Phycological Research 2008; 56: 33–38 Seasonal variation on size and chemical constituents of Sargassum sinicola Setchell et Gardner from Bahía de La Paz, Baja California Sur, Mexico Yoloxochitl Elizabeth Rodríguez-Montesinos,* Dora Luz Arvizu-Higuera and Gustavo Hernández-Carmona Interdisciplinary Center of Marine Science-IPN. PO Box 592, La Paz, Baja California Sur, 23000, México SUMMARY Investigation on seasonal variation in size and chemical constituents of Sargassum sinicola Setchell et Gardner from Bahía de La Paz, Baja California Sur, Mexico, was carried out from a control bed and compared with an experimental bed with artificial nutrients added. No significant differences were found between the control and experimental thalli for size or chemical composition, except for iodine and raw fiber. For control thalli the results were: size 7.5–56.0 cm, alginate yield 7.2–13.7%, viscosity 58.7–191.7 millipascal seconds (mPa s), mannitol 2.9–8.1%, raw fiber 5.5–7.5% and iodine 0.020–0.141%; while in the experimental thalli the size ranged from 7.5 to 80.3 cm and the alginate yield was 7.8–10.4%, viscosity 41.4–163.4 mPa s, mannitol 2.9–8.3%, raw fiber 5.9–10.7% and iodine 0.021–0.098%. These variations were related to its natural growth cycle, and showed reductions during the senescence period. Results suggest that S. sinicola is not affected by relatively low nutrient concentrations, and could be considered as raw material for alginate production. Key words: alginate, chemical constituents, iodine, mannitol, raw fiber, Sargassum sinicola, size. INTRODUCTION Sargassum sinicola Setchell et Gardner is the most common and abundant seaweed in the Gulf of California (Casas-Valdez et al. 1993; Nuñez-López & CasasValdez 1997, 1998; Pacheco-Ruíz et al. 1998). Potential uses of Sargassum species are as animal food complement, fertilizer, and as a source of antibacterial substances and alginates (Chapman & Chapman 1980; Ang 1987; Padmini-Sreenivasa-Rao et al. 1988; Marín et al. 2003). Total biomass estimated for all the species of the genus Sargassum from the western coast of the Gulf of California is 154 000 wet tons (Pacheco-Ruíz et al. 1998) and 18 900 wet tons in Bahía de La Paz (Hernández-Carmona et al. 1990; Fajardo-León 1994). However, in spite of its wide distribution and abundance, Sargassum species have not been harvested commercially in the Gulf of California. Some macroalgae (e.g. Macrocystis pyrifera (Linnaeus) C. Agardh) are severely affected by high water temperature and low nutrient concentrations as occur during the El Niño Southern Oscillation (ENSO). Experimental studies have demonstrated that adding artificial nutrients helps to maintain M. pyrifera during ENSO conditions. In contrast, other species such as Eisenia arborea J.E. Areschoug is more resistant to low nutrient concentrations (Hernández-Carmona et al. 2001). However, the effects of nutrients on the chemical constituents of S. sinicola are unknown. In addition, studies on the natural variation of the main chemical constituents of the alga could determine the potential use of the algae. The goal of this study was to examine the seasonal variation in the content of four of the main chemical constituents (alginate, mannitol, raw fiber and iodine) of S. sinicola under two conditions; without artificial nutrients (control) and with artificial nutrients added (experimental). Study area Bahia de La Paz, Baja California Sur, Mexico is a coastal water body adjacent to the Gulf of California with a length of 90 km and a width of 60 km. The climate is very dry and warm. The average annual rainfall in the area is 188 mm and the wettest month (62 mm) is September (Jiménez-Illescas et al. 1997). The sea surface water temperature in winter (December–February) ranges from 19 to 21°C, in spring (March–May) from 21 to 24°C, in summer (June– August) from 24 to 28°C and in autumn (September– November) from 28 to 18°C (González-Navarro & Saldierna-Martínez 1997). El Sauzoso, located to the *To whom correspondence should be addressed. Email: yrodriguez@ipn.mx Communicating editor: D. Fujita. Received 10 June 2006; accepted 21 May 2007. doi: 10.1111/j.1440-1835.2008.00482.x Y. E. Rodríguez-Montesinos et al. 34 Table 1. Average total length (cm) of experimental Sargassum sinicola thalli with and without (control) nutrients added, from Bahía de La Paz, Baja California Sur, Mexico. Data are mean ⫾ standard error (n = 30) Treatment Experimental Control Season Autumn Winter Spring Summer 7.5 ⫾ 2.8 7.5 ⫾ 2.8 40.3 ⫾ 19.5 38.3 ⫾ 22.1 56.3 ⫾ 29.8 80.3 ⫾ 28.9 11.0 ⫾ 2.3 16.5 ⫾ 5.1 west of the bay at 24°18′ 26′-N and 110°38′ 17′-W, is a semiprotected site having moderate surge, with a shallow slope (Cruz-Ayala et al. 1998). MATERIALS AND METHODS This experiment is part of a broader study of S. sinicola in El Sauzoso. We specifically examined the effect of nutrient availability on a population recruited on three quadrats of one square meter, where artificial nutrient (Osmocote: N 14%, P 14%, K 14%) was dispensed using PVC pipes attached to concrete blocks of 56 W ¥ 36 L ¥ 12 H cm (Hernández-Carmona et al. 2001). To avoid bias in sampling, quadrats were placed at random along the coast line from where S. sinicola thalli were sampled. We used another three quadrats for sampling thalli in areas without nutrient enrichment (control). Thirty thalli were tagged in each quadrat with numbered plastic tags and the total length was measured each month to determine changes in thallus size. S. sinicola was sampled from the control and experimental quadrats seasonally. Nitrate was determinate from bottom seawater, near the quadrats (1 m2), using the cadmium reduction technique (Strickland & Parson 1972). In alginate extraction, the method described by Arvizu-Higuera et al. (2002) was used. Twenty grams of the dried and milled algal materials (30 mesh with a sieve size of 0.5 mm) were hydrated overnight with 0.1% formaldehyde and then washed with HCl at pH 4. The alginate was extracted with Na2CO3 at pH 10 in a water bath at 80°C for 2 h. The paste was diluted and filtered under vacuum and the clarified solution was precipitated with ethanol at a proportion of 1:1. The alginate fibers were then dried at 50°C for 12 h. Alginate yield was expressed as percentage/dry weight. The apparent viscosity of the alginate was determined in 1% (w/v) solution at 22°C with a viscometer (Brookfield LVT, Middleboro, MA, USA), at 60 r.p.m. (6.28 rad·s-1) using the appropriate spindle, adding 0.5% sodium hexametaphosphate as calcium sequester. Mannitol, iodine and raw fibers were determined using the techniques described by Larsen (1978). Mannitol was determined by a fast oxidation of the polyol, titrating with Na2S2O3.5H2O, with starch solution as an indicator; iodine, by fusion in the presence of NaOH to release it, adding Br and titrating with Na2S2O3.5H2O; and raw fiber by extraction of the soluble material in acid and alkali at high temperature, and determining the organic part remaining by the weight lost. Data were computed to obtain the average and standard error (⫾1 SE) with the significance level at 95%. ANOVA was used to detect significant differences among seasons and among treatments (P < 0.05), and Tukey test to detect significant differences between means (Zar 1999). RESULTS At the beginning of the experiment, the average seawater nitrate concentration was not significantly different between the treatments with (0.62 mM) and without nutrients (0.25 mM). During winter, the concentration was significantly higher in quadrats with nutrients (8.76 mM) than without (2.55 mM). During spring, there was a trend for higher nitrate concentrations in quadrats with nutrients (6.03 mM) than the controls (2.57 mM), but this trend was not significantly different. Near the end of the experiment in summer, nitrate concentrations were not significantly different, although concentrations were still higher with nutrients (6.83 mM) than without nutrients (4.34 mM). The average initial size of S. sinicola thalli was 7.5 cm high (autumn) in both control and experimental thalli. Maximum average sizes reached in spring were 80.3 cm in control thalli and 56 cm in experimental thalli (Table 1). Size showed significant seasonal variation, but it was not significantly different between treatments (Table 2). Sodium alginate yield (Fig. 1a) and viscosity (Fig. 1b) were significantly different between seasons but not significantly different between treatments (Table 2). The highest yield was obtained in control thalli during spring (13.7%), and the highest viscosity (191.7 mllipascal seconds (mPa s)) in winter. Alginate viscosity showed the same trend for control and experimental thalli. The minimum viscosity occurred in spring with 58.7 mPa s in control thalli and 41.4 mPa s in experimental thalli. Minimum mannitol value was 2.9% in autumn in both treatments and increased to a maximum value in winter with 8.1% in control thalli and 8.3% in the experimental thalli. Mannitol decreased in spring and Constituents of Sargassum sinicola 35 Table 2. ANOVA for significant differences between seasons and treatments in size, viscosity and chemical composition from Sargassum sinicola Size Yield Viscosity Mannitol Raw fiber Iodine Effects d.f. effect MS effect F P Season Nutrients S vs N Season Nutrients S vs N Season Nutrients S vs N Season Nutrients S vs N Season Nutrients S vs N Season Nutrients S vs N 3 1 3 3 1 3 3 1 3 3 1 3 3 1 3 3 1 3 4 613.9800 283.5900 210.5900 24.2287 32.9720 12.7741 57 767.1731 133.9582 4 620.1986 58.3993 0.7151 7.0916 12.8379 19.1330 15.1331 0.0257 0.0103 0.0057 17.92 1.10 0.82 2.96 4.03 1.56 70.47 0.16 5.64 39.66 0.49 4.82 29.04 43.28 34.23 36.03 14.51 8.06 0.000* 0.309 0.502 0.042* 0.051 0.212 0.000* 0.688 0.002* 0.000* 0.489 0.005* 0.000* 0.000* 0.000* 0.000* 0.000* 0.000* *P < 0.05. d.f., degrees of freedom; MS, mean square. summer in both treatments (Fig. 1c). Differences were significant between seasons, but not between treatments (Table 2). In raw fiber, minimum values occurred in summer (5.5 and 5.9%) in both treatments. In contrast, the maximum values were in spring for the control thalli (7.5%) and autumn (10.7%) for experimental thalli (Fig. 1d). Significant differences were obtained between seasons and treatments (Table 2). Iodine in control plants were lower from autumn to winter and increased significantly in spring and peaked in summer (0.141%). Experimental thalli had similar iodine values as control thalli from autumn to spring and decreasing significantly in summer (0.027%) (Fig. 1e). Significant differences between seasons and treatments were obtained (Table 2). DISCUSSION Although nutrient concentration was higher in the experimental quadrats during winter, thalli were not significantly different in size. The thalli length from autumn represents the average size of all populations at the beginning of the experiment. During spring, in which thalli reached their maximum size of 56 cm with nutrients and 80 cm without nutrients, it was possible that high nitrate levels during spring (15 mM) (PaúlChávez pers. comm. 2005) in experimental quadrats might have lowered its basic metabolic process (Lobban & Harrison 1994), resulting in lower growth rates and smaller thalli. In summer, all thalli deteriorated due to natural senescence. The large variation of thallus length during winter and spring was due to the mix of old thalli, that lost part of the fronds, with new thalli that regenerated or germinated from zygotes. Seasonal variation in alginate yield from S. sinicola may be related with changes in light, wave exposure, currents, temperature (Espinoza-Avalos & RodríguezGarza 1986; Espinoza & Rodríguez 1987) or others. However our results suggest that nutrient concentration could not be the main factor affecting the size and chemical constituents and that the variation could be related to thallus growth. In control quadrats, the thalli collected from autumn to winter were juveniles with low content of alginate (11.7%). As they matured in the spring, the cell wall and intercellular matrix increased with alginate and reached a maximum of 13.7%. In summer, senescent thalli with residual fronds had lower levels of alginate (7.2%). Similar results were obtained by Aponte de Ataola et al. (1983) for Sargassum species from Puerto Rico with yields of 7–12% in January (winter) and 17.5–20% in May (spring) for Sargassum vulgare C. Agardh and Sargassum polyceratium Montagne, respectively. Also Sargassum swartzii C. Agardh and Sargassum tenerrium J. Agardh from India had alginate yields of 19.7 and 11%, respectively (Chauhan 1970). Hernández-Carmona (1985) found values of 16.5% in winter and 27% in spring for S. sinicola from La Paz, Baja California Sur, Mexico; Pérez-Reyes (1997) reported Sargassum spp. values of 6.6% in January (winter) and 11.8% in May (spring) from the same location as the present study. The experimental thalli exposed to nutrients followed a similar trend to control thalli. 36 Y. E. Rodríguez-Montesinos et al. Yield (%) (a) Viscosity (mPa s) (b) 18 16 14 12 10 8 6 4 2 California Sur, Mexico. 䊐, experimental thalli; 䊏, control thalli. Vertical bars indicate the standard error. 䉳 Autumn Winter 2000 Spring Summer 2001 Autumn Winter 2000 Spring Summer 2001 Autumn Winter 2000 Spring Summer 2001 Autumn Winter 2000 Spring Summer 2001 Autumn Winter 2000 Spring Summer 2001 250 200 150 100 50 0 (c) Fig. 1. Seasonal variation in average yield (a) and viscosity of 1% sodium alginate solution (b), mannitol (c), raw fiber (d) and iodine (e) of Sargassum sinicola collected from Bahía de La Paz, Baja 10 Mannitol (%) 8 6 4 2 0 (d) 12 Raw fiber (%) 10 8 6 4 2 0 (e) 0.20 Iodine (%) 0.16 0.12 0.08 0.04 0.00 Alginate viscosity was highly variable during the year. The lower values in the control thalli from spring to summer are correlated with senescence of thallus (Shah et al. 1970). The highest alginate viscosity of 191.7 mPa s in a 1% solution falls into the low viscosity type. This value was higher than that reported by Alankararao et al. (1988) for S. vulgare in India with 60 mPa s and lower than in S. polycystum C. Agardh with 70.8 mPa s in a 0.5% alginate solution (Saraswathi et al. 2003). The alginates extracted from Sargassum species contain a higher amount of guluronic acid blocks that produce stronger gels, as compared with alginates from Macrocystis (Shyamali et al. 1984; McHugh 1987; Saraswathi et al. 2003); therefore this type of alginate could be used in applications requiring the formation of strong gels. Mannitol is a monomeric compound used by algae as a reserve and is remobilized from mature or old tissue to provide energy and carbon skeletons for growth in the meristems, and is influenced by osmotic potential (Lobban & Harrison 1994). Mannitol showed significant seasonal variation in thallus growth and reproductive condition. However, it was not significantly different between control and nourished thalli. Mannitol content in control thalli (2.9–8.1%) was, however, higher than those reported for Sargassum ilicifolium (Turner) C. Agardh 2–5% and Sargassum myriocystum J. Agardh (1.3–5.0%) in India (Chennubhotla et al. 1982). The fiber method determines the amount of nonextractable organic material in the sample (Larsen 1978). In this study, the fiber content was significantly higher in experimental (5.9–10.7%) than control thalli (5.5–7.5%). These values, however, were lower than those reported by Pérez-Reyes (1997) for Mexican Sargassum spp. with 10.3–11.3% and by Huerta-Múzquiz et al. (1998) for S. polyceratium with 12% fiber content. Iodine is located mainly in the stipe tissue (Kolb et al. 2004). Iodine content in S. sinicola ranged from 0.021 to 0.141%, and was higher in mature than in juvenile thalli. The iodine content for S. sinicola was higher than those obtained by Qing-xiang and Xiao (1998) for different species of Sargassum from China, including Sargassum vachellianum Greville (4.5 ¥ 10-3%), Sargassum hemiphyllum (Turner) C. Agardh (4.7 ¥ 10-3%), and Sargassum assimile Harvey (5.6 ¥ 10-3%). This suggests that S. sinicola has a higher capacity to store iodine than other Sargassum species. Constituents of Sargassum sinicola In the present study it has been shown that there is an inverse relationship between the mannitol and iodine content (mannitol increasing – iodine decreasing) in all seasons and this relationship changed only in summer in experimental thalli. Variations of the main chemical constituents may be related to the plant growth cycle and not strongly influenced by seawater nitrate concentrations. S. sinicola showed significant reductions during the senescence period. The results suggest that S. sinicola could be considered to be an important raw material for alginate production. Thus, Sargassum spp. should be harvested in spring, a period of maximum biomass and high yield. Although viscosity is lower in spring, gel strength could be high. ACKNOWLEDGMENTS We thank the Instituto Politécnico Nacional (CICIMARIPN) for financial support, the ‘Comisión para el Fomento de Actividades Académicas (COFAA-IPN)’ and the program ‘Estímulo al Desempeño de la Investigación (EDI-IPN)’ for the scholarship received. Also we thank Matthew Forrest and Kim Siewers for English language editing. REFERENCES Alankararao, G. S. J. G., Rajendra Prasad, Y. and Rama Rao, K. 1988. Alginic acid from Sargassum vulgare Børgesen. Phykos 27: 174–6. Ang, P. O. Jr. 1987. Use of projection matrix models in the assessment of harvesting strategies for Sargassum. Hydrobiologia 151/152: 335–9. Aponte de Ataola, N. E., Diaz-Piferrer, M. and Graham, H. D. 1983. Seasonal variations and anatomical distribution of alginic acid in Sargassum spp. found along the coasts of Puerto Rico. J. Agric. Univ. P.R. 67: 464–8. Arvizu-Higuera, D. L., Hernández-Carmona, G. and RodríguezMontesinos, Y. E. 2002. Parameters affecting the conversion of alginic acid to sodium alginate. 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