Aquaculture, 87 (1990) 381-393 381

Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands

Technical Paper

An Intensive Continuous Culture System Using

Tubular Photobioreactors for Producing
Microalgae

CHARLES M. JAMES and A.M. AL-KHARS

Kuwait Institute for Scientific Research, Mariculture and Fisheries Department,
P.O. Box 1638,22017 Salmiya (Kuwait)
(Accepted 3 October 1989)

ABSTRACT

James, C.M. and Al-Khans, A.M., 1990. An intensive continuous culture system using tubular
photobioreactors for producing microalgae. Aquaculture, 87: 381-393.

Growth and productivity of marine microalgae, Chlorella Strain MFD-1 and Nannochloropsis
Strain MFD-2, in an intensive continuous culture system using 200-l capacity translucent vertical
air-lift tubular photobioreactors is described. Using a culture surface irradiance of 330 PE rn-’
s-i and at a controlled pH of 6.5 and 7.0, the productivity varied between 109 and 264 g mm3dd ’
with a mean of 169.7 k 34.7 g me3 d-’ for Chlorellu Strain MFD-1 and between 32.5 and 95.3 g
rnd3 dd’ with a mean of 50.6 k 14.2 g m-’ dd’ for Nannochloropsis Strain MFD-2. The produc-
tivity of microalgae in relation to different light intensities is discussed. The fatty acid composi-
tion, especially the ~3 Highly Unsaturated Fatty Acids (~3 HUFA), of these microalgae is spe-
cies-dependent in the culture system. The total w3 HUFA and the essential fatty acid
eicosapentaenoic acid (EPA) content were significantly higher (P < 0.001) in Nannochloropsis
Strain MFD-2 compared to Chlorella Strain MFD-1, showing that the former is more suitable for
aquacultural purposes since EPA is mandatory for the feeding of marine Ash larvae. The algal
productivity obtained in the present culture system is considerably higher than with the conven-
tional methods described to date for aquacultural purposes.

INTRODUCTION

Production of microalgae is an integral part of aquaculture since these or-

ganisms are the basis of the food chain in many aquaculture operations. In
spite of all efforts to replace live microalgae by inert feeds, aquaculturists are
still dependent on the production and use of microalgae as live food for com-
mercially important fish, molluscs and crustaceans, during at least part of the
life cycle (De Pauw et al., 1984; Laing, 1987). Microalgae could also be an
important resource for special chemicals and other byproducts (Litchfield, 1979;
Aaronson et al., 1980; Oswald, 1980; Soeder, 1980; Batter et al., 1981). Species

0044-8486/90/$03.50 0 1990 Elsevier Science Publishers B.V.

382 CM. JAMES AND A.M. AL-KHARS

of microalgae belonging to the genera Chlorellu and Nannochloropsis have been

widely utilized in marine fish hatcheries in view of transferring the essential
fatty acids and other dietary components from the algae via the rotifers to fish
larvae (Watanabe et al., 1978, 1983; James et al., 1986, 1987; Lubzens, 1987;
James and Abu-Rezeq, 1988). It has been shown that the presence of Chlorellu
improves growth and survival of 40 species of fish studied (Jones, 1970). Mi-
croalgae, particularly Chlorek, are usually added together with rotifers to fish
larval rearing tanks when marine fish larvae are reared to fry or even to met-
amorphosis stage (Liao, 1975; Nash and Kuo, 1975; Howell, 1979; Pullin and
Kuo, 1981; Juario and Starch, 1984). Recent studies have identified the algae
known as ‘marine Chlorellu utilized in marine fish hatcheries as Nannochlo-
ropsis oculuta, a member of the Eustigmatophyceae (Maruyama et al., 1986).
The usual technique for microalgal production in hatcheries has been a mul-
tistep backup batch culture system, whereby small-scale cultures are grown
and used to inoculate larger scale cultures, a procedure which is troublesome
and costly in terms of space and manpower utilization. Also, outdoor algal
cultures, which are conventionally used in hatcheries, rapidly lead to collapse
of the culture or takeover by other species better adapted to the prevailing
outdoor conditions. As a result, outdoor cultures of several cubic meters usually
last for periods of time that rarely exceed a few weeks (De Pauw et al., 1984).
In recent years, semi-continuous, turbidostat algal cultures have been dem-
onstrated using polyethylene bags for the production of marine phytogflagel-
lates (Trotta, 1981; Laing and Hepper, 1983; Laing and Jones 1983 ). Further-
more, a number of closed photobioreactors such as horizontal serpentine tubular
reactors (Pirt et al., 1983; Gudin and Therpenier, 1986; Lee, 1986) and small-
scale vertical tubular reactors (Benemann et al., 1978; Miyamoto et al., 1988)
have been described. Yet, most of these systems are either of very high cost or
suffer from significant technical operating limitations, or both. For example,
serpentine tubular reactors are limited by the build-up of oxygen, among other
problems (Weissman et al., 1988). Although less sophisticated but effective
systems have been discussed for algal mass culture (Waine, 1966; Droop, 1974;
Canzonier and Brunetti, 1975; Palmer et al., 1975; Trotta, 1981; Miyamoto et
al., 1988), literature on reliable intensive indoor continuous culture systems
for the large-scale production of microalgae for aquaculture is scarce.
In order to develop an intensive automated continuous culture system for
producing microalgae, studies were carried out at the Mariculture and Fish-
eries Department, KISR, using 200-l capacity vertical translucent tubes (pho-
tobioreactors) under controlled environmental conditions. The studies were
aimed at investigating the production dynamics and nutritional quality of mi-
croalgae in terms of fatty acid composition in the continuous culture system.

MATERIALS AND METHODS

The microalgae Chlorella Strain MFD-1 and Nannochloropsis Strain MFD-

2 used during this investigation were isolated from local seawater using 1.5%
CONTINUOUS CULTURE SYSTEM FOR MICROALGAE 383

agar plates containing MFD and Miquel’s media (James et al., 1986). Axenic
stock cultures of microalgae were maintained in 250 and 500 ml Erlenmeyer
flasks containing MFD medium in a rotary shaker incubator at 100 rpm for
the preparation of seed cultures. The temperature was controlled at 25’ C and
the quantum scalar irradiance was maintained at 140 @ mm2s-l.
The continuous culture system (Fig. 1) consisted of 200-l capacity translu-
cent vertical tubes (30 cm diameter) made of corrosion-free fiberglass-rein-
forced polymer sheets (Solar Components Corporation, U.S.A. ) and equipped
with an inlet for nutrients and outflow for algal culture. Aeration was provided
at the bottom of the tubes through polyethylene tubing, which was also used
as a sampling port. Each continuous culture unit consisted of five such vertical
tubes (photobioreactors) with the nutrient and algal flow as indicated in Fig. 1.
Tubes l-2 and 3-4 were kept as continuous photobioreactors, and the fifth
tube (in the middle) was kept as a reservoir for daily harvesting. Two such
units kept in a temperature-controlled room to maintain the temperature at
23-24’ C were used for the experiments. The base medium was a combination
of commercial fertilizers and other micronutrients, in diluted sea water of 30%0
salinity (James et al., 1988)) delivered to the algal reactors from a 500-l capac-
ity nutrient reservoir using a multichannel metering pump. Photon energy was
provided by a vertical bank of daylight cool fluorescent lights. To determine
the effect of different light intensities and photoperiod on the productivity of
ChlmeUu Strain MFD-1 and Nunnochlmopsis Strain MFD-2, experiments were
carried out in different batches (14 days culture period) using a quantum sca-
lar irradiance of 140,210 and330 PE m-2 s-l and 12:12 h and 24 h photoperiod
cycles. Long-term production trials of more than 2 months of continuous cul-
ture were carried out at a light intensity of 330 PE me2 s-’ using the 24 h
photoperiod.

Fig. 1. Schematic diagram of the continuous culture system: 1-2 and 3-4 - tubular photobioreac-
tars; R - reservoir for daily harvest; N - nutrient tank; P - pump; FL - fluorescent lights; MC -
pH and temperature monitor and controller; SV - solenoid valve, AD - algal distribution pipe.
384 C.M. JAMES AND A.M. AL-KHARS

The algal pH was monitored and controlled by Foxboro chemi-monitors

(model 872 monitor, Foxboro Inc., U.S.A. ). The controller activated a solenoid
valve on a pressurized CO,-enriched line when a desired pH was exceeded,
bubbled gas mixed with compressed air (5% CO,), then entered the culture
through a port at the base of the culture, lowering the pH to the designated
level. According to a previous investigation on optimum algal pH requirement
(James et al., 1988), the pH was controlled at 6.5 for Chlorella Strain MFD-1
and at 7.0 for Nannochloropsis Strain MFD-2. Fluctuations did not exceed
? 0.15 pH units from the set value. The system was connected to a timer that
controlled both the light and the dark photoperiods and the dilution control
device. The dilution rate was adjusted every day, in relation to the instanta-
neous growth rate of algae in the culture system, which ranged from 0.4 to 0.6
d-l. Algal cell counts were monitored at 09.00 h in the morning every day using
a Neubauer improved haemacytometer. The instantaneous growth rate and
algal productivity were calculated according to James et al. (1988). To study
the lipid and fatty acid content in algae, they were centrifuged using a contin-
uous centrifuge (model Z61, New Brunswick Scientific Inc., USA) with a cen-
trifugal effect of 17 670 g and the clarified cells were lyophilized at - 73 oC and
analysed for the presence of fatty acids as discussed by James et al. ( 1987,1988,
1989). Data were analysed using one-way ANOVA (PC 0.05).

RESULTS

Light intensity and photoperiod

The productivity of ChloreZZa Strain MFD-1 increased significantly (P < 0.05)
with increasing light intensities in the culture system (Fig. 2a). Furthermore,
the algal productivity significantly increased (I’ < 0.001) when using a 24 h
photoperiod (continuous illumination) compared with a 12:12 h photoperiod.
Whenusinga 12:12 hphotoperiod, amaximumof 155.3 gme3 dd’ (dry weight)
with a mean of 131.18 + 15.54 g me3 dd’ was obtained at a light intensity of
330 PE m-’ s-l. However, a considerable increase in the productivity of Chla-
rellu Strain MFD-1 was observed at this light intensity (up to 319.7 g mP3 d-’
with a mean of 212.05 + 45.67 g mP3 d-‘) when using a 24 h photoperiod.
The results of the productivity of Nannochloropsis Strain MFD-2 in relation
to different light intensities show (Fig. 2b) no significant increase (P> 0.05)
between various light intensities tested when using a 12:12 h photoperiod.
However, when using a 24 h photoperiod, a significant increase (P< 0.01) in
algal productivity was observed between a light intensity of 140 and 330 PE
me2 s-‘. With a 12:12 h photoperiod, it was possible to obtain up to 84.0 g me3
d-l, with a mean of 58.51% 14.17 g rnM3d-l, at a light intensity of 330pE me2
S - ‘. At this light intensity using a 24 h photoperiod, it was possible to achieve
an algal productivity of up to 110.5 g m-3 d-‘, with a mean of 81.57 -t 16.73 g
mP3 dd’. No significant increase (P> 0.05) in algal productivity was observed
CONTINUOUS CULTURE SYSTEM FOR MICROALGAE 385

320 0
G--412h Photoperiod a
-24h Photoperiod
280

20

140 210 330

L ig ht ( p E me2 s-’ )

Fig. 2. Productivity of microalgae in relation to different light regimes and photoperiod. (a) Pro-
ductivityof Chlorella Strain MFD-1. Productivity at 12 h photoperiod=52.089+0.241~; r2=0.6585
and n=30. Productivity at 24 h photoperiod= -9.282+1.107x-0.001x2; ?=0.6262 and n=30.
(b) Productivity of Nannochloropsis Strain MFD-2. Productivity at 12 h photoper-
iod= 42.748 +0.054x; ?=0.1828 and n=30. Productivity at 24 h photoperiod=
-2.974+0.523x-0.001x2; r2=0.5108andn=30.

at light intensities between 210 and 330 PE me2 s-l, suggesting that photic
energy did not limit the production of Nannochloropsis Strain MFD-2 in the
culture system.

Long-term productivity
Fig. 3 shows that, during the 2-month culture period, the productivity of
Chlorella Strain MFD-1 ranged from 109.1 to 264.5 g mW3d-‘, with a mean of
169.67 + 34.7 g mW3d-l. The growth rate varied from 0.92 to 1.35 d-l, with a
mean of 1.09 t 0.42 d-l. The productivity of Nannochloropsis Strain MFD-2
varied from 32.5 to 95.3 g mm3d-l, with a mean of 50.62? 14.2 g me3 d-l. The
growth rate varied from 0.31 to 0.93 d-l, with a mean of 0.60 + 0.27 d-l. During
386 CM. JAMES AND A.M. AL-KHARS

280 Chlorella strain MFD-1

260 Nennochloropsis strain MFD-2

240 A
220

200

180

160

140

120

100

e0

60

40

20
II
0 ‘,,l,,,,,,,,,, ,‘,,
,,I III I,,,,,. L‘,,,,,,,,,,,,,

I.3

1.2

I.1

I.0

0.9

0.8

0.'

0.0

0.5

0.4

0.3

0.2

0.1
I
0.0
c

Fig. 3. (A) Productivity of Chtorella Strain MFD-1 and Nannochloropsis Strain MFD-2 in the
continuous culture system; (B) Growth rate of Chlorella Strain MFD-1 and Nannochloropsis
Strain MFD-2 in the continuous culture system.

the observation period, the culture cell density varied from 78 to 134 x lo6 cells
ml- ’ with a mean of 100.6 + 40.6 x lo6 cells ml-’ for CMorekz Strain MFD-1,
and from 34 to 63 x lo6 cells ml-’ with a mean of 43.3 5 19.1 x lo6 cells ml-’
for Nannochloropsis Strain MFD-2. The results of this investigation show that
the growth rate and productivity of ChZorella Strain MFD-1 is significantly
CONTINUOUS CULTURE SYSTEM FOR MICROALGAE 387

higher (P-C 0.001) than that of Nunnochloropsis Strain MFD-2 in the culture
system.

Fatty acid composition

The fatty acid composition of microalgae (Table 1) shows that the total 03
HUFA content in Nunnochloropsis Strain MFD-2 is significantly higher
(P-C 0.001) than that of Chbrellu Strain MFD-1 in the culture system. Among
03 HUFA, linolenic acid (18: 303) constituted the major component in Chlo-

TABLE 1

Fatty acid composition (area % ) of microalgae in the continuous culture system

Fatty acid Chlorella Strain MFD-I* Nannochloropsis Strain MFD-2*

Photoperiod Photoperiod
12:12 h 24 h 12:12 h 24 h

14:o 1.1 0.4 7.5 6.6

14: 109 _
15:o 0.1 0.3 0.5
16:0 17.3 18.7 20.2 18.7
16:1& 1.1 3.5 19.1 18.5
17:o 0.4 0.3 0.3 0.4
18:O 0.4 0.5 2.2e 0.2
18: 1~09 3.8 4.6 2.2
18:2& 15.0 22.4 5.2 6.8
18:3&
18:3w3 30.1 26.0 0.8 0.3
19:o 0.9 0.5
20:o
20:109 0.4
20:26x
20 : 3w3 4.1 4.2
20 : 4w3
20 : 5w3 37.8 28.1
22: lw5
22 : 3W3
22 : 4W3
2215~3
22 : 603
24:0
241106
Cw3 HUFA 31.4 26.3 42.7 32.6
Total lipid (% ) 18.6 21.5 14.5 18.8

*Each value is the mean of two replicates.

-, Not detected.
“18:O and 18: 1~9 together.
388 C.M. JAMES AND A.M. AL-KHARS

rellu Strain MFD-1 ( > 26%) while the long-chain eicosapentaenoic acid
(20 : 503) content was very low ( < 1% ). An opposite trend was observed in
Nannochloropsis Strain MFD-2, where the essential eicosapentaenoic acid
constituted the major component ( > 28.1% ) and the linolenic acid content
was very low ( < 1% ). Furthermore, variations in the c03 HUFA content in
microalgae were observed between the 12:12 h and 24 h photoperiods. How-
ever, no significant difference (P> 0.05) in the total 03 HUFA content was
observed in Chlorellu Strain MFD-1. Although no significant difference
(P> 0.05) in the essential fatty acid 20 : 503 content was observed in Nan-
nochloropsis Strain MFD-2, the total ~03HUFA content in the 12:12 h photo-
period was significantly higher (P < 0.01) than that in the 24 h photoperiod.

DISCUSSION

The productivity of microalgae in the continuous culture system shows an

increase with increasing light intensities. Also a significant increase (P-c 0.05)
in the productivity of microalgae was observed when using a 24 h photoperiod
compared to a 12:12 h photoperiod. This is due to the fact that algal growth in
mass cultures is almost exclusively photolithotrophic. Consequently, light en-
ergy is the determining factor for the maximum capacity of any algal culture
system (Shelef, 1968; Droop et al., 1982 ). The productivity of microalgae shows
that the marine ChZoreZZa Strain MFD-1 is considerably more productive than
the Nannochloropsis strain MFD-2, in relation to the light energy utilized in
the continuous culture system. This suggests that light utilization is more ef-
ficient in Chlorella strain MFD-1 than in Nannochloropsis strain MFD-2. Op-
timum light saturation and growth of microalgae depend on the species utilized
in the culture system (Spectorova et al., 1986). Furthermore, recent experi-
ments have demonstrated that the photosynthesis of algae is not only light-
dependent, but also time-dependent (De Roos and Flik, 1985); this is consis-
tent with the present observation, in which productivity was significantly higher
(PcO.05) using a 24 h photoperiod (at 140 and 330pE mm2s-l) than a 12:12
h photoperiod.
Long-term tests show that growth, cell density and productivity of microal-
gae is species specific. After initiating the culture, although growth and pro-
ductivity of Chlorella strain MFD-1 reached a steady state, fluctuations in pro-
ductivity (observed especially on days 11, 32 and 41) were due to settling of
Chlorella cells on the walls of the photobioreactors. On these days the tubes
were cleaned and the culture was continued. The productivity of Chlorellu Strain
MFD-1 obtained in the continuous culture system (up to 264.5 g m-’ d-’ with
a mean of 169.67 2 34.7 g m-’ d-’ ) is considerably higher than in any of the
conventional algal production systems reported to date for aquacultural pur-
poses. Furthermore, the productivity of Chlorella strain MFD-1 achieved under
long-term culture conditions considerably exceeds that reported by James et
CONTINUOUS CULTURE SYSTEM FOR MICROALGAE 389

al. (1988) while studying the pH-dependent growth of Chlorella in the contin-
uous culture system. The variation observed in the productivity may be due to
the short culture duration (14 days) in the previous study.
Data in the literature on algal productivity in ponds range from 5 to 65 g
m-’ d-’ (dry weight) with a typical long-term average of lo-20 g me2 d-l.
Theoretical maximum photosynthetic yield under outdoor conditions is esti-
mated at 30-40 g mm2d-’ (Benemann et al., 1978). Goldman (1980) indicated
that available sunlight places an upper limit on potential productivity, which
is 30-50 g me2 dd’ (dry weight) under outdoor culture conditions. Prokop and
Fekri ( 1984)) using a thin-layer outdoor intensive culture system for cultivat-
ing C. sorokinianu, reported productivity of 28-35 g m-’ d-’ under Kuwaiti
environmental conditions. While using a 2 m3 capacity outdoor semicontin-
uous production system for cultivating a marine Chlorellu sp. for aquaculture
in Kuwait, the productivity was about 20-26 g me2 dd’ on a dry weight basis.
Ortega and Roux (1986), studying the production of Chlorella biomass in dif-
ferent types of flat bioreactor in temperate zones, obtained a maximum pro-
duction of 24.3 g mm2 d-’ using rigid PVC panels; this is considerably lower
than the productivity recorded during this investigation. The higher produc-
tivity is due to the larger culture volume exposed to photic energy, since the
system is vertical, whereas outdoor culture methods occupy proportionally more
space (horizontal) relative to unit culture volume. Accordingly, based on the
unit area occupied by the vertical culture system, the present continuous cul-
ture system yields up to 193.2 g mm2d-’ (dry weight) with a mean of 135 + 27.8
g m-2 d-’ for Chlorellu Strain MFD-1 and up to 76.2 g rnp2 d-’ with a mean
of 40.5 g me2 d-l for Nunnochloropsis Strain MFD-2 in the algal chemostats,
demonstrating that much hatchery space is saved using such vertical algal cul-
ture systems. The higher algal productivity in the present culture system is
also due to the continuous dilution method, compared with the non-dilution
method as observed by Hare and Schmidt (1968) for C. pyrenoidosa.
Under indoor culture conditions, excluding ChZorelZa,Palmer et al. (1975)
obtained 5 g d-’ (dry weight) of Monochrysis from 40-l chemostat cultures
(i.e., 125 g mm3d-l), which is comparable with the yield obtained in our cul-
ture system. Using 50-l polyethylene bag cultures operated as turbidostats,
Trotta (1981) obtained yields of 20-30 g d- ’ (wet weight) for Tetraselmis,
which is considerably lower than the algal yield we observed. Laing and Jones
(1983)) discussing a turbidostat culture system using 80-l internally illumi-
nated vessels, reported about 12 g d- ’ (wet weight) of Isochrysis and 20 g d- ’
(wet weight) of Tetruselmis, which is also considerably lower than the produc-
tivity of our system. Among the high density indoor culture systems, Spekto-
rova et al. (1986) obtained 50 g m-2 d-’ using a 15 mm thick layer culture
system for ChZureZZu. The high productivity of ChZureZZa Strain MFD-1 ob-
served during this investigation is unique and is due to the continuous culture
system, in which the parameters are kept optimal through continuous dilution
390 C.M. JAMES AND A.M. AL-KHARS

with nutrient enrichment and control of pH. Other advantages of the vertical
tubular reactors involve their high surface to volume ratios, low cost and high
efficiency of using COz, as observed by James et al. (1988). The inclusion of
/3-glycerophosphate as a source of phosphate and carbon would also have en-
hanced the algal productivity, as observed by Panczakowa ( 1983 ).
James et al. (1988,1989) observed that the major 03 HUFA component of
Chlorelkz Strain MFD-1 is linolenic acid ( 18 : 303)) and that in Nunnochlorop-
sis Strain MFD-2 is eicosapentaenoic acid (EPA). This illustrates that the 03
HUFA profile in microalgae depends on the species utilized in a culture system
(Ben-Amotz et al. 1987; James et al. 1988,1989). The 03 HUFA content ob-
served during this investigation for Chlorella Strain MFD-1 and Nunnochlo-
ropsis Strain MFD-2 is higher than that found by James et al. (1989). This
may be due to the optimum conditions such as pH control and nutrient en-
richment adopted in the culture system (James et al., 1988). Depending on the
species or culture conditions, increasing light intensity may cause an increase
(Orcutt and Patterson, 1973 ) or a decrease (Iwamoto and Sugimoto, 1955) in
total lipids. In isolated studies in various species, lipid components and/or
synthesis pathways have also been shown to vary with light and dark periods
(Nicholas, 1965; Fisher and Schwarzenbach, 1978). However, Shifrin and
Chisholm (1981) showed that the total lipid fraction remains constant over
the cell cycle in synchronized cultures regardless of light regime. In our inves-
tigation, the total w3 HUFA content in Chlorellu Strain MFD-1 showed no
significant variation (P> 0.05) between 12:12 h and 24 h photoperiods. How-
ever, in Nunnochloropsis Strain MFD-2, although the EPA (20 : 503) content
did not show any significant variation (P>O.O5) between 12:12 h and 24 h
photoperiods, the total w3 HUFA content was significantly different (P-c 0.05 )
between 12 h and 24 h photoperiods. In general, the EPA as well as the total
03 HUFA showed a declining trend with increasing photoperiods for both the
algal species tested in the culture system.
The presence of long-chain 03 HUFA is known to be important for selecting
unicellular marine algae for aquacultural purposes (Watanabe et al., 1983;
James et al., 1983,1987,1988). The fatty acids, for example, the EPA observed
during this investigation, depend on the species of microalgae utilized in the
culture system. The high quantities of EPA observed in Nunnochloropsis Strain
MFD-2 indicate that it will be more suitable for use in marine fish hatcheries
than Chlorekz Strain MFD-1. Furthermore, the productivity of microalgae in
the continuous culture system is considerably higher than in any conventional
systems. The vertical tubular photobioreactors described here will also save
considerable space and manpower utilization in a hatchery, compared to batch
culture methods. Another advantage of vertical tubular photobioreactors is
that the oxygen they evolve (Weissman et al., 1988) could be used to enrich
the oxygen levels for intensive fish/shrimp larval rearing systems in the
hatchery.
CONTINUOUS CULTURE SYSTEM FOR MICROALGAE 391

ACKNOWLEDGEMENTS

This research was financially supported by the Kuwait Foundation for the
Advancement of Sciences, Kuwait, under the project code 86-04-02. Our sin-
cere thanks are due to Dr. Ziad H. Shehadeh, Program Element Manager,
Aquaculture, Kuwait Institute for Scientific Research, who provided invalua-
ble insight and guidance through all phases of this study. We are also thankful
to the staff of the Central Analytical Laboratory, KISR, for their assistance in
the analysis of fatty acids.

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