Chemical compositon of three microalgae species for possible use in mariculture

BIOLOGICAL AND APPLIED SCIENCES

Chemical compositon of three microalgae species for possible use in mariculture

Alfredo Matos Moura Junior^I; Egídio Bezerra Neto^II; Maria Luise KoeningIII, ^*; Enide Eskinazi Leça^IV

^IColégio de Aplicação; Universidade Federal de Pernambuco; Av. Acadêmico Hélio Ramos, s/n; Cidade Universitária; [e-mail]; Recife - PE - Brasil

^IIDepartamento de Química; Universidade Federal Rural de Pernambuco; Recife - PE - Brasil

^IIIDepartamento de Oceanografia; Universidade Federal de Pernambuco; Recife - PE - Brasil

^IVPrograma de Pós-Graduação em Botânica; Universidade Federal Rural de Pernambuco; Recife - PE - Brasil

ABSTRACT

The purpose of this study was to determine the chemical composition of Chaetoceros gracilis, Cylindrotheca closterium and Tetraselmis gracilis, species frequently used as food for aquaculture in the northeast of Brazil. The species were grown in f/2 medium, in carboys, aerated from one liter, with constant illumination and analyses were made in late exponential growth phase. Cellular density (8,447 x 10 ⁶ cell.L^-1), soluble carbohydrates (0.10±0.01 mg.L^-1) and magnesium (14.78±0.08 mg.L^-1) were the highest in C. gracilis. C . closterium had the highest amount of chlorophyll a (13.76±1.11 mg.L^-1), soluble proteins (0.62±0.04 mg.L^-1), total amino acids (23.82±0.84 mg .L^-1), nitrate (0.36±0.02 mg.L^-1), sodium (0.46±0.05 mg.L^-1) and phosphorus (2.80±0.38 mg.L^-1). Both the species had potassium levels of 0.02 mg.L^-1 and sulfur levels of 0.03 mg.L^-1. T. gracilis had the lowest values in almost all the analyzed variables, except for chlorophyll a. Therefore, among the analyzed species and tested conditions, C. closterium represented the best nutritional option for aquaculture projects.

Key words:Chaetoceros gracilis, Cylindrotheca closterium, Tetraselmis gracilis, chemical composition

RESUMO

Este trabalho objetivou a determinação da composição química de Chaetoceros gracilis, Cylindrotheca closterium e Tetraselmis gracilis, espécies de freqüente uso na aqüicultura no Nordeste do Brasil. As espécies foram cultivadas em meio f/2-Guillard, em garrafão de vidro, aeradas a partir de um litro, com iluminação constante e as análises foram realizadas no final da fase exponencial. C. gracilis apresentou maior densidade celular, chegando a 8.442x10⁶ cel.L^-1, o maior valor para carboidratos solúveis (0,10 ± 0,01 mg.L^-1) e magnésio (14,78 ± 0,08 mg.L^-1). C. closterium apresentou a maior concentração para clorofila a (13,76 ± 1,11 mg.L^-1), proteína solúvel (0,62 ± 0,04 mg.L^-1), aminoácidos totais livres (23,82 ± 0,84 mg.L^-1), nitrato (0,36 ± 0,02 mg.L^-1), sódio (0,46 ± 0,05 mg.L^-1) e fósforo (2,80 ± 0,38 mg.L^-1). Os teores de potássio (0,02 mg.L^-1) e enxofre (0,03 mg.L^-1) foram iguais para C. gracilis e C. closterium. A espécie T. gracilis apresentou os menores valores nas variáveis analisadas, exceto para clorofila a. Apresentando-se, portanto, entre as três espécies analisadas, C. closterium como a melhor opção nutricional, dentre as condições observadas, para projetos em aqüicultura.

INTRODUCTION

With the development of aquaculture in the world and the necessity to increase the animals survival in culture, studies have shown the importance of food production for cultivated species development. Knowledge of the natural feeding habits and digestive system of organisms is an essential factor for the attainment of high productivity in an aquaculture setting. This, in turn, depends on the food efficiency of different trophic levels (Sipaúba-Tavares and Rocha, 2001). The importance of the success of unialgae mass cultures consists in the great variety of fields, such as genetic, cytology, taxonomy and plant physiology, that can utilize this resource (Sukenik and Whanon, 1991; Fidalgo et al., 1998; Renaud et al., 1999).

Microalgae chemical composition is frequently determined with the objective to provide the necessary nutritional balance for the captive animals (Whyte, 1987; Brown et al. 1997, 1998 Southgate et al., 1998;; Caers et al., 1999; McCausland et al., 1999), and also to determine the biochemical variation of the microalgae composition with respect to the nutritional medium in which the microalgae are being cultivated (Antia et al., 1977; Fabregas et al., 1985a and b, 1986; Melo et al., 1993; Lourenço et al., 1997).

In Brazil, the studies about microalgae chemical composition are recent and limited to studies about alternative culture mediums (Koening et al., 1990a, 1998; Melo et al., 1993) or to the comparisons of the microalgae chemical composition in relation to the nutritional medium (Koening, 1990b; Lourenço et al., 1998). Considering the substantial growth of aquaculture activities in Brazil (8% increase per year), according to Sipaúba-Tavares and Rocha (2001), it is necessary to increase the studies about microalgae nutritional value. The purpose of this work was to determine the chemical composition of Chaetoceros gracilis, Cylindrotheca closterium and Tetraselmis gracilis, three widely used microalgae for the feeding of aquaculture fish and shrimp in the northeast region of Brazil. The results of this study will aid aquaculture farmers in choosing microalgae for its nutritional value.

MATERIAL AND METHODS

Chaetoceros gracilis, Cylindrotheca closterium and Tetraselmis gracilis were cultivated in triplicate. Cultures were placed in aerated carboys (8 L) filled with f/2-Guillard medium (Guillard, 1975). Two daylight fluorescent tubes of 20 W were used to maintain constant illumination and culture temperature was 22±1 ºC. The experiments started with microalgae culture in the exponential growth phase and were kept in 10 mL assay pipes, with the same light, temperature and culture medium conditions. The three species were inoculated in 250 mL glass bottles with f/2-Guillard medium. Every eight days, this volume was duplicated with autoclaved seawater (1 atm for 1 h) and enriched with the same components of the f/2 medium until the total of 8 liters of culture was reached. After eight days the cultures were harvested for the chemical analyses.

Cellular density was counted daily for the first eight days of the experiment to determine the growth of each microalgae. The final cellular density was determined at the end of the experiment. These analyses were performed using a binocular Zeiss microscope, with Neubauer chambers from samples preserved with 4 % buffered formaldehyde. The cellular density result was presented as cell.L^-1.

Chlorophyll a was determined according to Becker (1995). Three replicates from each species were filtered in Whatmann GF/F fiberglass filters (diameter = 47 mm), extracted with 90 % acetone and analyzed with a spectrophotometer. The remaining part of each culture was centrifuged at 7,000 rpm for 15 minutes. The centrifuged material was collected and dried in an oven (60 ºC) until constant weight, according to Bezerra Neto et al. (1994).

For soluble carbohydrate, soluble protein and nitrate determinations, 1.0 g of dried, preweighed microalgae and 10 mL of distilled water were used to prepare an extract. The soluble carbohydrate was determined according to Yemm and Wills (1954). For this, a 200 µL sample of the extract was transferred to an assay tube. Then, 2 mL of the anthrone reagent was added for the color development, with the tubes placed in a hot water bath at 100 ºC, for 10 minutes to develop the color. The samples were then analyzed at 620nm in a spectrophotometer and compared with standard glucose solution (concentration from 0 to 300 mg.L^-1).

Soluble protein was determined according to Bradford (1976). Samples (100 µL) were collected from the microalgae extracts and transferred to assay tubes, which contained 2.0 mL of the Coomassie brillant blue reagent. After color development, spectrophotometric readings at 595 nm were determined and compared with the results from solutions of bovine albumen standards (BSA) in concentration from 0 to 200 mg.L^-1. Free total amino acids were determined according to Yemm and Cocking (1955).

Nitrate analysis was carried out according to Cataldo et al. (1975). From each extract, 200 µL was transferred to assay tubes and 800 µL of salicilic acid reagent (5 % H₂SO₄) was added to each assay tube. Twenty minutes later, 18 mL of NaOH (2 N) was added to the sample. Spectrophotometric readings at 410 nm were determined. Results were compared with standard nitrate solution in concentration from 0 to 300 mg.L^-1.

Sodium and magnesium were determined by the atomic absorption spectrophotometry as described by Malavolta et al. (1989). Sulphur was determined by the turbidimetry according to Sarruge and Haag (1974). Phosphorus was measured by molibdo-vanadato of ammonium colorimetric method and potassium was determined in a flame photometer, according to Bezerra Neto et al. (1994).

Data were treated statistically by the analysis of variance (ANOVA) with µ= 0.05. Additionally, a Tukey test was performed and ran in the SANEST program (Sarriés et al., 1992).

RESULTS AND DISCUSSION

Cellular density

Chaetoceros gracilis, Cylindrotheca closterium and Tetraselmis gracilis have been used successfully as a nourishing source for a variety of clams and other organisms (Sukenik and Wahnon, 1991; McCausland et al., 1999). The growth curves for C. gracilis and T. gracilis from the present study are presented in Figure 1A and 1B. A cell count for C. closterium could not be accurately determined because of cell clumping. This difficulty was also noted by Brown (1991).

C. gracilis cellular density at the end of the culture cycle presented the best result, reaching 8,442±7.07x10⁶ cell.L^-1,while T. gracilis reached 562±19.45x10⁶ cell.L^-1, with a significant difference between species (P<0.05). Similar cellular density values for C. gracilis (95-6,415x10⁶ cell.L^-1) were also determined by Yamashita and Magalhães (1984) using FeNS as the growth medium. Lourenço et al. (1997) cultivating T. gracilis in Conway medium, found highest cellular density values (1,71x10⁹ cell.L^-1). The high densities were probably because Conway medium was more nutritive than f2-Guillard.

Chemical composition

One of the best parameters to monitor microalgae production is the estimation of growth, generally expressed in biomass and density increase, proteins, pigments, carbohydrates contents over a certain period of time (Becker, 1995). As it happens in any superior vegetable, microalgae biomass and chemical composition can vary according to environment conditions and the age of the culture (Lourenço et al., 1998; Renaud et al., 1999; Araújo, et al., 2005). In C. gracilis cultures, pH varied from 7.7 to 9.5 and from 7.5 to 10 in T. gracilis cultures.

The highest chlorophyll a mean value was found in C. closterium (13.76±1.1 mg.L^-1), followed by T. gracilis (7.12±0.61 mg.L^-1) and C. gracilis (3.28±0.82 mg.L^-1), with significant differences among the species (P<0.05) (Figure 2A). These values were higher than those found by Lourenço et al. (1997), who cultivated T. gracilis in Conway medium (1.51-3.57 mg.L^-1) and by Brown (1991), who cultivated C. gracilis in f/2 (0.78 pg.cel^-1 or 1.48 mg.L^-1). High chlorophyll a values were probably due to the high cell density, which diminished irradiation inside the carboy, leading to the increased production of chlorophyll a (López-Muñoz et al., 1992; Sauodi-Helis et al., 1999; Valenzuela-Espinoza et al., 2002).

C. gracilis had the highest amount of soluble carbohydrates, with a value of 0.1±0.01 mg.L^-1, followed by C. closterium (0.09±0.01 mg.L^-1) and T. gracilis (0.06±0.01 mg.L^-1), with significant differences between the first two (P<0.05) (Figure 2B). These values were considered low for the analyzed culture phase (final exponential phase where the species tended to accumulate carbohydrates), due to nitrogen limitation during the log phase for the protein synthesis, reducing the amount of soluble protein, as also observed by Brown (1991), Brown et al. (1997) and Renaud et al. (1999). This was also observed in this work in relation to soluble protein (Figure 2C), where the highest value was found in C. closterium (0.62±0.04 mg.L^-1), followed by C. gracilis (0.58±0.02 mg.L^-1) and T. gracilis (0.38±0.04 mg.L^-1). There was a significant difference between the first two and the third (P<0.05).

The total free amino acid also showed this trend (Figure 2D), with significant differences among the species (P<0.05) (C. closterium: 23.28±0.84 mg.L^-1, C. gracilis: 15.25±1.40 mg.L^-1, T. gracilis: 1.49±0.12 mg.L^-1). These values were lower than those obtained by Fabregas et al. (1985b), whose mass culture of T. suecica was found to have a maximum protein value of 306 mg L^-1 in logarithmic culture phase. Koening et al. (1990b) determined the chemical composition of T. tetrathele using organic fertilizer as the medium. They observed that under these conditions, the soluble protein amount was 310 mg.L^-1.

C. closterium had the highest values for nitrate (0.36±0.02 mg.L^-1), sodium (0.46±0.05 mg.L^-1) and phosphorous (2.80±0.38 mg.L^-1). Nitrate values for C. closterium were significantly different from the other two species (P<0.05) and sodium values were significantly different among the species (P<0.05). C. closterium phosphorus values were different significantly from T. gracilis (P<0.05). Potassium content (0.02 mg.L^-1) and sulphur content (0.03 mg.L^-1) were equal for C. gracilis and C. closterium. T. gracilis showed lower inorganic nutrient content, with a significant difference in potassium levels (P<0.05) (Table 2).

As the present study showed, there were differences in the chemical composition among these three microalgal species, even when cultivated under the same conditions. These differences could be related to specific differences in the cell metabolism and therefore resulted in a variation of the chemical balance of chlorophyll a, proteins, carbohydrates and minerals. These factors, when associated to other chemical components, such as vitamins, were essential in promoting the herbivorous growth (Chu et al., 1982; Webb and Chu, 1983; Brown et al., 1998).

CONCLUSION

The microalgae C. gracilis, C. closterium and T. gracilis, when cultured in f/2-Guillard medium, in carboy, showed quantitative differences in chemical composition and biomass. These variations were observed in chlorophyll a, soluble proteins, carbohydrates, free total amino acids, mineral content and cellular density. T. gracilis presented lower results, producing significantly lower values in the majority of the analyzed parameters. With regards to chlorophyll a, soluble protein and carbohydrates, C. closterium showed the highest results. It also showed a significantly higher nitrate amount. According to the chemical parameters analyzed and tested conditions, C. closterium was the best nutritional option for aquaculture.

ACKNOWLEDGEMENTS

We thank Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) for two years of scholarship for the first author and we appreciate the use of the laboratory facilities at Rural Federal University of Pernambuco and Federal University of Pernambuco.

Received: May 15, 2005;

Revised: May 15, 2006;

Accepted: March 23, 2007.

• Antia, N. J.; Berland, B. R.; Bonin, D. J. and Maestrini, S. E. (1977), Effects of urea concentration in supporting growth of certain marine microplanktonic algae. Phycol 16:(1), 105-111.
• Araújo, S. C. and Garcia, V. M. T. (2005), Growth and biochemical composition of the diatom Chaetoceros cf. wighamii brightwell under different temperatures, salinity and carbon dioxide levels. I. Protein, carbohydrates and lipids. Aquacul, 246, 405-412.
• Becker, E. W. (1995), Microalgae: biotechnology and microbiology, Cambridge University Press, New York.
• Bezerra Neto, E.; Andrade A. G.; Barreto L. P. (1994), Análise química de tecidos e produtos vegetais, Recife, UFRPE.
• Bradford, M. M. (1976), A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principles of protein-dye binding, Analy. Biochem 72, 248-254.
• Brown, M. R. (1991), The amino-acid and sugar composition of 16 species of microalgae used in mariculture, J. Exp. Biol. Ecol145, 79-99.
• Brown, M. R.; Jeffrey S. W.; Volkman J. K. and Dunstan G. A. (1997), Nutritional properties of microalgae for mariculture, Aquacul 151, 315-331.
• Brown, M. R.; Mc Mcausland, M. A. and Kowalski, K. (1998), The nutritional value of four Australian microalgal strains fed to Pacific oyster Crassostrea gigas spat. Aquacul165, 281-293.
• Caers, M.; Coutteau, P. and Sorgeloos, P. (1999), Dietary impact of algal and artificial diets, fed at different rations, on the growth and fatty acid composition of Tapes philippinarum (L.) spat. Aquacul 170, 307-322.
• Cataldo, D. A.; Harron, M.; Schrader, L. and Youngs, V. L. (1975), Rapid colorimetric determination of nitrate in plant and tissue by nitration of salicilic acid, Commun. Soil Science and Plant Analysis 6, 71-80.
• Chu, F. L. E.; Dupuy, J. L. and Webb, K. L. (1982), Polysaccharide composition of five algal species used as food for larvae of the american oyster, Crassostrea virginica Aquacul 29:(¾), 241-252.
• Fabregas, J.; Herrero, C.; Abalde, J. and Cabezas, B. (1985a), Growth, chlorophyll a and protein of the marine microalga Isochryrsis galbana in batch cultures with different salinities and high nutrient concentrations, Aquacul 50, 1-11.
• Fabregas, J.; Herrero, C.; Cabezas, B. and Abalde, J. (1985b), Mass culture and biochemical variability of the marine microalga Tetraselmis suecica (Kylin) Butch with high nutrients concentrations. Aquacul 49, 231-244.
• Fabregas, J.; Herrero, C.; Cabezas, B. and Abalde, J. (1986), Biomass production and biochemical composition in mass cultures of the marine microalga Isochrysis galbana Parke at varying nutrient concentrations. Aquacul 53, 101-113.
• Fidalgo J. P.; Cid, A.; Torres, E.; Sukenik, A. and Herrero, C. (1998), Effects of nitrogen source and growth phase on proximate biochemical composition, lipid classes and fatty acid profile of the marine microalga Isochrysis galbana Aquacul 166, 105-116.
• Guillard, R. L. (1975), Culture of phytoplankton for feeding marine invertebrates. Culture of marine invertebrate animal, p. 29-60.
• Koening, M. L.; Lacerda, S. R. and Passavante, J. Z. O. (1990a), Cultivo em laboratório de Tetraselmis chuii e Tetraselmis tetrathele (Chlorophyceae) com fertilizantes orgânicos. Arq. Biolog. Tecnolog 33, 91-103.
• Koening, M. L.; Maia, P. R. and Campos-Takaki, G. M. (1990b), Composição bioquímica de Tetraselmis tetrathele (West. G. S) Butcher (Chlorophyceae) cultivada com fertilizante orgânico. Revist. Biol. Bras. 2, 23-38.
• Koening, M. L.; Passavante, J. Z. O.; Bartolomeu, C. C. and Costa, K. M. P. (1998), O vinhoto no cultivo de microalgas. Gayana 45, 253-263.
• Lopes-Muñoz, I.; Abalde, J. and Hererro, C. (1992), Crescimiento y contenido de pigmentos de cuatro especies de microalgas marinas cultivadas com diferentes temperaturas e intensidades de luz. Nov. A. Cient. Comp 3, 59-65.
• Lourenço, S. O.; Marquez, U. M. L.; Mancini-Filho, J.; Aidar, E. and Barbarino E. (1998), Distribution of intracellular nitrogen in marine microalgae: basis for the calculation of specific nitrogen-to-protein conversion factors. J. Phycol 34, 798-811.
• Lourenço, S. O.; Marquez, U. M. L.; Mancini-Filho, J.; Barbarino, E. and Aidar, E. (1997), Changes in biochemical profile of Tetraselmis gracilis I. Comparision of two culture media. Aquacul 148, 153-158.
• Malavolta, E.; Vitti, G. and Oliviera, S. A. (1989), Avaliação do estado nutricional das plantas: Princípios e aplicações. Associação Brasileira para Pesquisa da Potassa e do Fosfato, Piracicaba.
• McCausland, M. A.; Brown, M. R.; Barrett, S. M.; Diemaar, J. A. and Heasmn, M. P. (1999), Evaluation of live microalgae and miroalgae pastes as supplementary food for juvenile Pacific oysters (Crassostrea gigas). Aquacul., 174, 323-342.
• Melo, G. N.; Sassi, R. and Araújo, T. F. H. (1993), Crescimento de Phaeodactylum tricornutum BOHLIN (Bacillariophyta) em água do mar enriquecida com soluções derivadas da decomposição de algas arribadas com meio de cultura. R. Nord. de Bio. 8, 45-53.
• Renaud, S. M.; Thinh, L. and Parry, D. L. (1999), The gross chemical composition and fatty acid composition of 18 species of tropical Australian microalgae for possible use in mariculture. Aquacul 170, 147-159.
• Sarriés, G. A.; Oliveira, J. C. V. and Alves, M. C. (1992), SANEST, Série didática nş 06, Centro de Informática da Universidade de São Paulo, Piracicaba.
• Sarruge, J. R. S. and Haag, H. P. (1974), Análises Químicas em Plantas, Piracicaba, USP-ESALQ.
• Sauodi-Helis, L.; Dubacq, J. P.; Marty, Y.; Samain, J. F. and Gudin, C. (1999), Influence of growth rate on pigment and lipid composition of the microalga Isochrysis aff. galbana clone T. isso. J. Apl. Phy. 6, 315- 22.
• Sipaúba-Tavares, L. H. and Rocha, O. (2001), Produção de plâncton (fitoplâncton e zooplâncton) para alimentação de organismos aquáticos Rimaeditora, São Carlos.
• Southgate, P. C.; Beer, A. C.; Duncan, P. F. and Tamburri, R. (1998), Assessment of nutritional value of three species of tropical microalgae, dried Tetraselmis and a yeast-based diet for larvae of the blacklip pearl oyster, Pinctada margaritifera (L.). Aquacul 162, 247-257.
• Sukenik, A. and Whanon, R. (1991), Biochemical quality of marine unnicellular algae with special emphasis on lipid composition: I. Isochrysis galbana Aquacul 97, 61-72.
• Valenzuela-Espinoza, E.; Millán Núnez, R. and Núnez Cebrero, F. (2002), Protein, carbohydrate, lipid and chlorophyll a content in Isochrysis aff. galbana (clone T-Iso) cultured with a low cost alternative to the f/2 medium. Aquacul. Eng. 25, 207-216.
• Webb, K. L. and Chu, F. E. (1983), Phytoplankton as a food source for bivalve larvae. In- Pruder, G. D. Langdon, C., Conklin, D. (ed.) Biochemical and physiological approaches to shellfish nutrition. Proceedings of the Second International conference on Aquaculture Nutrition, Louisiana State University, L. A., pp. 272-291.
• Whyte, J. N. C. (1987), Biochemical composition and energy content of six species of phytoplankton used in mariculture of bivalves. Aquacul 60, 231-241.
• Yamashita C. and Magalhães P. M. S. (1984), Meios de Cultura para a Alga Chaetoceros gracilis, Boletim de Pesquisa n. 7, EMPARN, Rio Grande do Norte.
• Yemm, E. W. and Cocking, E. C. (1955), The determination of aminoacids with ninhydrin. Analsyt. 80, 209-213.
• Yemm, E. W. and Wills, A. J. (1954), The estimation of carbohydrates by anthrone. Bioch. J 57, 508-514.

*

Author for correspondence

Publication Dates

• Publication in this collection
  31 Aug 2007
• Date of issue
  May 2007

History

• Reviewed
  15 May 2006
• Received
  15 May 2005
• Accepted
  23 Mar 2007