Advanced
Selection of Suitable Species of <italic>Chlorella</italic>, <italic>Nannochloris</italic>, and <italic>Nannochloropsis</italic> in High- and Low-Temperature Seasons for Mass Culture of the Rotifer <italic>Brachionus plicatilis</italic>
Selection of Suitable Species of Chlorella, Nannochloris, and Nannochloropsis in High- and Low-Temperature Seasons for Mass Culture of the Rotifer Brachionus plicatilis
Fisheries and aquatic sciences. 2011. Dec, 14(4): 323-332
Copyright ©2011, The Korean Society of Fisheries and Aquatic Science
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
  • Received : April 04, 2011
  • Accepted : November 11, 2011
  • Published : December 31, 2011
Download
PDF
e-PUB
PubReader
PPT
Export by style
Share
Article
Author
Metrics
Cited by
TagCloud
About the Authors
Jean Hee Bae
Sung Bum Hur
hurs@pknu.ac.kr
Abstract
To find seasonally optimal microalgae for mass culture of the rotifer Brachionus plicatilis , the growth rates of 12 microalgal spe-cies (two marine Chlorella spp., five marine Nannochloris spp., two marine Nannochloropsis spp., one estuarine Nannochloropsis sp., and two estuarine Chlorella spp.) were compared at 25℃ at 15 psu and 30 psu. Among these, six species showing high growth rates were chosen and examined again at high (30℃ and 32℃) and low (10℃) temperatures. Their amino and fatty acids and the dietary value of the rotifers that fed on each microalgal species were examined. Nannochloris sp. (KMMCC-119) and Chlorella vulgaris (KMMCC-120) showed the highest growth rates at temperatures over 30℃ and at 10℃, respectively. The growth rate of Nannochloris was higher than those of Chlorella and Nannochloropsis at high temperatures, but lower than those of the latter at low temperatures. The growth rate of rotifers fed on Nannochloropsis was highest and that of those fed on Chlorella was lowest. Levels of eicosapentaenoic acid and docosahexaenoic acid were highest in Nannochloropsis and lowest in Nannochloris . However, total amino acid content was highest in Nannochloris and lowest in Chlorella . In conclusion, Nannochloropsis sp. (KMMCC-33) was the best microalgal species for the mass culture of the rotifer. However, during high- or low-temperature seasons in which Nannochloropsis does not grow well, Nannochloris spp. (KMMCC-119, 395) and C. vulgaris (KMMCC-120) would adequately replace Nannochloropsis sp. (KMMCC-33).
Keywords
Introduction
The rotifer Brachionus plicatilis is prevalently used as an early larval food for the seed production of seawater fish, as it is small, with low motility, and is suitable for high-density culture. Since the rotifer was first used as feed for Pagrus ma-jor in Japan (Fukusho, 1989), microalgae and yeast have been widely used as feed in the mass production of the rotifer.
Examples of commonly used microalgae and yeast include the microalgae Nannochloropsis (Fukusho, 1989; Kosto-poulou and Vadstein, 2007; Ferreira et al., 2009), Chlorella (Maruyama et al., 1997; Cabrera et al., 2005; Zhou et al., 2009), and Nannochloris (Witt et al., 1981; Cabrera and Hur, 2001, Cho et al., 2007), and the yeasts bread yeast Saccharomyces cerevisiae (Gilberto and Mazzola, 1981; Sarma et al., 2002; Wang et al., 2009) and marine yeast Candida utilis (Kim et al., 2005).
Yeasts are more economical than microalgae, but contain insufficient amounts of unsaturated fatty acids, which are es-sential for the growth of rotifers (Watanabe et al., 1980; Kim et al., 2009). In an attempt to supplement the shortcomings of yeast, ω-yeast was developed using bread yeast infused with squid oil. However, this had lower nutritional value than microalgae and caused water pollution (Hirayama and Funa-moto, 1983). Thus, in spite of the high costs incurred on the mass culture of rotifers, live microalgae are still preferred by hatcheries (Hur, 1991; Borowitzka, 1997).
Chlorella , Nannochloris , and Nannochloropsis have been well-known to be easily mass-cultured and to have high con-tents of protein or unsaturated fatty acids (Chini Zittelli et al., 1999; Hu and Gao, 2003; Cho et al., 2007). However, they are also at high risk of sudden mortality at temperatures over 30℃, and their low growth rates in water temperatures under 10℃ are problematic (Fukusho et al., 1985; Hur, 1991).
Therefore, this study aimed to identify a new species among Chlorella , Nannochloris , and Nannochloropsis that would be specifically suitable for mass culture as rotifer feed during sea-sons of high and low temperature.
Materials and Methods
- Culture of microalgae
The microalgae used in this study, from the Korea Marine Microalgae Culture Center (KMMCC), included nine ma-rine species and three estuarine species. These consisted of four kinds of Chlorella , five kinds of Nannochloris , and three kinds of Nannochloropsis ( Table 1 ). Their growth rates were observed and compared to each other.
Microalgae in the log phase stage were inoculated in 100 mL of f/2 culture medium (Guillard and Ryther, 1962) in a 250-mL Ehrenmeyer flask at a density of 100 × 10 4 cells/mL. Microalgae were cultured in standing water at 25℃ and 15 and 30 psu, with continuous light of 100 ㎛ol m -2 s -1 , three times during 7 days. The salinity was adjusted by the mixture
List of microalgal species used in the studyKMMCC, Korea Marine Microalgae Culture Center; UTEX, The Culture Col-lection of Algae at the University of Texas at Austin.
PPT Slide
Lager Image
List of microalgal species used in the study KMMCC, Korea Marine Microalgae Culture Center; UTEX, The Culture Col-lection of Algae at the University of Texas at Austin.
of filtered seawater and distilled water.
To measure growth rate, cells were counted by hemacy-tometer regularly, four times a day. The specific growth rate (SGR) was calculated according to Guillard (1973) [SGR = 3.322 × log(N 1 /N 0 )/t, where t is culture days after inocula-tion, and N 0 and N 1 are cell density after inoculation or t days, respectively].
- Growth of six kinds of microalgae at high and low temperature
Six kinds of microalgae that showed high growth rates in the aforementioned experiment were cultured at high tempera-tures of 30℃ and 32℃ and a low temperature of 10℃ at 15 psu with 100 ㎛ol m -2 s -1 of continuous lighting, namely, Nan-nochloropsis sp. (KMMCC-33), Nannochloropsis oceanica (KMMCC-13), Nannochloris oculata (KMMCC-16), Nanno-chloris sp. (KMMCC-119), Nannochloris sp. (KMMCC-395), and Chlorella vulgaris (KMMCC-120). Their SGR was exam-ined using the previously described method for 10 days.
- The adequacy of the six microalgae as rotifer feed
The rotifer Brachionus plicatilis (R-4, L-type), provided by the Culture Collection of Useful Marine Plankton (CCUMP) at Pukyung National University in Korea, was used in this study. The amount of microalgae fed daily to an individual rotifer was in accordance with algal cell volume as follows. For the smallest cells, N. oculata , Nannochloris sp. (KMMCC-119), and Nannochloris sp. (KMMCC-395), 30 × 10 4 cells were provided as feed. For N. oceanica and Nannochloropsis sp. (KMMCC-33), 22 × 10 4 cells were fed. For the largest cells, C. vulgaris (KMMCC-120), 15 × 10 4 cells were supplied. The ro-tifer was inoculated at 10 individuals/mL in 100 mL of a 250-mL Ehrenmeyer flask and cultured in standing water at 26℃ and 15 psu under continuous lighting of 60 ㎛ol m -2 s -1 . Cul-ture was conducted in triplicate during 5 days. One milliliter of each culture group was arbitrarily drawn and placed in Lu-gol's solution and the number of rotifers in 1 mL was counted under a stereoscopic microscope three times a day. The SGR of the rotifers was calculated by the method described above.
- Analysis of the nutritional content of the microal-gae and rotifers
The amino and fatty acids of the six selected microalgae and the rotifers fed on three selected microalgae, which in-duced high growth rates in rotifers, were analyzed. They were cultured by the method described above. At the end of the log phase of growth, they were harvested and kept at -80℃ until analysis.
For the analysis of amino acids, 20 mg of sample infused with 15 mL of 6 N HCl was heated, sealed, and hydrolyzed at 110℃ for 24 h. The sample was then filtered and dried to remove HCl. Then 25 mL of the sample was set by sodium dilution buffer (pH 2.2) and a portion of the sample was ana-lyzed by the ninhydrin method using S433 (Sykam, Fürsten-feldbruck, Germany). Conditions of the analysis were as fol-lows: column size, 4 mm × 150 mm; absorbance level, 570 nm and 440 nm; reagent flow rate, 0.25 mL/min; buffer flow rate, 0.45 mL/min; reactor temperature, 120℃; reactor size, 15 m; and analysis time, 65 min.
For the analysis of fatty acids, 20 mg of sample in a 15-mL flask was added to 2 mL of 10% BF 3 -methanol. Nitrogen was added to the sample and heated at 85℃ for an hour and a half to draw methyl ester (Morrison and Smith, 1964; Budge, 1999). Cooled to 30-40℃, the sample was combined with wa-ter and hexane to draw fatty acids separately. The extracted fatty acids were analyzed with a HP GC 6890 Plus installed with a HP autosampler (Agilent Technologies, Santa Clara, CA, USA). The GLC used in this analysis was the DB-225 (20 m × 0.1 mm, i.d., 0.1 ㎛ film thickness; J&W Scientific, Agilent Technologies, Santa Clara, CA, USA). Conditions of the analysis were as follows: column temperature levels, 60-195℃ (25℃/min); temperature conditions, 195-205℃ (3℃/min), 205-230℃ (8℃ min); injector, 250℃; detector, 250℃; and carrier gas used, He (60 cm/s). Fatty acids were identified by comparison with known standards.
- Statistical analysis
The results of this study were analyzed by one-way ANO-VA, and Duncan's multiple range test (Duncan, 1955) was ap-plied for the significance level ( P < 0.05). The SPSS version 17 (SPSS Inc., Chicago, IL, USA) program was used for all statistical analyses.
Results
- Growth of marine microalgae
After 7 days culture of the nine marine microalgae at 30 psu and 15 psu, results ( Fig. 1 ) indicated that N. oculata and Nan-nochloris . sp. (KMMCC-395) in 30 psu showed the highest growth rates of 0.9734 and 0.9640, respectively (highest cell densities, 11,229 × 10 4 cells/mL and 10,733 × 10 4 cells/mL, respectively). The growth rates of C. salina and C. vulgaris at 0.7912 and 0.7812 were not significantly different from each other. The growth rates of N. oceanica and Nannochloropsis . sp. at 0.7866 and 0.7842 were as low as those of Chlorella , while Nannochloris showed a significantly higher growth rate than those of Chlorella and Nannochloropsis ( P < 0.05). The growth rate of Nannochloris sp. (KMMCC-58) was signifi-cantly lower than those of the other four kinds of Nannochlo-ris ( P < 0.05).
At 15 psu, the growths of N. oculata and the three kinds of Nannochloris (KMMCC-117, 119, and 395) were highest, in
PPT Slide
Lager Image
Specific growth rate of nine marine microalgal species at 30 psu (A) and 15 psu (B) 25℃ and 100 ㎛ol m-2 s-1 (KMMCC Korea Marine Microalgae Culture Center; KMMCC-79 Chlorella salina; KMMCC-95 C. vulgaris; KMMCC-58 Nannochloris sp.; KMMCC-16 N. oculta; KMMCC-117 Nannochloris sp.; KMMCC-119 Nannochloris sp.; KMMCC-395 Nannochloris sp.; KMMCC-33 Nannochloropsis sp.; KMMCC-13 N. oceanica). Different letters on the bar mean significantly difference (P < 0.05).
the range of 0.9393 and 0.9504 (highest cell density, 9,718 × 10 4 cells/mL to 10,145 × 10 4 cells/mL). Conversely, Nanno-chloris sp. (KMMCC-58) showed a significantly lower growth rate than other strains, similar to that of Chlorella . N. oceanica and Nannochloropsis sp., the two kinds of Nannochloropsis , showed significantly lower growth rates compared to the four kinds of Nannochloris , but significantly higher growth rates than Chlorella ( P < 0.05). The growth rates of Chlorella and Nannochloropsis tended to be higher at 15 psu than at 30 psu. However, the four kinds of Nannochloris , except for Nan-nochloris sp. (KMMCC-58), showed slightly higher growth rates at 30 psu.
- Growth of estuarine microalgae
At 30 and 15 psu, the growth rates of C. vulgaris (KMMCC-120) were 0.8495 and 0.8601 (6,166 × 10 4 cells/mL and 6,491 × 10 4 cells/mL), respectively, indicating the sig-
PPT Slide
Lager Image
Specific growth rate of three estuarine microalgal species at 30 psu (black bar) and 15 psu (white bar) 25°C and 100 ㎛ol m-2 s-1 (KMMCC Korea Marine Microalgae Culture Center; KMMCC-120 Chlorella vulgaris; KMMCC-137 Chlorella sp.; KMMCC-327 Nannochloropsis sp.). Different letters on the bar mean significantly difference (P < 0.05).
PPT Slide
Lager Image
Specific growth rate of six microalgal species at 15 psu 25℃ 100 ㎛ol m-2 s-1 (KMMCC Korea Marine Microalgae Culture Center; KMMCC-33 Nannochloropsis sp.; KMMCC-13 N. oceanic; KMMCC-16 Nannochoris oculata; KMMCC-119 Nannochoris sp.; KMMCC-395 Nannochoris sp.; KMMCC-120 Chlorella vulgaris). Different letters on the bar mean significantly difference (P < 0.05).
nificantly highest rate ( P < 0.05) ( Fig. 2 ). The growth rates of Chlorella sp. (KMMCC-137) and Nannochloropsis sp. (KMMCC-327) were in the range of 0.7781-0.8204, which were lower than that of C. vulgaris (KMMCC-120). The growth rate of Chlorella sp. (KMMCC-137) was significantly higher at 30 psu than at 15 psu. As a result, Chlorella sp. (KMMCC-137) was distinguished as a marine microalga. The growth rates of the other two kinds of Chlorella indicated no significant difference according to salinity ( P < 0.05).
In contrast to the estuarine C. vulgaris (KMMCC-120), which showed a higher growth rate than marine Chlorella (KMMCC-79 and 95), Chlorella sp. (KMMCC-137) and Nan-nochloropsis sp. (KMMCC-327) showed similar growth rates to those of marine microalgae.
- Analyses of the nutritional content and growth of six selected microalgae, including Nannochlorop-sis, Nannochloris, and Chlorella
The growth and nutritional content of the marine microal-gae N. oceanica , Nannochloropsis sp. (KMMCC-33), N. ocu-lata , Nannochloris sp. (KMMCC-119), and Nannochloris sp. (KMMCC-395) and the estuarine C. vulgaris (KMMCC-120) were studied.
Growth rates under the same culture conditions, 15 psu, 25℃, and 100 ㎛ol m -2 s -1 , are shown in Fig. 3 . Among the six kinds of microalgae, the growth rate of Nannochloris sp. (KMMCC-119) was highest at 0.8753 (highest cell density, 6,987 × 10 4 cells/mL). The growth rates of the two kinds of Nannochloropsis were significantly higher than that of Chlo-rella and lower than that of Nannochloris ( P < 0.05). In addi-tion, the growth rate of estuarine C. vulgaris (KMMCC-120), 0.7807 (highest cell density, 4,416 × 10 4 cells/mL), was the lowest compared to those of marine microalgae ( P < 0.05).
Nannochloris sp. (KMMCC-395) contained the highest percentage amino acid content at 72.97% and C. vulgaris (KMMCC-120) the lowest at 45.72% ( Table 2 ). The amino acid contents of N. oceanica and Nannochloris sp. (KMMCC-119) were lower than that of Nannochloris sp. (KMMCC-395) and
Amino acid composition (%) of six microalgal speciesKMMCC, Korea Marine Microalgae Culture Center ; KMMCC-33, Nanno-chloropsis sp.; KMMCC-13, N. oceanic; KMMCC-16, Nannochoris oculata; KMMCC-119, Nannochoris sp.; KMMCC-395, Nannochoris sp.; KMMCC-120, Chlorella vulgaris; EAA, essential amino acid; NEAA, non-essential amino acid.
PPT Slide
Lager Image
Amino acid composition (%) of six microalgal species KMMCC, Korea Marine Microalgae Culture Center ; KMMCC-33, Nanno-chloropsis sp.; KMMCC-13, N. oceanic; KMMCC-16, Nannochoris oculata; KMMCC-119, Nannochoris sp.; KMMCC-395, Nannochoris sp.; KMMCC-120, Chlorella vulgaris; EAA, essential amino acid; NEAA, non-essential amino acid.
higher than that of C. vulgaris . Among nonessential amino acids, glutamine and leucine contents were high. Essential amino acids were highest in Nannochloris sp. (KMMCC-395) at 32.51%.
Nannochloropsis , Nannochloris , and Chlorella indicated high contents of fatty acids at ratios of 14:0, 15:1, 16:0, and 16:1 ( Table 3 ). The contents of polyunsaturated fatty acid, PUFA, in Nannochloropsis sp. (KMMCC-33) and Nannochlo-ris sp. (KMMCC-119) were highest at 40.68% and 39.63%, respectively. The content of eicosapentaenoic acid (EPA,
Fatty acid composition (%) of six microalgal speciesKMMCC, Korea Marine Microalgae Culture Center ; KMMCC-33, Nanno-chloropsis sp.; KMMCC-13, N. oceanic; KMMCC-16, Nannochoris oculata; KMMCC-119, Nannochoris sp.; KMMCC-395, Nannochoris sp.; KMMCC-120, Chlorella vulgaris; EPA, eicosapentaenoic acid; DHA, docosah;exaenoic acid.
PPT Slide
Lager Image
Fatty acid composition (%) of six microalgal species KMMCC, Korea Marine Microalgae Culture Center ; KMMCC-33, Nanno-chloropsis sp.; KMMCC-13, N. oceanic; KMMCC-16, Nannochoris oculata; KMMCC-119, Nannochoris sp.; KMMCC-395, Nannochoris sp.; KMMCC-120, Chlorella vulgaris; EPA, eicosapentaenoic acid; DHA, docosah;exaenoic acid.
20:5n-3) in Nannochloropsis sp., 34.88%, was high com-pared to that in Nannochloris sp. (KMMCC-119), which was the lowest at 0.35%. The contents of docosahexaenoic acid (DHA, 22:6n-3) in Nannochloropsis sp. (KMMCC-33) and Nannochloris sp. (KMMCC-119) were as low as 0.29% and 0.02%, respectively. The content of EPA+DHA in Nannochlo-ropsis sp. (KMMCC-33), 35.17%, was the highest and in Nan-nochloris sp. (KMMCC-119), 0.37%, the lowest.
- Growth of Nannochloropsis, Nannochloris, and Chlorella in high and low water temperatures
The growth rates of six kinds of Nanno-chloropsis , Nannochloris , and Chlorella at high (30℃ and 32℃) and low (10℃) temperatures are shown in Fig. 4 . At 30℃, Nannochloris sp. (KMMCC-119) and N. oculata showed the highest cell den-sity within 9-10 days of culture at 7,950 × 10 4 cells/mL and 7,951 × 10 4 cells/mL, respectively. The growth rate of N. oce-anica was 2,991 × 10 4 cells/mL up to the sixth day of culture, but thereafter, the growth rate rapidly decreased. At 32℃, Nannochloris spp. (KMMCC-119 and 395) showed the high-est cell density among the microalgae at 6,475 × 10 4 cells/mL and 5,932 × 10 4 cells/mL, respectively. In comparison to the other microalgae, however, N. oceanica showed a much lower cell density, as it did at 30℃.
The growth rates of N. oculata and Nannochloris sp. (KMMCC-119) at 30℃ were significantly the highest: 0.6313 and 0.6281, respectively. Conversely, N. oceanica showed the lowest growth rate: 0.3483 ( P < 0.05). At 32℃, growth rates significantly differed according to the individual strain. The growth rate of Nannochloris sp. (KMMCC-119), 0.6017, was the highest and that of N. oceanica , 0.1521, was the lowest. In high water temperatures of 30℃ and 32℃, Nannochloris had a significantly higher growth rate than Chlorella and Nan-nochloropsis ( P < 0.05).
At 10℃, the growth rate of C. vulgaris (KMMCC-120) was significantly the highest at 0.5052 (highest cell density, 3,316 × 10 4 cells/mL) ( P < 0.05). Growth rates of the other marine microalgae were low, in the range of 0.0109-0.3303 (highest cell density, 107-986 × 10 4 cells/mL). The growth rate of N. oculata in particular was significantly the lowest at 0.0109 (highest cell density, 107 × 10 4 cells/mL).
- Dietary value of the six kinds of Nannochloropsis, Nannochloris, and Chlorella microalgae as feed for rotifers
The growth rates of rotifers fed on the six kinds of microal-gae are shown in Fig. 5 . In 5 days of culture, the growth rates of rotifers fed on Nannochloropsis sp. (KMMCC-33) and N. oceanica were significantly higher than those of the other ex-perimental groups at 0.6806 (highest density, 301 individu-als/mL) and 0.6605 (highest density, 272 individual/mL), re-spectively ( P < 0.05). Nannochloris sp. (KMMCC-395) and
PPT Slide
Lager Image
Cell density of six microalgal species at 30℃ (A) 32℃ (B) and 10℃ (C) and specific growth rate (D) under 15 psu and 100 ㎛ol m-2 s-1 (KMMCC Korea Marine Microalgae Culture Center; KMMCC-33 Nannochloropsis sp.; KMMCC-13 N. oceanic; KMMCC-16 Nannochoris oculata; KMMCC-119 Nannochoris sp.; KMMCC-395 Nannochoris sp.; KMMCC-120 Chlorella vulgaris). Different letters on the bar in each temperature mean significantly difference (P < 0.05).
PPT Slide
Lager Image
Specific growth rate of Brachionus plicatilis fed different microalgal species (KMMCC Korea Marine Microalgae Culture Center; KMMCC-33 Nannochloropsis sp.; KMMCC-13 N. oceanic; KMMCC-16 Nannochoris oculata; KMMCC-119 Nannochoris sp.; KMMCC-395 Nannochoris sp.; KMMCC-120 Chlorella vulgaris). Different letters on the bar mean significantly difference (P < 0.05).
C. vulgaris (KMMCC-120) showed the lowest growth rates at 0.5332 and 0.5376 ( P < 0.05), respectively. Nannochlo-ris spp. (KMMCC-16 and 119) indicated lower growth rates than Nannochloropsis sp. (KMMCC-33) and N. oceanica , but higher growth rates than C. vulgaris (KMMCC-120) and Nan-nochloris sp. (KMMCC-395) ( P < 0.05).
Nannochloris sp. (KMMCC-119), with a high growth rate at high temperature, C. vulgaris (KMMCC-120), with a high growth rate at low temperature, and Nannochlorop-sis sp. (KMMCC-33), as a nutritious feed for rotifers, were cultured in three separate groups. The amino acid contents of these groups were analyzed ( Table 4 ). The kinds of amino acids in the three experimental groups were similar to each other. The content of total amino acids in rotifers fed on C. vulgaris (KMMCC-120) was the highest at 57.15%, and on Nannochloropsis sp. (KMMCC-33) was the lowest at 50.54%. Rotifers showed relatively high contents of leucine and lysine among essential amino acids and of glutamine and aspartate among nonessential amino acids.
For the contents of fatty acids in rotifers fed on the afore-mentioned three kinds of microalgae ( Table 5 ), those fed on C. vulgaris (KMMCC-120) contained 41.62% of C18:2n9 compared to 11.0% and 24.60% in Nannochloropsis sp. (KMMCC-33) and Nannochloris sp. (KMMCC-119), respec-tively. The contents of C16:0 in all three experimental groups were similar, in the range of 12.63-15.94%. The total content of PUFA in rotifers fed on C. vulgaris (KMMCC-120) was the highest at 63.51%. The content of EPA was highest in the Nannochloropsis sp. (KMMCC-33) group at 15.27%, and the content of DHA in rotifers fed on C. vulgaris (KMMCC-120) was the highest at 9.39%.
Discussion
The growth of rotifers depends on the kind of microalgae used as feed (Hirayama et al., 1979; Cho et al., 2007). Various kinds of microalgae as feed for rotifers have been reported, with Nannochloropsis , Nannochloris , and Chlorella , which are highly nutritious and suitable for high density culture, be-ing the most widely used in mass culture.
One of the obstacles to the mass culture of rotifers comes from difficulties in the outdoor mass culture of microalgae during certain seasons. In summer, sudden cell mortality of-ten occurs, and in the winter, the cell growth rates tend to be very low. Thus, further developing microalgae that are highly adaptive to conditions in the two aforementioned seasons is
Amino acid composition (%) of Brachionus plicatilis fed different microalgal speciesKMMCC, Korea Marine Microalgae Culture Center ; KMMCC-33, Nannochlo-ropsis sp.; KMMCC-119, Nannochoris sp.; KMMCC-120, Chlorella vulgaris; EAA, essential amino acid; NEAA, non-essential amino acid.
PPT Slide
Lager Image
Amino acid composition (%) of Brachionus plicatilis fed different microalgal species KMMCC, Korea Marine Microalgae Culture Center ; KMMCC-33, Nannochlo-ropsis sp.; KMMCC-119, Nannochoris sp.; KMMCC-120, Chlorella vulgaris; EAA, essential amino acid; NEAA, non-essential amino acid.
essential (Watanabe et al., 1978; James and Abu-Rezeq, 1988; Hur, 1991).
This study aimed to identify microalgae among Nannochlo-ropsis , Nannochloris , and Chlorella , which are specifically adaptive to high- and low-temperature seasons in Korea. The optimal salinity for the culture of rotifers was 15 psu (Miracle and Serra, 1989; Kim et al., 2005). Since marine microalgae are also euryhaline, their growth rates were compared at sa-linities of 15 and 30 psu.
Nine kinds of marine microalgae showed similar growth rates to each other at 15 and 30 psu. Their growth, howev-er, exhibited slight differences according to microalgal type
Fatty acid composition (%) of Brachionus plicatilis fed different microalgal speciesKMMCC, Korea Marine Microalgae Culture Center ; KMMCC-33, Nannochlo-ropsis sp.; KMMCC-119, Nannochoris sp.; KMMCC-120, Chlorella vulgaris; vulgaris; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid.
PPT Slide
Lager Image
Fatty acid composition (%) of Brachionus plicatilis fed different microalgal species KMMCC, Korea Marine Microalgae Culture Center ; KMMCC-33, Nannochlo-ropsis sp.; KMMCC-119, Nannochoris sp.; KMMCC-120, Chlorella vulgaris; vulgaris; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid.
and salinity. Three estuarine microalgae also showed similar growth rates at 15 and 30 psu. Based on these results, the 12 kinds of microalgae studied in this research can be inferred to be suitable as feed for rotifers because they were euryhaline.
The growth rates of the six kinds of microalgae cultured at 30℃ and 32℃ for the high-temperature experiments were highest for Nannochloris and lowest for Nannochloropsis . The growth rate of Chlorella was much lower than that of Nannochloris , but higher than that of Nannochloropsis . In terms of the highest cell density at 30℃ and 32℃, Nannochlo-ris showed a 25% decrease in growth rate, while Chlorella and Nannochloropsis exhibited 40% and 35% decreases in their growth rates, respectively. These results highlight the fact that the two aforementioned microalgae are more prone than Nan-nochloris to mortality at temperature levels over 30℃.
Among the three kinds of Nannochloris , the growth rates of Nannochloris sp. (KMMCC-119) collected at Deukryang Bay and imported N. oculata (UTEX, 1998) were the highest at 30℃. The growth rate of Nannochloris sp. (KMMCC-395) collected at Puan was significantly lower ( P < 0.05). At 32℃, however, the growth rate of Nannochloris sp. (KMMCC-119) was the highest and that of N. oculata was the lowest. Nan-nochloris sp. (KMMCC-395) showed similar growth at both 32℃ and 30℃. Such growth traits can be explained by Nan-nochloris sp. (KMMCC-395) being adaptive to higher tem-peratures, as it originated from salt ponds in Puan.
At 10℃, for the low-temperature experiment, N. oculata and Nannochloris sp. (KMMCC-119), which were highly vital at high temperature, exhibited the lowest growth rate. However, the growth of C. vulgaris (KMMCC-120), which was isolated from brackish water, was significantly higher than those of the other microalgae, which were from marine water ( P < 0.05). Thus, it is considered suitable for mass cul-ture in low-temperature seasons.
At high temperature, N. oculata was found to have a higher growth rate than Chlorella ellipsoidea or Nannochloropsis sa-lina (James et al., 1989; Hur, 1991). Phaeodactylum tricornu-tum , which belongs to the Bacillariophyceae, shows a higher growth rate than C. ellipsoidea at low temperature. However, it is also reported to be inadequate as a rotifer feed because its dietary value is lower than that of C. ellipsoidea (Hur, 1991; Cho et al., 2007).
The essential amino acid contents of most microalgae are in-fluenced by factors including the intensity of lighting (Thomp-son et al., 1990; Brown et al., 1997), temperature (James et al., 1989; Thompson et al., 1992), pH (Guckert and Cooksey, 1990), culture medium (Wikfors et al., 1984), and harvesting times (Brown et al., 1997; Pernet et al., 2003). In this study, the fatty acid contents of the two kinds of Nannochloropsis , 16:0 and 16:1, were high, which is consistent with reports by Hodgson et al. (1991) and Volkman et al. (1993). Whyte and Nagata (1990) reported that the main components of fatty ac-ids in the marine Chlorella saccharophila were 16:0, 16:1n7, and 18:1n9. However, estuarine C. vulgaris (KMMCC-120) differed from marine C. saccharophila in its main component of fatty acids 17:1 and 20:0.
With regard to EPA and DHA, Volkman et al. (1993) re-ported that Nannochloropsis sp. cultured at 20℃ contained no DHA and 16.1-28.2% EPA. The result of the present study, on microalgae cultured at 25℃, slightly differs from that of Volk-man et al. (1993). Conversely, the report of James et al. (1989) on Nannochloropsis cultured at 25℃ containing 0.4% of DHA is similar to the result of the present study.
The fatty acid contents of rotifers fed on Nannochloropsis sp. (KMMCC-33), Nannochloris sp. (KMMCC-119), and C. vulgaris (KMMCC-120) were 16:0, 16:1, and 18:2, respec-tively, which were similar to the contents of fatty acids in the aforementioned microalgae. The growth rate of rotifers fed on Nannochloropsis sp. (KMMCC-33), which had the high-est EPA content, was high and its content of EPA was also high. Thus, the nutritional content of a feed can be concluded to directly affect the rotifer (Scott and Middleton, 1979; Ben-Amotz et al., 1987; Frolov et al., 1991).
Nannochloris spp. (KMMCC-119 and 395), isolated from Korean coastal waters, showed higher growth rates at 30℃ and 32℃ than the foreign species N. oculata (UTEX, 1998). Estuarine C. vulgaris (KMMCC-120) showed a high growth rate at 10℃, at which temperature most microalgae hardly sur-vived. However, their effectiveness as rotifer feed was lower than that of Nannochloropsis because of their low EPA and DHA contents.
In conclusion, Nannochloropsis sp. (KMMCC-33) is the best choice for the mass culture of rotifers. Nannochlo-ris spp. (KMMCC-119 and 395) and estuarine C. vulgaris (KMMCC-120) seem the most adequate species to replace Nannochloropsis sp. (KMMCC-33) during high- and low- temperature seasons, respectively.
Acknowledgements
This work was supported by the Marine Biomaterials Re-search Center grant from Marine Biotechnology Program funded by the Ministry of Land, Transport and Maritime Af-fairs, Korea and the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2010-0027713).
References
Ben-Amotz A , Fishler R , Schneller A 1987 Chemical composition of dietary species of marine unicelluar algae and rotifers with em-phasis on fatty acids. Mar Biol 95 31 - 36
Borowitzka MA 1997 Microalgae for aquaculture: opportunities and constraints. J Appl Phycol 9 393 - 401
Brown MR , Jeffrey SW , Volkman JK , Dunstan GA 1997 Nutri-tional properties of microalgae for mariculture. Aquaculture 151 315 - 331
Budge SM 1999 Fatty acid biomarkers in a cold water marine environ-ment. Ph.D. Dissertation Memorial University of Newfoundland St. John’s NF CA.
Cabrera T , Hur SB 2001 The nutritional value of live foods on the larval growth and survival of Japanese Flounder Paralichthys olivaceus. J Appl Aquac 11 35 - 33
Cabrera T , Bae JH , Bai SC , Hur SB 2005 Comparison of the nutri-tional value of Chlorella ellipsoidea and Nannochloris oculata for rotifer and Artemia nauplii. J Fish Sci Technol 8 201 - 206
Chini Zittelli G , Lavista F , Bastianini A , Rodolfi L , Vincenzini M , Tredici MR 1999 Production of eicosapentaenoic acid by Nan-nochloropsis sp. cultures in outdoor tubular photobioreactors. J Biotechnol 70 299 - 312
Cho SH , Ji SC , Hur SB , Bae J , Park IS , Song YC 2007 Optimum temperature and salinity conditions for growth of green algae Chlorella ellipsoidea and Nannochloris oculata. Fish Sci 73 1050 - 1056
Ducan DB 1955 Multiple range and multiple F tests. Biometrics 11 1 - 42
Ferreira M , Coutinho P , Seixas P , Fábragas J , Otero A 2009 En-riching rotifers with “Premium” microalgae. Nannochloropsis ga-ditana. Mar Biotechnol 11 585 - 595
Frolov AV , Pankov SL , Geradze KN , Pankova SA , Spektorova LV 1991 Influence of the biochemical composition of food on the bio-chemical composition of the rotifer Brachionus plicatilis. Aqua-culture 97 181 - 202
Fukusho K 1989 Biology and mass production of the rotifer Brachio-nus plicatilis. Int J Aquac Fish Technol 1 232 - 240
Fukusho K , Okauchi M , Tanaka H , Wabyuni SI , Kraisingdecha P , Watanabe T 1985 Food value of a rotifer Brachionus plicatilis cultured with Tetraselmis tetrathele for larvae of a flounder Parali-chthys olivaceus. Bull Natl Res Inst Aquac 7 29 - 36
Gilberto S , Mazzola A 1981 Mass culture of Brachionus plicatilis with an integrated system of Tetraselmis suecica and Saccharomy-ces cerevisiae. J World Maric Soc 12 61 - 62
Guckert JB , Cooksey KE 1990 Triglyceride accumulation and fatty acid profile changes in Chlorella (Chlorophyta) during high pH-induced cell cycle inhibition. J Phycol 26 72 - 79
Guillard RRL 1973 Division rates. In: Handbook of Phycological Methods: Culture Methods and Growth Measurements. Stein JR ed. Cambridge University Press Cambridge GB 289 - 311
Guillard RRL , Ryther JH 1962 Studies of marine plankton diatoms. I. Cyclotella nana Hustedt and Detonula confervacea (Cleve) Gran. Can J Microbiol 8 229 - 239
Hirayama K , Funamoto H 1983 Supplementary effect of several nutrients on nutritive deficiency of baker’s yeast for population growth of the rotifer Brachionus plicatilis. Bull Jpn Soc Sci Fish 49 505 - 510
Hirayama K , Tagagi K , Kimura H 1979 Nutritional effect of eight species of marine phytoplankton on population growth of the roti-fer Brachionus plicatilis. Bull Jpn Soc Sci Fish 45 11 - 16
Hodgson PA , Henderson RJ , Sargent JR , Leftley JW 1991 Patterns of variation in the lipid class and fatty acid composition of Nan-nochloropsis oculata (Eustigmatophyceae) during batch culture. I. The growth cycle. J Appl Phycol 3 169 - 181
Hu H , Gao K 2003 Optimization of growth and fatty acid compo-sition of a unicellular marine picoplankton Nannochloropsis sp. with enriched carbon sources. Biotechnol Lett 25 421 - 425
Hur SB 1991 The selection of optimum phytoplankton species for roti-fer culture during cold and warm seasons and their nutritional value for marine finfish larvae. In: Rotifer and Microalgae Culture Sys-tems Proceedings of a U. S. Asia Workshop. Fulks W and Main KL eds. The Oceanic Institute Honolulu HI US 163 - 173
James CM , Abu-Rezeq TS 1988 Effect of different cell densities of Chlorella capsulata and a marine Chlorella sp. for feeding the rotifer Brachionus plicatilis. Aquaculture 69 43 - 56
James CM , Al-Hinty S , Salman AE 1989 Growth and ω3 fatty acid amino acid composition of microalgae under different temperature regimes. Aquaculture 77 337 - 351
Kim HY , Kim JK , Park KJ , Bae JH , Hur SB 2005 Nutritional value of Candida utilis for rotifer and larval flounder Paralichthys oliva-ceus. J Fish Sci Technol 8 235 - 242
Kim HY , Kim JK , Hur SB 2009 Dietary value of Candida utilis for Artemia nauplii and Mytilus edulis larvae. J Aquac 22 68 - 73
Kostopoulou V , Vadstein O 2007 Growth performance of the roti-fers Brachionus plicatilis B. 'Nevada' and B. 'Cayman' under dif-ferent food concentrations. Aquaculture 273 449 - 458
Maruyama I , Nakao T , Shigeno I , Ando Y , Hirayama K 1997 Application of unicellular algae Chlorella vulgaris for the mass-culture of marine rotifer Brachionus. Hydrobiologia 358 133 - 138
Miracle MR , Serra M 1989 Salinity and temperature influence in rotifer life history characteristics. Hydrobiologia 186/187 81 - 102
Morrison WR , Smith LM 1964 Preparation of fatty acid methyl es-ters and dimethylacetals from lipids with boron fluoride-methanol. J Lipid Res 5 600 - 608
Pernet F , Tremblay R , Demers E , Roussy M 2003 Variation of lipid class and fatty acid composition of Chaetoceros muelleri and Isochrysis sp. grown in a semicontinuous system. Aquaculture 221 393 - 406
Sarma S , Larios-Jurad PS , Nandini S 2002 Population growth of Asplanchna sieboldi fed two Brachionus spp. (Rotifera) raised on green alga and baker's yeast. Hydrobiologia 467 63 - 69
Scott AP , Middleton C 1979 Unicelluar algae as a food for turbo (Scophthalmus maximus L.) larvae: the importance of dietary long-chain polyunsaturated fatty acids. Aquaculture 18 227 - 240
Thompson PA , Harrison PJ , Whyte JNC 1990 Influence of irradi-ance on the fatty acid composition of phytoplankton. J Phycol 26 278 - 288
Thompson PA , Guo MX , Harrison PJ 1992 Effects of variation in temperature. 1. On the biochemical composition of eight species of marine phytoplankton. J Phycol 28 481 - 488
Volkman JK , Brown MR , Dunstan GA , Jeffrey SW 1993 The bio-chemical composition of marine microalgal from the class Eustig-matophycea. J Phycol 29 69 - 78
Wang HH , Wu ZH , Liao YY 2009 High density cultivation of ro-tifer Brachionus plicatilis by baker’s yeast. Fish Sci 28 225 - 228
Watanabe T , Arakawa T , Kitajima C , Fujita S 1978 Nutritional evaluation of proteins of living feeds used in seed production of fish. Bull Jpn Soc Sci Fish 44 985 - 988
Watanabe T , Oowa F , Kitajima C , Fujita S 1980 Relationship be-tween dietary value of brine shrimp Artemia salina and their con-tent of ω3 highly unsaturated fatty acids. Bull Jpn Soc Sci Fish 46 35 - 41
Whyte JNC , Nagata WD 1990 Carbohydrate and fatty acid compo-sition of the rotifer Brachionus plicatilis fed monospecific diets of yeast or phytoplankton. Aquaculture 89 263 - 272
Wikfors GH , Twarog JW , Ukeles R 1984 Influence of chemical composition of algal food sources on growth of juvenile oysters Crassostrea virginica. Biol Bull 167 251 - 263
Witt U , Koske PH , Kuhlmann D , Lenz J , Nellen W 1981 Produc-tion of Nannochloris spec. (Chlorophyceae) in large-scale outdoor tanks and its use as a food organism in marine aquaculture. Aqua-culture 23 171 - 181
Zhou W , Tang X , Qiao X , Wang Y , Wang R , Feng L 2009 Inges-tion of Brachionus plicatilis under different microalgae conditions. China J Oceanogr Limnol 27 473 - 479