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.

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 × 10⁴ 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

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(N₁/N₀)/t, where t is culture days after inocula-tion, and N₀ and N₁ are cell density after inoculation or t days, respectively].

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 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 × 10⁴ cells were provided as feed. For N. oceanica and Nannochloropsis sp. (KMMCC-33), 22 × 10⁴ cells were fed. For the largest cells, C. vulgaris (KMMCC-120), 15 × 10⁴ 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.

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, Fursten-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% BF₃-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.

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.

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 × 10⁴ cells/mL and 10,733 × 10⁴ 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

the range of 0.9393 and 0.9504 (highest cell density, 9,718 × 10⁴ cells/mL to 10,145 × 10⁴ 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.

At 30 and 15 psu, the growth rates of C. vulgaris (KMMCC-120) were 0.8495 and 0.8601 (6,166 × 10⁴ cells/mL and 6,491 × 10⁴ cells/mL), respectively, indicating the sig-

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.

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 × 10⁴ 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 × 10⁴ 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

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,

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.

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 × 10⁴ cells/mL and 7,951 × 10⁴ cells/mL, respectively. The growth rate of N. oce-anica was 2,991 × 10⁴ 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 × 10⁴ cells/mL and 5,932 × 10⁴ 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 × 10⁴ 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 × 10⁴ cells/mL). The growth rate of N. oculata in particular was significantly the lowest at 0.0109 (highest cell density, 107 × 10⁴ cells/mL).

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

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%.

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

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

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.