Development of Economical Fertilizer-Based Media for Mass Culturing of <italic>Nannochloropsis oceanica</italic>
Development of Economical Fertilizer-Based Media for Mass Culturing of Nannochloropsis oceanica
Fisheries and aquatic sciences. 2011. Dec, 14(4): 317-322
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 ( which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
  • Received : April 20, 2011
  • Accepted : November 08, 2011
  • Published : December 31, 2011
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Jean Hee Bae
Sung Bum Hur

This study was conducted to develop economical agricultural fertilizer media for the mass culturing of Nannochloropsis oce-anica . Specific growth rates of N. oceanica cultured with differing concentrations of commercial compounds, urea fertilizers, and trace elements (Zn, Cu, Co, Mo) were compared with the growth rate in f/2 medium. Among the various added trace elements, CuSO 4 ·5H 2 O was most effective for high growth of N. oceanica . The main nitrogen source in the agricultural fertilizers was am-monium, which was unsuitable for the growth of N. oceanica . Thus, the fertilizer at a lower concentration infused with NaNO 3 as a nitrogen source was more effective than fertilizer at higher concentrations. In this study, the growth of N. oceanica cultured with an agricultural fertilizer medium composed of compound fertilizer (41.7 mg/L), urea fertilizer (34.4 mg/L), NaNO 3 (150 mg/L), and CuSO4·5H 2 O (0.0588 mg/L) was similar to that of N. oceanica cultured in f/2 medium.
In the past, aquaculture was the main industry using micro-algae commercially. Today, various businesses and industries, including those involved in supplementary health products, cosmetics, medicine, and bio-energy, are making extensive use of microalgae (Borowitzka and Borowitzka, 1988).
The advantages of using microalgae as commercial bio-materials include eco-friendly culture methods that allow for continuous reproduction and wide-ranging uses without pol-lutants. However, weaknesses include the sudden death of mi-croalgae, which often leads to high costs and low productivity (Chisti, 2007).
The cost of microalgae used as live food in artificial seed production of shellfish is nearly 30% of the total cost of seed production (Borowitzka, 1997). However, if stable and eco-nomical microalgae production can be developed, microalgae will likely become one of the most important materials in bio-industry.
For mass production of microalgae, reagents used for in-door culture would be inappropriate because of their high cost. Instead, more economical resources, such as agricultural fertilizers, are frequently used (López-Ruiz et al., 1995; Va-lenzuela-Espinoza et al., 2002; Pacheco-Vega and Sánchez-Saavedra, 2009). Using agricultural fertilizers only, however, leads to the problem of lower cell growth rates than in com-mon media such as f/2 (Guillard and Ryther, 1962).
Nannochloropsis oceanica is commonly used to culture rotifers for marine fishes (Cabrera et al., 2005; Kobayashi et al., 2005; Ferreira et al., 2009) and to create "green water" for nursery tanks (Cabrera and Hur, 2001) because they are nutritious and easy to mass-produce. Additionally, their high contents of vitamins (Brown et al., 1997), lipids (Patil et al., 2007; Seychelles et al., 2009), highly unsaturated fatty acids (Sukenik et al., 1993; Zittelli et al., 1999; Hu and Gao, 2003), protein (Volkman et al., 1993), and natural pigment (Lubián et al., 2000) distinguish N. oceanica as a prospective microalgal species to be further researched and developed for the marine conbio-industry (Becker, 1981; Harting et al., 1988; Cha et al., 2010). In this study, we sought to develop economical media that could effectively replace f/2 for the mass production of N. oceanica .
Materials and Methods
The agricultural fertilizers used in this study were as fol-lows: urea fertilizer containing 46% nitrogen and compound fertilizer (Nam-Hae Chemicals Inc., Yeosu, Korea) contain-ing 22% nitrogen, 12% phosphorus, 17% potassium, and 3% magnesium. The amount of the fertilizers used in this study followed the Schreiber medium standard (Schreiber, 1927) that consists of NaNO 3 (100 mg/L) and Na 2 HPO 4 ·12H 2 O (20 mg/L). Because 1 L of filtered seawater with 166.7 mg of compound fertilizer and 137.6 mg of urea fertilizer equals the concentrations of nitrate and phosphate in Schreiber medium, this standard was set as 1.0 times the basic fertilizer medium. The fertilizers were ground, dissolved in warm water, and fil-tered immediately before use.
The N. oceanica (KMMCC-13) used in this study were obtained from the Korea Marine Microalgae Culture Center (KMMCC) at Pukyong National University, South Korea. To culture N. oceanica , the following steps were conducted. First, 100 mL of autoclaved fertilizer media and 10 mL of culture stock were put into a 250 mL Erlenmeyer flask. Standing cul-tures were then kept at 25℃ under continuous lighting of 100 ㎛ol m -2 s -1 and 15 psu. The culture was conducted in tripli-cate. Cell density was assessed twice daily at the same times using a hemocytometer, and the daily specific growth rate (SGR) was measured by the Guillard method (1973): SGR = 3.322 × log(N 2 /N 1 )/(t 2 -t 1 ), where t 2 and t 1 are culture days after inoculation, and N 2 and N 1 are the cell density at t 2 , and t 1 , respectively.
- Culturing N. oceanica with differing concentra-tions of fertilizers and addition of trace elements
To find the optimal concentrations of fertilizers, cell growth was observed for 5 days in the following conditions: 1. f/2 medium as a control group, 2. fertilizer medium 1.0 times (166.7 mg/L of compound fertilizer and 137.6 mg/L of urea fertilizer), 3. fertilizer media 1.25 times (208.4 mg/L of com-pound fertilizer and 172.0 mg/L of urea fertilizer), and 4. fer-tilizer 1.5 times (250.1 mg/L of compound fertilizer, 206.4 mg/L of urea fertilizer).
For trace elements, those used in f/2 medium, such as CoCl 2 ·6H 2 O (0.110 mg/L), CuSO 4 ·5H 2 O (0.0196 mg/L), ZnSO 4 ·7H 2 O (0.044 mg/L), and Na 2 MoO 4 ·2H 2 O (0.012 mg/L), were added to the fertilizer medium at varying concentrations: 0.5, 1.0, 1.5, and 2.0 times. The growth rates in these media were observed for 7 days and compared with the growth rate in f/2 medium.
- Effects of the addition of NaNO3
The concentration of the previous fertilizer medium was reduced to 0.5, 0.25, and 0.17 times. Then, CuSO 4 ·5H 2 O (0.0588 mg/L) at three times the concentration in f/2 medium and NaNO 3 (150 mg/L) at the same concentration as in f/2 medium were added. This experiment involved eight groups, and daily growth in each of the following groups was mea-sured for 7 days: 1) f/2 medium; 2) fertilizer medium 1.25 times; 3) fertilizer medium 0.5 times (compound fertilizer 83.4 mg/L + urea fertilizer 68.8 mg/L + NaNO 3 ); 4) group 3 + CuSO 4 ·5H 2 O; 5) fertilizer medium 0.25 times (compound fer-tilizer 41.7 mg/L + urea fertilizer 34.4 mg/L + NaNO 3 ; 6) group 5 + CuSO 4 ·5H 2 O; 7) fertilizer medium 0.17 times (compound fertilizer 28.3 mg/L + urea fertilizer 23.4 mg/L+NaNO 3 ); and 8) group 7 + CuSO 4 ·5H 2 O.
- Growth comparison with laboratory and indus-trial reagents
To develop an economical fertilizer medium for the mass production of N. oceanica , the NaNO 3 and CuSO 4 ·5H 2 O re-agents were examined separately with laboratory reagents (NaNO 3 , Samchun Pure Chemical Co., Ltd., Pyeongtaek, Ko-rea; CuSO 4 ·5H 2 O, Shimakyu’s Pure Chemicals, Osaka, Japan) and industrial reagents (NaNO 3 , Rifa Ind. Co. Ltd., Ludwig-shafen, Germany; CuSO 4 ·5H 2 O, Young Poong Inc., Seoul, Korea). The N. oceanica were cultured for 7 days and their growth was measured using the methods above.
- Statistics analyses
Results were analyzed by one-way analysis of variance, and Duncan's multiple range test (Duncan, 1955) was used to detect significant differences at the level of P < 0.05. SPSS ver. 17 (SPSS Inc., Chicago, IL, USA) was used for all analy-ses.
- Growth according to concentration of fertilizers and addition of trace elements
The growths of N. oceanica cultured in f/2 and agricultural fertilizer media for 5 days are shown in Fig. 1 . The f/2 me-dium, as the control group, showed the significantly highest SGR of 0.3815. In fertilizer medium 1.25 times, the growth rate of 0.3407 was significantly lower than that in f/2 medium. However, this growth rate was significantly higher than those of the other experimental groups ( P < 0.05).
The results of the 7-day cultures of N. oceanica in fertil-izer medium 1.25 times plus trace elements, CoCl 2 ·6H 2 O, CuSO 4 ·5H 2 O, ZnSO 4 ·7H 2 O, and Na 2 MoO 4 ·2H 2 O at con-
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Specific growth rate of Nannochloropsis oceanica cultured with agricultural fertilizer media (1 f/2; 2 compound fertilizer 166.7 mg/L + urea fertilizer 137.6 mg/L; 3 compound fertilizer 208.4 mg/L + urea fertilizer 172.0 mg/L; 4 compound fertilizer 250.1 mg/L + urea fertilizer 206.4 mg/L).
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Specific growth rate of Nannochloropsis oceanica cultured with agricultural fertilizer media added with different concentrations of trace elements [1 f/2; 2 compound fertilizer 208.4 mg/L + urea fertilizer 172.0 mg/L; 3 2 + CoCl2 (0.055 mg/L); 4 2 + CoCl2 (0.110 mg/L); 5 2 + CoCl2 (0.165 mg/L); 6 2 + CoCl2 (0.220 mg/L); 7 2 + CuSO4·5H2O (0.0098 mg/L); 8 2 + CuSO4·5H2O (0.0196 mg/L); 9 2 + CuSO4·5H2O (0.0294 mg/L); 10 2 + CuSO4·5H2O (0.0392 mg/L); 11 2 + ZnSO4·7H2O (0.022 mg/L); 12 2 + ZnSO4·7H2O (0.044 mg/L); 13 2 + ZnSO4·7H2O (0.066 mg/L); 14 2 + ZnSO4?·7H2O (0.088 mg/L); 15 2 + Na2MoO4·2H2O (0.006 mg/L); 16 2 + Na2MoO4·2H2O (0.012 mg/L); 17 2 + Na2MoO4·2H2O (0.018 mg/L); 18 2 + Na2MoO4·2H2O (0.024 mg/L)].
centrations of 0.5-2.0 times were as follows. Higher con-tents of trace elements produced higher growth rates of N. oceanica ( Fig. 2 ). The fertilizer medium 1.25 times and the media infused with 0.5 times CoCl 2 ·6H 2 O (0.055 mg/L) and Na 2 MoO 4 ·2H 2 O (0.006 mg/L) showed the lowest growth rates in the range of 0.2903-0.2930.
The growth rates of the experimental groups infused with CuSO 4 ·5H 2 O and ZnSO 4 ·7H 2 O were 0.3096-0.3598 and 0.3042-0.3559, respectively. These growth rates were rela-tively high compared with those for other media infused with CoCl 2 ·6H 2 O or Na 2 MoO 4 ·2H 2 O, which showed rates of
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Specific growth rate of Nannochloropsis oceanica cultured with agricultural fertilizer media added with different concentrations of CuSO4 5H2O [1 f/2; 2 compound fertilizer 208.4 mg/L + urea fertilizer 172.0 mg/L; 3 2 + CuSO4 5H2O (0.0392 mg/L); 4 2 + CuSO4·5H2O (0.0588 mg/L); 5 2 + CuSO4·5H2O (0.0784 mg/L); 6 2 + CuSO4 5H2O (0.098 mg/L)].
In the experimental groups infused with either 1.5 (0.0294 mg/L) or 2 times (0.0392 mg/L) CuSO 4 ·5H 2 O and 2 times ZnSO 4 ·7H 2 O (0.088 mg/L), the growth rates of 0.3457-0.3598 were still significantly lower than the rate of 0.3726 for the control group in f/2 medium. However, the former groups showed higher growth rates than the rest of the experimental groups ( P < 0.05).
To test the exact concentration of CuSO 4 ·5H 2 O for infusion, 2-, 3-, 4-, and 5-fold increased concentrations (0.0392?0.098 mg/L) of CuSO 4 ·5H 2 O were added to fertilizer medium 1.25 times and cultures were grown for 8 days. The results indicated the significantly highest growth rate of 0.6446 in the control f/2 group ( P < 0.05) ( Fig. 3 ). When three-fold CuSO 4 ·5H 2 O was added, the growth rate was 0.5955. Although this growth rate was lower than that in f/2 medium, it was significantly higher than the other fertilizer media ( P < 0.05). For more than three-fold CuSO 4 ·5H 2 O infusion, as the concentration of copper was increased, the growth rate decreased significantly ( P < 0.05).
- Growth according to addition of NaNO3
On the basis of the findings in this study, growth differ-ences in f/2 and fertilizer media seemed to be correlated with the high content of ammonia and low content of nitrate. With the infusion of CuSO 4 ·5H 2 O (0.0588 mg/L) and NaNO 3 (150 mg/L), the amounts of fertilizers were reduced by 0.5 times (compound fertilizer, 83.4 mg/L; urea fertilizer, 68.8 mg/L), 0.25 times (compound fertilizer, 41.7 mg/L; urea fertilizer, 34.4 mg/L), and 0.17 times (compound fertilizer, 28.3 mg/L;
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Specific growth rate of Nannochloropsis oceanica cultured with low concentration of agricultural fertilizer media added with NaNO3 (150 mg/L) and CuSO4 (0.0588 mg/L) [1 f/2; 2 compound fertilizer 208.4 mg/L + urea fertilizer 172.0 mg/L; 3 compound fertilizer 83.4 mg/L + urea fertilizer 68.8 mg/L + NaNO3; 4 3 + CuSO4·5H2O; 5 compound fertilizer 41.7 mg/L + urea fertilizer 34.4 mg/L + NaNO3; 6 5 + CuSO4·5H2O; 7 compound fertilizer 28.3 mg/L + urea fertilizer 23.4 mg/L + NaNO3; 8 7 + CuSO4·5H2O].
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Specific growth rate of Nannochloropsis oceanica cultured with agricultural fertilizer media (compound fertilizer 41.7 mg/L + urea fertilizer 34.4 mg/L) added with laboratorial or industrial reagent of NaNO3 (150 mg/L) and CuSO4·5H2O (0.0588 mg/L).
urea fertilizer, 23.4 mg/L) the level in Schreiber culture me-dium. As a result ( Fig. 4 ), the growth rate of 0.3985 in fertil-izer medium 1.25 times was significantly the lowest rate ( P < 0.05). Fertilizer medium 0.25 times infused with CuSO 4 ·5H 2 O (0.0588 mg/L) and NaNO 3 (150 mg/L) showed a high growth rate of 0.4481, about 96% of that in f/2 medium. In fact, this result was not significantly different from that in f/2 medium ( P < 0.05).
- Growth rate comparison between laboratory and industrial reagents
From the previously mentioned fertilizer media 0.25 times (compound fertilizer, 41.7 mg/L + urea fertilizer 34.4 mg/L) infused with CuSO 4 ·5H 2 O 0.0588 mg/L and NaNO 3 150 mg/L, NaNO 3 and CuSO 4 ·5H 2 O were assessed separately with labo-ratory and industrial reagents. The growth rates of N. oceanica in fertilizer media with laboratory and industrial reagents were 0.4331 and 0.4383, respectively ( Fig. 5 ). These results indi-cated no significant difference compared with the growth rate (0.4549) in f/2 medium ( P < 0.05).
Culture media for microalgae should be economical, allow for high growth rates, satisfy the needs of the microalgae, and be easy to prepare. F/2 medium, the most commonly used me-dium for small-scale indoor culture, is costly and difficult to set up for outdoor mass culture (Fabregas et al., 1985). Thus, agricultural fertilizers are commonly used as a replacement for f/2 culture medium (Fabregas et al., 1987; Bae, 2004; Pache-co-Vega and Sánchez-Saavedra, 2009). However, cell growth rates in such fertilizer-based media have not yet reached that in f/2 culture medium. The slower rate is attributable to the presence of nitrogen and phosphorus, major components in fertilizer cultures (Ukeles, 1980; González-Rodriguez and Maestrini, 1984; Bae, 2004). Lack of trace elements and vita-mins necessary for the growth of microalgae are also reasons for lower growth rates (Stein, 1973).
Our aim was to develop media using economical and conve-nient agricultural fertilizers to replace f/2 medium for outdoor mass culture of N. oceanica , which has recently been in the commercial spotlight (Cha et al., 2010). Most common culture media for microalgae are based on Schreiber medium, con-taining NaNO 3 (100 mg/L) and Na 2 HPO 4 ·12H 2 O (20 mg/L) (Schreiber, 1927). Thus, we tested for optimal concentrations of agricultural urea and compound fertilizers in relation to these nutrients.
From the second and third days after inoculation, the growth stage of the cells was in log phase in f/2 media. On the other hand, fertilizer media showed a longer lasting lag phase, and the lower level of the ultimate highest cell den-sity was problematic. The following factors are believed to have caused such results: urea fertilizer, consisting of 46% nitrogen, and compound fertilizer consisting of 22% nitrogen, 12% phosphorus, 17% potassium, and 3% magnesium. Other causes may have been the lack of essential trace elements for the growth of N. oceanica and a nitrogen source consisting mostly of ammonia.
After adding the four trace elements (Co, Cu, Zn, Mo) used in f/2 medium to each fertilizer medium, growth rates of N. oceanica were observed. When CuSO 4 ·5H 2 O (0.0588 mg/L) three times the concentration in f/2 medium was added, the N. oceanica growth rate was 80% of that in f/2 medium. Cu is essential for the growth of microalgae especially for pho-gatosynthesis and enzymatic reactions (Andrade et al., 2004). However, Okauchi et al. (2008) claimed that zinc and cobalt had greater influence than copper on the growth of N. ocu-lata (see Guillard, 1973). In this study, however, copper was the most effective factor in the growth of Nannochloropsis . In Bae’s (2004) study, the concentration of Cu added to f/2 medium was increased 2 to 80 times, and the growth rate of N. oceanica began to decrease above 5 times concentration (CuSO 4 ·5H 2 O 0.0196 mg/L).
González-Rodriguez and Maestrini (1984) used 12 kinds of fertilizers for 16 microalgal species with Conway medium as a control (Walne, 1966). The result of their research, which was similar to our result, showed extremely low growth rates of Nannochloris oculata, Isochrysis galbana, Chlamydomo-nas palla , and Chaetoceros sp. However, for Phaeodactylum tricornutum, Skeletonema costatum, Tetraselmis striata , and Thalassiosira pseudonana , growth rates were similar to or higher than that in Conway medium, the control group. Such differences in growth rates could be due to the nitrogen source, requiring differing media for each kind of microalgae.
Bae (2004) cultured N. oceanica with agricultural fertilizer for 16 days and then analyzed the water. The results showed that the concentration of NH 4 -N (10.0 ppm) in agricultural fertilizer was approximately 154 times higher than that in f/2 culture medium (0.065 ppm); in addition, the concentration of PO 4 -P was nine times higher. These results imply that the growth of microalgae depends on their sensitivity to the am-monium concentration (1 mg atom N/L) (Kaplan et al., 1986). If the concentration is higher than 0.5 mg atom N/L, the growth of microalgae exposed to high intensities of light and pH is likely to decrease (Admiraal, 1977; Kalpan et al., 1986).
In this study, the growth of microalgae in the fertilizer me-dia was slower than that in f/2 medium in the early stages of the experiment. The low level of the highest cell density was also believed to be due to the high ammonium content.
Thus, to reduce the concentration of ammonia in the fertil-izer media, the amounts of fertilizers were reduced to 25% of Schreiber’s nitrate and phosphate concentrations. The growth of N. oceanica in fertilizer media infused with NaNO 3 (150 mg/L) and CuSO 4 ·5H 2 O (0.0588 mg/L) and that in f/2 me-dium showed no significant difference ( P < 0.05). Moreover, in the case of using NaNO 3 and CuSO 4 ·5H 2 O, which are less costly than industrial fertilizers, the growth rate of N. oceani-ca showed no significant difference compared with that in f/2 medium ( P < 0.05).
In conclusion, for 1 ton of filtered seawater, an optimal me-dium for the mass culturing of N. oceanica can be achieved with the following materials: urea fertilizer containing 22% nitrogen (34.4 g), compound fertilizer containing 22% nitro-gen, 12% phosphorus, 17% potassium, and 3% magnesium (41.7 g), and industrial reagent grade NaNO 3 (150 g) and CuSO 4 ·5H 2 O (0.0588 g).
This work was supported by the National Research Foun-dation of Korea(NRF) grant funded by the Korea government (MEST) (No. 2010-0027713) and a part of the project titled "Marine Biotechnology Program" funded by the Ministry of Land, Transport and Maritime Affairs, Korea.
Admiraal W 1977 Tolerance of estuarine benthic diatoms to high con-centrations of ammonia nitrite ion nitrate ion and orthophosphate. Mar Biol 43 307 - 315    DOI : 10.1007/BF00396925
Andrade LR , Farina M , Amado GM 2004 Effects of copper on En-teromorpha flexuosa (Chlorophyta) in vitro. Ecotoxicol Environ Saf 58 117 - 125    DOI : 10.1016/S0147-6513(03)00106-4
Bae JH 2004 Selection of seasonal optimum Chlorella and Nannochloris species and development of media for mass culture. Pukyong National University Busan KR. Ph.D. Dis-sertation
Becker EW 1981 Algae mass cultivation: production and utilization. Process Biochem 16 10 - 14
Borowitzka MA 1997 Microalgae for aquaculture: opportunities and constraints. J Appl Phycol 9 393 - 401    DOI : 10.1023/A:1007921728300
Borowitzka MA , Borowitzka LJ 1988 Micro-algal Biotechnology. Cambridge University Press New York US.
Brown MR , Jeffrey SW , Volkman JK , Dunstan GA 1997 Nutri-tional properties of microalgae for mariculture. Aquaculture 151 315 - 331    DOI : 10.1016/S0044-8486(96)01501-3
Cabrera T , Hur SB 2001 The nutritional value of live foods on the larval growth and survival of Japanese flounder Paralichthys oli-vaceus. J Appl Aquc 11 35 - 53
Cabrera T , Bae JH , Bai SC , Hur SB 2005 Effects of microalgae and salinity on the growth three types of the rotifer Brachionus plicati-lis. J Fish Sci Technol 8 70 - 75
Cha SH , Kim MJ , Yang HY , Jin CB , Jeon YJ , Oda T , Kim D 2010 ACE α-glucosidase and cancer cell growth inhibitory activities of extracts and fractions from marine microalgae Nannochloropsis oculata. Korean J Fish Aquat Sci 43 437 - 444    DOI : 10.5657/kfas.2010.43.5.437
Chisti Y 2007 Biodiesel from microalgae. Biotechnol Adv 25 294 - 306    DOI : 10.1016/j.biotechadv.2007.02.001
Duncan DB 1955 Multiple range and multiple F tests. Biometrics 11 1 - 42    DOI : 10.2307/3001478
Fabregas J , Herrero C , Cabezas B , Abalde J 1985 Mass culture and biochemical variability of the marine microalgae Tetraselmis sue-cica Kylin (Butch) with high nutrient concentrations. Aquaculture 49 231 - 244    DOI : 10.1016/0044-8486(85)90082-1
Fabregas J , Toribio L , Abalde J , Cabezas B , Herrero C 1987 Ap-proach to biomass production of the marine microalga Tetrasel-mis suecica (Kylin) butch using common garden fertilizer and soil extract as cheap nutrient supply in batch cultures. Aquac Eng 6 141 - 150    DOI : 10.1016/0144-8609(87)90011-2
Ferreira M , Coutinho P , Seixas P , Fábregas J , Otero A 2009 En-riching rotifers with "premium" microalgae. Nannochloropsis ga-ditana. Mar Biotechnol 11 585 - 595    DOI : 10.1007/s10126-008-9174-x
González-Rodriguez E , Maestrini SY 1984 The use of some agri-cultural fertilizers for the mass production of marine algae. Aqua-culture 36 245 - 256    DOI : 10.1016/0044-8486(84)90240-0
Guillard RRL 1973 Division rates. In: Handbook of Phycological Meth-ods. Culture Method and Growth Measurement. Stein JR ed. Cam-bridge 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    DOI : 10.1139/m62-029
Harting P , Grobbelaar JU , Soeder CJ , Groeneweg J 1988 On the mass culture of microalgae: a real density as an important factor for achieving maximal productivity. Biomass 15 211 - 221    DOI : 10.1016/0144-4565(88)90057-1
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    DOI : 10.1023/A:1022489108980
Kalpan D , Richmond AE , Dubinsky Z , Aaronson S 1986 Algal nutrition. In: Handbook of Microalgal Mass Culture. Richmond A ed. CRC Press Boca Raton FL US 147 - 197
Kobayashi T , Nagase T , Kurano N , Hino A 2005 Fatty acid com-position of the L-type rotifer Brachionus plicatilis produced by a continuous culture system under the provision of high density Nan-nochloropsis. Nippon Suisan Gakkaishi 71 328 - 334    DOI : 10.2331/suisan.71.328
López-Ruíz JL , García-García RG , Soledad M , Almeda F 1995 Marine microalgae culture: Chaetoceros gracilis with zeolitic product Zestec-56 and a commercial fertilizer as a nutrient. Aqauc Eng 14 367 - 372    DOI : 10.1016/0144-8609(94)00011-O
Lubián LM , Montero O , Moreno-Garrido I , Huertas IE , Sobrino C , González-del Valle M , Parés G 2000 Nannochloropsis (Eu-stigmatophyceae) as source of commercially valuable pigments. J Appl Phycol 12 249 - 255    DOI : 10.1023/A:1008170915932
Okauchi M , Yamada T , Ozaki A 2008 Optimum media for outdoor large-scale and indoor small-scale batch style culture of Nanno-chloropsis oculata. Aquac Sci 56 147 - 155
Pacheco-Vega JM , Sánchez-Saavedra MDP 2009 The biochemical composition of Chaetoceros muelleri (Lemmermann Grown) with an agricultural fertilizer. J World Aquac Soc 40 556 - 560    DOI : 10.1111/j.1749-7345.2009.00276.x
Patil V , Källqvist T , Olsen E , Vogt G , Gislerød HR 2007 Fatty acid composition of 12 microalgae for possible use in aquaculture feed. Aquacult Int 15 1 - 9    DOI : 10.1007/s10499-006-9060-3
Schreiber E 1927 Die Reinkultir von marinem phytoplankton und deren bedeutung fur die erforschung der produktionsfahigkeit des meerwassers. Wiss Meersuntersuch NF 16 1 - 34
Seychelles LH , Audet C , Tremblay R , Fournier R , Pernet F 2009 Essential fatty acid enrichment of cultured rotifers (Brachionus pli-catilis Muller) using frozen-concentrated microalgae. Aqauc Nutr 15 431 - 439    DOI : 10.1111/j.1365-2095.2008.00608.x
Stein JR 1973 Handbook of Phycological Methods: Culture Methods and Growth Measurement. Cambridge University Press Cam-bridge GB.
Sukenik A , Zmora O , Carmeli Y 1993 Biochemical quality of ma-rine unicellular algae with special emphasis on lipid composition. II. Nannochloropsis sp. Aquaculture 117 313 - 326    DOI : 10.1016/0044-8486(93)90328-V
Ukeles R 1980 American experience in the mass culture of microalgae for feeding larvae of the American oyster Crassostrea virginica. In: Algae Biomass: Production and Use. Shelef G and Soeder CJ eds. Elsevier/North Holland Biomedical Press Amsterdam NL 287 - 306
Valenzuela-Espinoza E , Millán-Nûnez R , Núnez-Cebrero F 2002 Protein carbohydrate lipid and chlorophyll a content in Isochrysis aff. galbana (clone T-Iso) cultured with a low cost alternative to the f/2 medium. Aquac Eng 25 207 - 216    DOI : 10.1016/S0144-8609(01)00084-X
Volkman JK , Brown MR , Dunstan GA , Jeffrey SW 1993 The bio-chemical composition of marine microalgal from the class Eustig-matophyceae. J Phycol 29 69 - 78    DOI : 10.1111/j.1529-8817.1993.tb00281.x
Walne PR 1966 Experiments in the large-scale culture of the larvae of Ostrea edulis L. Fish Invest London Ser II 25 1 - 53    DOI : 10.1111/j.1529-8817.1993.tb00281.x
Zittelli GC , 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    DOI : 10.1016/S0168-1656(99)00082-6