Absorption of metals and characterization of chemical elements present in three species of Gracilaria (Gracilariaceae) Greville: a genus of economical importance

Absorption of metals and characterization of chemical elements present in three species of Gracilaria (Gracilariaceae) Greville: a genus of economical importance

Angela P. Tonon^*,I; Mariana C. Oliveira^II; Eliane M. Soriano^III; Pio Colepicolo^I

^IDepartamento de Bioquímica, Instituto de Química, Universidade de São Paulo, Av. Prof. Lineu Prestes, 748, Butantã, 05508-900 São Paulo-SP, Brazil

^IIDepartamento de Botânica, Instituto de Biociências, Universidade de São Paulo, Rua do Matão, Trav. 14, nº 321, Cidade Universitária,, 05508-900 São Paulo-SP, Brazil.

^IIIDepartamento de Oceanografia e Limnologia, Universidade do Rio Grande do Norte, Praia de Mãe Luiza s/n, Via Costeira, 59014-100 Rio Grande do Norte-RN, Brazil

ABSTRACT

Gracilaria Greville is a genus of seaweed that is economically explored by the cosmetic, pharmaceutical and food industries. One of the biggest problems associated with growing Gracilaria is the discharge of heavy metals into the marine environment. The absorption of heavy metals was investigated with the macroalga Gracilaria tenuistipitata Zhang et Xia, cultivated in a medium containing copper (Cu) and cadmium (Cd). In biological samples, EC50 concentrations of 1 ppm for cadmium and 0.95 ppm for copper were used. These concentrations were based on seaweed growth curves obtained over a period of six days in previous studies. ICP-AES was used to determine the amount of metal that seaweeds absorbed during this period. G. tenuistipitata was able to bioaccumulate both metals, about 17% of copper and 9% of cadmium. Basal natural levels of Cu were found in control seaweeds and in G. tenuistipitata exposed to Cd. In addition, the repertoire of other important chemical elements, as well as their concentrations, was determined for G. tenuistipitata and two other important seaweeds, G. birdiae Plastino & Oliveira and G. domingensis (Kützing) Sonder ex Dickie, collected in natural environments on the Brazilian shore.

Keywords: copper, cadmium, Gracilaria, metal absorption, bioactive, bioremediation

Introduction

The genus Gracilaria (Greville), a red macroalgae of the family Gracilariaceae (Rhodophyta) is extensively used in industry, due to its production of secondary metabolites of economic impact (Lee et al., 1999). This genus has high commercial value as a source of agar, a sulphated hydrocolloid (McHugh, 1991; Falcão et al., 2008). In addition, many secondary metabolic compounds have been isolated from Gracilaria and applied as antitumorals, antimicrobials and antioxidants. The majority of these are terpenes, but several fatty acids are also common as well as nitrogenous compounds (Van Alstyne & Paul, 1988; Cardozo et al., 2007; Boobathy et al., 2010; Zandi et al., 2010; Jain et al., 2010; Falcão et al., 2010). One of the greatest difficulties in Gracilaria cultivation is the gradual increase in the discharge of heavy metals and other pollutants into the environment. Marine organisms, including phytoplankton and macroalgae, are directly exposed to these chemical elements, which, due to their various degrees of toxicity, constitute an impediment to the development of the organisms, leading to a decrease in both growth and biodiversity in the environment (Pinto et al., 2003; Torres et al., 2008).

Brazil has several natural seaweed beds and various species of Gracilaria of economic importance. This, together with an extensive coastline, provides opportunities for their cultivation. Therefore, a screening of the concentrations of various chemical elements was undertaken for G. birdiae Plastino & Oliveira and G. domingensis (Kützing) Sonder ex Dickie in their natural environments. Because these species are already being studied and commercialized, they are worthy of special note.

In the present work, we estimated the absorption of copper (Cu) and cadmium (Cd) and the content of chemical elements in three species of Gracilaria: Gracilaria tenuistipitata, cultivated in our laboratory and exposed to Cu and Cd, and G. birdiae and G. domingensis collected in their natural environments.

Materials and Methods

Growth conditions

Specimens of G. tenuitipitata were cultivated in seawater enriched with von Stosch solution (Edwards, 1970) at a temperature of 23 ºC, with a plant density of 10 g.L^-1, under a light/dark cycle of 12-12 h at an irradiance of 100 µmol photon.m^-2 s^-1, provided by a fluorescent lamp. The culture medium was renewed every six days and presented a pH 7.5.

EC50 determination for Cu and Cd

The EC50 (Median Effective Concentration) was used in the toxicity-bio assays of these two metals. The methodology employed for seaweed exposure to metals and for the estimation of toxicological parameters was performed according to international protocol (EPA, OECD). The EC50 values were 0.95 ppm for Cu and 1 ppm for Cd. These values were encountered based on previous studies of the growth curve of these seaweeds during six days of treatment (Neto, 2008).

Experimental conditions

In order to evaluate the effects of Cu and Cd on G. tenuistipitata, the starting point was six days of exposure to EC50 of the metals. Samples of seaweed were added to the culture medium (von Stosch medium, 60% of filtered and sterilized sea water and 40% of MilliQ water), together with concentration of each metal equivalent to its EC50. The salts used were CdCl₂ and CuSO₄. The amount of 10 g of seaweed was inoculated in 2 L of culture medium with the metal solution already added. The two treatments and the control were performed in triplicate. The conditions of light and temperature were similar to the conditions used in the laboratory culture.

After six days of exposure to the metals, triplicate samples of the seaweeds were collected for each of the three experimental conditions: control (normal culture conditions), exposure to Cu and exposure to Cd. In addition, three culture medium controls were used; thus, culture medium diluted in sea water was measured 1) before the addition of the metals, 2) after the addition of the metals, and 3) after six days of exposure of the algae to the metals. Thus, it was possible to measure the amount of Cd and Cu in the culture medium before and after adding the metals and at the end of the exposure of the seaweeds. Aliquots were analyzed in triplicate for each of the controls (1, 2 and 3, above).

Inductively Coupled Plasma Atomic Emission Spectroscopy (ICP-AES)

Metal analysis was performed with an atomic emission spectrophotometric system with an inductively linked plasma source (ICP-AES, Genesis, SOP model). Five standard were used to so obtain complete adjustment of the apparatus. The standard curves were obtained using concentrations of 0, 1, 2, 5 and 10 ppm for both Cu and Cd to convert from counts [cps] to concentration [ppm] (Figure 1). Data were analyzed by using Smart Analyzer software.

The other chemical elements Al, Ca, Fe, Mg, P, Si, Mn, B, K, Na, Ba, Ni, Zn, Sr, Cr, Co and As were also analyzed by ICP-AES. The same procedure mentioned above was used to calibrate the response for each element. Two species of Brazilian Gracilaria (G. birdiae and G. domingensis) were used for comparison with G. tenuistipitata. The Brazilian species were collected in their natural coastal environments at Cotovelo (05°56'S-35°09'W) and Rio do Fogo beaches (05°16'S-35°22'W), Rio Grande do Norte (RN).

Results

Absorption of Cu and Cd

Figure 2 shows the atomic emission spectrum (counts per second [cps] versus wavelength λ [nm]) for Cu. Spectra 1 and 2 represent, respectively, the 1 ppm Cu standard and the seaweed exposed to Cu. The basal concentrations of Cu in seaweed exposed to Cd and in the control are indicated by spectra 3 and 4, respectively. In the atomic emission spectrum for Cd (Figure 3), no other representative emissions were detected in spectra 1 or 2 besides those of Cd in the 1 ppm standard and in algae exposed to Cd, respectively.

From the analysis of the ICP-AES results, the means and standard deviations of the concentrations of metals absorbed by seaweeds treated with Cu, Cd and control were obtained from experiments performed in triplicate. In the case of seaweed exposed to Cu, concentrations of Cd were also measured to serve as a control in the experiment. The same was done for seaweed exposed to Cd, in which Cu was measured. The concentrations of both metals (Cu and Cd) were also measured for the control alga. The data presented in Table 1, based on the calibration curves in Figure 1, were extracted from an analysis of the ICP-AES spectra. The amount of Cu absorbed by the seaweed was obtained by subtracting the basal amount from the total amount for a 10 g sample, resulting in 0.325 mg (Table 1). This result is in accord with the value obtained from the difference between the initial and final amounts of Cu in the medium (0.34 mg). The same analysis, carried out for Cd, gave an absorbed amount of 0.204 mg.

Figure 4 shows the comparison between the amount of metals found in the culture medium before and after exposure of the algae, as well as the values of the amounts absorbed by the alga (calculated from the analysis of the ICP-AES results, Table 1). The percentage of metal absorption is also shown. Note that G. tenuistipitata is able to absorb about two-fold more Cu as compared with Cd.

Elemental composition of three species of Gracilaria

The results of the screening for chemical elements detected by ICP-AES can be observed in Figure 5; the mean and standard deviation of each chemical element are shown in Table 2. For the three Gracilaria species, the concentration levels were different for some of the elements analyzed. For example, the concentration of aluminum (Al) in G. tenuistipitata was less than half of that in the other two species. The concentration of calcium (Ca) was two-fold higher in G. domingensis than in the other two species, while the concentration of manganese (Mn) was two-fold higher in G. birdiae than in G. tenuistipitata and G. domingensis. Cu was found in basal concentrations in all three species analyzed, whereas Cd was absent in all seaweed samples. Even though the elements Ba, Ni, Zn, Sr, Cr, Co, As and Cd were analyzed, none were detected in the three species.

Discussion

After exposing G. tenuistipitata to Cd and Cu during six days, the bioaccumulation these metals by the alga became evident. G. tenuistipitata was able to accumulate higher concentrations of Cu as compared to Cd. A plausible explanation for this difference might be the capacity of Cu to be transported into the cell. Since it is essential for seaweed metabolism, there might possibly exist specific transporters for this metal. In Arabidopsis thaliana, five members of a family of Cu transporters (COPT1-5) have been described and, in Saccharomyces cerevisiae, a gene (CTR1) that codifies proteins of the plasmatic membrane that transports Cu into the cell has been characterized (Dancis et al., 1994; Grotz, 1998; Himelblau & Amasino, 2000; Sancenon et al., 2003; Markossian & Kurganov, 2003). Basal natural concentrations of Cu were present in the control seaweeds (with and without Cd). Even without deliberate exposure. Cu is an essential metal for G. tenuistipitata, both as an enzymatic co-factor (amine oxidase and cytocrome c oxidase) and as an electron carrier in the process of photosynthesis (plastocyanin) (Gledhill et al., 1997; Collen et al., 2003). It is noteworthy that Cu is not a component of the von Stosch medium, i.e., the basal concentrations of this metal presumable reflect accumulation from sea water. Nonetheless, studies with terrestrial plants and green unicellular algae demonstrated that Cu in excess becomes toxic to these organisms, leading to production of Reactive Oxygen Species (ROS), which are detrimental to plant-growth (Charrier et al., 2008). Therefore, upon exposition of G. tenuistipitata to Cu in excess, cellular, biochemical and molecular responses are expected, leading to acclimatization of the seaweed to this condition.

In contrast, Cd has no known biological function in the metabolism of G. tenuistipitata. Stohs & Bagchi (1995) demonstrated that there is evidence of its transport into the cells. In terrestrial plants, it has been demonstrated that Cd competes for divalent ion carriers and can be transported with protons and type P ATPases. Studies on the biological effects of Cd are scarce (Hirschi et al., 2000). Stohs & Bagchi (1995) suggest that Cd ions dislocate zinc and iron from proteins. The liberation of iron ions might then induce the Fenton reaction, generating ROS and leading to total oxidative stress, with deleterious consequences for seaweed growth. Many toxic heavy metals, such as Cd, mercury (Hg) and lead (Pb), are able to compete for essential metal carriers during absorption (Guerrinot, 2000). Nevertheless, our results indicate that G. tenuistipitata, is a metal bio-accumulating organism.

The differences between the three species in the concentrations of the various elements in Table 2, may be due to physiological, biochemical or genetic differences between the seaweeds or to different acclimatization events that occurred in their environments or microenvironments (Figure 5). For example, Mn was present in a higher concentration in G. birdiae than in the other two species. Ca and K concentrations were higher in G. domingensis than in G. tenuistipitata and G. birdae. These results are relevant for future studies of the cultivation of macroalgae, both in tanks and the open sea, and for ecological, physiological, biochemical and molecular studies of bioremediation and bioprospection. The ICP-AES technique proved to be efficient for determining trace and ultra-trace amounts of metals, making it possible to compare the repertory of chemical elements necessary for the survival and growth of the three algal species investigated.

Acknowledgements

The authors thank Dra. Franciane Pellizzari for English suggestions. This work was supported by Fundação de Amparo à Pesquisa do Estado de São Paulo, Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Conselho Nacional de Desenvolvimento Científico e Tecnológico, Ministério da Saúde, Ministério de Ciência e Tecnologia and CNPq-INCT-Redoxoma.

References

Boobathy S, Soundarapandian P, Prithivraj M, Gunasundari V 2010. Biochemical characterization of protein isolated from seaweed, Gracilaria edulis. Curr Res J Biol Sci 2: 35-37.

Cardozo KHM, Guaratini T, Barros MP, Falcão VR, Tonon AP, Lopes NP, Campos S, Torres MA, Souza AO, Colepicolo P, Pinto E 2007. Metabolites from algae with economical impact. Comp Biochem Physiol C-Toxicol Pharmacol 146: 60-78.

Charrier B, Coelho SM, Le Bail A, Tonon T, Michel G, Potin P, Kloareg B, Boyen C, Peters AF, Cock JM 2008. Development and physiology of the brown alga Ectocarpus siliculosus: two centuries of research. New Phytol 177: 319-332.

Collen J, Pinto E, Pedersen M, Colepicolo P 2003. Induction of oxidative stress in the red macroalga Gracilaria tenuistipitata by pollutant metals. Arch Environ Contam Toxicol 45: 337-342.

Dancis A, Haile D, Yuan DS, Klausner RD 1994. The Saccharomyces cerevisiae copper transport protein (Ctr1p). Biochemical characterization, regulation by copper, and physiologic role in copper uptake. J Biol Chem 269: 25660-25667.

Edwards P 1970. Illustrated guide to the seaweeds and sea grasses in the vicinity of Port Aransas, Texas. Contr Mar Sc 15: 1-128, apud Rev Brasil Bot 24: 491-500, 2001).

Falcão VR, Tonon AP, Oliveira MC, Colepicolo P 2008. RNA Isolation method for polysaccharide rich algae: agar producing Gracilaria tenuistipitata (Rhodophyta). J Applied Phycol 20: 9-12.

Falcão VR, Nyvall P, Collen J, Pedersén M, Oliveira MC, Colepicolo P 2010. Molecular characterization of nitrate reductase gene and its expression in the marine red alga Gracilaria tenuistipitata (Rhodophyta). J Applied Phycol 22: 613-622.

Gledhill M, Nimmo M, Hill SJ, Brown MT 1997. The toxicity of copper (II) species to marine algae, with particular reference to macroalgae. J Phycol 33: 2-11.

Grotz N, Fox T, Connolly E, Park W, Guerinot ML, Eide D 1998. Identification of a family of zinc transporter genes from Arabidopsis that respond to zinc deficiency. Proc Natl Acad Sci USA 95: 7220-7224.

Guerinot ML 2000. The ZIP family of metal transporters. Biochim Biophys Acta-Biomembr 1465: 190-198.

Himelblau E, Amasino RM 2000. Delivering copper within plant cells. Curr Opin Plant Biol 3: 205-210.

Hirschi KD, Korenkov VD, Wilganowski NL, Wagner GJ 2000. Expression of Arabidopsis CAX2 in tobacco. Altered metal accumulation and increased manganese tolerance. Plant Physiol. 124: 125-134.

Jain K, Sasidharan S, Darah I 2008. In vivo and in vitro antifungal activity of the methanol extract from Gracilaria changii. http://www.britannica.com/bps/additionalcontent/18/34252431/In-vitro-and-In-vivo-antifungal-activity-of-the-methanol-extract-from-Gracilaria-angii/fulltext, access February 2011.

Lee TM, Chang YC, Lin YH 1999. Differences in physiological responses between winter and summer Gracilaria tenuistipitata (Gigartinales, Rhodophyta) to varying temperature. BBAS 40: 93-100.

Markossian KA, Kurganov BI 2003. Copper chaperones, intracellular copper trafficking proteins. Function, structure, and mechanism of action. Biochem-Moscow 68: 827-837.

McHugh DJ 1991. Worldwide distribution of commercial resources of seaweeds including Gelidium. In Hydrobiologia International Worshop on Gelidium 1. ed. Dordrecht: Kluwer Academic Publishers, p.10-29.

Neto AMP 2007. Efeito de Poluentes Metálicos nos Níveis de Pigmentos Fotossintéticos Presentes em Algas marinhas e Avaliação do Papel Estrutural de Carotenos em Membranas Miméticas. São Paulo, 139 p. Tese de Doutorado. Instituto de Química, Universidade de São Paulo.

Pinto E, Sigaud-Kutner TCS, Leitao MAS, Okamoto OK, Morse D, Colepicolo P 2003. Heavy metal-induced oxidative stress in algae. J Phycol 39: 1008-1018.

Sancenon V, Puig S, Mira H, Thiele DJ, Penarrubia L 2003. Identification of a copper transporter family in Arabidopsis thaliana. Plant Mol Biol 51: 577-587.

Stohs SJ, Bagchi D 1995. Oxidative mechanisms in the toxicity of metal ions. Free Radic Biol Med 18: 321-36.

Torres MA, Barros, Marcelo P, Campos SCG, Pinto E, Rajamani S, Sayre RT, Colepicolo P 2008. Biochemical biomarkers in algae and marine pollution: a review. Ecotoxicol Environ Saf 71: 1-15.

Val Alstyne KL, Paul VJ 1988. The role of secondary metabolites in marine ecological interactions. Proc 6^th Int Coral Reef Symposium1:175-186. Townsville, Australia.

Zandi K, Tajbakhsh S, Nabipour I, Rastian Z, Yousefi F, Sharafian S, Sartavi K 2010. In vitro antitumor activity of Gracilaria corticata (a red alga) against jurkat and molt-4 human cancer cell lines. Afr J Biotechnol 9: 6787-6790.

Received 18 Jan 2011

Accepted 10 Feb 2011

References

• Boobathy S, Soundarapandian P, Prithivraj M, Gunasundari V 2010. Biochemical characterization of protein isolated from seaweed, Gracilaria edulis. Curr Res J Biol Sci 2: 35-37.
• Cardozo KHM, Guaratini T, Barros MP, Falcão VR, Tonon AP, Lopes NP, Campos S, Torres MA, Souza AO, Colepicolo P, Pinto E 2007. Metabolites from algae with economical impact. Comp Biochem Physiol C-Toxicol Pharmacol 146: 60-78.
• Charrier B, Coelho SM, Le Bail A, Tonon T, Michel G, Potin P, Kloareg B, Boyen C, Peters AF, Cock JM 2008. Development and physiology of the brown alga Ectocarpus siliculosus: two centuries of research. New Phytol 177: 319-332.
• Collen J, Pinto E, Pedersen M, Colepicolo P 2003. Induction of oxidative stress in the red macroalga Gracilaria tenuistipitata by pollutant metals. Arch Environ Contam Toxicol 45: 337-342.
• Dancis A, Haile D, Yuan DS, Klausner RD 1994. The Saccharomyces cerevisiae copper transport protein (Ctr1p). Biochemical characterization, regulation by copper, and physiologic role in copper uptake. J Biol Chem 269: 25660-25667.
• Edwards P 1970. Illustrated guide to the seaweeds and sea grasses in the vicinity of Port Aransas, Texas. Contr Mar Sc 15: 1-128,
• apud Rev Brasil Bot 24: 491-500, 2001).
• Falcão VR, Tonon AP, Oliveira MC, Colepicolo P 2008. RNA Isolation method for polysaccharide rich algae: agar producing Gracilaria tenuistipitata (Rhodophyta). J Applied Phycol 20: 9-12.
• Falcão VR, Nyvall P, Collen J, Pedersén M, Oliveira MC, Colepicolo P 2010. Molecular characterization of nitrate reductase gene and its expression in the marine red alga Gracilaria tenuistipitata (Rhodophyta). J Applied Phycol 22: 613-622.
• Gledhill M, Nimmo M, Hill SJ, Brown MT 1997. The toxicity of copper (II) species to marine algae, with particular reference to macroalgae. J Phycol 33: 2-11.
• Grotz N, Fox T, Connolly E, Park W, Guerinot ML, Eide D 1998. Identification of a family of zinc transporter genes from Arabidopsis that respond to zinc deficiency. Proc Natl Acad Sci USA 95: 7220-7224.
• Guerinot ML 2000. The ZIP family of metal transporters. Biochim Biophys Acta-Biomembr 1465: 190-198.
• Himelblau E, Amasino RM 2000. Delivering copper within plant cells. Curr Opin Plant Biol 3: 205-210.
• Hirschi KD, Korenkov VD, Wilganowski NL, Wagner GJ 2000. Expression of Arabidopsis CAX2 in tobacco. Altered metal accumulation and increased manganese tolerance. Plant Physiol. 124: 125-134.
• Jain K, Sasidharan S, Darah I 2008. In vivo and in vitro antifungal activity of the methanol extract from Gracilaria changii http://www.britannica.com/bps/additionalcontent/18/34252431/In-vitro-and-In-vivo-antifungal-activity-of-the-methanol-extract-from-Gracilaria-angii/fulltext, access February 2011.
• Lee TM, Chang YC, Lin YH 1999. Differences in physiological responses between winter and summer Gracilaria tenuistipitata (Gigartinales, Rhodophyta) to varying temperature. BBAS 40: 93-100.
• Markossian KA, Kurganov BI 2003. Copper chaperones, intracellular copper trafficking proteins. Function, structure, and mechanism of action. Biochem-Moscow 68: 827-837.
• McHugh DJ 1991. Worldwide distribution of commercial resources of seaweeds including Gelidium. In Hydrobiologia International Worshop on Gelidium 1. ed. Dordrecht: Kluwer Academic Publishers, p.10-29.
• Neto AMP 2007. Efeito de Poluentes Metálicos nos Níveis de Pigmentos Fotossintéticos Presentes em Algas marinhas e Avaliação do Papel Estrutural de Carotenos em Membranas Miméticas São Paulo, 139 p. Tese de Doutorado. Instituto de Química, Universidade de São Paulo.
• Pinto E, Sigaud-Kutner TCS, Leitao MAS, Okamoto OK, Morse D, Colepicolo P 2003. Heavy metal-induced oxidative stress in algae. J Phycol 39: 1008-1018.
• Sancenon V, Puig S, Mira H, Thiele DJ, Penarrubia L 2003. Identification of a copper transporter family in Arabidopsis thaliana. Plant Mol Biol 51: 577-587.
• Stohs SJ, Bagchi D 1995. Oxidative mechanisms in the toxicity of metal ions. Free Radic Biol Med 18: 321-36.
• Torres MA, Barros, Marcelo P, Campos SCG, Pinto E, Rajamani S, Sayre RT, Colepicolo P 2008. Biochemical biomarkers in algae and marine pollution: a review. Ecotoxicol Environ Saf 71: 1-15.
• Val Alstyne KL, Paul VJ 1988. The role of secondary metabolites in marine ecological interactions. Proc 6^th Int Coral Reef Symposium1:175-186. Townsville, Australia.
• Zandi K, Tajbakhsh S, Nabipour I, Rastian Z, Yousefi F, Sharafian S, Sartavi K 2010. In vitro antitumor activity of Gracilaria corticata (a red alga) against jurkat and molt-4 human cancer cell lines. Afr J Biotechnol 9: 6787-6790.

Publication Dates

• Publication in this collection
  15 Apr 2011
• Date of issue
  Apr 2011

History

• Received
  18 Jan 2011
• Accepted
  10 Feb 2011