J Appl Phycol (2008) 20:1–8 DOI 10.1007/s10811-007-9173-8 Spatial and temporal variation of Hypnea musciformis carrageenan (Rhodophyta - Gigartinales) from natural beds in Rio de Janeiro State, Brazil Renata Perpetuo Reis & Yocie Yoneshigue-Valentin & César Pereira dos Santos Received: 31 July 2006 / Accepted: 14 March 2007 / Published online: 4 May 2007 # Springer Science + Business Media B.V. 2007 Abstract Hypnea musciformis (Wulfen in Jacqu.) J. V. Lamour. is a native source of kappa-carrageenan, a commercially important galactan, exploited for decades on the northeastern Brazilian coast. Further studies to obtain information about the effects of abiotic factors on the carrageenan yield and quality of specimens from the southeastern Brazilian coast (Rio de Janeiro State) are needed. In this study, the carrageenan yield and quality of H. musciformis was analyzed over a period of 1 year in populations from Rio de Janeiro state. One epiphytic population was collected at a site situated at the entrance of the Sepetiba Bay (Praia Vermelha), another in the middle of the bay (Praia Grande) and a third (Praia Rasa) in a place exposed to the open sea. An epilithic population was also collected at Praia Rasa. In the sampling period, different changes were observed in the carrageenan yield and quality (viscosity and sulphate content) of the H. musciformis (August 1994 to June 1995) at the different sites and R. P. Reis (*) Instituto de Pesquisas Jardim Botânico do Rio de Janeiro, Ministério do Meio Ambiente, Rua Pacheco Leão, 915, CEP 22460-030 Rio de Janeiro, RJ, Brazil e-mail: rreis@jbrj.gov.br Y. Yoneshigue-Valentin Centro de Ciências da Saúde, Departamento de Botânica, Instituto de Biologia, Universidade Federal do Rio de Janeiro, Ilha do Fundão, CEP 21941-900 Rio de Janeiro, RJ, Brazil C. Pereira dos Santos Departamento de Bioquímica, Instituto de Química, Universidade Federal do Rio de Janeiro, Ilha do Fundão, CEP 21941-900 Rio de Janeiro, RJ, Brazil habitats (epilithic and epiphytic). Kappa carrageenan was the only one of its type detected. Only one population showed a correlation between viscosity and degree of sulphate content. Our analyses suggest that abiotic factors, such as water movement, desiccation stress, low salinity and extreme temperatures, were the main abiotic factors that influenced the changes in carrageenan of the populations studied. Thus, for a rational use of natural beds of H. musciformis, it is desirable to estimate the changes in carrageenan and algal biomass in the place to be exploited. Keywords Carrageenan yield . Hypnea musciformis . Sulphate content . Viscosity Introduction Carrageenan is red algal galactan used as a texturing agent, with gelling and thickening properties for food and non-food applications. The demand for this hydrocolloid has risen by 5% annually (McHugh 2003). In Brazil, the carrageenan raw material is supplied by importing algae, harvesting H. musciformis (Wulfen in Jacqu.) J. V. Lamour. in natural beds (Bulboa and Paula 2005), and by commercial cultivation of Kappaphycus alvarezii (Doty) Doty ex P. Silva planted 2 years ago. H. musciformis is the only source of kappa-carrageenan from native algae in Brazil and has been exploited for decades in the northeastern states (Oliveira 1998), but its production is sporadic and unable to supply national requirements (Saito and Oliveira 1990; Bulboa and Paula 2005). Attempts to cultivate H. musciformis were unsuccessful due to technical problems (Bulboa and Paula 2005), but this algae has a high 3.6 anhydro-galactose content, low sulphate content (Saito and Oliveira 1990), and high growth rate of 10% day−1 (Reis et al. 2005). This 2 species is found along much of the Brazilian coastline from Maranhão State, 4°S, 37°W, to Rio Grande do Sul’s State, 29°S, 49°W (Schenkman 1989). Information about this algae needs to be researched (Reis et al. 2005). A better understanding of the changes in the phycocolloid yield and quality of H. musciformis is fundamental for its exploitation (Saito and Oliveira 1990). To this end, studies were developed using some rheological parameters of the carrageenan (Humm and Willians 1948), while the seasonal variation in the carrageenan yield was studied in one population in Pernambuco (Guedes et al. 1985), São Paulo (Schenkman 1989) and in Bahia State (Wallner et al. 1992). But these studies did not compare the carrageenan production at different sites and habitats (epilithic and epiphytic), the viscosity was measured only at the Bahian site and the sulphate content was not analyzed. This present study was performed to investigate the spatial and temporal variation of H. musciformis carrageenan yield and quality (viscosity and sulphate content) in three populations along the coast of Rio de Janeiro State and in different habitats (epilithic and epiphytic populations at one of the sites). The possible environmental factors that can interfere with the carrageenan yield and quality of this species are also analyzed. Materials and methods Study area and sample site Samples were collected every 2 months from August 1994 to June 1995 in Rio de Janeiro State. Two epiphytic populations were collected at Sepetiba Bay (Praia Vermelha: 22°57′S, 44° 01′W; and Praia Grande: 22°57′S,43°54′W) and another one at a site exposed to the open sea (Praia Rasa: 22°43’S, 42°53’W). At Praia Rasa, an epilithic population was also collected to test differences between the epilithic habitat and the epiphytic habitat. The voucher specimem was deposited at the Herbarium of the Botanical Garden of Rio de Janeiro (RB 328.041). Abiotic factors, such as wind speed, precipitation, cloud cover, insolation, water and air temperature, length and period of waves, number of rainy days, clouds, evaporation, and air humidity, were obtained from data collected daily by the official Brazilian meteorological database: the National Meteorology Institute/Agricultural Ministry (Instituto Nacional de Meteorologia/Ministério da Agricultura e do Abastecimento - INMET) and the Oceanography Database/Marine Ministry (Banco de Dados Oceanográficos/Diretoria de Hidrografia e Navegação/Ministério da Marinha - BNDO/ DHN), using the meteorological stations of Marambaia at Sepetiba Bay and Macaé near Búzios, and data collected by boats navigating at coordinates near Sepetiba Bay and Búzios. The month before the sample period was used in consideration of the environmental time factors. J Appl Phycol (2008) 20:1–8 Carrageenan extraction and viscosity determination In the laboratory, seaweeds were washed with tap water to remove sand, debris and epiphytes. They were de-pigmented with methanol and dried in the shade at room temperature for 5 days, subsequently dried in an oven at 60°C until constant weight was reached. Two hundred ml of de-ionized water was added to 2 g of dried specimens, followed by extraction by continuously stirring for 1 h at 90±5°C. The extract was mixed with two parts of ethanol per volume of extract to precipitate the carrageenan. This precipitate was filtered using cheese cloth (surgical gauze) and dried in an oven at 60°C, until reaching constant weight. The carrageenan yield of the collected samples of H. musciformis was expressed as the percentage of carrageenan from a sample of algal dry mass (Saito and Oliveira 1990; Martinez and Buschmann 1996; Reis et al. 2005), according to the formula: yield = (Wc-Ws) x100, where Wc is the extracted carrageenan dry weight and Ws is the dry seaweed weight used for extraction. During August 1994 to June 1995, Reis and YoneshigueValentin (1998) estimated the H. musciformis biomass through seven 0.25 m2 quadracts randomly positioned along the 20 m transect in the middle of the H. musciformis belt at the sites of this study, using the destructive sampling method (De Wreede 1985). These results were used to obtain the total carrageenan dry mass from H. musciformis obtained at each site in each sample. Carrageenan yield per m2 was calculated at each site based on the biomass obtained from each period of time and the percentage of carrageenan collected. The viscosity of the gel obtained from H. musciformis carrageenan were measured in Brookfield Model DV-III, Programmable Rheometer (Brookfield Engineering Laboratories), using spindle LV SC4-18 and the software Brookfield Rheocalc for Windows. Triplicate samples of the above extracted carrageenan were dissolved in hot de-ionized water (1%) and cooled to 5°C. Samples were homogenized in a water bath at 40°C, stressed at 10, 20, 30 and 40 rpm at 10-s intervals. Viscosity was determined from 10 to 100 RPMs, with intervals, and then returned to 10 rpm. The unit used was centipoise (cP). Carrageenan sulphate content The carrageenan sulphate content was obtained by infrared analysis in an infrared Spectrometer (Nicolet 760 Magna IR Spectrometer). The absorbance bands were obtained using triplicate samples made by 1 mg of dry powder of carrageenan milled with 99 mg of potassium chloride. Infrared spectrometry has been the most used technique to J Appl Phycol (2008) 20:1–8 3 obtain important information about the chemical nature and molecular structure (Ingle and Crouch 1988). The carrageenan sulphate content was expressed as a degree of substitution, which is the mean number of sulphate groups per disaccharide repeat unit in carrageenan according to the method of Rochas et al. (1986). The relation of sulphate to total carbohydrate was estimated from the ratio of absorbance at 1,250, 930, 845 cm−1 to the band at 2,920 cm−1 used to represent the total sugar content (Rochas et al. 1986). Statistical analysis 2 1 0 Nov Jan Mar Insolation (hours and decimes) 5 4 3 o Nov Jan Mar 29 25 23 21 19 Nov Jan Mar 5 Nov Jan Mar May d 170 120 Jul 27 Sep Sep 220 May e Jul 50 0 3 2 1 0 Jan Mar May f 26 24 22 20 Jul 4 Nov 28 May g Sep o Sep 100 270 6 Jul 150 Jul c 7 b 250 200 May Water temperature ( C) Cloud cover (0-10) Sep Sep Nov Jan Mar 8 Wave period (seg) -1 Precipitation (mm) 3 2 Air temperature ( C) 300 a 4 Jul Wave length (m) Fig. 1 Monthly variation of environmental factors over the study period at Búzios (black bars) and at Sepetiba Bay (white bars) obtained from INMET (A wind speed, B precipitation, C cloud cover, D insolation and F air temperature) and from BDO/ DHN (E water temperature, G length and H period of waves). Arrows = no data collected Wind speed (Km.h ) The normality and homogeneity assumptions of the variances were tested using the Shapiro Wilk’s test and Cochran test respectively. When necessary logarithmic transformation was employed [x = log(x+1)] and retested (Zar 1996). After that one-way analysis of variance (ANOVA) was carried out to test for significant differences between means of carrageenan yield and quality (viscosity and sulphate content) in the sampling period, and the Tukey’s multiple means comparison test was used to separate those differences. Letters in the graphs show that difference. When date was non-parametric, the Kruskal Wallis Test was used (Zar 1996). The non-parametric Spearman coefficient of correlation was used to measure the intensity of association between viscosity and sulphate content of H. musciformis. The tests were carried out at p=0.05 level of statistical significance. Data are expressed as mean ± standard deviation (SD). May h 6 4 2 0 Jul Sep Nov Jan Mar May Jul Sep Nov Jan Mar May Fig. 2 Seasonality of H. musciformis carrageenan yield (percentage of carrageenan in relation to dry biomass extracted) from Praia Rasa at Búzios Town and from Praia Grande and Praia Vermelha, both at Sepetiba Bay. The vertical lines at the mean value bar represent standard deviations J Appl Phycol (2008) 20:1–8 Carrageenan (%) 4 52 47 42 37 32 27 22 52 47 42 37 32 27 22 Praia Rasa (epilithic) b a a a a a a Praia Grande a a b b b 52 47 42 37 32 27 22 Aug Oct Dec Feb Apr Jun 1994 Praia Rasa (epiphytic) 52 47 42 37 32 27 22 Praia Vermelha a a a a b b Aug Oct Dec Feb Apr Jun 1994 1995 1995 Results Carrageenan yield, viscosity and sulphate content Environmental factors The carrageenan yield varied from 21 to 48% in the four H. musciformis populations studied (Fig. 2). The higher carrageenan yield of epilithic specimens from Praia Rasa (one-way ANOVA, F=10.78, p<0.001, Tukey test) occurred in the summer (February 1995), and in the population of Fig. 3 Seasonality of the H. musciformis carrageenan viscosity from Praia Rasa at Búzios Town and from Praia Grande and Praia Vermelha, both at Sepetiba Bay. The vertical lines at the mean value bar represent standard deviations Viscosity (cP) Monthly changes in environmental factors (wind speed, precipitation, cloud cover, insolation, water and air temperature, length and period of waves) are shown in Fig. 1. 60 50 40 30 20 10 0 60 50 40 30 20 10 0 c Praia Rasa (epilithic) b a a a a Praia Grande b a a a b a Aug Oct Dec Feb Apr Jun 1994 1995 60 50 40 30 20 10 0 60 50 40 30 20 10 0 Praia Rasa (epiphytic) b b a a a a Praia Vermelha a a a b Aug Oct Dec Feb 1994 a b Apr Jun 1995 J Appl Phycol (2008) 20:1–8 5 Fig. 4 Total carrageenan dry mass calculated from H. musciformis carrageenan yield per m2 from each site based on the biomass obtained at each period of time and the percentage of carrageenan in this period collected at Praia Rasa at Búzios Town and Praia Grande and Praia Vermelha both at Sepetiba Bay. August 1994 to June 1995 shows the carrageenan means of this period Praia Grande (one-way ANOVA, F=19.57, p<0.001, Tukey test) in the winter (August 1994) and in the spring and summer of December 1994 and February 1995. Working with the population of Praia Vermelha (one-way ANOVA, F=12.61, p<0.001, Tukey test), carrageenan yield dropped twice, once in the spring (December 1994) and again in the fall (June 1995). The carrageenan yield of epiphytic specimens from Praia Rasa maintained itself constantly over the year (one-way ANOVA, F=1.41, p=0.29). Fig. 5 Sulfate content of H. musciformis carrageenan from epilithic and epiphytic specimens from Praia Rasa at Búzios Town and specimens from Praia Vermelha and Praia Grande at Sepetiba Bay. The vertical lines at the mean value bar represent standard deviations In relation to the viscosity of the gel obtained from this carragenophyte it varied from 4 to 53 cP (Fig. 3). The epilithic specimens from Praia Rasa varied during the sampling period (Kruskal-Wallis, H=12.2, p=0.02). The viscosity at the end of the spring (December 1994) was higher than the one obtained at the beginning of the season (October 1994). The epiphytic specimens from Praia Rasa showed two increases in viscosity (one-way ANOVA, F=43.86, p<0.001, Tukey test, log transformation), one in 6 the beginning of spring (October 1994) and another in the summer (February 1995). The specimens from Praia Grande (Kruskal-Wallis, H=13.54, p=0.02) showed an increase in the summer (February 1995) until spring (April 1995). At Praia Vermelha, two increases were observed (one-way ANOVA, F=9.14, p<0.001, Tukey test), one in the summer (February 1995) and another at the end of spring (June 1995) (Fig. 3). Considering the H. musciformis carrageenan yield per m2 calculated from the biomass and percentage of carrageenan obtained at each site and period of time (Fig. 4), usually the mean of carrageenan obtained through the year in Praia Grande and Praia Rasa was close to 10 g m−2, except in June at Praia Grande and from spring to summer at Praia Rasa. Only in autumn were the yields of specimens of Praia Vermelha more than that. Comparing the means from August 1994 to June 1995, the specimens from Praia Grande presented the highest mean (12.94 g m−2) followed by the specimens from Praia Rasa (9.39 g m−2) and Praia Vermelha (7.23 g m−2). In the infrared spectroscopy analysis of the samples of H. musciformis the absorbance bands were detected at 1.250, 930, 845 cm−1. The epilithic specimens from Praia Rasa (one-way ANOVA, p>0.05; Fig. 5) and the epiphytic specimens from Praia Vermelha (one-way ANOVA, p> 0.05; Fig. 5) did not present significant variations in this study. Meanwhile., in the specimens from Praia Grande (one-way ANOVA, p<0.001, Tukey test; Fig. 5) the sulphate content varied during the year. An increase in the ratio 930/2.920 cm −1 was observed in the summer (February 1995). The sulphate content in epiphytic specimens from Praia Rasa differed (one-way ANOVA, p< 0.001, Tukey test, Fig. 5), a decrease was observed in October 1994. Epiphytic specimens from Praia Rasa presented carrageenan sulphate content negatively correlated with viscosity of H. musciformis as observed in the ratio at 845/ 2.920 cm−1 (r=−0.59 p=0.009, n=18), at 930/2.920 cm−1 (r=−0.54, p=0.02, n=18) and at 1.250/2.920 cm−1 (r= −0.51, p=0.02, n=18). Discussion The carrageenan yield and quality of H. musciformis varied in the sites and sampling period searched in accordance with the hypothesis that yield and physical properties of carrageenan is affected by change in the environmental conditions (Durako and Dawes 1980; Bird et al. 1981). This fact shows the necessity of understanding the effect of these abiotic factors on algae responses to obtain good results in its exploitation. J Appl Phycol (2008) 20:1–8 The specimens with highest carrageenan yield and viscosity were collected at the site exposed to the open sea (Praia Rasa). This site when compared with the populations from Sepetiba Bay has the highest degree of wave movement (Reis and Yoneshigue-Valentin 1998). Durako and Dawes (1980) obtained similar results with two populations of H. musciformis from the east and west coasts of Florida. They suggested that specimens submitted to more exposed water movement produced more carrageenan to increase the flexibility of the thallus to face the strong water movement while John and Asare (1975) suggested that the maturity of the thallus was responsible for the lower carrageenan yield since higher values were obtained in young specimens and lower values in specimens with different ages (more stable sites). We believe that our result was related more to the necessity of flexibility and not the age of the algae. At this site, the epilithic and epiphytic specimens grew together in a place exposed to high wave motion in addition the epiphytic specimens with higher carrageenan yield which were more exposed to the strong ebb and flow of water in the fissure of the rocky shore. The difference in the carrageenan yield and viscosity of the H. musciformis from specimens growing on Praia Rasa in different habitats (epilithic and epiphytic populations) seems to be a consequence of their exposure to different environmental factors that also influenced the morphology of this algae (personal observation). At this site, a decrease in the H. musciformis epilithic biomass occurred at the end of spring and summer (Reis and Yoneshigue-Valentin 1998) that could be related to the increase in the carrageenan viscosity in spring (October and December 1994), when the abiotic factors in this period (low waves and high temperature) induced desiccation. Apparently, they constitute ecotypes with the epiphytic specimens producing more carrageenan to support high water motion, which causes fragmentation of the thallus, and the epilithic specimens were more viscous to be able to support desiccation in accordance with the hypothesis that carragenophyte produces more carrageenan to support local stress (Percival 1979; Durako and Dawes 1980; Kloareg and Quatrano 1988). Rees (1969) reported that polysaccharide conformation is under metabolic control. In this way, abiotic factors such as temperature and salinity (water precipitation) may be responsible for the changes in the viscosity and yield of H. musciformis carrageenan. In summer, all populations of H. musciformis showed an increase in carrageenan yield or viscosity probably due to unfavorable environmental conditions for its growth (water and air temperatures were high accompanied by heavy water precipitation). This result was observed in the Brazilian population in Bahia State (Wallner et al. 1992) but not in São Paulo State (Schenkman 1989). High temperatures were J Appl Phycol (2008) 20:1–8 considered negative to the growth of H. musciformis (Reis and Yoneshigue-Valentin 1998), and some authors suggested that carrageenan is very important for the survival of the plant in saline sites and that it is responsible for the ionic equilibrium of the cell (Percival 1979), due to the cation– anion balance to the negatively charged polysaccharides (Mariani et al. 1990). The higher total carrageenan yield obtained from the availability of total biomass of Hypnea m. collected from Praia Grande showed that the best commercial carrageenan yield should not be estimated without an analysis of changes in algal biomass at the site to be exploited. The negative correlation between the degree of sulphate content and carrageenan viscosity was observed only in epiphytic specimens from Praia Rasa. This inverse relationship is similar to the model correlating gel strength with low sulphate content and the presence of more units of 3.6 anhydrogalactose in the polymer chain (Yaphe and Duckworth 1972). These results also agree with those obtained with specimens in Senegal (Mollion 1979) and with cultivated specimens from ponds in Israel (Friedlander and Zelikovitch 1984). On the other hand, the H. musciformis population from Praia Vermelha and Praia Rasa epilithic specimens presented sulphate content constant during the year and fluctuations in carrageenan yield and viscosity. These results do not fit the model mentioned above (Yaphe and Duckworth 1972; Mollion 1979; Friedlander and Zelikovitch 1984), but they agree with the results obtained with other carragenophyte, Agardhiella subulata, that showed parallel behavior in sulphate and 3.6 anhydrogalactose content (Craigie 1990) and were identical to the results obtained with Gracilaria tikvshiae Mc Lachlan (Bird et al. 1981). Only the characteristic kappa carrageenan absorbance bands at 1.230, 930, 845 cm−1 (McCandless and Gretz 1984) were detected in H. musciformis by the infrared spectroscopy analysis and the band at 805 cm−1 associated to iota carrageenan (Rochas et al. 1986) was not detected in our samples. No spatial and temporal variations of the carrageenan type were detected in the populations studied here. This is in accordance with the results of H. musciformis analyzed along the Brazilian coastline by Saito and Oliveira (1990) which are in disagreement with the results of Senegal specimens (Mollion 1979). In summary, we suggest that water movement, desiccation (caused by the frequency with specimens is emerged), low salinity (water precipitation) and extreme water and air temperatures were the main abiotic factors that influenced the viscosity and yield of carrageenan of H. musciformis from Rio de Janeiro. We also propose that there was a balance between carrageenan yield and quality produced by the algae for their protection against undesirable environmental factors and when the algae needs more protection the carrageenan 7 produced is more viscous. 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