PHYLOGENETIC RELATIONSHIPS OF GELIDIALES SPECIES IN COSTA RICA

Despite a general paucity of studies on marine algae in Costa Rica, within the red algal order Gelidiales, 9 species are reported from the Caribbean, and 6 species from the Pacific. In this study 5 species from the southeast Caribbean coast and 2 species from the northwest Pacific coast of Costa Rica were collected for DNA sequence and morphological analyses. At the time of this writing, morphological observations of four Caribbean taxa have identified them as Gelidium microdonticum , Pterocladiella capillacea , P. melanoidea , and Gelidiella acerosa. In rbc L sequence analyses, Gelidium microdonticum is resolved within a clade of small Gelidium species predominantly found in the south Atlantic. Two collections representing different forms of Pterocladiella melanoidea sensu Dawson were found to have identical rbc L sequences. These samples were resolved in rbc L trees with a taxon from Texas identified as P. bartlettii and not P. melanoidea from Europe. Comparisons with type material are being made to verify these identifications so that the status of P. melanoidea in the Caribbean may be determined. The unidentified Caribbean taxon is resolved as a distinct species within the Pterocladiella clade as well. Gelidiella acerosa from Caribbean Costa Rica is closely allied with other Caribbean G. acerosa samples in rbc L trees. The two pacific taxa are small intertidal turf species and have not yet been identified. Sequence analyses resolve these taxa within a large clade of Indo-Pacific and Caribbean Gelidium species.     

Chemical characteristics and gelling properties of agar from two Philippine Gracilaria spp. (Gracilariales, Rhodophyta)

The chemical structure of agars extracted from Philippine Gracilaria arcuata and G. tenuistipitata were determined by NMR and infrared spectroscopy. Agar with alternating 3-linked 6-O-methyl-β-D-galactopyranosyl and 4-linked 3,6-anhydro-2- O-methyl-α-L-galactopyranosyl units was isolated from G. arcuata, while the agar from G. tenuistipitata possesses the regular agarobiose repeating unit with partial methylation at the 6-position of the D-galactosyl residues. Both agars exhibit sulphate substitution at varying positions in the polymer. Chemical analyses reveal higher 3,6-anhydrogalactose and lower sulphate contents in alkali-modified than in native agar from both samples. Also, alkali modification enhanced agar gel strength and syneresis. Native G. arcuata agar produces a viscous solution (2000 cP at 75 °C) with a high gelling point (>60 °C) that forms a soft gel even after alkali modification (gel strength: <300 g cm⁻²). On the other hand, the agar from G. tenuistipitata exhibits gel qualities typical of most Gracilaria agars. This revised version was published online in June 2006 with corrections to the Cover Date.     

Preparation of superior quality products from two Indian agarophytes

The effect of alkali pre-treatments on the properties of agar obtained from Gelidiella acerosa and Gelidium pusillum growing along the West and South coast of India was studied in order to evaluate its potential for industrial use. Agar samples were extracted by hot water extraction and termed native agar, and alkali pre-treatment was carried out with various NaOH concentrations e.g. 4%, 6%, 8%, 10% and 15%, to optimise the alkali concentration for preparation of alkali-treated agar. Gelidiella acerosa and Gelidium pusillum produced superior quality agar polymers having greater gel strengths 2,000 ± 50 and 1,400 ± 50 g cm⁻² and lower sulphate contents ≤0.30% and ≤0.50%, respectively, as opposed the low gel strength and high sulphate content agars reported in the literature. Furthermore, the physicochemical properties of agars were identical with the commercially available agars. The results of this study revealed that Indian agarophytes may be used for production of superior quality agar and agaroses, which may be used for food and biological applications.     

Farming of agarophytes in India—a long-time sustainability for the industry and preserving wild stocks

In India, food-grade agar is produced from Gracilaria edulis while bacteriological-grade agar is extracted from Gelidiella acerosa. Agarose is directly obtained from Gracilaria dura. Seaweeds for agar production mainly come from wild stocks located in reefs of the Gulf of Mannar, southeast coast. Landings of Gr. edulis were peak (982 dry t) during 1990 while landings of Ge. acerosa reached maximum (665 dry t) during 2002–2003. Overexploitation is leading to a decrease in biomass of these two algae at an alarming rate for the last decade. Commercial cultivation of agarophytes is yet to increase in India in spite of growing pressure on wild stocks triggered by increase in demand of raw material by the industry. Taking this into consideration, a number of efforts have been initiated to develop feasible cultivation methods for agarophytes. The present paper summarizes various methods developed for commercial cultivation of Gr. edulis, Gr. dura, Gracilaria verrucosa and Ge. acerosa in the open sea. We also discuss the challenges in the development of commercial farming of agarophytes in India.     

First record of the twostripe goby,Valenciennea helsdingenii (Gobiidae,Gobiiformes) from the?southeast?coast of India

Two specimens of Valenciennea helsdingenii (Bleeker, 1858) were collected off Punnakayal coast, from Gulf of Mannar, southeast coast of India in November 2012. The morphometric and meristic characters of the recorded specimens are described and discussed. This is the first record of the species from the Indian waters that is a range extension of its known range within the Indian Ocean.     

On the properties of agar gel containing ionic and non-ionic surfactants

Rheological and thermal properties of agar sol and gel in presence of various cationic, anionic and non-ionic surfactants are reported. The agar used was from the red seaweed Gelidiella acerosa. The gel strength, viscosity, rigidity (G'), gelling temperature and melting temperature were observed to decrease in presence of non-ionic surfactants whereas these were enhanced in presence of ionic surfactants. TGA studies showed that 1.5% agar gels containing non-ionic surfactants lose water at a lower temperature than the control agar gel whereas gels containing ionic surfactants hold on to water more tenaciously. DSC studies, on the other hand, show that the gel to sol transition occurs at lower temperatures in presence of non-ionic surfactants and at higher temperature in presence of ionic surfactants when compared with the control gel. The non-ionic surfactants, Triton X-100 and Brij 35, enabled relatively concentrated agar extractive to be filtered readily, as a result of which water usage in the process could be reduced by 50%. The surfactant was subsequently removed through freeze-thaw operations to restore the gelling capacity of the agar. The finding that 0.3-0.4% (w/v) sodium lauryl sulfate (SLS) lowers the sol-gel transition temperature from 41 to 36 degrees C without adversely affecting gel strength is another useful outcome of the study that may enable better formulations of bacteriological agar to be prepared.     

3,6 anhydrogalactose, sulfate and methoxyl contents of commercial agarophytes from different geographical origins

The chemical composition of agars extracted from six economically important species of Gracilaria from different geographical sources (Argentina, Brazil, Indonesia, China and Turkey) and one species of Gracilariopsis (G. lemaneiformis) collected from two different Japanese localities was investigated. Agar was extracted by pretreatment with various concentrations of NaOH (3%, 5%, 7%, 10%) for 2 h at 80°C. The sulfate, 3,6-anhyd, rogalactose and methoxyl contents of each agar extract were analyzed. High sulfate and 3,6 anhydrogalactose contents were found in non-alkali treated agar from Turkish Gracilaria gracilis (3.4%) and from Chilean G. chilensis (54.3%) after alkali treatment concentration of 5% NaOH, respectively. High methoxyl contents (4.9%) were obtained from non-alkali treated agar from Chinese G. tenuistipitata. This revised version was published online in August 2006 with corrections to the Cover Date.     

Seasonal variation in the physical properties of agar and biomass of Gracilaria sp. (chorda type) from Tosa Bay,southern Japan

Plants of Gracilaria sp.(chorda type), which grow along the coast of Uranouchi Inlet in Tosa Bay, southern Japan, showed the highest biomass in the summer (26 °C to 31 °C) and spring season (15.1 °C to 24.9 °C). Maximum biomass was 6952 g m^–2 in July, but gradually decreased in the autumn (30.5 °C in September to 20 °C in November) and winter (19.5 °C in December to 14.9 °C in February). Variation in yields and gel strength of the agars, were shown to depend on the time in the season. After alkali treatment (5% NaOH, 2 h) at three different temperatures (70, 80, and 90 °C), the agars showed gel strengths essentially that of commercial grade agars, with the best gel obtained at 80 °C. Maximum gel strength (1455 g cm^–2 of 1.5% agar gel) occurred in winter when the biomass and agar yield were low. Minimum gel strength was in spring. Gel strength was inversely correlated with agar yield, but was positively correlated with apparent viscosity. Maximum viscosity was 40 cP. in December. Gelling temperatures, pH of 1.5% agar gel, and moisture content in agars showed little variation.     

Development of an eco-friendly agar extraction technique from the red seaweed Gracilaria lemaneiformis

The red seaweed, Gracilaria lemaneiformis growing as an aquaculture bioremediator along the coasts of Liaodong Peninsula, China, was investigated for the agar production. An eco-friendly method called agar photobleaching extraction process was developed for the benefit of workers' health and safety of the environment. The native agar (NA), alkali-modified agar (AA), chemical-bleached agar (CA) and photobleached agar (PA), which were extracted using different processes, were evaluated for their physical and chemical properties. The PA showed most desirable performances in terms of gel strength, gelling temperature, sulfate content and 3,6-anhydro-l-galactose content. Among the different processed agars, PA gel strength was 1913 g/cm2, the highest among the different processed agars, which increased 8.6% on the basis of the AA. Further we applied this new technique to extract agars from Gracilaria asiatica, and similar results were obtained with that of G. lemaneiformis. This indicates that the agar photobleaching extraction process is a feasible method for Gracilaria species and has a potential application. During the whole agar photobleaching extraction process the pigment content of G. lemaneiformis declined gradually and the TOC concentration in photobleaching solution increased along with the increase in the irradiation time. The mechanism of agar photobleaching could be elucidated by the photolysis theory.     

Seasonal variation in the biomass, quantity and quality of agar from Gelidiella acerosa (Forsskal) Feldmann et Hamel (Gelidiales, Rhodophyta) from the Gulf of Mannar Marine Biosphere Reserve, India

The Gulf of Mannar on the southeast coast of India, which has recently been declared as biosphere reserve, is the main harvesting place of Gelidiella acerosa for the Indian agar industry. The biomass harvested from natural stocks was estimated to be over 10 000 tonnes dry weight (DW) in the last two decades and is a serious cause of concern on long term sustainability of this valuable resource from this region. The present study reports the seasonal variation in the biomass and agar yield and quality from G. acerosa in four different habitats such as Rameswaram (rocky intertidal), Ervadi (lower intertidal), Sethukarai (subtidal) and Krusadai Island (lagoon reef area). A maximum biomass of 260 ± 26 g DW m^?2 was recorded from Ervadi, whereas Sethukarai showed the highest percentage cover (69.83 ± 4.83%) and density (208.20 ± 30.16 plants m^?2). Biomass, percentage cover and density were lowest in Rameswaram and significantly lower (P < 0.001) than the other three stations. A single peak in biomass was observed with autumn maxima in Ervadi and Rameswaram and southwest monsoon maxima in Sethukarai and Krusaidai Island. Length of G. acerosa was maximum (9.65 ± 0.25 cm) in Ervadi and the branch index value was maximum (24.70 ± 4.01 br^?1 g^?1 DW) for Rameswaram. Agar yield (37.24 ± 7.59%) and gel strength (448.66 ± 6.50 g/cm²) were higher in Sethukarai plants. The agar yields attained a peak in the northeast monsoon and decreased in summer, whereas a reverse trend was observed for gel strength. Evidently, agar yield showed a strong negative correlation with gel strength (P < 0.0001) and gelling (P < 0.0001) and melting temperatures (P < 0.01). The single annual peak in biomass observed in the present study, as compared with two peaks reported earlier, are attributed to the continuous overharvesting that would have had an effect on the overall annual biomass production. The findings of this study reveal that a single harvest during the autumn months (January to March) could yield optimum biomass with moderate agar and that would be the best for sustainable usage and conservation of this resource from this region.     

Agars fromGracilaria chilensis (Gracilariales)

The chemical composition of the agars extracted with water at 95 °C fromGracilaria chilensis of Estero de Lenga (43.2% of yield) and of Bahia de Coquimbo (43.4% of yield) were determined. The gel strength, gel melting and gelation temperature of the agars were also measured. Polysaccharides obtained by extraction with cold water and by extraction of the alkali pre-treatedG. chilensis of Bahia de Coquimbo were also analyzed. The physical constants and the contents of sulfate and 3,6-anhydrogalactose of the alkali-modified agar fromG. chilensis of Estero de Lenga were comparable to those of commercial agar. This algae represents an excellent potential commercial resource of agar.G. chilensis of Bahia de Coquimbo gave hydrocolloids with low gel strength values more suitable for use in the food industry. Anion exchange chromatography of the agars obtained by extraction with water at 95 °C indicated that the whole polysaccharides and all of the acidic fractions were not homogeneous. Neutral fractions comprised only about 25% of the agars.     

Structure and properties of agar from two unexploited agarophytes from Venezuela

The red seaweeds Gelidiella acerosa and Gracilaria mammillaris growing along the coast of Venezuela were investigated as potential economic sources of agar. Agar extracted from Gracilaria mammillaris accounted for 27% of the algal dry weight but had quite a poor gelling ability because of the presence of alkali-stable sulphate groups on the D-galactose residue. However, the gel strength of its aqueous solutions was considerably enhanced by the addition of potassium, sodium and calcium ions (up to 1N). On the contrary, the galactan from Gelidiella acerosa (yield of about 20% w/w) exhibited quite good properties. In particular, the gel strength was comparable to that of commercial agaroses. Sulphate esters were not detectable by chemical methods and NMR spectroscopy revealed an agarose backbone with a high degree of methylation on both D and L-galactose residues.     

Extraction and characterization of agar from Australian Pterocladia lucida

Polysaccharides were sequentially extracted from Australian Pterocladia lucida at 50 °C, to give the Warm Water (WW) fraction, and at 95 °C. The 95 °C extract was further separated into gelling (GF) and thaw water (TW) fractions by freezing-thawing. Fourier Transform infrared (FTIR) spectroscopy, compositional and linkage analyses, and physico-chemical properties indicated that the GF contained an agar with nearly idealized repeating structure and low levels of sulfate and pyruvate substitution. By contrast, the WW and TW contained heterogeneous, highly sulfated galactans with relatively low levels of 3,6-anhydrogalactose and higher levels of pyruvate and glycosyl branching and impurities, such as starch and protein. The properties of the gels formed from the GF and two commercially available agars (Sigma High Gel Strength agar and Sigma Type A agar) were investigated with a texture analyser. The GF from P. lucida had a gel strength intermediate between that of the commercial agars. The gel setting temperature of a 0.8% (w/v) solution formed from the GF was 2 °C below that of comparable solutions of the two commercial agars. This revised version was published online in August 2006 with corrections to the Cover Date.     

Chemical composition of agars from a newly reported Japanese agarophyte,Gracilariopsis lemaneiformis

The chemical composition of agars from Gracilariopsis lemaneiformis, newly reported from Japan, was investigated. Native agars were isolated by a sequential extraction of plants in water at 22 °C and 100 °C, and in boiling 20, 40 and 60° ethanol. Agars in each extract were analyzed by chemical methods, ¹H, ¹³C NMR; and IR spectroscopy. The highest yield of agar (total carbohydrate) was obtained from the 40° ethanol extract (55°). Highest sulfate content was attained in non-alkali treated agars extracted with hot water (4.81°, DS 0.2). The 3,6-anhydrogalactose content was highest in the 40° ethanol extract (36.1° in non-alkali treatment, 40.3° in alkali treatment). The highest methoxyl content (6.51°, DS 0.66) was obtained in the 60° ethanol extract. The G. lemaneiformis agar is composed of the biological precursor to agarobiose repeating units and agarobiose containing 6-O-methyl agarobiose and a small amount of 2-O-methyl-α-l-galactopyranose residues. Alkali treatment improved the chemical quality of the agar fractions, which was comparable with Japanese commercial agar and agarose.     

Isolation of Vibrio cholerae no-01 in the Gulf of Nicoya, Costa Rica]

In a bacteriological study on samples of bivalves, mud and surface waters from the Gulf of Nicoya, Costa Rica, 18 strains of non-01 Vibrio cholerae and 50 of V. mimicus were isolated. The samples were enriched in alkaline peptone water, and streaked on MacConkey and inositol-brilliant green bile agars. Biochemical and serological tests were used for their identification. Both species were isolated from all sampling sites (Lepanto, Jicaral and Puntarenas) with either of the two agar media, even though these were not specific for vibrios.     

Selective agars for the isolation of Streptococcus iniae from Japanese flounder, Paralichthys olivaceus, and its cultural environment

Two kinds of selective agar were developed for the isolation of Streptococcus iniae, the causal agent of streptococcosis, from Japanese flounder (Paralichthys olivaceus) and from culture tanks in flounder farms. The selective agars were heart infusion agar with added thallium acetate and oxlinic acid (TAOA), and colistin sulphate and oxolinic acid (CSOA). For samples containing various bacterial flora, selective agars were supplemented with defibrinated horse blood in order to distinguish beta-haemolytic colonies of Strep. iniae. Streptococcus iniae was quantitatively isolated from the brain and kidney of diseased flounders in pure culture. Two-thirds of isolates picked up from selective blood agars inoculated with intestinal samples were identified as Strep. iniae. The bacterial colony numbers of deposits and water from culture tanks on selective blood agars were about 10-10(5) times smaller than those on control heart infusion agar; Strep. iniae was isolated from few deposit and water samples.     

Effects of season on the yield and quality of agar from Gracilaria species (Gracilariaceae, Rhodophyta)

The effect of season on yield and physical properties of agars extracted from Gracia gracilis and G. bursa-pastoris were determined. The agar yield from G. gracilis was maximum during spring (30%) and minimum during autumn (19%). In G. bursa-pastoris, the agar yield was greatest in summer (36%) and lowest in winter (23%). Agar yield from G. bursa-pastoris was positively correlated with temperature (r=0.94; P<0.01) and salinity (r=0.97; P<0.01) and negatively with nitrogen content (r=−0.93; P<0.01). Agar gel strengths fluctuated from 229 to 828 g cm⁻² and 23 to 168 g cm⁻² for G. gracilis and G. bursa-pastoris, respectively. The gelling temperature showed significant seasonal variation for both species. Chemical analysis of agar from the two seaweeds indicated variation in 3,6-anhydrogalactose and sulfate content (P<0.01). Furthermore, there was an inverse correlation between the two chemical variables. In general, agar extracted from G. gracilis possessed better qualities than agar extracted from G. bursa-pastoris and can be considered a candidate for industrial use.     

Agar from the red seaweed,Laurencia flexilis (Ceramiales,Rhodophyta) from northern Philippines

The worldwide production of the gelling agent agar mainly rely on the red algae of the order Gracilariales and Gelidiales for raw material. We investigate here the potential of a species from another red algal order, Ceramiales as an agar source. The agar from Laurencia flexilis collected in northern Philippines was extracted using native and alkali treatment procedures and the properties of the extracts were determined using chemical, spectroscopic and physical methods. The native agar, 26% dry weight basis, forms a gel with moderate gel strength (200 g cm^?2). Alkali‐treatment did not enhance the gel strength, indicating insignificant amounts of galactose‐6‐sulfate residue, the precursor of the gel‐forming 3,6‐anhydrogalactose (3,6‐AG) moieties. Furthermore, the Fourier transform infrared and chemical analysis showed low sulfate and high 3,6‐AG levels, not affected significantly by the alkali treatment. Nuclear magnetic resonance spectroscopic analysis revealed 3‐linked 6‐O‐methyl‐D‐galactose and 4‐linked 3,6‐anhydro‐L‐galactose as the major repeating unit of the native extract, with minor sulfation at 4‐position of the 3‐linked galactose residues. The native and alkali treated agars have comparably high gelling and melting temperatures, whereas the former exhibits higher gel syneresis. Laurencia flexilis could be a good source of agar that possesses physico‐chemical and rheological qualities appropriate for food applications. Due to the inability of alkali treatment to enhance the key gel qualities of the native extract, it is recommended that commercial agar extraction from this seaweed would be done without pursuing this widely‐used industrial procedure.     

Physicochemical Properties of Biodegradable Polyvinyl Alcohol–Agar Films from the Red Algae <Emphasis Type="Italic">Hydropuntia cornea</Emphasis>

Agar obtained from the red alga Hydropuntia cornea was blended with polyvinyl alcohol (PVOH) in order to produce biodegradable films. In this study, we compare the properties of biopolymeric films formulated with agars extracted from H. cornea collected at different seasons (rainy and dry) in the Gulf of Mexico coast and PVOH as synthetic matrix. The films were prepared at different agar contents (0%, 25%, 50%, 75%, and 100%) and their optical, mechanical, thermal, and morphological properties analyzed. The tensile strength of PVOH–agar films increased when agar content was augmented. The formulation with 50% agar from rainy season (RS) had a significant higher tensile strength when compared to those from dry season (DS; p < 0.05). Tensile modulus also displayed an increasing trend and likewise, for 50% and 75% agar blends from RS showed higher values than those from DS (p < 0.05). In contrast, elongation at break decreased as the agar content increased, independently of the season. Environmental scanning electron microscopy images of PVOH–agar 75% biofilms from RS showed a homogeneous structure with good interfacial adhesion between the two components. The changes evidenced in the FTIR spectrum of this blend suggest that hydrogen bonding is taking place between the agar ether linkages (C-O-C) and the hydroxyl groups (OH) of the PVOH. Based on the above mentioned results, blends of PVOH and 75% agar from H. cornea collected in rainy season showed good properties for applications in the biodegradable packaging industry.     

Agar from some Hawaiian red algae

From describing the agars of Gelidiella acerosa Forssk., Gelidium pluma Loomis, G. pusillum (Stackh.) Lejolis, Gracilaria abbottiana Hoyle, G. bursapastoris (Gmelin) Silva, G. canaliculata (Kützing) Sonder, G. coronopifolia J.Ag., G. epihippisora Hoyle, Pterocladia caerulescens (Kützing) Santelices and P. capillacea (Gmelin) Born. & Thur. as found in Hawaiian samples of these species, it is concluded that the species of Gelidium and especially Pterocladia and Gelidiella may merit more consideration for usage due to their agar gel strengths.The nature of the gel from Gracilaria abbottiana suggests the generic status might well be reexamined. The agars from the Gelidiella and the other Gracilaria species should be studied further for their prospective values to the food industry other than gel strength. Mixtures of the agars from G. bursapastoris and G. coronopifolia would merit attention for the taste texture of their mixtures.