Long-term preservation of Tetraselmis suecica: influence of storage on viability and fatty acid profile

ELSEVIER

Aquaculture 134 (1995) 81-90

Long-term preservation of Tetraselmis suecica: influence of storage on viability and fatty acid profile E. Montaini a, G. Chini Zittelli a, M.R. Tredici a, E. Molina Grima b, J.M. Fern6ndez Sevilla b, J.A. Sinchez Perez bp* a Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche dell’(lniversit~ di Firenze e Centro di Studio dei Microrganismi Autotroji de1 CNR, P.le delle Cascine, 27-50144 Florence, Italy b Grupo de Investigacick de Biotecnologia de Microalgas Marinas, Facultad de Ciencias Experimentales, Universidad de Almeria, E-04071 Almenh, Spain

Accepted9 February 1995

Abstract Aquaculture production could be improved by using preserved microalgal biomass as feedstuff for marine animal larvae and juveniles. The present paper reports the effects of preserving Tetraselmis suecica by freezing with and without cryoprotectant, freezing in liquid nitrogen and maintenance of concentrated cultures at 4°C on viability and fatty acid profile. For long-term preservation (as long as 21 months), freezing can keep the fatty acid profile unaltered although leading to complete loss of cell viability. On the other hand, concentrated cultures kept in darkness at +4”C show a strong capacity for survival closely correlated with cell concentration. At 4 g. l- ‘, residual photosynthetic activity (ca. 6% of the initial) was still present after 150 days of storage. Furthermore, oxygen availability affected cell survival. Cultures stored in hermetically sealed vials lost their viability much more rapidly than those kept in cotton-plugged vials. Dry weight percentages of all fatty acids slightly increased in stored biomass in comparison with fresh biomass, because of reserve material consumption. Nonetheless, the fatty acid profile on a total fatty acid basis remained unchanged over storage time regardless of viability. This is an important fact to bear in mind as fatty acids play an important role in the quality of the cell as food in aquaculture. Keywords: Long-term preservation; Algae; Tetraselmis suecica; Fats and fatty compounds; Viability

1. Introduction The use of microalgae as the traditional diet for many cultured aquatic organisms is confronted with the need for live microalgae. An important breakthrough for aquacultural * Corresponding author. Tel. (34) (50) 215314; Fax (34) (50) 215070. 0044-8486/95/$09.50

0 1995 Elsevier Science B.V. All rights reserved

SSDIOO44-8486(95)00034-8

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research and commercial hatcheries could be the successful storage of algae in algal production facilities which could supply hatcheries (Holliday et al., 1991). Researchers have been focusing their attention on preserved microalgae as alternative diets and different preservation techniques are being investigated: e.g., frozen concentrated cultures, freeze-, spray-, or air-dried biomass and refrigerated algal concentrates (for references see Corder0 Esquivel et al., 1993). In this context, Tetraselmis suecica is a widely studied organism. Concentrates of T. suecica stored at 4°C for a minimum of 7 days and a maximum of 14 days before use were evaluated as a food source for Sydney rock oyster (Saccostrea commercialis) larvae (Nell and O’Connor, 1991). Laing and Millican ( 1992) described promising results obtained in the indoor nursery cultivation of juvenile bivalve molluscs using diets of spray-dried T. suecica and Cyclotella cryptica. Nutritional value of microalgal species for larval stages and juveniles of marine animals is related to polyunsaturated fatty acid (PUJ?A) content (Langdon and Waldock, 198 1; Chu and Webb, 1984). Therefore, preservation methods must be devised that keep the fatty acid content unchanged or that involve modifications that do not decrease the nutritional value of the biomass. In the present study, the effects of different preservation techniques on the viability and fatty acid content of T. suecica are described. The preservation methods tested were freezing with and without cryoprotectant, freezing in liquid nitrogen, and maintenance of concentrated cultures at 4°C. 2. Materials and methods Organism and culture conditions The strain of Tetraselmis suecica used in this work was obtained from the Centrale dell’ENEL, which operates an experimental aquaculture station in Civitavecchia (Italy). The microalga was cultivated under continuous lighting in a “bubble column” flat photobioreactor with a surface area of 0.30 m2 devised by Tredici et al. ( 1991). Temperature was maintained at 27 + 1°C and a photon flux density (PAR) of 270 pmol . rn-*. s- ’ was provided by a 1000-W mercury halide lamp. The culture pH was regulated at 7.8 + 0.5 by the addition of pure CO* which was supplied by a pa-stat system. The dilution rate adopted was 0.4. day-‘. The culture medium was synthetic seawater (Tropic Marine Salt, Euraquarium, Bologna, Italy) at 2.9% salinity, enriched with: K,HPO,, 35 mg el-r; NaN03, 700 mg.l-‘; EDTA, 29.75 mgel-r; FeS04, 24.5 mgel-‘; H3B03, 2.86 mg.l-‘; MnC12, 1.81 mg.l-‘; ZnSO,, 222 pg*l-‘; CoC12, 35 pg+l-‘; CuSO,, 80 pg.l-‘; and NaMo04, 230 pg. 1- *. Further, K,HPO, and NaN03 were added according to the phosphorus and nitrogen needed for growth. Steady-state biomass was used for the experiments reported here. When required, the cultures were harvested by centrifugation at 12 000 rpm and either stored as paste or resuspended in a fresh medium to obtain the required concentration before storage. Analytical procedures Cell concentration was evaluated by determining the biomass dry weight. The cells were collected by filtration through a pre-weighed Sartorius (Gottingen, Germany) membrane filter (5 pm), which was then washed with distilled water and dried at 105°C to constant

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weight. Cell number was estimated by counting cells under a light microscope in a Btirker haemacytometer. Chlorophyll content of algal cells was estimated spectrophotometrically following the procedure described by Parson and Strickland ( 1963). Fatty acid analysis For testing, 2 samples at a time were taken from each type of stored biomass, and subjected to fatty acid analysis. Fatty acid methylation was done by direct transesterification with acetyl chloride/methanol ( 1:20) following the method of Lepage and Roy (1984). The analysis of methyl esters was carried out by gas chromatography using a 30-m capillary column of fused silica (X2330, Supelco, Bellefonte, PA, USA), internal diameter of 0.25 mm, 0.20 pm standard film, split ratio lOO:l, and a flame ionization detector. Supelco PUFA-1, PUFA-2 and PUFA-3 patterns were used for the determination of the retention times. Nonadecanoic acid was used as an internal standard to quantify fatty acid content in dry weight biomass. Results given as percentages of total fatty acid content were calculated taking as a basis the sum of all the fatty acids detected. Dry weight content of a given fatty acid is thus obtained as the product of its percentage over total fatty acids by the total fatty acid dry weight. Viability assays Viability of cells during storage was estimated by determining both the percentage of motile cells (4 replicates per sample) and the photosynthetic activity (in duplicate) on aliquots of the preserved cultures. Before being used in viability tests, all the samples were diluted with fresh medium to a final chlorophyll concentration of 6-7 pg 1ml- ’ and incubated at 27 + 0.5”C under a photon flux density (PAR) of 60 pm01 *m- * *s - ’ for 2 (cultures preserved at 4°C) or 24 (frozen cultures) h. The percentage of motile cells was determined by counting non-motile cells, before and after the sample was treated with 1% formaldehyde. Photosynthetic activity was evaluated by measuring the O2 evolution rate of a culture sample in a Biological Oxygen Monitor mod. 5300 (Yellow Springs Instrument Co., Inc., Yellow Springs, OH, USA) equipped with a Clark-type electrode. During measurement, the sample was maintained at 27 f 0. 1°C under a photon flux density (PAR) of 500 pm01 . rn-*. SC’ provided by a 150-W metal halide lamp. Viability was calculated by dividing the number of motile cells (or the photosynthetic activity) in the treated sample by the number in the control sample and expressed as a percentage of the control. Preservation

methodr

Freezing at - 18°C The paste obtained by centrifugation (72% moisture) was placed in 2 Petri dishes and cooled to - 18”C, without addition of cryoprotectant. After 6 days of storage, 4 samples of the frozen paste (2 per Petri dish) were thawed at room temperature and resuspended in fresh medium for the viability assay. Freezing at - 196°C For freezing in liquid nitrogen, eight 0.9 ml samples taken from 8 g (dry wt) . I-’ cultures were placed in cryovials with 0.9 ml of a 20% (v/v) solution of glycerol or DMSO (dimethyl sulfoxide) for a 10% final cryoprotective agent concentration. The vials were

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incubated at room temperature for 10 min (vials with glycerol) or 30 min (vials with DMSO) and then either directly plunged into liquid nitrogen at -196°C .or first pre-cooled ( - 1°C. min- ‘) to - 30°C and then transferred to -196°C. All treatments were performed in duplicate. Viability was tested after 2 days of storage. For viability testing, cells were thawed at room temperature until the ice had completely melted, and then diluted and centrifuged to remove the residual concentration of the cryoprotectant. The pellet was resuspended in fresh medium and incubated under standard conditions for 24 h prior to use in viability assays. Preservation at + 4°C Preservation at +4” C in darkness was tested on cell suspensions at 3 different cell concentrations (4, 20, 60 g dry wt. l- ‘) and on paste. In a first experiment, each cell suspension was stored in a 250 ml flask and the paste in a Petri dish. Both the flasks and the Petri dish were sealed with Parafilm@ and were opened periodically to withdraw the samples (2 for each treatment) for viability tests. In a second experiment, the cell suspensions were stored in 25-ml vials containing 8 ml of culture and 17 ml of air. On the whole, 96 vials ( 16 vials per treatment) were prepared, and 2 vials per treatment were used at each determination. Half of the vials were closed with cotton plugs in order to allow free exchange with the atmosphere and half were hermetically sealed with rubber caps. Each vial was disposed of after use.

3. Results Post-thaw viability of T. suecica stored at sub-zero temperatures Direct freezing of T. suecica paste at - 18°C without addition of cryoprotectant led to the complete loss of cell viability (Table 1). The same results were obtained when a concentrated suspension (4 g. 1-l) of T. suecica with 10% cryoprotectant (DMSO or glycerol) was plunged directly into liquid nitrogen ( - 196°C). When storage in liquid nitrogen was preceded by slow cooling ( - 1°C.min-‘) down to - 3O”C, some post-thaw viability ( 1 l-13% of the control when measured through motility, and 24% when measured through photosynthetic activity) was retained with both cryoprotectants (Table 1) . Table 1 Post-thaw viability of T. suecica stored at sub-zero temperatures Preservation

method

Freezing at - 18°C Freezing at - 196°C Direct plunging lO%DMSO 10% glycerol Slow cooling 10% DMSO 10% glycerol Control (untreated culture)

Motility (% of motile cells)

Photosynthetic

0

0

0 0

0 0

10.2 11.5 90.2

0.20 0.11 5.4

activity (~10,.

mg Chl-

’ . h- ’ )

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Time (days) Fig. 1. Changes in motility (A) aad photosynthetic activity (B) over time of concentrated cultures and paste of T. sue&a stored at + 4°C. Mean values f s.d. are shown. Errorbars not shown when smaller than the dimensions of the symbols. (0) 4 g.I-‘; (A) 20 g.l-‘; (0) 60 g.l-‘; (v) paste.

Viability ofT. suecica stored at + 4°C: effect of cell concentration Fig. 1 shows the variation of viability over time, measured as motility (Fig. 1A) and photosynthetic activity (Fig. 1B ). of concentrated cultures (4, 20, 60 g dry wtal- ‘) and paste of T. suecica stored at +4”C in darkness. As can be seen, loss of viability during storage was strongly correlated with cell concentration: the higher the cell concentration, the lower the survival rate. The paste obtained by centrifugation maintained full viability for 4 days, but there was a high rate of mortality afterwards and after 2 weeks of storage, viability was nil. Cell suspensions at 20 and 60 g *1- ’ maintained high viability (over 70% when measured through photosynthetic activity) for approximately 10 days, whereupon viability decreased rapidly to total mortality after 2 1 days at 60 g. 1-l and 24 days at 20 g. l- ‘. At 4 g. 1-l no significant reduction in viability was observed during the first 50 days of storage, and after 80 days only a 35% reduction in photosynthetic activity and 50% reduction in motility were found. Cell motility was completely lost after 115 ‘days, while residual photosynthetic activity (about 6% of the initial) was still present after 150 days of storage. In general, T. suecica cells showed a more rapid loss of motility than of photosynthetic activity during storage at low positive temperature. Viability of T. suecica stored at + 4°C: influence of the type of storage vial closure (oxygen availability) Motility and photosynthetic activity of cultures stored at +4”C in darkness in both hermetically sealed and cotton-plugged vials were periodically evaluated for about 2 months (Fig. 2). In addition to the influence of cell concentration noted in the previous experiment

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6( Time (days) Fig. 2. Changes in motility (A) and photosynthetic activity (B) of concentrated cultures of T. suecica stored at +4’C in cotton-plugged (solid symbols) and hermetically sealed (open symbols) vials. ( q.m) 4 g. 1-l; (A, A ) 20 g.l-‘; (0.0) 60 g.l-‘. 7%Total fatty acids 14 _

Fatty acid

q October, 1992

[13July, 1994

Fig. 3. Comparison of the T. suecica fatty acid profile measured at the beginning of the experiment and 21 months later frozen at - 18°C.

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Table 2 Influence of preservation factors (time, type of closure and biomass concentration) (percent of total fatty acids f s.d.) of the biomass for samples stored at 4°C Fatty acid

Control sample

on the fatty acid profile

Concentration 60 g.1-’ Cotton

4 g.1-’

plugged

Hermeticallysealed Days of storage 51 15

0

15

43

14:o 16:O 16:ln-7 18:O lS:ln-9 18:ln-7 18:2n-6

0.53 f 0.04 22.32kO.30 1.12*0.01 0.38 f 0.01 26.40f 0.21 3.20+0.01 9.47+0.14

18:3n-6 8:3n-3 20: ln-3 18:4n-3 20:4n-6 20:4n-3 20:5n-3 Total d.wt.

0.67 f 0.04 20.72kO.18 1.30f0.01 7.24 f 0.07 1.10*0.02 0.58 f 0.02 5.15 f0.06 9.75 f 0.81

0.50*0.07 22.53kO.48 0.95kO.18 0.34*0.24 26.45f0.56 1.55k2.19 10.67*0.28 2.96*0.06 19.87f0.44 1.35*0.04 5.34kO.16 1.23kO.05 0.77&0.08 5.25YcO.12 12.11*0.59

0.59kO.30 0.60*0.03 22.9OkO.12 24.89rt0.46 1.08*0.02 1.14*0.04 0.35f0.01 0.00~0.00 25.92f0.17 25.33f0.16 3.28*0.01 3.75kO.02 11.93~tO.03 11.59+0.05 2.45kO.11 2.68kO.01 17.77f0.19 18.08f0.23 1.34kO.05 1.23*0.04 4.58rtO.08 4.48f0.09 1.42kO.01 1.64*0.03 0.77&0.02 O.OOf0.00 4.71kO.04 4.97+0.11 13.08*1.12 11.61&0.29

22

0.55f0.01 0.57rtO.01 22.77f0.28 22.74kO.45 0.85kO.02 1.02f0.02 O.OO+O.OO0.00*0.25 25.44* 0.37 27.35kO.68 3.00*0.05 1.59f0.21 9.2OkO.13 10.11~0.30 2.46&-0.01 2.30f0.03 21.00~0.06 20.40*0.60 1.06*0.07 1.32kO.03 6.11*0.11 5.86kO.17 1.30*0.06 1.17kO.05 0.00~0.00 0.61*0.03 5.8O~tO.02 5.20f0.18 12.71*0.50 12.52*0.40

Hermetically sealed 90 0.61*0.02 23.75kO.31 1.27f0.02 0.25kO.18 24.85f0.56 3.2OkO.02 9.30&-0.10 2.2lf0.081 20.25*0.09 1.37ItO.09 5.28kO.09 1.44*0.08 0.00~0.00 5.90f0.08 10.25kO.55

(the pattern of survival previously observed in the hermetically sealed cultures was repeated in this experiment), the type of storage vial closure also clearly affected survival. Cultures stored in hermetically sealed vials lost their viability much more rapidly than those kept in cotton-plugged vials, suggesting that a limited supply of oxygen or the accumulation of a gaseous metabolite (e.g., CO*) or both were involved in causing an increased rate of mortality. The higher the cell concentration of the preserved culture, the greater the influence of the type of closure. Variation of the fatty acid projile in frozen and low-positive temperature stored T. suecica biomass A frozen sample of T. suecica analysed after 21 months at - 18°C (July, 1994) for lipid content, was found to retain its fatty acid profile (Fig. 3) unchanged in comparison to that determined at the beginning of the experiment (October, 1992), total fatty acid content being 14.4% in 1992 and 13.5% in 1994. The slight differences between both profiles could be attributed to changes in chromatographic conditions after such a long period of time. The fatty acid profiles of fresh T. suecica biomass and biomass stored at low positive temperature for up to 90 days at 2 different concentrations are given in Table 2. The fatty acids 18:ln-9, 16:0 and 18:3n-3 make up more than 20% of the total fatty acids, followed by unsaturated acid 18:2n-6 and polyunsaturated fatty acids (PUPAS) 18:4n-3 and 20:5n3 (EPA) with less than 10% of the total. Dry weight percentages of all fatty acids increased

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slightly in stored biomass at 60 g. l- ’ in comparison with fresh biomass, because of reserve material consumption. Nonetheless, the fatty acid profile expressed as percentage of total fatty acid remained unchanged over storage time regardless of viability. At 4 g-1-i after 90 days of storage, the fatty acid profile had not changed as percentage of total fatty acid although on a dry weight basis a slight decrease in fatty acid content was observed.

4. Discussion Freezing at either - 18°C or at - 196°C has proven to be a suitable preservation method for T. suecica as long as the main object of the process is to maintain the biochemical composition of the biomass (e.g., fatty acid profile) unchanged. In this sense, frozen biomass of the marine microalga, Zsochrysis galbana, kept at - 20°C for 1 month was reported to maintain its fatty acid profile unchanged over time (Molina Grima et al., 1994). For longterm preservation, data presented in Fig. 3 show how freezing can keep microalgal biomass unaltered as a source of fatty acids for aquaculture nutrition, even after a period of 21 months, making surplus stocking possible. Only when storage in liquid nitrogen was preceded by slow cooling down to - 30°C was a maximum of 13% viability retained after thawing. DMSO and glycerol did not show any significant difference in increasing viability during storage at sub-zero temperatures. These results are contradictory to a recent study made by Fenwick and Day ( 1992) who found a high (over 70%) post-thaw viability of T. suecicu CCAP 66/4 stored at - 196°C with 10% glycerol. The difference between the data reported here and those of Fenwick and Day ( 1992) could be explained by the difference in growth conditions of the algae from which each study started. Growth phase and growth conditions have been reported to significantly affect the cell membrane structure and biochemical composition and hence the response of cells to freezing (Ben Amotz and Gilboa, 1980; Ben Amotz and Rosenthal, 198 1) . In the present study, T. suecicu was grown in a semi-continuous culture at a relatively high growth rate (0.4. day- ’ ) , and steady-state biomass was used to carry out the experiments. Exponential or linear-phase cells have weaker cell membranes than stationary-phase cells. Fenwick and Day ( 1992) used late log phase (stationary phase?) cultures, suboptimal growth of which could have induced tolerance to freezing (Ben Amotz and Rosenthal, 1981). If viability of T. suecica at subzero temperatures was very limited or nil, the survival capability showed by this alga in darkness at low positive temperature was surprisingly high. Storage at low positive temperature has been proposed by Ben Amotz and Gilboa ( 1980) and by Ben Amotz and Rosenthal ( 1981) to increase tolerance to freezing. Umebeyashi ( 1972) has reported that several marine diatoms could be maintained for many months at + 5°C. However, in that study, cells were exposed to short periods of light several times a day. In our experiments with T. suecicu, viable cells could still be found after 5 months of storage in complete darkness. Evidently, this alga has large amounts of reserve material which can be efficiently metabolized to provide the energy required to maintain cell integrity and active movement. To produce enough energy in darkness, cells must rely upon a respiratory metabolism of stored material, and indeed significant oxygen consumption rates have been measured in T. suecicu cultures stored in darkness at +4”C. This

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explains why cultures stored in hermetically sealed vessels (i.e., under conditions which prevent exchange with the atmosphere) or at high cell concentration (i.e., under conditions which reduce the supply of oxygen to the single cell) showed limited survival. The positive effect of an adequate oxygen supply during storage was confirmed through an experiment in which some of the vials were flushed with N, before being hermetically sealed. The complete substitution of the air contained in the vial with N2 before storage reduced viability by 50% after 15 days of storage, while no reduction had yet been observed at that time in the non-flushed hermetically sealed vials. An oxygen consumption rate of between 0.5 and 1 ~1 Oz. mg( dry wt) - ’ . h-i (about 40 times less than the consumption rate at 25°C but still significant) was measured in T. suecica cultures stored in darkness at +4”C. Although the role of oxygen was demonstrated to be relevant, the existence of some additional factor which increases the rate of mortality during storage, such as the lowering of the pH due to the accumulation of CO2 or to the production of organic acids via fermentation, cannot be ruled out. These results assume a relevant practical significance. Live T. suecica biomass must be shipped from production installation to hatchery in non-concentrated cultures in non-hermetically sealed vessels unless transport and storage are of limited duration (a few days). At the 3rd week of storage, photosynthetic activity of cells stored at 60 g. 1-l in hermetically sealed vials was nil, although in cotton-plugged vials 66% of the initial photosynthetic activity remained. However, this difference in viability was not reflected in their fatty acid profiles (Table 2). Nonetheless, there was a general increase in percent dry weight content of all fatty acids over the initial profile. This suggests that during maintenance, energy was supplied by consumption of reserves other than lipids (carbohydrates), causing the fatty acid portion in dry weight to rise. When this comparison is extended to the fatty acid composition of the biomass stored at 4 g. l- ’ for 3 months, during which time cellular activity was prolonged, a decrease in content of 16:0, 18:ln-9 and 18:2n-6 dry weight was found, indicating that these fatty acids could be located in storage lipids. Nonetheless, EPA content remained unchanged as could be expected since polyunsaturated fatty acids are mainly located in structural lipids. This is an important fact to bear in mind as EPA is the main PUPA of interest for aquaculture found in T. suecica. The same trend was found for 9 g. 1-l concentrates of Zsochrysis galbana stored at 4°C with continuous illumination in which fatty acids located in storage lipids ( 16:0, 16:l) decreased over time and polyunsaturated fatty acids (EPA, DHA) remained unaltered (Molina Grima et al., 1994).

Acknowledgements This research was carried out within the framework of the project “Microalgae biomass from photobioreactors as food for fish and shellfish larvae” EEC AIRl-CT92-0286.

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Benz-Amotz, A. and Rosental, H., 1981. Cryopreservation of marine unicellular algae and early life of fish for use in mariculture. Eur. Maricult. Sot., Special Publication, 6: 149-162. Chu, F.L.E. and Webb, K.L., 1984. Polyunsaturated fatty acids and neutral lipids in developing larvae of the oyster Crassostrea uirginica. Lipids, 19: 815-820. Corder0 Esquivel, B., Voltolina Lobina, D. and Cornea Sandoval, F., 1993. The biochemical composition of two diatoms after different preservation techniques. Comp. Biochem. Physiol., 105B: 369-373. Fenwick, C. and Day, J., 1992. Cryopreservation of Tetraselmis sue&a cultured under different nutrient regimes. J. Appl. Phycol., 4: 105-109. Holliday, J.E., Allan, G.L. and Frances, J., 1991. Cold storage effects on setting of larvae of the Sydney rock oyster Saccosrrea commercialis and the Pacific oyster Cassostrea gigas. Aquaculture, 92: 179-l 85. Laing, I. and Millican, P.F., 1992. Indoor nursery cultivation of juvenile bivalve molluscs using diets of dried algae. Aquaculture, 102: 231-243 Langdon, C.J. and Waldock, M.J., 1981. The effect of algal and artificial diets on the growth and fatty acid composition of Cassostrea gigas spat. J. Mar. Biol. Assoc. UK, 61: 431438. Lepage, G. and Roy, C.C., 1984. Improved recovery of fatty acid through direct transesterification without prior extraction or purification. J. Lipid Res., 25: 1391-1396. Molina Grima, E., Sanchez Perez, J.A., Garcia Camacho, F., Acien Femandez, F.G., L6pez Alonso, D. and Segura de1 Castillo, C.I., 1994. Preservation of the marine microalga Isochrysis galbana; influence on the fatty acid profile. Aquaculture, 123: 377-385. Nell, J. and O’Connor, W., 1991. The evaluation of fresh algae and stored algal concentrates as a food source for Sydney rock oyster, Saccostrea commercialis (Iredale and Roughley) larvae. Aquaculture, 99: 277-284. Parson, T.R. and Strickland, J.D.H., 1963. Discussion of spectrophotometric determination of marine plant pigments, with revised equations for ascertaining chlorophylls and carotenoids: J. Mar. Res., 21: 155. Tredici, M.R., Carlozzi, P., Chini Zittelli, G. and Materassi, R., 1991. A vertical alveolar panel for outdoor mass cultivation of microalgae and cyanobacteria. Bioresource Technol., 38: 153-159. Umebeyashi, 0.. 1972. Preservation of some cultured diatoms. Bull. Tokai Regional Fish. Res. Lab., 69: 5562.