Effect of dark and salinity treatment in the yield and quality of agar from Gracilaria cornea ( Rhodophyceae )

The alkali treatment used previous to agar extractions from the Gracilaria genus reduces, among other reactions, the sulphate content and improves the gel strength; however, at an industrial level it requires expensive effluent processing to reduce its polluting charge. The red alga Gracilaria cornea was cultivated under dark and salinity treatments to replace this alkali treatment. The different treatments tested were: (a) darkness and 33‰ salinity for 8 days, [dark treatment]; (b) darkness and 50‰ salinity for 4 days, followed by darkness and 25‰ salinity for 4 days, [4+4]; and (c) darkness and 50‰ salinity for 8 days, followed by darkness and 25‰ salinity for 4 days, [8+4]. The treatment [4+4] increased the agar yield in 26% (from 36.6 to 46.1%). All treatments reduced the sulphate content of the agar in approximately 24% when compared with agar obtained without any treatment. For G. cornea the [4+4] treatment might become in the future an additional treatment to a mild alkali treatment using less alkaline reagents for agar extraction.

Efecto del tratamiento de oscuridad y salinidad en el rendimiento y calidad del agar de Gracilaria cornea (Rhodophyceae) Effect of dark and salinity treatment in the yield and quality of agar from Gracilaria cornea (Rhodophyceae)
Gracilaria cornea J. Agardh, a red seaweed from the Yucatan coast of Mexico, has recently been recognised as a potential source of agar (Freile-Pelegrín and Robledo, 1997a).The native agar of this species gives a high yield (40%) but due to its high sulphate content (5%) and low gel strength (50-100 g cm -2 it has been recognised as an agaroid (Freile-Pelegrín and Robledo, 1997a).The improvement of the agar quality in this species has been carried out successfully using an alkali treatment prior to the extraction which reduced the sulphate content and increased the gel strength through the conversion of the alkali labile sulphate 4-L-galactose-6-sulphate to 3,6-AG (Freile-Pelegrín and Robledo, 1997b).However, this treatment promotes a slight reduction on the agar yield (≈16 %).
Despite the advantage of the alkali treatment for increasing agar quality, it requires expensive industrial effluent processing before releasing to nature, in order to reduce its polluting charge mainly due to sodium agaropectinates (Armisén, 1995).
An alternative method to induce the conversion of the alkali labile sulphate to 3,6-AG and to increase the yield in Gracilaria has been developed in Sweden (Ekman and Pedersén, 1990;Ekman et al., 1991;Rincones et al., 1993).This method involves the management of certain algal enzyme processes by means of algal treatment under hyper-and hyposaline conditions and darkness.Under these conditions the activation of the enzymes sulphohydrolase and sulphotransferase produces a desulphation of the agar molecule increasing the 3,6-AG levels and therefore the agar quality.
On the other hand, the phosphorylase and the α- glucanlyase are two enzymes responsible for the degradation of the floridean starch, a carbon storage pool in red algae that is considered an impurity of agar (Yu and Pedersén, 1991).These enzymes are reported to maintain their activity during darkness in Gracilaria (Rincones et al., 1993).An increase in the low molecular-weight carbon source, the floridoside, in G. sordida Nelson (now called G. chilensis Bird, McLachlan et Oliveira) has been observed under darkness and hypersaline conditions (Ekman et al., 1991).Under these conditions floridean starch is converted to floridoside that serves as an organic osmolyte (Macler, 1986).The α-galactosidase activity, a floridoside degrading enzyme, increases when Gracilaria is exposed to hyposaline conditions (Yu andPedersén, 1990a, 1990b;Ekman et al., 1991).Ekman et al. (1991) suggests that the released carbon from the degradation of floridean starch and floridoside during darkness and hyper and hyposaline conditions can be mobilised towards the agar synthesis (fig.1).condiciones hipo-salinas (Yu y Pedersén, 1990a, 1990b;Ekman et al., 1991).Ekman et al. (1991) sugieren que el carbono resultante de la degradación del almidón florideano y del florodósido durante estas condiciones de oscuridad y choque osmótico pueden movilizarse hacia la síntesis de agar (fig.1).
En este estudio se llevó a cabo una investigación del efecto del tratamiento de oscuridad y salinidad sobre el rendimiento y la calidad del agar nativo de Gracilaria cornea en cultivos de laboratorio.El objetivo de este estudio fue eliminar los grupos sulfatos inestables de la molécula de agar para aumentar la fuerza de gel en el agar nativo de G. cornea y al mismo tiempo incrementar el rendimiento.
In this work we studied the effects of dark and salinity treatments on the yield and quality of native agar from Gracilaria cornea in laboratory cultures.The aim of the study was to eliminate labile sulphate groups of the agar molecule and at the same time to increase both gel strength and yield.

Plant material and culture conditions
Gracilaria cornea was collected from a natural population at Dzilam de Bravo (21°23'N, 88°57'W) in September 1998.In order to assure the same internal nutrient status, they were cultivated in the laboratory with filtrated (45 µm) seawater (33‰) and enriched according to Provasoli (1968), but modified with a phosphorous concentration of 82 µM and without vitamins (MPES).Algae were cultivated for seven weeks in 12 aerated 80-L Plexiglass cylinders with an irradiance of 100 µmol m -2 s -1 , temperature between 22 and 27°C, and a 16:8 h (L:D) photoperiod.The initial plant density was 3 g L -1 fresh weight (fw) and the mean daily growth rate was 2% d -1 .

Experimental treatments
The dark and salinity treatments were carried out by placing 200 g fw of Gracilaria cornea in aerated lid-covered buckets containing 16 L of natural filtrated seawater kept at 27 ± 1°C.The buckets were painted outside with black paint to avoid light passing through.In order to determine the salinity values for the osmotic shock treatment, preliminary experiments of salinity tolerance for G. cornea were performed.The salinities used were 50 and 25‰ and were achieved with the addition of natural sea salt or fresh water respectively.To avoid algae using their nutrient pool as osmotic regulators, water was enriched with MPES.All treatments were carried out with three replicates with algae randomly selected from the 12 cultivation cylinders.
Before experimental treatments were performed, 400 g fw of algae were taken out, washed thoroughly with tap water, oven dried at 60°C and stored in sealed plastic bags at ambient temperature until agar extraction.These samples without any treatment were used as control.
After the treatments mentioned above, algae were washed with tap water, dried, weighed and stored in the same way as control samples.

Agar extraction
For the agar extraction, 10 g of dry algae were used.The algae, previously hydrated overnight in distilled water, were submerged in 400 mL distilled water adjusting pH at 6.3-6.4 and boiling them at 100-105ºC for 1.5 h.At the time of boiling, pH was adjusted again to 6.3-6.4 with a pH-meter provided with a temperature compensated pH electrode.The extract was mixed with 8 g of Celite and pressure filtered.The agar was allowed to gel during 24 h at room temperature then frozen during 2 days and thawed at room temperature.Finally the agar was dried for 24 hours at 60ºC, cooled in a dessicator and weighed to determine the agar yield, which was expressed as percentage of algae dry weight.The extractions were made in three replicates per treatment.

Physical properties
The agar gel strength, and its melting and gelling temperatures were measured by triplicate in an agar solution (1.5% w/v).To avoid evaporation, the gel was covered with a thin water layer and allowed to cool down at room temperature.The gel strength and melting temperature were measured after the gel had stabilised at room temperature for 24 hours.The gel strength was measured by the Nikan Sui method, that is based in the force (g cm -2 ) driven by a round plunger (1-cm 2 cross-section) to break a standard gel in 20 s (Armisén and Galatas, 1987).The gelling temperature was measured by pouring 10 mL of agar solution into a test tube with a glass bead.The tube was tilted in a water bath at 50°C and the temperature of the solution was recorded with a precision thermometer when the glass bead ceased from moving.The melting temperature of the gel was measured by placing an iron bead (9 mm in diameter) on the surface of the gel in a test tube.The tube was clamped in a water bath with temperature slowly raising from 50 to 100ºC.The melting temperature was measured with a precision thermometer at the moment that the bead sank into the solution.

Chemical properties
The agar sulphate content was analysed using the spectrophotometric method described by Jackson and McCandless (1978), using K 2 SO 4 as standard.Agar samples (50 mg) where oven dried at 105ºC and hydrolysed in 2 mL 1M HCl for 6 h in

Análisis estadístico
Las diferencias entre los controles y los tratamientos experimentales fueron analizadas con un ANOVA de una vía y una prueba t.Para determinar la correlación entre las propiedades del agar se utilizó el test de correlación de Pearson.
a sand bath at 110ºC.The hydrolyte was centrifuged and quantitatively transferred and diluted twenty times, 2.2 mL of this were mixed with 2.4 mL 8% trichloric acid and 1.2 mL 0.01% agarose solution with 0.5 g of barium chloride.A commercial agar from Hispanagar (ref.A-127/94) with known sulphate content was used as control.Absorbance was read at 500 nm and sulphate content expressed as percentage of agar dry weight.
The 3,6-AG content was determined following the spectrophotometric method described by Yaphe and Arsenault (1965), using D-fructose as standard.Agar samples (10 mg) were dissolved in 25 mL distilled water for 3 h in a water bath at 80ºC.The solution was diluted 16 times and 2 mL of this were mixed thoroughly with 10 mL resorcinol-acetal reagent.After heating, the absorbance was read at 550 nm and the 3,6-AG content was expressed as percentage of agar dry weight.All analyses were done in three replicates per treatment.

Statistical analysis
Differences between non-treated algae and the dark and salinity treatments were tested using a one way ANOVA and a t-test.A Pearson's correlation test was used to determine the correlation between agar properties.

Results
In general, dark and salinity treatments increased the agar yield of G. cornea (fig.2a).The increase was significant (P < 0.01) for algae under the [4+4] treatment with an increase from 36.6% obtained in the control to 46.1%, representing a 26 % increase in the agar yield.
None of the treatments led to a significant change in the 3,6-AG content (fig.2b); however, all treatments reduced sulphate content in approximately 24% when compared to untreated algae (P < 0.001, fig.2c), resulting in sulphate contents of 3.2%.
The [4+4] treatment did not affect significantly the gel strength, although both, the [8+4] and the [darkness] treatments reduced gel strength from 251 to 125 g cm -2 and 188 g cm -2 , respectively.The treatments did not significantly affect the gelling and melting temperatures of the extracted agars.Only the dark and the [4+4] treatments caused a small decrease in the agar gelling temperature from 41.8 to 39.1°C and to 39.5°C, respectively.

Discussion
The agar yields for non-treated G. cornea were high when compared to other tropical species of Gracilaria (Cote and Hanisak, 1986;Aponte-Diaz and Lemus, 1989;López-Bautista and Kapraun, 1995).According to the results obtained in the present study, the [4+4] treatment led to higher yields (46.1%) than those reported in the studies mentioned above.The increase in the agar yield after the dark and salinity treatment of G. cornea could be due to a redistribution of the metabolic carbon towards the agar synthesis as found in other Gracilaria sp.(Ekman and Pedersén, 1990;Ekman et al., 1991).The increase in yield was not evident for algae subjected to the dark treatment, indicating that the synthesis of new agar was primarily stimulated by the salinity shock.Furthermore, agar Tabla 1. Fuerza de gel y temperaturas de gelificación y fusión del agar de Gracilaria cornea después de los tratamientos experimentales.directamente con el peso molecular del agar (Murano, 1995).En este sentido, la disminución que se observa en ambos parámetros para el tratamiento de [8+4] puede indicar un acortamiento no deseado del polímero.Los tratamientos probados en este estudio incrementaron el rendimiento y disminuyeron el contenido de sulfatos del agar nativo de G. cornea, aunque no aumentaron siginificativamente la fuerza de gel.Sin embargo, para que estos tratamientos puedan ser tomados como un método alternativo al tratamiento alcalino se requieren más estudios, a fin de probar y optimizar los diferentes tratamientos en cultivo.Hasta ahora, los tratamientos presentados aquí podrían ser usados como métodos aditivos a tratamientos alcalino ligeros, que podrían proveer agares con propiedades similares a los obtenidos con concentraciones más elevadas de álcali.

Agradecimientos
Este trabajo fue financiado por el proyecto J2839-B de CONACYT y por el programa Marine Macroalgae STINT Fellowship que hizo posible la colaboración entre México y Suecia.Los autores agradecen de igual forma la ayuda prestada en el laboratorio por Cresencia Chávez y María Luisa Zaldivar.yield diminished during the extended exposition to high salinity ([8+4]) probably due to a carbon loss through respiration or degradation.The different treatments reduced successfully the agar sulphate content, indicating that this process was affected primarily by the absence of light.The observed decrease agrees with previous results in Gracilariopsis lemaneiformis by Rincones et al., (1993) and in Gracilaria chilensis by Hemmingson and Furneaux (2000).Hemmingson and Furneaux (2000) suggest that by inhibiting photosynthesis, the balance is shifted to the formation of 3,6-AG from Lgalactose-6-sulfate due to the activation of sulfohydrolases.
The sulphate reduction did not have any direct effect on the gel strength because a decrease in alkali-labile sulphate, along with an increase in the 3,6-AG content, increases the gel strength only if the reaction is carried out under those conditions whene there is not any severe reduction of the agar molecule weight (Armisén and Galatas, 1987).
There was a significant correlation (r = 0.84) between the gel strength and the melting temperature for agar from cultivated algae.This has also been documented for alkalitreated agars from G. cornea (Freile-Pelegrín and Robledo, 1997b).On the other hand, the melting temperature as well as the gel strength are positively correlated with the molecular weight of the agar polymers (Murano, 1995).In this way, the decrease observed in both parameters for the [8+4] treatment may indicate an undesired shortening of the agar polymer.
All treatments tested in this study increased the agar yield and decreased the sulphate content in the native agar from Gracilaria cornea although no significant increase in the gel strength was observed.In order to use these treatments as an alternative to the alkali treatment, more studies are needed to evaluate and optimize them in culture.At present, these treatments might be used as an additive method to a milder alkali treatment that could provide agars with similar properties to those obtained with higher alkali concentrations.

Figure 1 .
Figure 1.Carbon pathway to the formation of agar from floridean starch in Gracilaria spp.

Table 1 .
Gel strength and gelling and melting temperatures of Gracilaria cornea after experimental treatments.