Purification, characterization and application of a novel extracellular agarase from a marine Bacillus megaterium

A marine, gram positive, aerobic, spore forming, and non flagellated bacterium which degrades low melting point (LMP) — agarose was isolated from the west coast of India and identified as Bacillus megaterium based on its morphological, biochemical, and molecular characterization. This bacterium produced clear haloes or zone of clearance on agar containing plates which was a clear indication of its agarolytic property. The extracellular agarase thus obtained was purified 8.8 and 78 fold from the culture supernatant by ammonium sulfate precipitation and gel filtration, respectively. Molecular mass by gel filtration and SDS-PAGE gave values of 15 and 12 kDa, respectively. The optimum temperature and pH for maximum agarase activity were 40°C and 6.6. The activity of agarase was drastically reduced by addition of metal ions in the assay system. This agarase, gave a K _m and V _max value of 4 mg/mL and 2.75 μmol/min/mg. The isolation of protoplast from agarophyte like Gelidiella acerosa using indegenous agarase is reported for the first time.     

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.     

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.     

Studies on Gracilaria changii (Gracilariales,Rhodophyta) from Malaysian mangroves

Gracilaria changii, recorded from Malaysia and Thailand, is one of the more abundant agarophytic seaweeds found in Malaysia. A wild population of Gracilaria changii growing in mangroves was monitored for seasonal variation in agar content and gel strength as well as spore production. Agar yield and gel strength ranged from 12 to 25% dry weight and 294 to 563 g cm^–2, respectively, over a 15-month period. Gel strength but not yield was positively correlated with amount of rainfall. Cystocarps were observed throughout the study, but use of sporetraps showed that spore release peaked around July–August and January–February after the two monsoon periods.Mariculture of Gracilaria changii in shallow ponds in the mangroves, in an irrrigation canal and in a shrimp farm pond was conducted. Average growth rate of cuttings tied to monofilament lines was 3.3 ± 1.7% d^–1, 8.4 ± 1.8% d^–1 and 3.6 ± 1.6% d^–1 respectively. Cuttings were lost to siltation, wave action, predation and heavy epiphytisation.These studies show that Gracilaria changii has potential commercial application in the agar industry.     

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.     

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.     

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.     

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.     

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.     

Karyology and agar analysis of the agarophyteGelidiella acerosa (Forsskål) Feldmannet Hamel from the Philippines

Microspectrophotometry with the DNA-localizing fluorochrome DAPI demonstrated ploidy level differences in tetrasporophytic and presumptive gametophytic phases ofGelidiella acerosa from the Philippines. Comparison of mean nuclear DNA (I _f ) values to chicken erythrocytes (RBC) resulted in an estimate of 0.32 pg/2 C genome. Karyological studies with aceto-orcein revealed a chromosome complement of six bivalents during diakinesis of tetrasporocytes. The agar yield ranges from 13–24% dry weight, depending on the method of extraction. Agar extraction in 1 N NaOH resulted in an increased gel strength of 189 g cm^–2 at 1.5% concentration. Infrared spectroscopy indicated a relatively high sulfate content in native agar. The low (61 °) melting temperature is indicative of high sulfation and small molecular size.     

Gracilaria phuquocensis sp. nov., a new flattened Gracilaria species (Gracilariales,Rhodophyta), previously recognized as G. mammillaris,from the southern coast of Vietnam

Flattened Gracilaria species are widely distributed along the coasts of the South China Sea with more than 20 species recorded. Within the South China Sea, Gracilaria mammillaris has only been reported from Vietnam, but this species is likely restricted to the western Atlantic. This study aimed to reevaluate the taxonomic status of Vietnamese specimens of ‘G. mammillaris’ using combined morphological and molecular data. Our data clearly indicated that Vietnamese specimens were morphologically and genetically distinct from authentic G. mammillaris from the western Atlantic, and also other described flat Gracilaria species. We, therefore, propose that specimens from Vietnam originally identified as G. mammillaris be designated as a new species, Gracilaria phuquocensis sp. nov. Morphologically, G. phuquocensis can be distinguished from other flat Gracilaria species by its small thallus size, narrower blades, many medullary layers, abundant basal nutritive filaments within mature cystocarps, and tetrasporangial nemathecium. Our rbcL sequence analyses showed that the new species was sister to Gracilaria rhodymenioides from Thailand, and these two species formed a clade with cylindrical Gracilaria species. Our study contributes to clarification of the taxonomic status of misidentified specimens attributed to the flattened Gracilaria species in the South China Sea.     

First report of Gelidiella ligulata (Gelidiales, Rhodophyta) in Japan

A gelidialean red alga that was newly found in Japanese waters is referred to as Gelidiella ligulata Dawson. It is characterized by erect lanceolate blades developing from a creeping axis that are relatively large in the genus Gelidiella. Among the 22 species currently recognized in the genus, Gelidiella indica Sreenivasa Rao is most similar to G. ligulata and a further comparative study is needed to elucidate the status of G. indica. Gelidiella ligulata and the type species of the genus, Gelidiella acerosa (Forsskal) Feldmann et Hamel, have the unicellular independent attachments that are common to the members of Gelidiella investigated to date. This type of attachment is unique in the Gehdiales and this feature may be a useful taxonomic criterion in distinguishing Gelidiella from other genera.     

Growth,photosynthesis and agar in wild-type strains of Gracilaria verrucosa and G. conferta (Gracilariales,Rhodophyta), as a strain selection experiment

The local species Gracilaria conferta and the foreign G. verrucosa were grown together under a wide range of photon flux density and temperature conditions. Gracilaria verrucosa showed a higher growth rate, especially under low temperatures, and higher photosynthetic performances as well as higher ribulose-1,5-bisphosphate carboxylase activity as compared with G. conferta. Gracilaria verrucosa also showed a better quality and yield of agar, suggesting that this species could be more suitable than G. conferta for outdoor cultivation in Israel and may improve winter growth in ponds. Growth rate and agar quality (gel strength) were rated as the most suitable characteristics influencing the preference of strains for outdoor cultivation.     

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.     

PHYSIOLOGICAL ECOLOGY OF GELIDIELLA ACEROSA (FORSSKAL) FELDMANN ET HAMEL1

The effects of different concentrations of salinity, pH, and nutritional and growth-promoting sub-stances on the production of proliferations in Gelidiella acerosa were investigated, with a view of increasing the production rule of the alga in its aquiculture.     

Superior quality agar from <Emphasis Type="Italic">Gracilaria</Emphasis> species (Gracilariales,Rhodophyta) collected from the Gulf of Mannar,India

Gracilaria edulis, G. crassa, G. foliifera, and G. corticata are naturally occurring agarophytes of Indian waters. These agarophytes were evaluated for their agar contents using an improved process recently reported by us (US Patent 2005/0267296A1). The effect of different concentrations of NaOH in the alkali treatment was studied for optimizing the extraction conditions. These Gracilaria species of Indian waters produced agars, both native and alkali treated, with different properties confirming the heterogeneity of the agar polymers in this genera, as one would expect. Among these, G. edulis and G. crassa produced agar polymers having high gel strengths of 490 ± 8.16 and 800 ± 15.4 g cm⁻², respectively, with 8% NaOH treatment as opposed the low gel strength agars that have been reported in the literature to date.     

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     

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).     

Profiling of cellulose content in Indian seaweed species

Cellulose contents were estimated in 12 seaweed samples belonging to different families e.g. red, brown and green, growing in Indian waters. Each cellulose sample was fractionated to yield alpha (α) and beta (β) celluloses. Characterization was done using various analytical tools and results were validated by comparison with those of the cellulose obtained from Whatman filter paper No. 4. The greatest yields of cellulose (crude), α- and β-cellulose were obtained from Gelidiella acerosa (13.65%), Chamaedoris auriculata (9.0%) and G. acerosa (3.10%). G. acerosa was also found to contain relatively high amount of α-cellulose (8.19%). The lowest cellulose contents were recorded from Kappaphycus alvarezii (2.00%) and Sarconema scinaioides (2.1%), while the latter contained the lowest α-, and β-celluloses (1.0% and 0.30%, respectively). It appears that agarophytic and alginophytic algae contain high cellulose and α-cellulose contents, while the carrageenophyte contains low cellulose. The brown algae, in general contain high cellulose as well as α- and β-celluloses.     

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.