Aquaculture 246 (2005) 405 – 412 www.elsevier.com/locate/aqua-online Growth and biochemical composition of the diatom Chaetoceros cf. wighamii brightwell under different temperature, salinity and carbon dioxide levels. I. Protein, carbohydrates and lipids Sirlei de Castro AraújoT, Virgı́nia Maria Tavano Garcia Laboratory of Ecology of Phytoplankton and Marine Microorganisms, Dept. of Oceanography, Federal University of Rio Grande, Avenida Itália, Km 8, Rio Grande-RS, Brazil, 96201-900 Received 29 November 2004; received in revised form 17 February 2005; accepted 18 February 2005 Abstract The marine diatom Chaetoceros cf. wighamii has been investigated for its potential use as food in mariculture. In this work, we investigated temperature (20, 25, and 30 8C), salinity (25 and 35) and carbon dioxide addition (air and air + CO2) effects on growth and biochemical composition of C. cf. wighamii, under laboratory conditions. C. cf. wighamii growth and biomass was primarily affected by carbon dioxide addition and to a lesser extent by temperature and salinity. In general, lipid and carbohydrate content were higher at lower temperatures (20 and 25 8C) while protein was unaffected. Carbon dioxide addition increased protein and lowered carbohydrates, but had no effect on lipid content. Carbohydrates were enhanced while lipids and protein decreased at the highest salinity (35). These results should be taken into consideration when evaluating the nutritional value of this microalga for marine invertebrate larvae. D 2005 Elsevier B.V. All rights reserved. Keywords: Chaetoceros wighamii; Growth; Composition; Temperature; Salinity; Carbon dioxide; Microalgae 1. Introduction Microalgae are the main food source for many marine animals and investigations to develop more productive culture techniques and improve their nutritional value are desirable (De Pauw and Persoone, 1988). Many specific characteristics are T Corresponding author. Tel.: +55 53 2336510. E-mail address: sirleicastro@yahoo.com.br (S. de Castro Araújo). 0044-8486/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.aquaculture.2005.02.051 thought to influence the nutritional value of microalgae, such as cell wall digestibility (Epifanio et al., 1981), cell size, toxic substances produced by algae and their biochemical composition (Fernández-Reiriz et al., 1989). The biochemical composition of microalgae can change with their growth rates and/ or environmental conditions and with the phase of their life cycle (Richmond, 1986). Two characteristics are important to evaluate the potential of a species to be used for aquaculture purposes: growth rates, in terms of cell numbers or biomass; and 406 S. de Castro Araújo, V.M.T. Garcia / Aquaculture 246 (2005) 405–412 (20, 25 and 25 8C), while two salinities (25 and 35) and two CO2 conditions (with (+) and without ( ) addition) were used for the other experiments. Carbon dioxide concentration was monitored by the free carbon dioxide method, which is based on the titration of dissolved carbon dioxide with NaOH (0.0227 N) with end-point reaction at pH 8.3 (Baumgarten et al., 1996). Cultures were grown aerated in f/2 medium (Guillard, 1975), in borosilicate flasks (6 l) and the culture temperature was controlled (F2 8C) by a water bath. Before each experiment, cultures remained at the determined experimental conditions for an adaptation period of approximately 4 generations. The cultures were started by inoculating a volume 5–10% of the total volume. Cell counts were performed daily to determine maximum cell density and specific growth rate (K 2), which was calculated by linear regression of the log2 cell concentration on time, at the exponential growth phase (Guillard, 1973). Each treatment was run in triplicate. Chlorophyll baQ concentration was determined at the beginning and end of the experiment, by filtering a known culture volume on GF/F filters and extracting the pigment in 90% acetone solution for 24 h at 20 8C. Fluorescence was then determined in the extract with a Turner DesignsR TD 700 fluorometter, according to Welschmeyer (1994). Light intensity was kept at 530 Amol m 2 s 1 under a photoperiod of 12 h:12 h light:dark. Experiments were made in bbatchQ cultures, which were grown until either late exponential phase or early stationary phase. Algal biomass was obtained by concentrating the three replicates from each treatment on GF/A WhatmanR filters, using a MilliporeR peristaltic pump. The samples retained on filters were dried at biochemical composition, which must be optimized in terms of essential nutrients. Several diatom species were isolated from Cassino Beach, Brazil (southern Atlantic Ocean waters), to establish mono-algal cultures and to determine their biochemical composition. Among them, Chaetoceros cf. wighamii was selected due to its apparent fast growth in culture, its adequate cell size as food for marine planktonic larvae (2–20 Am according to Brown et al., 1997) and to belong to the Bacillariophyceae group, mostly considered as adequate food to these animals (D’Souza and Lorenagan, 1999). The main factors controlling microalgae growth and chemical composition are light, nutrients, temperature and pH (Tzovenis et al., 1997; Zhu et al., 1997) but other factors can be important to some species, such as salinity (Chu et al., 1996). The aim of the present work was to determine the effect of different levels of temperature, salinity and Carbon dioxide addition on the biochemical composition and growth of the marine diatom C. cf. wighamii. This part of the work focuses mainly on the gross and relative contents of protein, carbohydrate and lipid. 2. Material and methods K2 (divisions. day -1) Mono-algal, non-axenic cultures of C. cf. wighamii, a species isolated from the southern Atlantic Ocean waters, were established and kept under controlled laboratory conditions. Three separate experiments were performed to test the effects of temperature, salinity and carbon dioxide concentration on the growth and gross biochemical constituents of this species. Temperature was tested at three levels 5 4 3 2 1 0 - + 20ºC - + 25ºC S 25 - + 30ºC - + 20ºC - + 25ºC S 35 - + 30ºC Fig. 1. Growth rate of C. cf. wighamii under different temperature, salinity (S25 and S35) and CO2 addition (with (+) and without ( )). Maximum cell density (106 cells mL -1) S. de Castro Araújo, V.M.T. Garcia / Aquaculture 246 (2005) 405–412 10 9 8 7 6 5 4 3 2 1 0 - + - 20ºC + - 25ºC S 25 + - 30ºC + - 20ºC + 407 - 25ºC S 35 + 30ºC Fig. 2. Maximum cell density of C. cf. wighamii under different temperature, salinity (S25 and S35) and CO2 addition (with (+) and without ( )). 60 8C until constant weight. The filters with algae samples were stored at 20 8C until chemical analysis. The biochemical composition of C. cf. wighamii was determined, in terms of total protein, total lipids and total carbohydrates. Total lipids were extracted according to Bligh and Dyer (1959) as modified by Whyte (1987). In the lipid extract residue, (polymeric fraction), total protein was determined with the Kjeldahl technique (Whyte, 1987). Samples retained in GF/A filters were hydrolyzed in 10 ml 80% sulfuric acid (Myklestad and Haug, 1972), and carbohydrates determined according to Dubois et al. (1956). Statistical analysis included one-way analysis of variance (ANOVA) and Tukey test. Total protein, total carbohydrates and total lipids and other constituents percentages were transformed using arcsine prior to statistical analysis (Vieira and Hoffmann, 1988). 3. Results and discussion 3.1. The effect of temperature, salinity and carbon dioxide addition on the growth of C. cf. wighamii Temperature had a significant effect ( P b 0.05) on the growth rate of C. cf. wighamii, under salinity of 25, but not at 35 (Fig. 1). The highest temperature (30 8C) caused the growth rate to be lower at salinity 25 with no addition of carbon dioxide. Temperature showed no significant effect ( P N 0.05) on maximum cell density (Fig. 2), although a visible trend of higher values at 25 8C was observed when carbon dioxide was added. Biomass (Fig. 3) showed lower values at 30 8C while chlorophyll per cell (Fig. 4) was not affected by temperature as well as by any of the factors tested. The effect of temperature on the growth rate of microalgae has been observed in other species. Significant Biomass (g of dry weight) 3.5 3 2.5 2 1.5 1 0.5 0 - + 20ºC - + 25ºC S 25 - + 30ºC - + 20ºC - + 25ºC S 3t5 - + 30ºC Fig. 3. Biomass of C. cf. wighamii under different temperature, salinity (S25 and S35) and CO2 addition (with (+) and without ( )). 408 S. de Castro Araújo, V.M.T. Garcia / Aquaculture 246 (2005) 405–412 Chlorophyll a (fg. cell -1) 1200 1000 800 600 400 200 0 - + 20ºC - + 25ºC S 25 - + - 30ºC + 20ºC - + 25ºC S 35 - + 30ºC Fig. 4. Chlorophyll a per cell of C. cf. wighamii under different temperature, salinity (S25 and S35) and CO2 addition (with (+) and without ( )). for C. cf. wighamii is between 20 and 25 8C, under the conditions used in these experiments. Salinity (25–35) had no significant effect ( P N 0.05) on C. cf. wighamii growth, maximum cell density, biomass and chlorophyll per cell (Figs. 1–4) although a tendency of higher growth and biomass and lower cell density was observed at the lower salinity. This contrast between higher growth rate and lower maximum cell density could be explained by a limitation in some nutritional factor not determined, at the lowest salinity (25) as the medium of this salinity was obtained by dilution of the seawater. Tests must be performed in order to prove this hypothesis and or to determine the causes of these results. Higher growth rates of C. cf. wighamii occurred when carbon dioxide was added to the cultures (Fig. increases in growth rate of Chaetoceros pseudocurvisetus, Skeletonema costatum, Skeletonema hantzschii, with maximum at 25 8C was observed by Yoshihiro and Takahashi (1995). Renaud et al. (2002) attributed the higher growth rate of Chaetoceros sp. (Clone CS256) to increase in temperature from 25 to 30 8C. The effect of temperature on the gel:liquid phase transition of photosynthetic membrane lipids has been proposed by Quinn and Williams (1983). Fogg and Thake (1987) stated that lower microalgae growth rate could be a result of the increase in respiration due to rise in temperature above the species optimum level. It is possible that all these effects are related with the results observed in growth rate, and therefore in cell density and biomass in this work. As the results suggest, the adequate temperature Total lipids (mg g-1 dry weight) 250 200 150 100 50 0 - + 20ºC - + 25ºC S 25 - + 30ºC - + 20ºC - + 25ºC S 35 - + 30ºC Fig. 5. Total lipids of C. cf. wighamii under different temperature, salinity (S25 and S35) and CO2 addition (with (+) and without ( )). S. de Castro Araújo, V.M.T. Garcia / Aquaculture 246 (2005) 405–412 409 Total carbohydrates (mg g-1 dry weight) 250 200 150 100 50 0 - + 20ºC - + 25ºC S 25 + 30ºC - + 20ºC + 25ºC S 35 + 30ºC Fig. 6. Total carbohydrates of C. cf. wighamii under different temperature, salinity (S25 and S35) and CO2 addition (with (+) and without ( )). 1), demonstrating that, although other factors may be sufficient, this nutrient can limit microalgae growth. Increases in growth rate have been observed in other microalgae species, with carbon dioxide addition (Olaizola et al., 1991). Another effect of carbon dioxide addition on C. cf. wighamii was to prolong exponential phase, an important result for aquaculture, as it is in this phase that microalgae have the highest nutritional value for aquatic animals (Fabregas et al., 2001). 3.2. The Effect of temperature, salinity and carbon dioxide addition on the biochemical composition of C. cf. wighamii Temperature was the main factor influencing C. cf. wighamii composition (Figs. 5–8). At temperatures of 20 and 25 8C, lipids and carbohydrates were higher than at 30 8C (Figs. 5 and 6). Protein was not significantly affected by temperature, but a tendency for lower values was observed at 25 8C (Fig. 7), augmenting below and above this temperature. Renaud et al. (1995) observed that, in general, maximum lipid content coincides with optimal range in growth temperature in many species, and this content is lower at temperatures below and above this range. Other investigations showed higher lipid content at 25 8C for Chaetoceros sp. (clone CS256) than in higher temperatures, while for other species (Rhodomonas sp., Cryptomonas sp. and Isochrysis sp.) higher concentrations were observed between 27 and 30 8C (Renaud et al., 2002). All the species studied showed significantly lower protein content at temperatures above 27 8C. Carbohydrates were significantly higher between 25 and 30 8C in Chaetoceros sp. and other species tested, and became lower at higher temperatures. Renaud et al. (2002) also observed a clear correlation between ash content and temperature (25–35 8C). In general, the results of biochemical Total protein (mg g-1 dry weight) 800 600 400 200 0 - + 20ºC - + 25ºC S 25 + 30ºC - + 20ºC + 25ºC S 35 + 30ºC Fig. 7. Total protein of C. cf. wighamii under different temperature, salinity (S25 and S35) and CO2 addition (with (+) and without ( )). 410 S. de Castro Araújo, V.M.T. Garcia / Aquaculture 246 (2005) 405–412 Other constituents (mg g-1 dry weight) 600 500 400 300 200 100 0 - + 20ºC - + 25ºC S 25 + 30ºC - + 20ºC - + 25ºC S 35 + 30ºC Fig. 8. Other constituents of C. cf. wighamii under different temperature, salinity (S25 and S35) and CO2 addition (with (+) and without ( )). composition of C. cf. wighamii are in agreement with other works. Lipids and carbohydrates are considered cellular fuel, besides their important function as structural constituents of membranes (Thompson et al., 1992). Hence, their decrease can negatively affect growth and metabolism of cells. Data in this work suggest that temperatures between 20 and 25 8C could be used to optimize nutritional value of C. cf. wighamii due to the higher lipid and carbohydrate and adequate protein content under these conditions. Higher levels of carbohydrates are reported to produce higher growth of juvenile oysters (Ostrea edulis; Enright et al., 1986) and larval scallops (Patinopecten yessoensis; Whyte et al., 1989). On the other hand, a high dietary protein provided the best growth for juvenile mussels (Mytilus trossulus; Kreeger and Langdon, 1993). The effect of salinity in C. cf. wighamii composition can be seen in Figs. 5–8. Protein content (Fig. 7) was lowered at salinity of 35, while other constituents, represented mainly by mineral fraction increased (Fig. 8). Although many species of microalgae are tolerant to great variations in salinity, their chemical composition can be affected (Brown et al., 1989; Roessler, 1990). Protein, lipids and carbohydrates seem slightly affected by a wide range of salinity for most microalgae species (Richmond, 1986). However, in some species, increases in ash and lipid content were observed at higher salinity (Ben-Amotz et al., 1987). Other authors (Fabregas et al., 1985) observed a decrease in protein content with increase in salinity in other species. The apparent increase in other constituents (mainly mineral fraction) in Chaetoceros can be related to cellular adjustments to osmotic stress due to the high salinity (Richmond, 1986). According to results in this work, a salinity of 25 seems more adequate to the growth and chemical composition in terms of protein, lipids and carbohydrates, for C. cf. wighamii. The effect of carbon dioxide on C. cf. wighamii biochemical composition is showed in Figs. 5–8. It was noted an increase in protein content (Fig. 7) and a decrease in carbohydrates (Fig. 6), as observed for other species when carbon dioxide is added to cultures. Brown et al. (1997) for example, noticed increases (i 100%) in protein content when cultures were enriched with 1% carbon dioxide, in many species in different groups of microalgae. Lipids and carbohydrates on the other hand were not affected. In Phaeodactylum tricornutum, protein content was increased with carbon dioxide addition (Chrismadha and Borowitzka, 1994). Chu et al. (1996) observed increases in lipids and carbohydrates at protein expenses in Nitzschia inconspicua, when culture was enriched with 5% (v/v) of carbon dioxide. In the present work, C. cf. wighamii apparently directed the extra-assimilated carbon mainly to protein synthesis, indicating a positive effect on cell physiology. 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