capacity for resource translocation, meaning that trade-offs between growth and defence, for example, should be localised rather than systemic. This may change the predictions of chemical defence theories for seaweeds. We developed a model that mimicked the simple growth pattern of the red seaweed Asparagopsis armata which is composed of repeating clusters of somatic cells and cells which contain deterrent secondary chemicals (gland cells). To do this we created a distinct growth curve for the somatic cells and another for the gland cells using empirical data. The somatic growth function was linked to the growth function for defence via differential equations modelling, which effectively generated a trade-off between growth and defence as these neighbouring cells develop. By treating growth and defence as separate functions we were also able to model a trade-off in growth of 2-3% under most circumstances. However, we found contrasting evidence for this trade-off in the empirical relationships between growth and defence, depending on the light level under which the alga was cultured. After developing a model that incorporated both branching and cell division rates, we formally demonstrated that positive correlations between growth and defence are predicted in many circumstances and also that allocation costs, if they exist, will be constrained by the intrinsic growth patterns of the seaweed. Growth patterns could therefore explain contrasting evidence for cost of constitutive chemical defence in many studies, highlighting the need to consider the fundamental biology and ontogeny of organisms when assessing the allocation theories for defence.

Biomass, lipid and fatty acid production in large-scale cultures of the marine macroalga Derbesia tenuissima (Chlorophyta).

Biomass productivity was quantified for the marine macroalga Derbesia tenuissima cultivated outdoors at seven stocking densities from 0.25 to 8 g L(-1) for 5 weeks. Total lipids and fatty acid quantity and quality was measured from samples that were freeze-dried, dried by oven (75 °C), food dehydrator (60 °C), or outdoor in the sun (40 °C) or shade (38 °C). Stocking densities of 0.25 to 2 g L(-1) yielded the highest biomass productivities (>20 g dry weight m(-2) day(-1)) with no effect on total lipid quantity (11 %), or fatty acid quantity (5.3 %) or quality at any density tested. However, there was an interactive effect of stocking density and drying technique, with a decrease of up to 40 % in polyunsaturated fatty acids in sun-dried compared to freeze-dried biomass. Notably, while fatty acid and biomass productivity may be inseparable in macroalgae, cultivation conditions have a significant carryover effect in the post-harvest delivery of high-quality bio-oils.

Enzyme-assistant extraction (EAE) of bioactive components: a useful approach for recovery of industrially important metabolites from seaweeds: a review.

Over the years, the biological activities of seaweeds could have gained a considerable research interest because of their specific functional compounds, which may not be available in land plants. Thus, efforts at discovery of novel metabolites from seaweeds over the past years have yielded a considerable amount of new active compounds. In addition, studies about the extraction of active compounds from natural products have attracted special attention in the last recent years. Potent biologically active compounds of seaweeds have been demonstrated to play a significant role in prevention of certain degenerative diseases such as cancer, inflammation, arthritis, diabetes and hypertension. Therefore, seaweed derived active components, whose immense biochemical diversity looks like to become a rich source of novel chemical entities for the use as functional ingredients in many industrial applications such as functional foods, pharmaceuticals and cosmeceuticals. Thus, the interest in the extraction of active compounds from seaweeds is obvious. However, the physical and chemical barriers of the plant material become the key drawbacks of such extraction process. Therefore, enhanced release and recovery of active compounds attached to the cells have been addressed. Taken together, the aim of this communication is to discuss the potential use of enzyme treatment as a tool to improve the extraction efficiency of bioactive compounds from seaweeds.

Cytochemical characterization and ultrastructural organization in calluses of the agarophyte Gracilariopsis tenuifrons (Gracilariales, Rhodophyta).

The culture and physiology of red macroalgae calluses are well documented. To date, however, no report has either performed a cytochemical analysis or characterized the ultrastructural organization of calluses at different stages of development and under the effect of plant growth regulators. Therefore, to undertake such analyses, this work studied the red seaweed Gracilariopsis tenuifrons (Bird et Oliveira) Fredericq et Hommersand. Morphology studies suggested three types of calluses: a) terminal callus having an irregular amorphous shape and filamentous projections originating from the cortical region of the thallus; b) apical callus growing on apical branches and having an elongated semispherical shape; and c) intercalary callus developing along the intermediary region of the thallus and having the appearance of small declivities with irregular edges. The abundance of intercalary calluses over terminal and apical calluses is most likely a result of a major cortical surface that would support the cellular growth required to generate calluses. Callus development was initially observed as a matrix of cellular disorganization with filamentous projections; then, the cellular mass seemed to become more compact with spherical uncolored aspect. The presence of starch grains in the inner part of the explant could be explained by absorption from the culture medium and by proper biosynthesis during callus development. Cell wall reaction to staining suggested cellulose and agar composition with acidic polysaccharides. Results suggest that none of the three morphological types of calluses showed any significant differences on the basis of either cytochemistry or ultrastructural organization.

Discovery of lignin in seaweed reveals convergent evolution of cell-wall architecture.

Lignified cell walls are widely considered to be key innovations in the evolution of terrestrial plants from aquatic ancestors some 475 million years ago. Lignins, complex aromatic heteropolymers, stiffen and fortify secondary cell walls within xylem tissues, creating a dense matrix that binds cellulose microfibrils and crosslinks other wall components, thereby preventing the collapse of conductive vessels, lending biomechanical support to stems, and allowing plants to adopt an erect-growth habit in air. Although "lignin-like" compounds have been identified in primitive green algae, the presence of true lignins in nonvascular organisms, such as aquatic algae, has not been confirmed. Here, we report the discovery of secondary walls and lignin within cells of the intertidal red alga Calliarthron cheilosporioides. Until now, such developmentally specialized cell walls have been described only in vascular plants. The finding of secondary walls and lignin in red algae raises many questions about the convergent or deeply conserved evolutionary history of these traits, given that red algae and vascular plants probably diverged more than 1 billion years ago.

Characterization of thermostructural damages observed in a seaweed used for biosorption of cadmium: effects on the kinetics and uptake.

The effect of drying Sargassum filipendula on the kinetics and uptake of cadmium was studied. The maximum uptake was not reduced when ovendried biomass was used for cadmium concentrations from 10.0 to 500.0 mg/L. Kinetics indicated better performance of the in natura biomass. Drying at 333 K affected the uptake capacity. Results fit the Langmuir model better than the Freundlich. This process followed pseudo-second-order kinetics. Thermogravimetric and infrared analysis confirmed that no structural damage occurred after drying, and no differences between the biomasses were observed. Temperatures from 303 to 328 K affected cadmium uptake capacity.

Antioxidant metabolism in the intertidal red seaweed Stictosiphonia arbuscula following desiccation.

Seaweeds grow in distinct vertical bands on the seashore and it is well known that their ability to recover physiological processes following desiccation is correlated to their shore position. Despite this, little is known of the cellular mechanisms by which intertidal seaweeds limit membrane damage during desiccation and subsequent rehydration. In this study, specimens of the intertidal red seaweed Stictosiphonia arbuscula were placed in sealed tanks and maintained at different relative humidities (control, RH 90-100%; moderate desiccation, RH 70-80% and severe desiccation, RH 40-50%) for 12, 24 or 48 h. Membrane damage and antioxidant metabolism was examined immediately following specimen rehydration. Amino acid leakage, through the plasmalemma, was greater for desiccated low-band specimens than high-band specimens, indicating greater membrane damage. In addition, low-band specimens produced more hydrogen peroxide and lipid hydroperoxides than high-band specimens. This indicates that, upon rehydration, high-band populations have a greater ability to reduce the build-up of hydrogen peroxide, limit lipid peroxidation and hence membrane and protein damage, than low-band populations. The greater ability to prevent or reduce the production of reactive oxygen species was not due to a larger antioxidant pool, but rather increased activity of the enzymes required to regenerate ascorbate and glutathione. These findings suggest that antioxidant metabolism is one of the defence mechanisms that protect S. arbuscula from cellular damage due to desiccation.

Characterization of the egg vesicular components in the seaweed, Fucus serratus L. (Fucales, Phaeophyta), using enzyme histochemistry and vital staining: the search for a lysosome-like body.

Fucus serratus eggs were examined for evidence of the existence of a lysosome-like body using enzyme histochemical and vital staining techniques. Simultaneous coupling azo-dye techniques for lysosomal acid phosphatase proved inappropriate owing to endogenous phenolic binding artefacts. The large number of alginate polysaccharide and polyphenolic egg vesicles interfered with vital staining techniques for lysosomes. Lysosomal esterase activity was detected in the abundant egg lipid bodies. The role of the egg lipid body as an equivalent lysosome-like body of higher plants, the spherosome, is discussed in relation to egg fertilization and early zygote development.

Cortical asymmetries direct the establishment of cell polarity and the plane of cell division in the Fucus embryo.

External gradients, such as unilateral light, applied to apolar zygotes of Fucus result in a cortical asymmetry expressed as the actin-dependent translocation of existing plasma membrane molecules (e.g., DHP receptors) to the shaded side (Fig. 1a). This process corresponds to the alignment of the polar axis. The localized cortical domain identified by the accumulation of DHP receptors, F-actin, and free calcium forms a target site for Golgi vesicle (F granule) secretion. Localized secretion of F granules is essential to stabilize the polar axis (Fig. 1b), and to complete a structural complex at the site for polar growth, postulated to span the plasma membrane, from the actin cytoskeleton to the cell wall (Fig. 2). Furthermore, targeted secretion of the contents of F granules into the plasma membrane and/or cell wall appears to provide localized positional information required to orient the first cell division plane and to differentiate the rhizoid and thallus cells of the two-celled embryo (Fig. 1c). Our cytological approaches using Fucus zygotes point to the importance of directed vesicle movement and secretion in creating asymmetries in the plasma membrane/cell wall during embryogenesis, which appear to have a critical role in cell morphogenesis. Conclusions drawn from these results may provide a useful paradigm for the study of cell morphogenesis and pattern formation in higher plant embryos and vegetative tissues.