Journalof Applied Phycology 3: 43-53, 1991. 1991 Kluwer Academic Publishers. Printed in Belgium.

43

Effect of light intensity on the proximate biochemical and fatty acid

composition of Isochrysis sp. and Nannochloropsis oculata for use in

tropical aquaculture
S.M. Renaudla, D.L. Parryla, Luong-Van, Thinh b, C. Kuo 2 , A. Padovanla.b & N. Sammy 3 i a. School of Chemistry and Earth Sciences, b. School of Biological Sciences, Faculty of Science, Northern Territory University, P.O. Box 40146, Casuarina, N.T. 0811, Australia;2Department of Primary Industry and Fisheries, G.P.O. Box 990, Darwin, N.T. 0801, Australia; 3 Office of the Minister for Conservation, G.P.O. Box 3146, Darwin, N.T. 0801, Australia.
Received 28 November 1990; accepted 12 December 1990

Key words: microalgae, fatty acids, mariculture, nutrition, capillary gas chromatography

Abstract The total protein, carbohydrate, lipid and ash compositions, and fatty acid contents of two species of marine microalgae, the eustigmatophyte Nannochloropsisoculata (formerly 'Chlorella sp., Japan') and the chrysophyte Isochrysis sp. (Tahitian) used in tropical Australian mariculture, were studied. The microalgae were grown under a range of culture conditions (4 1 and 60 1 laboratory culture, 300 1 bag culture, and 8000 1 outdoor culture) and four light regimes (100 to 107 ktE m - 2 - , 240 to 390 E m- 2 s - ', 340 to 620 iE m- 2 s -1, and 1100 to 1200 ,tE m-2 s- 1 respectively) to determine the effect of light intensity on the chemical composition of large scale outdoor cultures. Laboratory and bag cultures were axenic and cultured in Walne medium while outdoor cultures were grown in a commercial medium designed for optimum nutrition in tropical outdoor aquaculture operations. Change in growth medium and photon flux density produced only small changes in the proximate biochemical composition of both algae. N. oculata and Isochrysis sp. both showed a trend towards slightly lower carbohydrate and higher chlorophyll a in shaded outdoor culture. Isochrysis sp. showed significant concentrations of the essential polyunsaturated fatty acid 22:6(n-3) (docosahexaenoic acid) from 5.3 to 10.3% of total fatty acid, and 20: 5(n-3) (eicosapentaenoic acid) ranged from 0.6 to 4.1%. In contrast, N. oculata had high concentrations of 20: 5(n-3) (17.8 to 39.9%) and only traces of 22: 6(n-3). The fatty acid composition of Isochrysis sp. grown at high photon flux density (1100-1200 #tE m-2 s- ') under outdoor culture showed a decrease in the percentage of several highly unsaturated fatty acids, including 20: 5(n-3), and an increase in 22: 6(n-3). N. oculata showed a similar decrease in the percentage of 20: 5(n-3). High light intensity caused a decrease in the ratio of total C, 6 unsaturated fatty acids to saturated 16: 0 in N. oculata, and a decrease in the ratio of total C,8 unsaturated fatty acids to saturated 18 : 0 together with a decrease in the ratio of total unsaturated fatty acids to total saturated fatty acids in both microalgae. Introduction Microalgae are the primary source of feed for at least some stages in the life cycle of most aquaculture organisms, in spite of attempts by aquaculturists to substitute artificial food sources. The nutritional value of the microalgae is related to their biochemical composition, especially the lipid

44 and fatty acid compositions (Ackman & Tocher, 1968; Scott & Middleton, 1979; Chu & Dupuy, 1980; Watanabe et al., 1983). Optimum nutritional value of microalgal species is associated with levels of some essential, highly unsaturated fatty acids (HUFA), in particular two members of the 'omega-3' group, eicosapentaenoic acid (EPA), 20:5(n-3) and docosahexaenoic acid (DHA), 22:6(n-3), although the importance of other unsaturated fatty acids is also under study. The importance of these fatty acids has been demonstrated in the growth of oyster larvae (Langdon & Walcock, 1981; Chu & Webb, 1984; Wikfors etal., 1984; Enright etal., 1986), queen conch larvae, Strombus gigas (Pillsbury, 1985), and barramundi fingerlings, Lates calcarifer (Rimmer et al., 1988). DHA was the most important HUFA for the development of the mud crab, Eurypanopeus depressus (Levine & Sulkin, 1984). Sorgeloos et al. (1988) reported that survival of seabass larvae, Dicentrarchuslabrax, was associated with increased concentration of EPA in the diet, while growth was associated with increased DHA content. Many researchers have shown that microalgae undergo biochemical changes when the culture conditions are varied. The greatest biochemical changes are associated with low levels of nitrogen in the culture medium causing a large decrease in microalgal protein and large increases in lipid and carbohydrate (Spoehr & Milner, 1949; Iwamoto etal., 1955; Collyer & Fogg, 1955; Myklestad, 1974; Darley, 1977; Dortch, 1982; Varum & Myklestad, 1984; Millie, 1985; Fabregas etal., 1986; Wikfors, 1986; Suen etal., 1987). Silicate deficiency led to the increase of lipid composition in diatoms (Coombs etal., 1967; Shifron & Chisholm, 1981; Roessler, 1988), high light intensity lowered the carbohydrate levels of the diatom Skeletonema costatum (Varum & Mycklestad, 1984) and higher incubation temperature caused an increase in lipid and protein concentrations in the chrysophyte Ochromonas danica (Aaronson, 1973), the green alga Scenedesmus (Materassi et al., 1980) and the diatom Nitzschia palea (Opute, 1974). Amino acid composition can be altered by changing the culture conditions (Dortch, 1982; James etal., 1989), as can fatty acid composition (Ackman et al., 1968; Fisher & Schwarzenbach, 1978; Teshima etal., 1983; Watanabe etal., 1983; Ben Amotz etal., 1985; James et al., 1989; Thompson et al., 1990). It is this modification of the biochemical composition which allows the selection of culture conditions which will produce microalgal feeds of the appropriate nutritional value for brine shrimps, rotifers, bivalves, penaeid prawns, shrimps or fish species under culture. Of particular interest in aquaculture projects is the fatty acid composition of large volume cultures of microalgae. Early work with laboratory cultures suggested that increased light intensity encouraged the formation of polyunsaturated C16 and C1 8 fatty acids, mainly 16: 2(n-6), 16: 3(n-6), 16: 4(n-3), 18: 2(n-6) and 18: 3(n-3) in Chlorella vulgaris and Euglena gracilis (Nichols, 1965; Pohl & Zurheide, 1979). More recent studies have reported decreases in the percentages of unsaturated fatty acids in microalgae grown at high photon flux density (PFD), especially 20: 5(n-3) in Cylindrothecafusiformis(Opute, 1974), 16: 2(n-6) and 18: 3(n-3) in two species of the green alga Scenedesmus, (Materassi et al., 1980), 16: 2(n-7), 18: l(n-9), 18: 2(n-6), 18: 4(n-3), 20: 4(n-6) and 22: 6(n-3) in the diatom Chaetoceros gracilis (Mortensen etal., 1988), and 20:4(n-6) and 20: 5(n-3) in the eustigmatophyte Nannochloropsis sp. (Sukenik et al., 1989). Thompson et al. (1990) reported that there was no consistent, species specific correlation between light intensity and the concentration of a particular fatty acid in eight species of marine phytoplankton. However, they found that most species had the greatest proportion of 20: 5(n-3) at low levels of light and that 22: 6(n-3) generally decreased with decreasing light intensity. Further evidence for this trend was found by Harrison et al. (1990) working with Chaetoceros calcitrans, Isochrysis galbana Green, and Thalassiosirapseudonana. We report here an investigation of total protein, carbohydrate, lipid, chlorophyll a and ash composition, and fatty acid compositon of two species of marine microalgae, Nannochloropsis oculata, (Eustigmatophyceae)formerly'marineChlorella sp.,

45 Japan' (Maruyama et al., 1986) and Isochrysis sp. (Chrysophyceae), also known as 'Tahitian Isochrysis', used in tropical Australian mariculture. The microalgae were grown under a range of culture conditions, including laboratory culture, bag culture, and large scale outdour culture, under different light regimes. We consider that one factor that may be manipulated in tropical aquaculture projects, where, typically there are many hours of sunlight throughout the day for most of the year, is the photon flux density that reaches the surface of the culture tank. Our aim was to determine whether the modification in chemical composition associated with changes in light intensity, observed in laboratory cultures, could be demonstrated in large scale outdoor cultures. enriched with commercial fertilisers. The commercial fertilizer was potassium nitrate-based for the culture of Isochrysis sp., and ammonium sulphate-based for the culture of N. oculata. Laboratory 41, 601 and 300 1 cultures were illuminated with 18 watt cool-white fluorescent lights on a 12: 12 h light/dark cycle at 25 + 1 C. Photon flux density (PFD) was measured with a Licor Model Li 1000 datalogger. PFD at depth was measured using an underwater quantum sensor. Outdoor cultures were shaded using openweave polypropylene shade-cloth supported on perspex sheeting. Constant mixing was achieved by use of an air lift system from the bottom of each tank. Darwin (12 25' S, 130 52' E) has an average of twelve hours of daylight with maximum PFD 2100 to 2200 uE m - 2 s - , and twelve hours of darkness. The temperature within each tank ranged from a minimum of 25 C to a maximum of 29 C over a 24 h period. Microalgal cells were harvested in late logphase of growth by centrifugation using a Beckman Model J2-21M/E refrigerated centrifuge. Samples were lyophilized and stored at - 80 C prior to chemical analysis.

Materials and methods Organisms Isochrysis sp. (Tahitian), T.ISO. (CS177) and Nannochloropsis oculata (CS179) were obtained from Dr S. Jeffrey, CSIRO Algal Culture Collection, C.S.I.R.O. Division of Fisheries, Hobart, Tasmania.

Analytical methods Growth conditions The experimental culture conditions, including cell count at harvest, for each of the cultures are shown in Table 1. Microalgae were cultured in 4 1 Erlenmeyer flasks, 60 1rectangular aquaria, 300 1 polythene bags, or 8000 1circular (diameter 3.8 m, depth 0.8 m) fibre glass tanks. Cell counts were made daily, using a Neubauer haemacytometer, and algal cells harvested at late log-phase. The salinity of all culture media was adjusted to 25 ppt with distilled water before sterilization. Salinity of outdoor cultures was readjusted daily. Axenic laboratory culture media were obtained by autoclaving at 103.4 kPa for 15 minutes. Culture bags were sterilized by chlorination followed by sodium thiosulphate rinse. Outdoor culture media was prepared using 0.2 pm filtered seawater, Total protein was determined using the Buchi semi-micro kjeldahl system with potentiometric endpoint detection, and protein calculated as nitrogen x 6.25 (Horwitz, 1980). Carbohydrates were analysed by the colorimetric phenol-sulphuric acid method with the measurement of absorbance at 485 nm following hot water extraction of the algal cells for 1 h on a steam bath (Dubois et al., 1956), using a Varian DMS 200 UV/Visible spectrophotometer. Total lipids were determined gravimetrically after extraction of the microalgae with chloroform-methanol (2: 1) using the method of Folch etal., (1957) as modified by Bligh and Dyer (1959). Chlorophyll a was determined in the pigments extracted from 10 mg of lyophilized cells by sonication for 5 min in 1 ml of methanol, AR. This

46
Table 1. Algal identification, culture conditions and cell counts at harvest. Species & i.d. CSIRO culture Volume (1) Medium Cells mlat harvest 6 ( x 10 ) 5.08 11.26 12.06 4.52 2.21 0.41 3.81 0.78 1.24 1.11 0.98 1.27 1.44 Photon flux density (E m- 2 s- ) 135 140 140 107 107 1200 1200 620 620 620 390 390 390

Isochrysis sp. A B C D E F G H I J K L M Nannochloropsis oculata A B C D E F G H I J K L M

aWalne,

CS177

4 4 4 60 300 8000 8000 8000 8000 8000 8000 8000 8000

Walnea Walne Walne Walne Walne Comm. Ib Comm. I Comm. I Comm. I Comm. I Comm. I Comm. I Comm. I

CS179

4 4 4 60 300 300 8000 8000 8000 8000 8000 8000 8000

Walne Walne Walne Walne Walne Walne Comm. Comm. Comm. Comm. Comm. Comm. Comm.

IIF II II II II II II

8.49 5.69 49.1 59.0 8.50 10.49 6.55 9.17 8.22 7.97 5.88 4.56 6.40

107 100 100 100 107 107 1100 340 340 340 243 243 243

1966. Commercial I Potassium nitrate (100-150 mg 1- ), sodium dihydrogen phosphate (10-15 mg 1- ), ferric chloride (5 mg 1-'), EDTA (5 mg 1- ) + Walne medium trace metals. c Commercial II Ammonium sulphate (150 mg 1- ), urea (7.5 mg 1-), superphosphate (25 mg 1-), ferric chloride (5 mg 1- ), EDTA (5 mg 1-').
b

was followed by 24 h extraction in the dark at 4 C after the addition of 10 ml of acetone, AR, and 10 mg of magnesium carbonate, AR. Chlorophyll a was determined spectrophotometrically and calculated using the equations of Jeffrey and Humphrey (1975). Total ash was determined gravimetrically after ashing at 540 C. Fatty acid methyl esters (FAME) were prepared by transesterification of the saponified lipids with 14% methanol-BC13 at 60 C for

15 minutes. FAME samples were analysed using a Varian 6000 gas chromatograph with FID detector using a fused-silica column (SGE BP225, 50% cyanopropyl 50% phenyl dimethylsiloxane, 25 m x 0.22 mm i.d.). Data were collected and manipulated using the Delta chromatography data system. Fatty acids were identified by comparison with retention times of known standards obtained from Sigma Chemical Co. and Aldrich Chemical Co. and using cod liver oil as a secondary standard. Identities were verified using

47 a second column (Alltech RSL150, polymethylsiloxane, 25 m x 0.35 mm i.d.) and calculations of relative retention (Ackman, 1986). The molecular weights of fatty acids were confirmed by GC-MS analysis with an HP5890 GC and 5790 MSD using the SGE, BP225 capillary column. The shorthand notation used in fatty acid identification is L: B(n-x), where L is the chain length, B is the number of double bonds, and x is the position of the ultimate double bond from the terminal methyl group.
Table 2. Fatty acid composition of Nannochloropsis oculata cultured in two different nitrogen-based media, at PFD 1100#E m- 2 s- l Fatty acid (% of total fatty acids) Outdoor culture 80001 Commercial I NO3 1.7 6.3 0.1 0.2 26.0 21.7 1.3 4.0 11.6 5.2 0.2 nd 3.1 nd 3.6 13.0 nd 30.3 Commercial II NH4 0.6 6.4 0.3 2.0 30.0 21.4 3.4 2.2 6.5 2.8 0.5 0.3 0.4 0.2 5.1 17.8 0.2 36.6

Results and discussion

Surface irradiance The surface irradiance (PFD) of microalgal cultures, measured in late log-phase when cell densities and self-shading were at a maximum, is given in Table 1. Irradiance at the bottom of all 8000 1 outdoor cultures averaged 5.68 + 1.48 /ME
m-2 s-.

12:0 14:0 14: 1 15:0 16:0 16: (n-9) 16:2 18:0 18: 1(n-9) 18: 2(n-6) 18: 3(n-6) 18: 3(n-3) 18:4(n-3) 20:0 20: l(n-9) 20: 5(n-3) 22: 6(n-3) Total lipid, % OW

% OW percentage of organic matter. Culture solutions nd not detected.

Axenic laboratory cultures grown under optimum conditions in potassium nitrate-based Walne medium (Walne, 1966) provided the baseline for our study of the effect of increased culture volume, commercial growth media and natural sunlight in larger scale out-door culture. Growth media derived from fertilizers that are readily available commercially were used in the large scale outdoor cultures. Outdoor cultures of Isochrysis sp. were grown in a potassium nitratebased medium (Commercial I). Large scale cultures of Nannonochloropsisoculata were grown in an ammonium sulphate-based medium (Commercial II). This ammonium sulphate-based culture solution was chosen for outdoor culture of N. oculata over a nitrate-based culture medium due to results of preliminary outdoor trials (Table 2) which showed a higher total lipid content (36.6% of organic weight compared with 30.3% OW) and higher percentage composition

of the essential, highly unsaturated fatty acid eicosapentaenoic acid, 20:5(n-3) (17.8% OW compared with 13.0% OW) when the microalga was grown in the Commercial II medium.

Proximate cellular chemical composition The proximate chemical composition of Isochrysis sp. and N. oculata are shown in Tables 3 and 4. a. Comparison of laboratory cultures with out-door cultures grown at low irradiance Laboratory cultures of Isochrysis sp. and large scale outdoor cultures, grown at lowest PFD (K, L and M in Table 3), showed little difference in gross biochemical composition, except that the concentrations of chlorophyll a in laboratory cultures were an average of 1% (organic weight) lower than those of the out-door cultures. The

48
Table 3. Chemical composition, expressed as percentage of organic matter and as mass per cell, of Isochrysis sp. cultured at different photon flux densities (PFD). Sample PFDa (E m- 2 S I) Protein (%OW) A B C D E 135 140 140 107 107 38.7 34.0 37.6 39.3 38.4 Mean 37.6 F G 1200 1200 Mean H I J 620 620 620 Mean K L M 390 390 390 Mean
a b

CHOb (pgcell ') 10.6 8.9 9.0 9.6 9.9 9.6 12.9 9.1 11.0 6.2 6.9 10.1 7.7 7.8 6.7 10.9 8.4
-

Lipid (pg cell ') 3.2 2.9 2.2 1.3 2.9 2.5 4.0 4.3 4.2 1.5 1.0 2.2 1.6 1.9 1.8 1.8 1.8
-

Chi a (pg cell ') 9.4 10.5 9.9 8.7 7.9 9.3 10.8 9.7 10.3 6.4 6.8 8.5 7.2 7.5 6.7 7.0 7.1
-

Ash (pg cell-') 0.4 0.4 0.4 0.4 0.5 0.4 0.5 0.4 0.4 0.4 0.4 0.7 0.5 0.5 0.6 0.6 0.5 (pg cell - ') 1.9 1.8 1.4 2.2 2.2 1.9 1.5 1.5 1.5 1.7 1.7 2.7 2.0 2.0 1.7 1.6 1.8

(% OW) 11.5 11.0 9.2 5.2 11.1 9.6 13.0 16.4 14.7 8.3 9.2 7.8 8.4 8.8 9.6 9.5 9.3

(% OW) 34.2 40.3 41.4 35.5 30.9 36.4 34.8 36.6 35.7 35.3 35.9 30.7 33.9 34.8 35.8 36.9 35.8

(% OW) 1.44 1.59 1.69 1.71 1.83 1.65 1.60 1.37 1.49 2.18 2.11 2.43 2.24 2.13 3.12 2.95 2.74

41.7 34.5 38.1 34.2 36.4 36.3 35.6 36.2 35.7 38.1 36.7

Photon flux density at surface of culture. Carbohydrate.

N. oculata laboratory cultures had much higher levels of lipid (average 13.7% OW higher) and lower levels of carbohydrate (average 1% OW lower) when compared with out-door, low light intensity cultures. (K, L and M in Table 4) grown in the ammonium sulphate based Commercial II medium. b. Out-door cultures grown at different irradiance The proximate chemical composition of large scale, outdoor cultures of Isochrysis sp. showed a trend towards increasing concentrations of chlorophyll a (average increase 1.25 % OW), and a 5.4% OW decrease in carbohydrates with decreasing PFD; total protein and lipid showed no differences (Table 3, F to M). Out-door cultures of N. oculata, showed small increases in chlorophyll a and decreases in total lipid (7.3% OW) and carbohydrate (1% OW) with decreasing PFD. The carbohydrate content was

at a minimum at the lowest PFD (Table 4, G to M).

Fatty acid composition a. Laboratory cultures The actual fatty acid composition of the lipid fractions of the microalgae used as food species is of greatest importance to the growth and survival of aquaculture organisms. The fatty acid contents of Isochrysis sp. and N. oculata are shown in Tables 5 and 6 respectively. The major fatty acids of Isochrysis sp. were 14: 0, 16: 0, 18: l(n-9), 18:4(n-3) and 22: 6(n-3), and of N. oculata were 16: 0, 16: l(n-9) and 20: 5(n-3). Eicosapentaenoic acid, 20: 5(n-3), was the major component of the fatty acid composition of N. oculata in laboratory culture (mean for laboratory cultures 32.0% of total fatty acids). This

49
Table 4. Chemical composition, expressed as percentage of organic matter and as mass per cell, of Nannochloropsis oculata cultured at different photon flux densities (PFD). Sample PFDa
(E m-2 s1

Protein
)

CHO b (pgcell-') 2.6 2.7 2.8 1.7 2.1 2.7 2.4 2.6 2.1 2.8 3.1 2.7 2.4 2.4 2.3 2.4 (% OW) 3.00 2.49 2.30 2.00 2.28 2.32 2.40 4.30 4.04 4.29 5.26 4.53 3.44 3.69 3.18 3.44 (pgcell-') 0.2 0.1 0.1 0.1 0.2 0.2 0.1 0.3 0.2 0.3 0.4 0.3 0.2 0.2 0.2 0.2

Lipid (% OW) 37.9 36.5 33.5 36.5 37.2 33.4 35.8 30.4 26.3 22.9 25.8 25.0 19.7 23.9 22.8 22.1 (pgcell-') 2.2 2.1 2.0 1.6 2.4 2.2 2.1 1.5 1.3 1.5 1.7 1.5 0.9 1.3 1.3 1.2

Chl a (% OW) 3.49 2.95 2.64 2.70 2.83 3.34 2.99 2.25 2.05 2.55 3.15 2.58 2.04 2.40 2.70 2.38 (pgcell 0.2 0.2 0.2 0.1 0.2 0.2 0.2 0.1 0.1 0.2 0.2 0.2 0.1 0.1 0.2 0.1
-l

Ash ) (pgcell-') 0.4 0.4 0.4 0.4 0.7 0.7 0.5 0.6 0.5 0.8 0.7 0.7 0.6 0.7 0.7 0.7

(% OW) A B C D E F 107 100 100 100 107 107 Mean G H I J 1100 340 340 340 Mean K L M 243 243 243 Mean
a b

44.6 46.8 46.4 38.6 33.4 40.9 41.8 44.0 44.7 43.8 45.5 44.7 47.5 45.2 41.6 44.8

Photon flux density at surface of culture. Carbohydrate.

average value is higher than the value of 27.8% reported by Watanabe et al. (1983) for the same species, identified as 'marine Chlorella sp.' and compared favourably with values for 20: 5 (n-3) in other microalgae currently recommended for aquaculture (Volkman etal., 1989; Ben-Amotz et al., 1987). Docosahexaenoic acid, 22: 6(n-3) was detected only in trace amounts in all cultures of N. oculata. Isochrysis sp. had an average 22: 6(n-3) value of 7.9% in laboratory culture which is comparable to the value of 8.3% reported by Volkman et al. (1989) for the same clone grown in a different culture medium, f2 . EPA was present in small amounts in laboratory culture (mean 1.7%). b. Effect of photon flux density on large-scale outdoor cultures The results show a trend towards increasing saturation of the fatty acids of both Isochrysis sp. and N. oculata, grown outdoors in the tropics, with

increasing photon flux density (Table 5, Samples F to M and Table 6, Samples G to M). However, it is difficult to hold all variables constant in outdoor, large scale culture. During this study the salinity was constant, the culture solution temperature range was 27 + 2 C and the tropical daylight-dark cycle was close to 12 h: 12 h, but the cultures were not axenic and the actual photon flux density reaching the microalgal cells was influenced by the effect of self-shading. Shading effects were minimised by the use of shallow tanks (culture depth 0.65 m) and identical airlift systems to acheive similar mixing rates in each tank. At the time of harvest, there was a 97.7-99.5% drop in PFD between the surface and the bottom of each culture, but microalgal cells in each tank spent a similar proportion of time at the surface of the tank where irradiance was at a maximum. The percentages of the highly unsaturated fatty acids 18: 4(n-3), 18: 3(n-6) and 18: 3(n-9) were lower in Isochrysis sp. cells grown at highest PFD,

50
Table 5. Fatty acid composition of Isochrysis sp. under four different culture conditions and four light regimes. Fatty acid (% of total fatty acids) Laboratory culture 41 A B C 60 1 D 300 1 E Outdoor culture 8000 1 F G H I J K L M

1200IE m-2 s-' 12: 0 14:0 14:1 15:0 16:0 16: 1(n-7) 16: 2(n-4) 18:0 18: (n-9) 18: 2(n-6) 18: 3(n-6) 18: 3(n-3) 18: 4(n-3) 18: 5(n-3) 20: 1(n-9) 20: 5(n-3) 22: 5(n-6) 22: 6(n-3) 16 unsat./716:0 I18 unsat./i18 :0 20:5/22:6 Z unsat./Z sat. tr = trace. nd = not detected. tr 21.5 0.6 1.1 11.2 1.8 5.1 0.8 13.3 3.5 0.8 5.5 20.8 3.4 1.0 0.7 tr 8.7 0.61 60.1 0.08 3.05 0.6 21.6 0.5 0.7 13.3 3.8 1.2 0.6 11.1 3.8 1.0 5.4 22.7 3.8 0.6 0.6 1.1 7.7 0.39 83.9 0.08 3.28 tr 17.5 tr 0.8 9.1 6.0 4.3 0.8 10.6 5.0 1.3 5.7 23.9 2.8 1.0 0.9 1.3 9.0 1.13 63.1 0.10 2.54 0.4 18.5 0.4 0.4 9.1 8.2 0.8 0.8 8.3 9.8 3.5 5.0 20.5 2.6 1.3 4.1 0.7 5.3 0.97 59.9 0.77 2.39 tr 14.0 0.5 1.7 10.1 6.0 2.1 2.2 10.1 2.8 tr 6.8 28.4 tr 0.8 2.0 1.4 8.7 0.80 22.2 0.23 2.53 3.1 15.3 1.3 2.6 16.6 6.4 3.1 2.9 9.2 3.0 2.1 4.1 16.7 7.6 nd 1.5 nd 5.5 0.57 12.0 0.28 1.74 1.9 15.9 1.5 1.9 16.3 8.7 3.2 2.0 6.3 3.2 1.6 4.7 14.3 7.2 nd 1.6 nd 10.0 0.73 18.7 0.13 1.65

620uEm-2 s - ' tr tr 0.1 14.8 14.6 15.0 1.1 0.9 0.4 nd nd nd 8.3 9.1 9.2 6.3 6.6 6.8 3.6 2.8 4.7 2.2 1.8 0.6 9.2 9.6 7.4 5.1 5.6 4.3 1.6 1.8 1.8 5.7 7.0 6.0 24.7 24.0 24.9 4.9 6.8 6.1 nd nd nd 1.6 1.2 1.3 nd nd nd 8.2 9.2 10.3 1.06 1.13 1.27 48.2 160 24.3 0.08 0.16 0.15 2.41 2.92 2.76

390pE m-2 s tr tr 0.3 17.7 19.2 18.9 0.5 0.1 0.4 nd nd nd 8.6 9.1 9.2 7.8 7.4 8.4 4.1 3.8 3.5 0.5 0.7 0.4 8.8 9.1 9.2 8.3 6.6 7.2 2.0 2.8 3.5 5.1 5.2 4.8 21.0 24.4 20.6 5.1 3.2 4.7 nd nd nd 1.1 0.8 0.9 nd nd nd 7.6 7.7 9.0 1.39 1.22 1.29 95.0 76.3 120 0.14 0.08 0.12 2.49 2.65 2.33

1200 E m - 2 s -1 . In particular, the concentration of 18: 4(n-3) was decreased by 6.5% and the concentration of 18: 2(n-6) was decreased by 4.3% of total fatty acids. Concentrations of the saturated fatty acids 16: 0 and 18: 0 increased with increasing irradiance by 7.5% and 2% respectively. There were small increases in the concentrations of two highly unsaturated fatty acids, 22: 6(n-3), and 18: 5(n-3) associated with increasing irradiance (2% and 2.9% respectively). Eicosapentaenoic acid, 20: 5(n-3), had a small but constant concentration, suggesting that it was uneffected by irradiance. Harrison et al. (1990) found similar results for 20: 5(n-3) and a slightly larger increase (4%) in the concentration of

22: 6(n-3) in laboratory cultures of Isochrysis galbana Green (Tahitian Isochrysis) grown at 50 Em-2s - ' and 255)uEm-2s -1, and a decrease of 1% between cells grown at 1 . The per125p E m- 2 s-1 and 255, Em - 2 scentage differences in fatty acid composition in our study are similar to those reported by Thompson et al. (1990) for the Tahitian Isochrysis aff. galbana grown in laboratory culture with and of 225 E m - 2 -1 irradiances 125 /E m- 2 s -1. One difference was that we did not find an increase in 18: l(n-9) concentration with increased PFD. Cells of N. oculata grown at high irradiance (1100 E m - 2 s-l) had decreased concentrations of the highly unsaturated fatty acids

51
Table 6. Fatty acid composition of Nannochloropsis oculata under four different culture conditions and four light regimes. Fatty acid (% of total fatty acids) Laboratory culture 41 A B C 601 D 300 1 E F Outdoor culture 80001 G 1100 12: 0 14:0 14: 1 15:0 16:0 16: (n-9) 16: 2(n-4) 18:0 18: (n-9) 18: 2(n-6) 18: 3(n-6) 18: 3(n-3) 18: 4(n-3) 20: 0 20: 1(n-9) 20: 5(n-3) 22: 6(n-3) l16 unsat./16:0 1l8 unsat./18 :0 E unsat./E sat. tr = trace. nd = not detected. 0.5 5.7 0.3 0.3 21.2 25.9 1.5 0.3 2.2 2.2 0.4 0.7 tr tr 4.5 34.3 nd 1.29 18.4 2.58 0.5 5.3 0.6 0.4 20.5 25.0 0.9 0.3 3.7 2.4 0.6 0.3 tr tr 4.8 34.8 nd 1.27 24.0 2.71 0.7 6.8 1.0 tr 23.9 28.4 0.7 0.5 4.2 3.3 0.5 tr tr tr 5.6 24.5 nd 1.22 17.8 2.15 0.6 5.2 0.2 0.7 18.6 26.1 1.1 0.4 3.1 2.4 0.9 0.4 0.3 0.4 4.2 35.5 nd 1.46 19.3 2.93 0.8 8.2 0.3 0.6 16.0 23.0 1.1 0.4 2.8 3.0 1.1 0.9 2.0 0.4 4.1 35.0 0.6 1.51 26.9 2.78 0.5 5.8 0.5 0.7 18.3 29.8 2.1 0.8 3.9 1.9 0.5 0.4 0.8 0.3 5.9 27.7 nd 1.74 9.68 2.82 0.9 5.4 tr 1.4 18.4 19.9 7.6 1.1 5.0 2.5 1.0 0.6 1.0 tr 5.1 33.8 0.2 1.28 9.18 2.81 H 340 tr 5.7 tr 1.1 14.7 22.5 4.3 0.7 3.7 2.7 tr 0.7 tr tr 6.5 37.2 nd 1.82 9.53 3.51 tr 4.3 0.2 tr 14.7 22.4 5.3 0.9 4.2 2.7 1.2 0.8 tr tr 5.9 37.7 tr 1.89 10.4 4.05 0.4 4.4 0.2 0.4 18.0 22.5 3.8 0.7 4.3 2.9 0.6 0.1 0.5 0.2 6.6 37.0 tr 1.41 11.48 3.16 I J K L M

243/pE m-2 s' 1.0 4.9 0.4 0.3 14.5 19.4 5.6 0.9 5.0 3.3 tr 0.6 tr tr 5.1 39.1 nd 1.72 9.44 3.62 0.63 4.96 tr 0.40 14.21 21.62 4.61 0.69 4.27 2.19 tr 0.71 0.18 0.58 6.05 38.90 nd 1.85 10.6 3.79 0.9 4.9 0.6 1.1 14.0 19.3 6.4 1.0 3.1 2.6 tr 0.8 tr tr 5.6 40.0 nd 1.85 6.79 3.59

16: 2(n-4), 18: 3(n-3), and 20: 5(n-3). The concentration of the major fatty acid, 20:5(n-3), decreased by 5.5% while concentrations of the saturated fatty acid 16: 0 increased by 4% at high photon flux densities. Sukenik etal. reported similar results with a reduction in the percentage concentration of 20: 5(n-3) (from 10% to 5% of total fatty acids) in laboratory cultures of Nannochloropsis sp. grown at irradiances of 290btEm- 2 s- and 550/pEm-s-2 respectively. High ratios of (n-3) to (n-6) polyunsaturated fatty acids have been used as an index of high nutritional value to aquaculture animals (Watanabe et al., 1983). The total unsaturation ratios, the ratios of total unsaturated C 16 fatty acids to 16: 0, and the ratios of total unsaturated C18 fatty acids to 18: 0 are shown in Tables 5 and 6. Fatty

acid composition of Isochrysis sp. grown at high PFD (1200pEm-2s - 1 ) in outdoor culture showed decreases in the ratio of total C18 unsaturated fatty acids to saturated 18:0, total C1 6 unsaturated fatty acids to 16: 0 and total unsaturated fatty acids to total saturated fatty acids. N. oculata showed similar decreases in these ratios when grown at high PFD in outdoor culture. These indexes may be used in conjunction with consideration of which fatty acids are present to indicate the nutritional value of microalgal cells to aquaculture animals (Volkman et al., 1989). Increase in temperature may influence the fatty acid composition of microalgae. James et al. (1989) reported that Nannochloropsis strain MFD-2 grown at 35 C showed a decline of 4% in the content of 20.5(n-3) when comparing cul-

52 tures grown at 25 C and 30 C. Temperatures of the outdoor cultures in our study, ranging from 25 C to 29 C have not caused a decrease in the percentage of 20: 5 (n-3) and we found little difference in the percentage of 20: 5(n-3) between laboratory cultures grown at 25 + 1 C and the outdoor cultures. The optimum surface photon flux density for producing the highest percentages of unsaturated fatty acids and highest ratios of unsaturated to saturated fatty acids is <620 #E m-2 s- for Isochrysis sp. and <490 E m - 2 s - 1 for N. oculata. Optimum PFD corresponds to 73 to 77% shading by the polypropylene shade cloth. These PFDs are also necessary for optimisation of the essential highly unsaturated fatty acids 20: 5(n-3) and 22: 6(n-3). The algae under culture had a light gradient from the surface to the bottom of the tank, but continuous circulation of the algae ensured that algal cells were frequently exposed to maximum irradiance. At present hatcheries using microalgae have given little attention to the possibility of modifying the culture conditions to optimize the fatty acid composition. Our findings show that the optimization of fatty acid composition by control of the photon flux density of tropical sunlight is possible in commercial-scale aquaculture projects and indicates optimum light intensities for two commonly used microalgae, Isochrysis sp. (Tahitian) and Nannochloropsis oculata.
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Acknowledgements This project was funded by the Rural Credits Development Fund of the Reserve Bank (DLP, TLV, & NS) and N.T. University (SR). We thank Mr R. McFarlane of the Biochemistry Unit, Royal Darwin Hospital for the use of the Hewlett-Packard GC/MS system.

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