Cultivation of Gracilaria verrucosa (Gracilariales,Rhodophyta) Strain G-16 for agar

The agarophyte, Gracilaria verrucosa Strain G-16, has been grown in sustained cultivation over a period of five years. During this period, a number of experiments were conducted to examine the productivity, agar yield and agar gel strength of this strain. Productivities range from 3–31 g dry wt m^–2 d^–1 and are generally highest in the summer when annual water temperatures and daily irradiances are highest. In the summer months agar yield from Strain G-16 appears to be lowest whereas the gel strength of the agars was highest (> 750 g cm^–2).     

Agar quality of commercial agarophytes from different geographical origins: 1. Physical and rheological properties

Six economically important species ofGracilaria, from a number of commercial sources around the world, andGracilariopsis lemaneiformis, collected from two Japanese localities, were used as the sources of raw material for the evaluation of agar quality. Agar-agar was extracted by pretreatment with various concentratrions of NaOH (0%, 3%, 5%, 7%, 10%) incubated at 80 °C for 2 h. Agar yield, viscosity, dynamic gelling and melting temperature and gel texture were determined for 1.5% agar gels. The highest agar yield was obtained fromG. gracilis from Argentina (39.5%), while the lowest was from BrazilianG. gracilis (13.37%). Dynamic gelling temperature was highest in the agar fromG. gracilis from Turkey (59 °C) and lowest in the non-alkali treated agar isolated fromG. edulis from Indonesia (46 °C). Melting temperature ranged from 96 °C in the agars from the JapaneseGracilariopsis andG. chilensis from Chile to 69 °C in the non-alkali treated agar fromG. edulis from Indonesia. In general, all species produced an agar with high gel strength after treatment with 5% NaOH, except forG. chilensis and the twoGracilariopsis species, which produced an agar with high gel strength after treatment with 3, 7 and 10% NaOH. The highest gel strength (2056 ± 13.6 cm^–2) and hardest gel (261 ± 19.89 g mm^–2) were obtained fromG. lemaneiformis from Japan (Oita Prefecture) after treatment with 7 and 10% NaOH respectively. The lowest gel strength (351 ± 93 cm^–2) was obtained fromG. gracilis from Brazil after treatment with 3% NaOH. The softest gel (66.31 ± 9.63 g mm^–2) was isolated fromG. tenuistipitata from China, after treatment with 3% NaOH. The most flexible gel (11.62 ± 0.31 g mm^–2 × 10²) was obtained fromG. chilensis from Chile after treatment with 3% NaOH.Author for correspondence     

Genetic diversity of <Emphasis Type="Italic">Gracilaria changii</Emphasis> (Gracilariaceae,Rhodophyta) from west coast,Peninsular Malaysia based on mitochondrial <Emphasis Type="Italic">cox</Emphasis>1 gene analysis

Mitochondrial cytochrome c oxidase subunit I (cox1) was employed to investigate the intraspecific genetic diversity of Gracilaria changii collected from various localities distributed along the west coast of Peninsular Malaysia. Gracilaria changii is an agarophyte with potential for commercialization in Malaysia as it has high yields of good quality agar with high gel strength for the production of food grade agar and agarose. The phylogeographic aspect of G. changii has not been studied despite its abundance and potential commercialization. In this study, six mitochondrial haplotypes (C1–C6) were revealed from 62 specimens varying by 0–3 bp over 923 bp. Results indicate that haplotype C1 is the common ancestor and the most widespread haplotype due to its prevalence in Morib, Gua Tanah, Middle Banks, Batu Besar, Batu Tengah, Sungai Pulai, and Kuala Sungai Merbok. In this study, Morib was suggested as contributing the highest intra-population diversity with the identification of three haplotypes. The mitochondrial marker cox1 is a highly divergent mitochondrial marker and is applicable for studies on species identification and assessment of genetic diversity of G. changii.     

Growth rates and agar properties of three gracilarioids in suspended open-water cultivation in St. Helena Bay, South Africa

Relative growth rates (RGRs), yields and agar characteristics of threegracilarioid isolates (Gracilariopsis sp. from St. Helena Bay, and Gracilaria gracilis isolates from Langebaan Lagoon and Saldanha Bay) weremeasured to assess the suitability of a site in St. Helena Bay for suspendedcultivation. The gracilarioids were grown on polypropylene ropes and`netlon' lines, and the RGRs were 4.0–11.0% d<sup>-1</sup> and 5.0–7.0%d<sup>-1</sup>, respectively. The RGR of the Langebaan isolate of G. gracilis grown on ropes was significantly higher than the RGR of otherisolates. The mean net yield for the Langebaan isolate grown on `netlon'lines was 2.6 ± 0.9 kg wet wt m^-2 30 day^-1. Thecultured gracilarioids were extracted for native and alkali treated agars. Themean native agar yield over the entire period was 39.0% dry wt. Alkalipretreatment reduced the yield by 55%, but significantly increased gelstrength. High gel strengths (>750 g cm^-2) were measured inagars from Gracilariopsis sp. and Saldanha Gracilaria gracilis inmid-summer and winter. The dynamic gelling and melting temperatures ofnative and alkali treated agars varied among the gracilarioids. The meangelling and melting temperatures of agars were about 39.0 ^°C and86.0 ^°C, respectively. The 3,6-AG content ranged from 29% to38% for native agars and 34–45% for alkali treated agars. While theseresults indicate that this site is suitable for gracilarioid cultivation, occasionallow-oxygen events in St. Helena Bay lead to production of hydrogensulphide in the sea water (`black tides'). Such events killed most inshorebiota (including seaweeds) in 1994 and 1998. This frequency (on average1–2 per decade) and duration (maximum 2 weeks) would have to beconsidered in planning commercial seaweed farming in St. Helena Bay.     

Optimisation of RNA extraction from Gracilaria changii (Gracilariales, Rhodophyta)

RNA extraction from seaweed tissues is problematic due to the presence of polysaccharides and polyphenolic compounds upon cell disruption. Besides, a successful RNA isolation from seaweed tissues can sometimes be strain- and species-specific. Four different methods were used to extract RNA from Gracilaria changii (Gracilariales, Rhodophyta), collected from the mangrove area at Morib, Selangor, Malaysia. An optimised and modified total RNA extraction method was developed for this recalcitrant species. The use of sand in tissue grinding, and the incorporation of phenol extraction at the initial stage resulted in the highest RNA yield (0.65–1.14 g g^–1 fresh weight) with high quality (A_260:280 ratio 1.80–2.05). The RNA obtained is suitable for cDNA synthesis and future functional genomic studies.     

Does storage time influence yield and agar properties in the tropical agarophyte Gracilaria cornea?

The agar yield and quality characteristics of Gracilaria cornea from Yucatán, Mexico, werestudied during 18 months of storage. Biomass wasstored at a temperature of 22.1 ± 0.9 ^°Cand humidity of 59.8±3.6%. The agar contentvaried erratically, but the average value waspractically constant over the storage period with an average of 20.1 ± 1.5±. Gel strength, gelling and meltingtemperatures were negatively affected by the totalstorage time. No significant changes were found duringthe first five months for these characteristics withmean values of 1134 ± 57 g cm^-2, 40.8 ±0.4 ^°C and 91.2 ± 0.9 ^°Crespectively. Agar degradation was evident after thefifth month and accounted for a 17± loss in gelstrength and 7± in gelling and meltingtemperatures. Nevertheless, gel strength valuesremained around 930 ± 23 g cm^-2 with nosignificant changes until the end of the storageperiod. The decrease in gel strength showed asignificant relationship with decrease in3,6-anhydrogalactose but not variation in sulphatecontent. This was probably due to agar hydrolysiscaused by enzymatic processes of endogenous and/ormicrobial origin. These results suggest that thetropical G. cornea had a similar resistance todegradation during storage to that observed for G. chilensis, a cold water species. Agar qualityand yield in G. cornea after one and a half yearof storage are within the range of food grade agar.     

Effects of sulfate starvation on agar polysaccharides of Gracilaria species (Gracilariaceae,Rhodophyta) from Morib,Malaysia

The effects of sulfate starvation on the agar characteristics of Gracilaria species was investigated by culturing two red algae from Morib, Malaysia, Gracilaria changii and Gracilaria salicornia in sulfate-free artificial seawater for 5 days. The seaweed samples were collected in October 2012 and March 2013, periods which have significant variation in the amount of rainfall. The agar yields were shown to be independent of sulfate availability, with only 0.60–1.20 % increment in treated G. changii and 0.31–1.40 % increment in treated G. salicornia while their gel strengths did not increase significantly (approximately 5–7 %) after sulfate starvation for both species. The gelling and melting temperatures did not vary between control and treated samples from both species, except for the treated G. changii collected in March 2013. The gel syneresis index of G. salicornia collected in March 2013 increased significantly after sulfate deprivation. Sulfate starvation introduced some variations in the content of 3, 6-anhydrogalactose and total sulfate esters, but the changes did not have a pronounced effect on the physical properties of agar.     

Comparison of growth rate in culture,dry matter content,agar content and agar quality of two New Zealand red seaweeds,Gracilaria chilensis Bird,McLachlanet Oliveira andGracilaria truncata Kraft

Plant growth rates and agar characteristics were compared for two agarophytes,Gracilaria chilensis (Gracilariales, Rhodophyta) andG. truncata, so that the suitability ofG. truncata for mariculture could be assessed.G. chilensis plants grew steadily in the laboratory culture system at rates of 5–8% day^-1 (mean Relative Growth Rate) throughout the 6 week experimental period, with no decline in health.G. truncata plants grew at rates of 2–4% day^-1 for 5 weeks, but during the sixth week their apical tips became necrotic and growth rates fell to zero. There was no significant difference in the dry matter content (as a percentage of their wet weight) between the two species ofGracilaria, with values falling in the range of 16–19%. Slightly higher agar yields were obtained from alkali-treatedG. chilensis (17–20% dry matter) than from untreatedG. truncata (16–18%) although the agar fromG. truncata formed softer gels from which it was more difficult to recover. The gel strength of untreated agar extracted fromG. chilensis was very low (ca. 100 g cm^-2 for a 1% gel) but when an alkali treatment step was included in the extraction process, it increased to 520 g cm^-2 for a 1% gel. Contrary to an earlier report, untreatedG. truncata agar also had a very low gel strength (ca. 100 g cm^-2 for a 1.5% gel), but it rose to only 167 g cm^-2 after alkaline treatment.     

Comparison of three gracilarioids: growth rate, agar content and quality

Three gracilarioid species, Gracilariopsis bailiniae and Gracilaria tenuistipitata from Vietnam and Gracilaria gracilis from Russia, were studied in order to determine whether Gracilaria gracilis might be a superior species for cultivation in brackish-water ponds for agar production compared with the Vietnamese species. The effects of different salinity levels on the growth rate and agar production as well as agar properties of three gracilarioid species were compared in controlled laboratory experiments. Gracilaria tenuistipitata and G. gracilis were tolerant to low salinity (∼10‰), whereas Gp. bailiniae died under these conditions. G. tenuistipitata showed superior growth among the three species examined. Gracilaria gracilis had the highest agar content [36.8–46.6% dry weight (dw)]. Agar yield from Vietnamese gracilarioids did not exceed 30% dw. Gel strength of native agar from Gracilaria gracilis was two-fold higher that from Vietnamese species (278 g cm⁻² vs 130 g cm⁻²). Alkali pretreatment increased gel strength significantly for Gracilaria gracilis (1.4-fold), and G. tenuistipitata and Gp. bailiniae (2.3-fold) compared with native agar. The results suggest that Gracilaria gracilis may be a suitable species for production of reasonably good quality agar.     

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.     

Agar yield and quality of Gracilaria chilensis (Gigartinales,Rhodophyta) in tank culture using fish effluents

Tank cultivation of Gracilaria using fish effluents has permitted a production of 48 kg m^–2 yr^–1 and can reduce the dissolved nitrogen loads in the seawater. We report the yield, gel strength, gelling and melting point of agar from Gracilaria cultivated in tanks with seawater previously utilized in intensive, land-based salmon cultures and compared to a control using directly pumped seawater, over a study period of 22 months. The results show that the highest agar yield (20 to 22%) was obtained when Gracilaria was cultivated with pure seawater as compared to the fish effluents. The gel strength, gelling and melting point were higher in the agar obtained from algae cultured with fish effluents. During the spring, the gel strength, gelling and melting point increased in tanks with fish effluents and decreased in tanks with a supply of pure seawater.     

Agar properties of two species of Gracilariaceae from the Gulf of California,Mexico

Agar properties of two potentially commercial important seaweeds from the Gulf of California were studied. Maximum yield in Gracilaria vermiculophylla (45.7%) occurred during the summer months, coinciding with high water temperatures (31°C) whereas minimum yields (11.6%) were obtained during the coldest months of the year when populations of this species diminish in the bay. Gracilariopsis longissima showed two yield peaks, one in spring and another in fall, before the maximum and minimum seawater temperatures. Gel strength in native agar from the two species was low (<22.5 g cm⁻²) for most of the year. G. vermiculophylla native agar showed a slight increase in gel strength from June to August, which were the hottest months. Maximum value was 85 g cm⁻¹ in August. Maximum gel strength in G. longissima was observed in October (91 g cm⁻¹), and an unusual native agar with no detectable gel strength was observed in March and April samples. Gelling temperatures range from 27.7 to 36.5°C in G. vermiculophyla and from 26.6 to 34.9°C in G. longissima, meanwhile melting points were 73.9 – 53.5°C and 75.5 – 56.6°C, respectively. Sulfate content was high, 6.3–13.9% in G. vermiculophylla and 1.9–11.9% in G. longissima, and on the other hand 3,6 anhydrogalactose content was low 12.1–26.7% and 9.1–23%, respectively compared to other species. Results obtained showed that mean native agar yields of Gracilaria vermiculophylla and Gracilariopsis longissima from the Gulf of California are comparable to other tropical Gracilaria. However, the low gel strength, high sulfate content and low 3,6 anhydrogalactose content observed in the native agar extracted from these species make this an agaroid, thus alternative methods of extraction should be used to evaluate the possibility of commercial utilization of both species.     

Seasonal agar yield and quality inGelidium canariensis (Grunow) Seoane-Camba (Gelidiales,Rhodophyta) from Gran Canaria,Spain

The seasonal effects on yield and gel properties ofGelidium canariensis agar were investigated at two intertidal populations at the northern coast of Gran Canaria. Physical and rheological properties were measured in 1.5% w/v solutions after treatment with alkali. No significant differences were found on agar characteristics between the two sites studied. The highest yields were obtained during summer with a maximum in June (27.8%) and minimum during late autumn and winter (18–18.6%). Overall quality was highest in winter (November–January), when gel strength peaked above 850 g cm^–2. The results showed an agar of industrial quality.     

Experimental investigations for the mariculture ofGracilaria in Saldanha bay,South Africa

Rope rafts were used to evaluate the growth of localGracilaria gracilis at Saldanha Bay, on the west coast of South Africa, over four years. The plants were grown on horizontally-suspended ropes or netting lines. Relative Growth Rates (RGR) of plants on ropes with low stocking weights often exceeded 10% d^–1 in (austral) summer, and fell to between 6 and 7.5% d^–1 in winter. Commercial-style lines of plastic netting stocked at 400 g m^–1 and placed 0.75 m apart yielded a mean of about 2 kg m^–2 30 d^–1, with a mean RGR of about 5% d^–1 over a two-year period. Various methods of improving yields were tested, including attaching floats to individual lines and optimising stocking weight. In Saldanha Bay in late summer, warm, oligotrophic water can severely reduce growth. Growth was optimised by growing the plants as close as possible to the surface (0.2 m), where water motion, and by implication, nutrient uptake, are higher. Agar content and gel strength generally remained high all year round. The potential for commercial production is evaluated in the light of these results.     

Inhibition of Pigments and Phycocolloid in a Marine Red Alga Gracilaria Edulis by Ultraviolet-B Radiation

Vegetative fragments of the subtidal macroalga Gracilaria edulis (Gmel.) Silva cultured under field conditions at Thonithurai were subjected in the laboratory to UV-B radiation (280 – 320 nm). UV-B inhibited the accumulation of chlorophyll and phycobiliproteins, and lowered agar yield (23 to 43%) and its gel strength (22 to 36%) under 12 to 72 h exposure. The longer the exposure to UV-B radiation the more significant impact on pigments and phycocolloid properties of Gracilaria edulis was observed.     

The effect of phosphate concentration on growth and agar content of Gelidium robustum (Gelidiaceae,Rhodophyta) in culture

Phosphate concentration of the growth medium was found to affect the growth rate and agar yield of a clone of Gelidium robustum grown in the laboratory. To study differences in growth we used phosphate concentrations from 0 to 200 µM. To determine the effect of phosphate on agar yield and its properties we used concentrations from 0 to 20 µM. Growth rates generally increased with increasing phosphate concentration, with the highest growth rate (21% d^–1) obtained at 150 µM. Agar yield as percentage of fresh weight was highest (10%) in the algae grown with low phosphate concentrations, but agar yield as percentage of dry weight was highest(43%) at 20 µM of phosphate. Gel strength increased with phosphate concentration with a maximum of 160 g m^–2 for 0.75% gels for the cultures at 20 µM. Melting and gelling temperatures of the gels were also affected by phosphate concentration of the growth medium. Starch yield was highest in algae grown in low phosphate concentrations.     

Studies on the yield and gel strength of agar from Gracilaria domingensis Sonder ex Kuetzing (Gracilariales,Rhodophyta) following the addition of calcium

Studies were carried out on the seasonal variation in yield and gel strength of agar from Gacilaria domingensis with and without the addition of calcium chloride. Extraction was done with and without treatment with 1% hydrochloric acid. The results showed an increase in yield and gel strength when an alkaline solution of calcium was used, but the gel strength was low. For commercial use, Gracilaria domingensis should be mixed with better quality Gracilaria species because of its low gel strength.     

The yield,physical and chemical properties of agar gel from Gracilaria species (Gracilariales,Rhodophyta) of the Kenya coast

Six species of Gracilaria, G. corticata J. Agardh, G. crassa Harvey, G. millardetii J. Agardh, G. salicornia (J. Ag.) Dawson, G. verrucosa (Huds.) Papenfuss and Gracilaria sp, collected from different stations along the Kenya coast were studied. The yield of hot water native agar extract ranges from 8.1–30% of dry weight, with G. verrucosa and G. salicornia having the greatest and the least yield, respectively. The gel-strength of 1.5% agar solution was highest in G. verrucosa (220 g cm^–2) and lowest in G. corticata (< 60 g cm – 2) whereas the highest gelling temperature was recorded for Gracilaria sp. (40.4 °C) and the lowest in G. verrucosa (28.9 °C). 3,6 anhydrogalactose content was the highest in G. verrucosa and the lowest in G. corticata while sulphate content was higher in the latter.     

Seasonal changes in the chemical and gelling characteristics of agar from Gracilaria verrucosa collected in Turkey

The chemical and gelling properties of agar from G. verrucosa collected from Izmit bay in Turkey at different months of the year were studied. Purification of agar was performed by using amylase treatment and isopropyl alcohol precipitation. The phycocolloid content was between 24.0–43.0% of the algal dry weight and was maximum in summer collected algae. Relative total sulfate and 3,6-anhydrogalactose content in the agar were determined from the ratios of infrared spectroscopy band intensities at 1250/2920 cm^–1 and 930/2920 cm^–1, respectively. 3,6-anhydrogalactose and sulfate contents were the highest in agar from algae collected from June until November and January until July, respectively. The gel strength of native agar were the highest from in autumn collected algae and increased to about 1250 N m^–2 after alkali treatment. Thus, this study demonstrated that G. verrucosa from Turkey produces an agar with optimal chemical and gelling properties after alkali-treatment in fall and winter collected algae and may be used for industrial agar production.