Introduction

Microalgal culture constitutes a key procedure in shellfish hatcheries, but this activity is far from being optimized and several problems remain to be solved, because the conventional microalgal culture system in hatcheries could be inadequate in qualitative or quantitative terms, and the quality of biomass produced could be subject to a nutritional drift (Ponis et al. 2006). In particular, the number of good quality microalgae currently available in hatcheries is limited, and several species which were previously used in commercial hatcheries have now been discarded due to their poor nutritional value (e.g., Dunaliella sp., Phaeodactylum tricornutum, Tetraselmis sp.) (Coutteau and Sorgeloos 1992; Robert and Gérard 1999). Therefore, substitutes for fresh microalgae produced on-site have been sought and many experimental studies have been carried out to find the best alternatives.

Microalgae, the primary food source for bivalves, provide essential sterols and highly unsaturated fatty acids, such as 20:4(n − 6), 20:5(n − 3), and 22:6(n − 3) (Soudant et al. 2000; Rivero-Rodríguez et al. 2007). Vitamin E was originally considered as a dietary factor of animal nutrition, which has an importance in reproduction. In aquaculture, vitamin E is used for the fortification of feed to improve the growth and resistance to stress and disease, as well as for survival of fish and shrimp (Vismasa et al. 2003). As in higher vertebrates, vitamin E deficiency affects reproductive performance, causing immature gonads and lower hatching rate and survival of offspring (Izquierdo et al. 2001). However, the amounts of these components vary with environmental conditions, including temperature and growth phase (Richmond 1986; Renaud et al. 1995; Soudant et al. 2000). Especially, temperature has a major effect on the biochemical composition of some microalgae (Thompson et al. 1992; Zhu et al. 1997; Renaud et al. 2002). Many microalgal species respond to decreased growth temperature by increasing the ratio of unsaturated to saturated fatty acids (Thompson et al. 1992; Renaud et al. 1995, 2002; Oliveira et al. 1999). However, the response to growth temperature varies from species to species, with no overall consistent relationship between temperature and fatty acid unsaturation (James et al. 1989; Thompson et al. 1992; Renaud et al. 1995). James et al. (1989) observed that the temperature yielding the highest Nannochloropsis growth rate was between 15 and 20°C. Renaud et al. (1995) concluded that the optimum temperature range for reasonable growth rate together with maximum lipid production and high levels of the essential fatty acids was found to be 20–30°C for Isochrysis sp.

There is no study on the effect of temperatures on the biochemical composition of D. vlkianum. Donato et al. (2003) and Ponis et al. (2006) studied biochemical composition of this alga at only 18 and 19–20°C, respectively. Therefore, this study has a novelty in that it was carried out to determine the effect of temperatures and growth phase on the biochemical composition (α-tocopherol, fatty acids and pigments) in batch culture of D. vlkianum at both 18 and 26°C. Additionally, the knowledge obtained from this study is important for the optimization of marine aquaculture programs.

Materials and methods

Microorganisms

The microalga Diacronema vlkianum belongs to the AQ/INIAP (Portugal) collection of living microalgae, and this study was done in the Aquaculture laboratory at AQ/INIAP.

Culture conditions

The microalgae were grown in 100-l polyethylene bags (165 × 25 cm). Three replicates were used in all trials. All cultures were carried out in a batch-culture system in enriched sterilized seawater with Wallerstein and Miquel medium (3:1). The salinity was 2.5 g l−1. Cultures were kept at 18 ± 1°C in one room and 26 ± 1°C in another room at constant temperatures. Three polyethylene bags were placed in each room and for each bag continuous illumination were made with fluorescent lamps (Philips TLM 40 W/54RS). Photon flux density at the surface of bags was 196 μmol m−2 s−1 (Li-Core 195). Cell number was measured using an electronic particle counter (Coulter EPICS XL; Beckman Company, USA) and instantaneous growth rates (μ) were calculated using this Eq. 1.

$$ \mu = \frac{{{ \ln }\,(N_{1} ) - (N_{0} )}}{{t_{1} - t_{0} }} $$
(1)

where N 1 is the cell number at time t 1 and N 0 is the cell number at time t 0.

Harvesting process

The cultures kept at 18 and 26°C were harvested at days 17 and 34, with sample cultures at early exponential phase and late exponential phase, respectively, at day 17, and late exponential phase and stationary phase, respectively, at day 34. Harvesting of the microalgae was done by flocculation with FeCl3 as 10-l samples taken each time from each bag, followed by centrifugation as reported by Batista and Martins (1991). Each pellet obtained was freeze-dried and each analyzed separately.

α-Tocopherol

Alpha-tocopherol was immediately analyzed after freeze-drying. The extraction was carried out following a method adapted from Chen et al. (1998). The organic phases were pooled and a 20-μl aliquot was injected in a HPLC JASCO model 980 (Japan) equipped with an automatic injector (JASCO Model AS-950-10 (Japan)) and a fluorescent detector [JASCO Model FP-1520 (λ exc = 290 nm and λ em = 300 nm)]. The separation was carried out in a Lichrosorb Si 60-5 (250 mm × 3 mm i.d.) column from Chrompack (USA) protected by a silica pre-column S2-SS (10 mm × 2 mm i.d.) from Chrompack (USA). The mobile phase was a mixture of n-hexane and isopropanol (99.3:0.7 v/v) degassed in the Gastor Model GT-104 System (Japan) and eluted at a constant flow of 1 ml/min. The data was recorded and analyzed using Borwin chromatographic software (version 1.21, France).

Fatty acids

Fatty acid methyl esters were prepared according to Lepage and Roy (1986) modified by Cohen et al. (1988). The analysis was performed in a gas chromatograph Varian Star 3400 Cx (USA) equipped with an auto-sampler and fitted with a flame ionization detector at 250°C. Separation was done in a polyethylene glycol capillary column DB-WAX 30 m in length, 0.25 mm i.d., and 0.25 μm film thickness from J&W Scientific (USA). The column was subjected to a temperature program starting at 180°C for 5 min, heating at 4°C/min for 10 min, and held at 220°C for 25 min. The injector (split ratio 100:1) temperature was kept constant at 250°C during the 40-min analysis.

Pigments

Total carotenoid content in the algae was determined spectrophotometrically after extraction with acetone (Choubert and Storebakken 1989). Carotenoids are expressed using extinction coefficients (\( E_{{1\,{\text{cm}}}}^{1\% } \)) of 2,150 for algal pigments at their absorption maximum in acetone (Gouveia et al. 1997). Individual pigments were detected by HPLC (Perkin Elmer, USA), reversed-phase, with a μ-Boundapak C18 column and a detector UV/VIS Waters 481 (λ = 460 nm), with methanol:acetonitrile:water (65:35:2) as eluent. Methanol and acetonitrile were HPLC-grade reagents, used without further purification other than filtration and degassing. The pigments were eluted over 20 min with a flow rate of 1 ml/min.

All reagents except otherwise stated are from Merck (Germany), and standards are from Sigma-Aldrich (USA).

Statistical analysis

Th Kolmogorov–Smirnov test was used to verify the normality and homogeneity of variances, and data were analyzed using an ANOVA. Having demonstrated a significant difference somewhere among the groups with the ANOVA, a Tukey test was applied to find out where those differences were (Zar 1999), using the software SPPS (ver. 12.0 for Windows).

Results

Growth

The variations in cell density of D. vlkianum maintained at 18 and 26°C during the growth phases are shown in Fig. 1. The cell density of D. vlkianum at 18°C increased rapidly to 11.9 × 106 cells ml−1 on day 39 without any apparent lag phase, and maximum specific growth rate was recorded 0.20 division day−1. On the other hand, the cell density at 26°C showed a gradual increase in the density, and maximum cell number was 2.3 × 106 cells ml−1. The maximum specific growth rate was recorded at day 11 as a 0.12 division day−1. So, the lowest cell concentrations always occurred in the 26°C temperature cultures.

Fig. 1
figure 1

Cell number of Diacronema vlkianum at different temperatures (A 18°C, B 26°C). The cultures were harvested at early exponential phase and late exponential phase, respectively, at day 17, and late exponential phase and stationary phase, respectively, at day 34 (↑). Values are means from three replicates with standard deviations

Full size image

α-Tocopherol

The variations in the amount of α-tocopherol detected in D. vlkianum during growth phases are shown in Table 1. At 18°C, there is a significant increase (P ≤ 0.05) in α-tocopherol between early exponential phase and late exponential phase, detected as 167.4 μg g−1 DW and 257.7 ± 21.6 μg g−1 DW, respectively. At 26°C, the tocopherol levels were lower than for 18°C, decreasing from 80.9 ± 9.4 μg g−1 DW to 75.8 ± 22.8 μg g−1 DW for late exponential phase and stationary phase, respectively.

Table 1 α-Tocopherol, total carotenoid, chlorophyll a, astaxanthin and lutein of D. vlkianum at different temperatures (18 and 26°C), when the cultures were harvested at 17 and 34 days
Full size table

Fatty acids

The proportion of total polyunsaturated fatty acids considerably decreased, with simultaneous increase of saturated fatty acids and monounsaturated fatty acids, when the temperature increased from 18 to 26°C, (Table 2). 14:0 and 16:0 were in the highest percentages of saturated fatty acids. The highest level recorded at 26°C was 20.8% in stationary phase for 14:0 and 10.1% in early exponential phase for 16:0 at 18°C. 16:0% was not significantly affected at both temperatures (P ≥ 0.05). Palmitoleic acid 16:1(n − 7) was the main detected monounsaturated fatty acid for both temperatures, decreasing from 18.2 to 15.5% at 18°C in the early exponential phase and late exponential phase, respectively, and 14.9–13.7% at 26°C in the late exponential phase and stationary phase, respectively. There was significant decrease between phases (P ≤ 0.05) in the percentages of total monounsaturated fatty acids at 18°C, but there was no significant change between phases (P ≥ 0.05) in the percentages of total monounsaturated fatty acids at 26°C. The maximum level attained for total monounsaturated fatty acids at 18 and 26°C was similar (P ≥ 0.05). There was no significant change in the percentages of total polyunsaturated fatty acids at 18 and 26°C in all the groups of phases (P ≥ 0.05). The highest value of 20:5(n − 3) was 20.4% at 18°C in late exponential phase and 16.5% at 26°C in the late exponential phase (P ≤ 0.05).

Table 2 Main fatty acids profile of D. vlkianum at different temperature (18 and 26°C), when the cultures were harvested at 17 and 34 days
Full size table

Pigments

Total carotenoids and chlorophyll levels are shown in Table 1. The total carotenoid recorded values were higher at 18°C than at 26°C (P ≤ 0.05). Chlorophyll levels revealed a decrease at 18°C, falling from early exponential to late exponential phase. Astaxanthin and lutein were detected as the main pigments. The highest value of astaxanthin was 50.4 ± 18.3% DW at 26°C in the stationary phase and 47.6 ± 13.0% DW at 18°C in the early exponential phase. The highest lutein levels was recorded at 18°C in the early exponential phase (41.0 ± 2.0% DW), while lutein was not determined at 26°C.

Discussion

Growth

The results show that a compromise between the nutritional properties and growth kinetics could be achieved at 18°C which allows for high values for specific growth and biomass productivity. In production of biomass with certain desired characteristics, the culture temperature is a fundamental factor.

α-Tocopherol

Huo et al. (1997) observed that α-tocopherol was the major tocopherol in several microalgae samples. Vitamin E level was evaluated in D. vlkianum and it was recorded that maximum α-tocopherol was 257.7 ± 21.6 μg g−1 in the DW of samples at 18°C. α-Tocopherol results of this algae were higher than for Dunaliella salina and Tetraselmis suecica recorded by Vismasa et al. (2003) as 153.2 and 157.7 μg g−1, respectively. Concerning other microalgae, Fábregas and Herrero (1990) obtained at the end of the exponential phase values of 421.8, 58.2, 116.3, and 669.9 μg g−1 of α-tocopherol for T. suecica, Isochrysis galbana, Dunaliella tertiolecta, and Chlorella stigmatophora, respectively. Maximum α-tocopherol levels obtained by using different extraction methods were 222, 692, 289, and 302 μg g−1 from Chlorella sp., Chaetoceros sp., Tetraselmis sp., and Isochrysis sp., respectively (Huo et al. 1997). In addition, the increase of α-tocopherol of D. vlkanum during growth phases was also observed by Donato et al. (2003), and these algae produced 69.3 μg g−1 in the exponential phase and 551.3 μg g−1 in the decay phase. This variability is not only inherent in the species but also depends on environmental conditions, e.g., temperature and light (Huo et al. 1997). These results can be due to the high instability of this compound and the important role of this antioxidant in the oxidation reactions during the ageing process.

Fatty acids

In the present work, polyunsaturated fatty acids were the predominant group detected in these algae at 18°C, reaching 39.5% of the total fatty acids in late exponential phase. Among polyunsaturated fatty acids, 20:5(n − 3) reached the highest percentage (20.4% in late exponential phase). Donato et al. (2003) obtained 21.1, 20.5, 21.4, and 17.2% 20:5(n − 3) at 18°C for 1st exponential phase, 2nd exponential phase, stationary phase, and decay phase, respectively. Total polyunsaturated fatty acids and 22:6(n − 3) results of the study in the late exponential phase were higher than those recorded by Donato et al. (2003): 33.3 and 4.2% in the stationary phase, respectively.

Carotenoids and chlorophylls

Total carotenoid amount of D. vlkanum during growth phases at 18°C was approximately similar to those obtained by Donato et al. (2003). However, the carotenoid results decreased when the temperature was high at 26°C, and this alga produced 2.1 mg g−1 DW in the late exponential phase and 1.7 mg g−1 DW in the stationary phase. Chlorophyll a content decreased in response to the reduction in nitrogen load (Sukenik and Wahnon 1991). Therefore, the decrease in chlorophyll at high temperature, observed in this study, can be related to a lower photosynthetic activity of the growth phases.

Applications to aquaculture

Gross composition may not always correlate directly with nutritional value, but when other specific essential nutrients (e.g., essential fatty acids, vitamins, minerals) are in adequate proportion the differences may become important (Rivero-Rodríguez et al. 2007). This suggests that the relative food values of the D. vilkianum tested is mainly caused by whether or not they contain certain essential nutrients and at what concentration these nutrients are effective and present. This finding could prove beneficial to the developing hatchery industry of D. vilkianum since the alga can be an alternative to the standard diet used so far; a combination of Chaetoceros calcitrans and Isochrysis galbana. When the nutritional value of alga could be optimized, D. vlkianum improved the growth and survival of the larvae and oyster culture (Ponis et al. 2006). The present study suggested that the nutritional value (fatty acids, α-tocopherol, and pigments) of D. vlkianum culture is determined to be optimal at a temperature of 18°C.

Conclusion

Diacronema vlkianum is very sensitive to growth phase and growth temperature with the highest biomolecule production at 18°C during late exponential phase, and the results of the current study showed that there occurred considerable increases in biochemical composition and content of microalgae depending on temperature and growth phase. These current findings indicate that there exists an opportunity to maximize the biochemical composition (fatty acids, α-tocopherol, sterol, and carotenoid) of microalgae D. vlkianum at 18°C with the potential to improve animal growth and to attain increased success in breeding alternative fish species in mariculture operations.