J. Exp. Mur. Bioi. Ecol., 1989, Vol. 128, pp. 219-240 Elsevier

219

JEM 01260

Fatty acid and lipid composition of 10 species of microalgae used in mariculture

J. K. Volkman’, S. W. Jeffrey2, P. D. Nichols’, G. I. Rogers’ and C. D. Garland3

CSIRO Division of ~ceano~uphy, Hobart, Tasmania, Australia; ‘CSIRO Di~,is~o~~ of Fisheries, Hobart, Tasnlania, Australia; 3Department of A~tcultural Science, Universit_y of Tasmania, Hobart, Tasmania.

Australia

(Received 14 October 1988; revision received 9 March 1989; accepted 18 March 1989)

Abstract: Total fatty-acid composition, lipid classes, Chl a contents and ceil volumes of 10 species of microalgae commonly used in mariculture were examined. The microalgae were grown under defined conditions and harvested during log phase. Significant concentrations of the polyunsaturated fatty acid 20: 5(n-3) (eicosapentaenoic acid) were present in each of the four diatoms Chaetoceros calcitrans, Chaetocerosgracilis, Skeletonema costatum and Thalassiosirapseudonana (4.6 - 11.1% of the total fatty acids), the cryptomonad Chroomonas sahna (10.9, 11.9%) and the prymnesiophyte Pa&vu lutheri (19.7%). Very small amounts were found in another prymnesiophyte, Zsochrysis sp., and the green algae Nunnochiorjs atomus and Tetraselmis sue&a but none was detected in the green alga Dunalief~a ierttolecta. In contrast, high concentrations of 22 : 6(n-3) (d~s~exaenoic acid) were present only in the two prymnesiophyte algae and Chroomonussulina. Chl a and total fatty-acid contents were not related to cell volume. Fatty acids were four to six times more abundant than Chl a in most species. Relative concentrations of intact lipids were determined for each species using Iatroscan thin-layer chromatography-flame-ionisation detection. Polar lipids predominated in all species but triacyglycerols were abundant only in Chaetoceros gracihs, Isochrysis sp. and Chroomonas sp. Free fatty acids were particularly abundant in the two Chaetoceros species. The importance of these data for determining the nutritional value of the microalgae as food for animals in mariculture is discussed.

Key words: Capillary gas chromatography; Fatty acid; Lipid; Mariculture; Microalga; Nutrition

The number and diversity of marine and estuarine animals being reared commercially for human consumption and other purposes increases each year. Microalgae have an important role in mariculture as food for larval stages of crustaceans and fish, for all stages of bivalves and as food for zooplankton (rotifers, copepods and brine shrimp) which are fed to late larval and juvenile fish and crustaceans. Since it is important that the nutritional quality of the microalgae is optimal for the animals being reared (Brown et al., 1989), we are studying the biochemical composition of microalgae commonly used

Correspondence address: J. K. Volkman, CSIRO Division of Oceanography, GPO Box 1538, Hobart, Tasmania 7001. Australia.

0022-0981/89~SO3.5~ 0 1989 Elsevier Science Publishers B.V. (Biomedical Division)

in mariculture, together with the environmental parameters that modify the nutritional quality of these algae, The 10 microalgal species studied here are strains held in the CSIRO Algal Culture Collection (Jeffrey, 1980) which are made available to the Australian mariculture industry to rear larval and juvenile mussels, edible oysters, pearl aysters, clams, abalone, barramundi, dolphin fish, penaeid prawns, mud crabs and other species (Jeffrey & Garland, 1987; Garland, 1988).

Many *narine animals appear to have a limited ability to synthesize the poly- unsaturated fatty acids (PUFA) 20:5(n-3) and 22 :6(n-3) from precursor fatty acids such as linolenic acid (Kanazawa et al., 1979). Examples where these PUFA are considered essential include commercially important species of oysters (Langdon & Waldo&, 1981; Enright et al., 1986a,b), penaeid prawns and shrimps (Kanazawa, 1985). Some animals may not have an absolute dietary requirement for 20:5(n-3) and 22:6(n-3) but grawth rates and larval survival usually increase when these fatty acids are included in the diet (Webb & Chu, 1983; Pillsbury, 1985; Rodgers & Barlow, 1987).

The fatty acids of many species of microalgae have been examined previously but too few studies have used defned culture conditions or harvested the microalgae at a specified stage of growth. Cells in exponential phase may have a different biochemical composition to those in stationary phase (e.g., Ballantine et al., 1979; Webb & Chu, 1983; Borowitzka, 1988). Changes in other culture conditions, such as light intensity, nutrient status of the medium, length of the light : dark cycle and temperature, can also change the lipid composition (e.g., Orcutt & Patterson, 1974; Brown et al., 1989). In this study, microalgae were grown under well-defined light, temperature and nutrient conditions and harvested in mid-exponential phase to ensure healthy cells.

Detailed analyses of lipids and fatty acids can now be carried out on very small ~subrniIli~~~ samples. With the use of polar and nonpolar high-resolution capillary columns, almost all of the different double-bond isomers found in microalgae can be identified. Simple derivatisation techniques, such as formation of dimethyldisui~de (DMDS) adducts (DunkelbIum et al., 1985) which can then be identified by gas chro- matography-mass spectrometry, are also available to confirm double-bond positions in monounsaturated fatty acids. Such information can be particularly valuable in chemotaxonomic studies and can be used in mariculture studies to follow the uptake of dietary lipids by the animal. Submicrogram amounts of lipid classes can also be determined by a technique that combines thin-layer chromato~aphy with flame- ionisation detection (Tanaka et al., 1980; Parrish & Ackman, 1983; Volkman et al., 1986).

We report here the lipid composition, chlorophyll content and total fatty acids, including positional isomers of unsaturated fatty acids, of 10 species of microalgae used extensively by the mar&culture industry. Details of amino acid, carbohydrate and vitamin contents will be described elsewhere.

FATTY ACID AND LIPID COMPOSITION IN MICROALGAE 221

MATERIALS AND METHODS

MICROALGAL CULTURES

Microalgae (Table I) were obtained from R.R.L. Guillard, Bigelow Laboratory for

Ocean Sciences, Maine, U.S.A., and maintained in the CSIRO Algal Culture Collection

(Jeffrey, 1980). Culture media used were medium f2 (Guillard & Ryther, 1962) medium

fu containing EDTA (Jeffrey, 1980), or medium G, (Loeblich & Smith, 1968).

Nonaerated cultures were grown in 160 ml culture medium in 250-ml Erlenmeyer flasks

at 20 + 0.5 “C on glass shelves illuminated from beneath with 70-80 PE 9 me2 s ’

white fluorescent light (Philips daylight tubes) on 12 : 12 h light: dark cycles. Light

intensities were measured with a Biospherical Optics light meter. Cells were harvested

towards the end of log phase (see Table I for cell densities at harvest). A lo-ml aliquot

was removed from each culture for cell counts and Chl a determination and the

remainder (z 150 ml) was used for lipid and fatty acid analyses.

In a second experiment performed 6 months later, three of the species were cultured

under similar conditions to test whether similar compositional data would be obtained.

Cells were harvested after the same growth period; the cell densities were slightly less

than before (Table I).

Cell counts were determined with a Neubauer haemocytometer, the percentage error

in six replicate cell counts ranged from 5.6 to 11.9% (Table I). Cell volumes were

estimated according to Smayda (1978). For volume measurements, cells were not fixed

in Lugol’s or other preservative since these cause significant change in cell volume. The

length, width or diameter of at least five representative living cells in each culture was

measured. Volumes were calculated according to the basic geometry of the cell: e.g.,

sphere (Chaetocerosgracilis, Chaetoceros calcitrans), oblate ellipsoid (Chroomonas salina, Tetraselmis suecica, Dunaliella tertiolecta, Pavlova lutheri, Nannochloris atomus, Isochrysis sp.) and rectangular box (Skeletonema costatum, Thalassiosira pseudonana). Mean vol-

umes and ranges are given in Table I.

Cultures of microalgae were tested for bacterial contamination by inoculating 0.1 ml

culture medium into seawater broth or onto seawater agar containing vitamins and

checking for bacterial growth after incubation at 25 “C for 5-7 days (Lewis et al., 1986).

Nine of the species were found to be axenic, the only exception being Chaetoceros gracilis, CS-176.

CHLOROPHYLL DETERMINATION

Cells were harvested from 3-5 ml culture by centrifuging at 2000 x g for 3-5 min.

Pigments were extracted from the cell pellet of most species in 90 % acetone, followed

by sonication for 5 min in a Bransonic S2 sonicator bath. The extracts were then left

for 30 min in the dark for pigment extraction to take place. Since 90% acetone does

not extract the pigments from the green flagellates Tetraselmis or Nannochloriq these

algae were first treated with 0.2 ml dimethylsulfoxide (DMSO) with sonication for 5 min

TABLE I

Spec

ies

used

fo

r lip

id

and

fatty

ac

id

anal

ysis

w

ith

cell

coun

ts

and

cell

volu

mes

at

ha

rves

t.

Cla

ss

and

spec

ies

CSI

RO

D

epos

ition

or

C

ultu

re

No.

or

igin

Sp

ecie

s co

de

used

A

xeni

c C

ultu

re

med

ium

Cel

ls * m

l -

’ at

harv

est

Mea

n ce

ll vo

lum

e an

d ra

nge

(pm

’)

Chl

orop

hyce

ae

(gre

en

flag

ella

te}

Dun

alie

lla

tert

iole

cta

Nan

noch

lori

s at

omus

Pr

asin

ophy

ceae

(g

reen

fl

agel

late

) T

etra

selm

is s

ueci

ca’

Bac

illar

ioph

ycea

e (d

iato

m)

Cha

etoc

eros

cal

citr

ans

Cha

etoc

eros

gra

cili

s Sk

elet

onem

a co

stat

um

Tha

lass

iosi

ra p

seud

onan

a ’

Prym

nesi

ophy

ceae

Is

ochr

ysis

sp.

(T

ahiti

an)

Pav

lova

luth

eri’

C

rypt

ophy

ceae

C

hroo

mon

as s

alin

a

cs-1

75

cs-1

83

CS-

187

CS-

I78

CS-

176

cs-1

81

cs-1

73

cs-1

77

cs-1

82

cs-1

74

WH

O

I D

UN

25

1/4B

WT

ET

C.C

AL

C

HA

RA

SKE

L

3H

TIS

O

MO

NO

3c

DU

N

NA

N

TE

T

CC

AL

C

.GR

A

SKE

L

TH

AL

TIS

O

PAV

CH

RO

Yes

Y

es

Yes

Yes

N

o Y

es

Yes

Yes

Y

es

Yes

f2

1.39

x

106

295

(203

-441

) f*

5.

72

x lo

6 57

(3

7-88

)

fi

6.25

x

10’

640

(441

-814

)

f*

3.92

x

IO6

5 (l

-12)

fi

2.

15

x IO

6 38

(1

2-66

) fz

1.

94

x 10

6 15

4 (1

11-2

25)

G,

6.32

x

lo6

32

(17-

61)

fi

2.81

x

IO6

51

(28-

122)

f2

2.

04

x 10

6 91

(5

3-25

0)

t 8.

05

x 10

5 34

0 (2

26-5

35)

Prev

ious

ly

know

n as

’ P

laty

mon

as s

ueci

ca,

2 C

yclo

tell

a na

na a

nd

’ Mon

ochr

ysis

lut

heri

. 4

Perc

enta

ge

erro

r in

ce

ll co

unt

rang

ed

from

5.

6 to

11

.9%

.

FATTY ACID AND LIPID COMPOSITION IN MICROALGAE 223

and then extracted with 90% acetone (2.8 ml) as for the other algae. The extracts were then centrifuged and Chl a determined spectrophotometrically in the clear supernatant, using the appropriate equations of Jeffrey & Humphrey (1975).

LIPID EXTRACTION AND FRACTIONATION

Cells were harvested from 150 ml culture medium by filtering through a 47-mm diameter glass-tibre filter (Whatman GF/F, nominal pore size 0.7 pm) and extracted immediately, using ultrasonication with propan-2-01 (7 ml, 10 min) to minimise transesteri~cation, and then with chloroform-meth~ol (2: 1, 3 x 10 ml). The com- bined extracts were partitioned with purified water (ME-Q system) to remove salts and water soluble material. Lipids were recovered in the lower chloroform phase. The solvents were then removed under vacuum and the lipids stored under N, at - 20 “C.

The modified Bligh & Dyer (1959) procedure, originally designed for extraction of fish lipids, gave excellent recovery of lipids in most species with the exception of Nunnochloris

(a)

n C.CAL

FFA TG

0 1.0 Rt

(b) PAV

TG

I 1

0 1.0 Rf

Fig. I. Typical Iatroscan TLC-FID chromatograms of lipids in microalgae. (a)diatom Chcretoceros calcirrans, (b) prymnesiophyte Pavfova lutheri. POL, polar lipids; *, pigment and unknown lipids; ST, sterols, MS, Pmethyl sterois; FFA, free fatty acids; TG, triacylglycerols; HC, hydrocarbons (and wax esters if

present).

224 J. K. VULKMAN ET AL.

atomus. Cells of this alga were still slightly green after three extractions with chloro- form : methanol : water, so an additional extraction was performed with dimethyl- sulfoxide followed by three extractions with chloroform : methanol : water as before. This yielded an additional 15% of fatty acids.

The concentrations of major lipid classes were determined by analysing a portion of the total lipid extract with an Iatroscan Mk III TH-10 TLC-FID analyser (Iatron Laboratories, Japan) as described by Volkman et al. (1986). The solvent system used far the lipid separation was hexane-diethyl ether-acetic acid (60/ 17iO.5, v/v/v) which resolves triacylglycerols and free fatty acids from other common neutral lipid classes. Typical TLC-FID chromatograms are shown in Fig. I. Total fatty acid methyl esters (FAME) were furmed directly by treating an aliquot of the total extract with BE;,-methanol at 80 “C for 90 min. This procedure transesterifies all common algal lipids and also esterifies free fatty acids. Extracts were stored at - 20 “C until analysis which usually occurred within 2 days,

ANALYSES OF FATTY ACIDS BY CAPILLARY GAS CHROMATOGRAPHY

All samples were analysed with a Shimadzu 9A gas chromatograph equipped with an FID and cool WI-3 on-column injector (SGE, Australia). Samples were dissolved in chloroform to which a known amount of methylnonadecanoate (19 : 0) was added as an internal standard. Samples were injected at 40 “C onto a nonpolar methyl silicone

10

9

8

7

s P

0

f8 20 22 24 26 28

Time (minuted

Fig. 2. Part of a reconstructed ion chromatogram of fatty acid methyl esters from Tetraselmis xuecica obtained from gas chromatography-mass spectrometric analysis using a 50 m x 0.2 mm id methyl silicone

fused-silica capillary column.

FATTY ACID AND LIPID COMPOSITION IN MICROALGAE 225

fused-silica capillary column (50 m x 0.32 mm id, Hewlett Packard). After 1 min, the oven temperature was raised to 120 “C at 30 “C . min - ’ and then to 3 10 “C at

4 ‘C * min _ ‘. H, was used as the carrier gas. The detector temperature was 340 ‘C. FAME were further analysed using a polar carbowax (BP-20) fused-silica columns (25 m x 0.25 mm id, 50 m x 0.22 mm id; SGE) using a Hewlett Packard 5890 gas chromato~aph, GC conditions were similar, except that 240 “C was the upper limit of the second temperature ramp. A typical chromatogram obtained using the nonpolar column is shown in Fig. 2. Peak areas were quantified with a Shimadzu C-R3A combined computing integrator and plotter.

ANALYSES OF FATTY ACIDS BY GAS CHROMATOGRAPHY-MASS SPECTROMETRY

(GC-MS)

GC-MS analyses were performed with an HP 5890 GC and 5190 MSD fitted with a direct capillary inlet. The nonpolar column, injector and chromatography conditions were the same as those described above with the exception that He was used as the carrier gas. Electron impact mass spectra were acquired and processed with a 59970A Computer Workstation. Typical MSD operating conditions were: electron multiplier 2000 V, transfer line 310 “C, an electron impact energy of 70 eV, 0.8 scan. s ‘, mass range 40-600 Da.

Fatty acid identifications were confirmed by comparing mass spectra and retention data with those previously reported and with those obtained from commercial stan- dards. The identification of positional isomers was verified from the observation that on the non-polar column (n-6) and (n-3) PUFA eluted as just-resolved pairs where the second isomer has one additional double bond, i.e., three C atoms closer to the methyl end of the molecule. This characteristic elution order is illustrated in Fig. 2 for the pairs 16: 2(n-6)/16: 3(n-3), 16: 3(n-4)~16:4(n-3), 18:2(n-6)/18:3(n-3), 20:4(n-6)~20:5(n-3), 22:5(n-6)/22:6(n-3).

Dimethyldisulfide (DMDS) adducts of monounsaturated FAME were formed to determine double bond position and geometry, using methods previously described (Dunkelblum et al., 1985; Nichols et al., 1986a). Samples were analysed by GC and CC-MS using the nonpolar capillary column as described above. Mass spectra of the adducts showed major ions attributable to fragmentation between the two CH,S groups located at the original site of unsaturation. Cis and tram isomers in the original monounsaturated fatty acids were determined from the fact that the erythro adduct (originally the tram fatty acid isomer) elutes after the threo isomer (originally the cis fatty acid isomer). Isomers differing in the position of the double bond were well separated under the conditions used in our study.

Fatty acids are designated as “total number of C atoms: number of double bonds (n-x)” where “x” is the position of the ultimate double bond from the terminal methyl group. This is equivalent to the earlier omega nomenclat~e. The sufBxes c and t indicate cis and tram geometry. PUFA is an abbreviation for poly~saturated fatty acids. Double bonds in PUFA are separated by a methylene group.

226 J. K. VOLKMAN ET AL

RESULTS AND DISCUSSION

CELL VOLUMES AND CHLOROPHYLL CONTENTS

All microalgal cultures were harvested during the exponential phase of growth and

contained healthy viable cells with normal morphology. Cell volumes ranged from 5 pm3

for the diatom Chaetoceros calcitrans to 640 pm’ for the green alga Tetraselmis suecica (Table I). The range of cell volumes found in each culture shows the considerable

natural variation that occurs in nonsynchronous cultures (Table I). Chl a contents

ranged from 0.12 pg. cell- ’ for the green alga Nannochloris atomus to 1.85 pg. cell- ’

for Chroomonas salina (Table II). There was little correlation with cell volume:

Chaetoceros calcitrans, the smallest cell, had the highest chlorophyll content per unit

volume (Table II). Note that these chlorophyll contents refer specifically to the con-

ditions used in this study and would vary with different culturing conditions, particularly

different light regimes.

TABLE II

Concentrations of Chl a and total fatty acids (pg. cell - ‘).

Chl a FA Chl a,prn -’ FA,prn-’ FA.Chla-’ (pg.cell-‘) (pg. cell ‘) (x 102) (x 10’)

Chaetoceros calcitrans 0.16 0.74 32 148 4.6 Chaetoceros gracilis no. I 0.36 2.2 9.5 58 6.1 Chaetoceros gracilis no. 2 0.27 2.4 7.1 63 8.9 Skeletonema costatum 0.64 1.5 4.2 9.7 2.3 Thalassiosira pseudonana 0.21 1.0 6.6 31 4.8 Isochrysis sp. 0.20 1.2 3.9 24 5.8 Pavlova lutheri 0.80 3.2 8.8 35 4.1 Dunaliella tertiolecta 1.36 7.4 4.6 25 5.4 Nannochloris atomus 0.23 0.41 4.0 7.1 1.8 Tetraselmis suecica no. I 0.56 2.7 0.9 4.2 4.8 Tetraselmis suecica no. 2 1.1 4.5 1.7 2.6 4.1 Chroomona salina no. I 1.85 7.8 5.4 23 4.2 Chroomona salina no. 2 1.75 8.2 5.1 24 4.7

FA. total fatty acids. Nos. 1 and 2, cultures of same strain grown 6 months apart (see text).

LIPID COMPOSITION

The relative proportions of intact lipids were determined by Iatroscan TLC-FID

(Table III). There was considerable variation between classes and even between species

within the same class, even though similar growth conditions were used. Polar lipids,

consisting of glycolipids, phospholipids, chlorophylls and other lipids, were the major

constituents of the lipid extracts with values > 65 y0 for all but the diatom Chaetoceros gracilis which has an unusually high content of triacylglycerols and free fatty acids.

FATTY ACID AND LIPID COMPOSITION IN MICROALGAE 221

TABLE III

Percentage composition of lipid classes determined by Iatroscan thin-layer chromatography-flame ioni-

zation detection.

HC TG FFA ST POL Others

Chaetoceros calcitrans 0.4 8.4 11.4 6.1 12.8 0.9 Chaetoceros gracilis 1.3 34.0 14.4 6.0 44.2 Skeletonema costatum 0.8 1.7 8.5 1.1 84.6 1.3 Thalassiosira pseudonana 1.2 14.4 1.1 2.8 80.4 Isochrysis sp. 0.4 2.8 TR 0.2 83.0 13.5*

Pavlova lutheri 0.2 4.0 TR 6.3 78.3 1 I .o** Dunalieila tertiolecta TR 1.9 0.9 2.1 94.3 2.1 Nannochloris atomus TR TR 0.4 1.0 98.6 _

Tetraselmis suecica 1.8 3.3 0.8 1.9 91.5 0.7 Chroomonas salina 3.5 21.9 1.9 4.9 67.8

HC, hydrocarbons and wax esters; TG, triacylglycerols; FFA, free fatty acids; ST, sterols and alcohols;

POL, polar lipids and chlorophylls. TR < 0.2%. * Mainly C,,-C,, unsaturated methyl and ethyl ketones (Volkman et al., 1981; Marlowe et al., 1984). ** Mainly 4-methylsterols and compounds of unknown structure.

Individual polar lipids were not quantified. Triacylglycerols were minor components of

the three green algae but values from 8.4 to 34% were found in the other classes of

microalgae; highest contents were in Chaetoceros gracilis, Isochrysis sp. and Chroomonas salina.

Free fatty acids were very minor constituents in all species, except for the diatoms

Chaetoceros gracilis, Chaetoceros calcitrans and Skeletonema costatum. Both cultures of

C. gracilis had high levels of free fatty acids which suggests that this is typical of the

alga when grown in f2 medium. Volkman & Hallegraeff (1988) also noted high values

in another diatom Thalassiosira oceanica cultered under similar conditions. In other

species, large amounts of free fatty acids are more usually associated with lipid break-

down in stressed or dying cells but this was not the case in the present work since

cultures were harvested at mid-log phase. Sterol content varied from 0.2 to 6.3 y0 of total

lipids (Table III) with no consistent trends within or between algal classes.

Hydrocarbons were minor constituents of the lipids of all 10 species. The major

constituent was n-heneicosahexaene (n-C,, : 6), except in the green algae Nannochforis atomus and Dunaliella tertiolecta where it was not detected. The mass spectrum of this

compound was the same as that presented by Youngblood et al. (1971) for

3,6,9,12,15,18-heneicosahexaene which is common in many microalgae (Blumer et al.,

1970). n-Heptadecane and smaller amounts of a C,, monoene and diene which are

common in green algae were detected in Dunaliella tertiolecta. Two microalgae,

Tetraselmis suecica and Chaetoceros gracilis, contained smaller quantities of an alkene

tentatively identified from its mass spectrum as n-heneicosapentaene (n-C,, : 5). This

alkene eluted just after n-C,, : 6 on the nonpolar column and just before it on the BP-20

polar column. Isochrysis sp. contained an unusual C,, diunsaturated alkene with minor

228 J. K. VOLKMAN ET AL.

amounts of other alkenes that were not identified. An alkene with the same mass spectrum and retention index has previously been identified in the related alga fsochrysis g~~~~~~ (Volkman et al., 1981).

TOTAL FATTY ACIDS

Total fatty acids ranged from 0.41 pg 1 cell - ’ for Nannochloris atomus to 8.2 pg * cell - 1

for C~roomona~ salina (Table II). ~una~ieIla terti~~e~ta also had a high concentration of total fatty acids (7.4 pg - cell - ‘) whereas the diatoms had values that were three to seven times lower (Table II). Total fatty acid content showed little correlation with cell volume. However, the ratio total fatty acids : Chl a was remarkably similar in most species the range was 4-6 (Table II).

The percentage composition of individual fatty acids for the four diatoms and two prymnesioph~~~ shown in Table IV. Data for the green algae and two cultures of Chroomonas salina are shown in Table V. Values are expressed as a percentage of total fatty acids; these values can be converted to concentrations * cell- 1 from the total fatty acid content given for each alga (Table II). Relative percentages of C,, and C,, PUFA, 20: 5 and 22: 6 PUFA, and (n-3) and (n-6) PUFA are shown as histograms in Fig. 3a-c. The few peaks in each chromato~am that could not be identi~ed represented < 3 y0 of the total fatty acids.

TOTAL FATI-Y ACIDS IN DIATOMS

The fatty acids of diatoms have been the most extensively studied of all the classes of microalgae. Almost without exception, the dist~butions show high concentrations of 16: l(n-7) and 16: 0 with variable, but usually high, concentrations of 14: 0 and 20: 5(n-3). These four acids accounted for 62-70x of the total fatty acids in the diatoms studied. C,, and C,, PUFA were minor constituents (Table IV, Fig. 3a) which is common in most diatoms (e.g., Ackman et al., 1968; Chuecas & Riley, 1969; DeMort et al., 1972, Orcutt & Patterson, 1975; Moreno et al., 1979; Volkman et al., 1980a; Nichols et al., 1986b; Ben-Amotz et al., 1987).

C,, PUFA were particularly abundant compared with other species (Fig. 3a). The major components in the four diatoms were identified as 16: 2(n-7), 16: 2(n-4), 16 : 3(n-4) and 16 : 4(n-1) which are synthesized by further desaturation of 16 : l(n-7) on either side of the A9 double bond. The high propo~ion of these fatty acids accounts for the overall lower abundance of (n-3) and (n-6) fatty acids compared with the other microalgae studied (Fig. 3~). Green microalgae (Chlorophyta) synthesize mainly 16 : 2(n-6), 16 : 3(n-3) and 16 : 4(n-3) as shown in Table V. This difference in double- bond positions can be exploited in ecological studies to estimate the relative abundances of diatoms and green algae in mixed algal samples (Volkman & Johns, 1977).

Most diatoms contain low levels of 22 : 6(n-3) (Fig. 3b) but T. ps~M~onana contains significant amounts as noted in a previous analysis of this species (Volkman & Hallegraeff, 1988). However, Orcutt & Patterson (1975) did not detect 22 : 6 in the clone

FATTY ACID AND LIPID COMPOSITION IN MICROALGAE

TABLE IV

Percentage composition of fatty acids in diatoms and prymnesiophytes.

229

Diatoms

CCAL CGRA no. 1 CGRA no. 2 SKEL THAL

Saturates l2:O TR

l4:O 17.5

15:o 0.8

l6:O 10.7

l7:O 0.3

18:0 0.8 20: 0 TR

22:o TR 24:0 0.1

Sum “/, 30.2

Monounsaturates 16: l(n-10) -

16: l(n-9) - 16 : 1 (n-7) 30.0 16 : l(n-5) 0.1 16: l(n-13)f 0.7

18 : l(n-10) -

18: l(n-9) 2.8

18: l(n-7) 0.2

20: l(n-9) -

Sum 9,, 33.8 Po!lunsaturates 16 : 2(n-7) 3.5

16 : 2(n-6) -

16 : 2(n-4) 1.6

16 : 3(n-6) -

16: 3(n-4) 8.0

16 : 3(n-3) -

16:4(n-3) - 16:4(n-1) 0.3

18 : 2(n-9) 0.8

I8 : 2(n-6) 0.8

18: 3(n-6) 0.4 18 : 3(n-3) TR

18 : 4(n-3) 0.5 18 : 5(n-3) - 20 : 4(n-6) 5.7

20 : 4(n-3) 0.2 20 : 5(n-3) 11.1

22 : 5(n-6) -

22 : 6(n-3) 0.8

Sum 0; 33.7 Others 2.3

Total 100.0

TR TR TR TR TR 0.3 8.8 11.6 20.1 14.3 16.0 11.5 1.0 1.2 1.2 0.8 0.5 0.5

23.3 17.8 16.5 11.2 14.5 21.3 0.3 0.2 0.6 0.1 TR 0.2 4.1 3.1 0.8 0.7 0.2 1.3 0.3 0.2 TR 0.1 0.3 0.3 0.6 0.6 TR TR 0.6 0.3 0.3 0.8 TR TR TR 0.2

38.7 35.5 39.2 21.2 32.2 35.9

33.4 26.8 28.6 18.0 0.1 0.2 0.6 0.3 i.2 1.6 1.3 0.4

3.6 1.7

40.0

6.0 3.9

TR

38.5

1.4

0.1

32.0

0.5 20.1

0.1 1.3

0.2 0.2

19.5 26.1

2.9

1.7

2.3

TR 4.2

0.7 1.1

2.6

2.0

0.5 0.8

0.2 1.2

2.2 0.3

0.3 2.2

2.3 TR

0.4 0.2

0.1 5.3

4.5 6.2 TR TR

4.6 5.7

TR 6.0

0.3

0.3 19.3

0.3 0.4 2.0 3.9 19.8 24.8 26.1 52.6

1.5 1.2 2.7 1.3

100.0 100.0 100.0 100.0

2.4 3.3 _

3.5

2.7 0.5

0.7 4.5 0.7

0.2 _

0.2

2.2 3.7 12.7 1.4 0.4

2.5 2.4

3.6 17.4

2.5

0.4 1.5 0.4

1.8 6.0 _

TR

0.2 19.7

1.8 2.0

8.3 9.4

41.3 42.0

0.5 1.8

100.0 100.0

Prymnesiophytes

T.ISO PAV

0.3 4.2

_

16.8 TR

0.3 1.7 1.4

0.2 20.4

C.CAL, Chaetoceros calcitrans; C.GRA, Chaetoceros gracilis; SKEL, Skeletonema costatum; THAL, Thalassiosira pseudonana; T.ISO, Isochrysis sp.; PAV, Pavlava lutheri.

230 J. K. VOLKMAN ET AL.

TABIX V

Percentage composition of fatty acids in green algae and C~r~o~onus.

DUN _

Saturam

12:o

14:o 15:o 16:O

17:o 18:O

20 : 0 22:o

24:0

Sum “/, ~~~lo~~~aturate~ 16: l(n-10) 16: I(n-9)

16 : t (n-7) 16: l(n-5) 16: l(n-13)t

18: l(n-10) 18 : I (n-9) 18 : 1 (n-7)

20 : 1 (n-9)

Sum T;, P#~~:u~~a~rates 16 : 2(n-7)

16: 2(n-6)

16 : 2(n-4) 16: 3(n-6)

16: 3(n-4) 16 : 3(n-3)

16 : 4(n-3) 16:4(n-t)

18 : 2(n-9) 18 : 2(n-6) 18 : 3(n-6)

18: 3(n-3) 18 : 4(n-3) 18: 5(n-3) 20 : 4(n-6)

20 : 4(n-3) 20 : 5(n-3)

22 : 5(n-3) 22 : 6(n-3)

Sum “; Others

Total Y,

TET no. 2 ^._ _ ___~

TR TR 0.1 0.2 0.6 0.6

TR 0.1 0.3 14.7 20.1 20.3 0.1 TR TR 0.4 1.1 0.9

TR 0.1 TR TR TR 0.2 TR 0.1 TR 15.4 22.1 22.3

0.7

0.9 0.3

24.0

0.3

0.6

TR

26.8

0.1 TR

8.6 8.2

0.3 0.2

15.1 12.9

0.4 0.2

0.9 0.7

TR TR 0.1 TR

TR TR

25.5 22.2

0.1

0.1 -

2.7

2.0 0.3

5.2

0.5

1.3 0.6

0.9

0.3

1.2 0.3

0.2

0.1

0.5

0.2

0.6

8.9 1.5 0.8

4.9 0.4

12.0 14.5

0.4 1.1

1.6 2.6 16.7 20.5

1.3 1.2 0.1 0.4 2.9 2.3

3.5 3.2

16.6 8.6 7.9

0.7 4.2 I.1 1.8

0.9 4.6 6.0

4.2 21.0

14.4 TR 0.5 13.7 7.9 -

4.8 10.3 13.8 13.9 11.6 10.5

2.7 TR 0.7 2.7 3.0 2.6

43.5 21.7 11.1 4.6 11.9 14.2

1.0 2.7 8.4 4.8 19.8 21.3

0.5 1.5 2.1

1.1 0.3 0.1

3.2 4.3 5.3

- TR TR TR 17.9 59.0 59.5 49.7

1.5 2.3 1.5 3.0

100.0 100.0 100.0 100.0

1.0 0.9 0.9 1.0

10.9 11.9

TR 0.3

5.7 5.2 64.8 67.9

1.1 2.0 100.0 100.0

NAN TET no. I

Green algae Cryptomonad --._.- - CHRO no. 1 CHRO no. 2

DUN, Dunaliella tertiolecta; NAN, Nannochloris atomus; TET, Tetraselmis suecica; CHRO, Chroomonas salina.

FATTY ACID AND LIPID COMPOSITION IN MICROALGAE

Diatoms Prymnesiophytes GreeI%i CiYDtomonad

1

231

Fig. 3. (a) Relative amounts ofC,,- and C,,-polyunsaturated fatty acids (PUFA) in microalgae; (b) relative amounts of 20: 5(n-3) and 22: 6(n-3) PUFA in microalgae; (c)relative amounts of (n-3) and (n-6) PUFA

in microalgae. Species abbreviations are given in Table I.

232 J.K. VOLKMAN ET AL.

of Thalussiosira pseudonana they analysed and we did not detect the unusually high amounts of 20 : 0 that were recorded by Epifanio et al. (1981).

The fatty acids of Chaetoceros calc~t~ans and Chaetoce$os g~a~iIis are very similar (Table IV) as might be expected in taxonomically related species. However, C. calcitrans contains a higher proportion of polyunsaturated fatty acids, particularly 16 : 3(n-4) and 20 : S(n-3), which may partly explain the higher larval growth rates obtained with this species (e.g., Waldock & Nascimento, 1979). Similar data for ~haetoce~o.~ g~ac~~~s were reported by Ben-Amotz et al. (1987). C. calcitrans and C. graciiis were the only species that contained significant amounts of arachidonic acid [20:4(n-6)]. Small amounts were also detected in the green alga T. suecica and the cryptomonad C. saliva (Tables IV, V). Bell et al. (1986) have suggested that salmonids, and other marine fish, require arachidonic acid from their diet to produce prostaglandins and eicosanoids.

TOTAL FATTY ACIDS IN PRYMNESIOPHYTES

The two prymnesiophytes ~sochrysis sp. Tahitian strain (T.ISO) and Pavlova l~theri have very different fatty acid distributions (Table IV) although both are rich in PUFA. One unusual feature is the lack of 16 : l(n-13)t which occurs in the phosphatidyl glycerols of almost all other classes of microalgae (Nichols et al., 1965). P. lutheri also contains 4-methyl sterols and unusual unidenti~ed compounds (Fig. 1) not found in Isochrysis sp.

The fatty acid distribution of P. lutheri exhibits a predominance of 16 : 0, 16 : 1 (n-7) and 20 : 5(n-3) which is quite different from that of most prymnesiophytes and may be of use in chemot~onomi~ studies (Table IV). Diatoms have similar fatty acid compo- sitions (Table IV) but most contain more C,, PUFA 16: 2(n-7), 16: 3(n-4) and 16 : 4(n-1) (Table IV). Previous analyses of this alga (Chuecas & Riley, 1969; Chu & Dupuy, 1980; Langdon & Waldock, 1981) are similar although the clone analysed by Chuecas & Riley (1969) contained less 22 : 6(n-3). P. lutheri is widely used as an algal feed in mariculture and it is significant that it contains the highest proportions of 20 : 5

and 22: 6 of the 10 algae studied (Fig. 3b). The major fatty acids of Zsochr-ysis sp. clone T.ISO are 14: 0, 16 : 0, 18: l(n-9),

18 : 4(n-3) and 22 : 6(n-3). Similar distributions have been reported in other prymnesio- phytes (Volkman et al., 1981). Pillsbury (1985) and Enright et al. (1986b) studied the fatty acids of this clone although the latter presented few data. The results of Pillsbury (1985) are very similar to ours, even though the culture conditions differed slightly. A distinctive feature of the fatty acid composition of this strain is the very low abundance

of 20 : 5(n-3) (Fig. 3b). Zsochrysis sp. clone T.ISO has largely replaced ~soch~ysisgaZ~a~a as a food for bivalve

molluscs (Helm & Laing, 1987). The two species are thought to be closely related and have similar biochemical compositions including unusual long-chain alkenes and unsaturated ketones (Volkman et al., 1980b; Marlowe et al., 1984). It has been assumed that the two have similar nutritional qualities but the proportions of major fatty acids

FATTY ACID AND LIPID COMPOSITION IN MICROALGAE 233

are quite di~erent. Also, major v~iations in the fatty acid compositions of ~~~~c~~s~~

galbana have been reported (Chuecas & Riley, 1969; DeMort et al., 1972; Watanabe & Ackman, 1974; Waldock & Nascimento, 1979; Chu & Dupuy, 1980; Volkman et al., 1981; Ben-Amotz et al., 1987).

The percentage of 20: 5(n-3) in 7 analyses of I. ~~~~u~~ ranges from 0.7 to 14.4?‘0 whereas the content of 22 : 6(n-3) exhibits an even broader range of values from “trace constituent’ to 18.9% of the total fatty acids. The proportion of C,, PUFA is also particularly variable with only two analyses reporting high levels of 18 : 2 and two reporting high levels of 18 : 3. Neither acid is abundant in the TISO clone, perhaps because there is a greater conversion of these acids to 18 : 4(n-3), (19.7”/,, Table IV). Small amounts of the pentaunsaturated fatty acid 18 : 5(n-3) occur in the TISO clone and in a clone of Isochrysis gulbuna from the Plymouth Culture Collection (Volkman et al., 1981). It is possible that this fatty acid can be elongated to 20: 5(n-3), so its presence may well increase the nutritional value of algae containing it.

Given such differences in composition, it is hardly surprising that there is disagree- ment about the value of I. galbana as a mariculture feed. Although it is now usually replaced by Zsochrysis sp. clone TISO (Helm & Laing, 1987), it should be noted that some clones of 1. g~Ibunu have higher levels of polyunsaturated fatty acids. Whether these biochemical variations are due to differences in culture conditions, or age of the culture at harvest, or to genetic differences between strains of the same species is not clear. These variations in composition compound the difficulties faced by the maricultu- rist in choosing an algal species solely on the basis of literature reports of its fatty acid composition.

TOTAL FATTY ACIDS IN GREEN ALGAE

Fatty acid data for the three green algae (two chlorophytes and one prasinophyte) are shown in Table V. The distributions are typical of most green algae, with C,, and C,, PUFA being most abundant (DeMort et al., 1972; Prahl et al., 1984; Ben-Amotz et al., 1985, 1987). The major acids in the three species were identified as 16 : 0, 16 : 4(n-3), 18 : 2(n-6) and 18 : 3(n-3). C,, PUFA consisted of 16 : 2(n-6), 16 : 3(n-3) and 16 : 4(n-3) which are probably synthesized by chain-shortening of C,, PUFA or possibly by progressive desaturation of 16 : 1 (n-9). Small amounts of C,, PUFA were detected in Nannochloris atomus and Tetraseimis sue&a but not in Dunaliellu tertiolectu. None of the species contained measurable amounts of 22 : 6(n-3) (Fig. 3b). The low abundance of C,,, PUFA and 22 : 6(n-3) is common to most green algae, with the notable exception of some marine Chlorefla species which can have up to 30% 20 : 5(n-3) (Watanabe et al., 1983). D. tertiolectu contained the highest proportion of (n-3) fatty acids of the 10 algae studied ( = 70 %, Fig. 3~).

The fatty acid abundances of D. tertiolecta clone DUN reported by Piilsbury ( 1985) are mostly similar to those reported here, except that the present culture contains more 18: 3(n-3) and 16:4(n-3) (43.5 cf. 31.2% and 26.0% cf. “trace”) and none of the

234 J.K. VOLKMAN ET AL.

unusual acid 16 : 3 (n- 1) reported by Pillsbury (1985). The fatty acid data for D. tertiolecta

presented by Joseph (1977) and Langdon & Waldock (198 1) are also similar to our data, except for the abundances of 16: 4(n-3) and 18: 3(n-3). The unusual fatty acid 16: 3(n-1) is not mentioned in either study nor is it mentioned in the limited data presented by Evans et al. (1982), so it is possibly a misident~cation by Pillsbury (1985).

The prasinophyte Tetraselmis suecica contains the same suite of fatty acids as the two chlorophytes, but the proportions of major constituents differ. T. suecica contains much more 18 : l(n-9) and 18 : 4(n-3), less 18 : 3(n-3) and more 20 : 5(n-3) (Table V). A gas chromatogram obtained from GC-MS analysis using the non-polar methyl silicone column (Fig. 2) shows that the elution order of the C,, PUFAs 18 : 2(n-6), 18 : 3(n-6) 18 : 3(n-3) and 18 : 4(n-3) is identical with that of the corresponding C,, PUFAs, thus supporting the identi~cations made, p~icularly that of 16 : 4(n-3). The compositional data match closely the data presented by Langdon & Waldock (198 1), except for a lower abundance of 18 : l(n-9) and higher abundance of 18 : 2(n-6) in our two samples. DeMort et al. (1972) however, found low levels of 16 : 0 (10.7%) and high levels of 18:3 (23.8%) 20: 1(18.2x) and 20:5 (11%).

The fatty acid composition of Nannochforis atomus shows some distinctive features not seen in the other two green algae. The major constituents are 18 : 3(n-3) 16 : 0, 16: 3(n-3) and 18 : 2(n-6) (Table V). The high abundance of 16: 3(n-3) and almost complete lack of 16 : 4 fatty acids suggests that this alga lacks the ability to further desaturate triunsaturated C,6 fatty acids. Another unusual feature is the high abundance of 16 : l(n-13)t fatty acid which was almost 9% of the total fatty acids.

The fatty acid distribution found here is very different from that of other species of this genus. Several have high levels of 16 : 1 and 20 : 5 (Watanabe et al., 1983; Ben- Amotz et al., 1985), neither of which is abundant in N. atomus. Another has significant levels of 10 : 0 and low levels of C,, PUFA (Hamdy & Martin, 1986). It seems from these limited data that species of this genus do not have a characteristic fatty acid composition.

TOTAL FATTY ACIDS IN CHROOMONAS SAUNA

The major fatty acids of Chroomonas sulina are 18 :4(n-3), 16: 0, 18: 3(n-3), 18 : 2(n-6) and 20 : 5(n-3). The high proportion of C,, PUFA and 20 : 5 and the very low abundance of C,, PUFA (Fig. 3a) were also noted by Beach et al. (1970) in their study of 13 cryptophyte algae. Chuecas & Riley (1969) reported similar proportions of the same major acids in four species, as well as significant amounts of 20 : 1 which has not been noted in more recent studies. The proportion of (n-3) PUFA in Chroomonas

salina is particularly high and only slightly less than in Dunaliella tertiolecta (Fig. 3~). The fatty acid composition of a 50-day-old culture of Chroomonas salina grown

photoheterotrophic~ly with glycerol was reported by Antia et al. (1974). These authors found very high concentrations of lipids (44 “/, dry weight), most of which were wax esters. PUFA were not detected which contrasts markedly with our data (Table IV).

FATTY ACID AND LIPID COMPOSITION IN MICROALGAE 235

Lipid-compositional data from TLC-FID (Table III) also show that wax esters are not

typical constituents of this alga. A Chroomonas sp. analysed by Beach et al. (1970)

contained the same major constituents: 16:0 (16%) 18: 3(n-3) (23%) 18 :4(n-3)

(23%) and 20 : 5(n-3) (14%).

VARIABILITY IN FATTY ACID COMPOSITION

The marked differences in fatty acid content and composition noted for some species

grown in different laboratories, as exemplified by data for Isochrysis gulbana and other

species mentioned above, raises the question whether consistent results can be obtained

for the same species cultured under the same conditions. The amount of total fatty acids

in the repeat cultures of Chaetoceros gracilis and Chroomonas salina agree very closely

(to within 4%) with the values obtained for these two species grown 6 months previously

(Table II). The relative proportions of major fatty acids are also very similar (Tables

IV, V). We have also obtained similar data for the relative proportions of fatty acids in

cultures of T. pseudonana, P. lutheri and N. atomus grown at different times in our

laboratories (unpubl. obs.). A few species show greater variation as exemplified by

Tetraselmis suecica which contained almost twice as much chlorophyll and fatty

acids . cell- ’ in the second culture (Table II). The proportions of some polyunsaturated

fatty acids also varied (Table V). Despite these differences, the ratio of total fatty

acids : Chl a concentrations was reproducible to within k 20% (Table II).

LONG-CHAIN SATURATED FATTY ACIDS

Most reports of fatty acids in microalgae found no saturated fatty acids with chain

lengths > C,,. By using sensitive mass spectrometric analysis, we found that most of

the species studied contain ~2% of C,,-C,, saturated fatty acids (Tables IV, V).

Clearly, some chain elongation of saturated fatty acids occurs but in most species these

acids are not then desaturated to long-chain unsaturated fatty acids. These data confirm

suggestions by Nichols et al. (198613) that small amounts of long-chain saturated fatty

acids in the marine environment can be contributed by microalgae.

BACTERIAL SIGNATURE LIPIDS

Although starter cultures of microalgae are axenic in most hatcheries, microalgal mass

cultures often contain bacteria that can, if pathogenic, lead to losses in animal

production. Most bacterial species have distinctive fatty acid distributions and lack

polyunsaturated fatty acids. Bacterial fatty acids include iso- and anteiso-branched C,,

and C,,-saturated and monounsaturated fatty acids, vaccenic acid [ 18 : l(n-7)], cyclo-

propane and mid-chain branched fatty acids and trans-monounsaturated fatty acids

which are easily identified by capillary GC and GC-MS. Fatty acid distributions can

thus provide a quick semiquantitative indication of the extent of the bacterial load of

mass cultures.

236 J. K. VOLKMAN ET AL.

Bacterial fatty acids were not detected in the nine axenic cultures, as expected, but iso- and anteiso-brushed represented x: 1% of the total fatty acids in C~#e~~ce~~~ grads and the relative amount of vaccenic acid was considerably higher than in Chaetoceros caicitruns. The culture of C. grucilis contained 1 x IO6 bacteria. ml _ ’ which would be sufftcient to account for the elevated levels of 16 : 0, 18 : 0, 18 : 1 (n-7) and to a small extent 16 : 1 (n-7). Bacteria contribute at least 5 y0 of the total fatty acids in this culture.

UNUSUAL DOUBLE-POND ISOMERS

Small amounts of the unusual fatty acids 16 : l(n-10) and 18 : l(n-10) were detected in the green alga Nannochloris atomus and the cryptomonad Chroomonas salina. 18 : l(n-10) was also detected in Puvlova lutheri. We are not aware of any other reports of these fatty acids in microalgae but they might have been overlooked in studies where derivatisation techniques, such as formation of DMDS adducts, were not used. One possible mode of synthesis is desaturation of 16 : 0 by a A6-desaturase which is common in higher piants (Stearns, 1970), followed by chain elongation to 18 : l(n-10).

The ratio of 16 : 1 fatty acid isomers showed significant variations according to the taxonomic position of the alga. Diatoms contained very high concentrations of 16 : 1 (n-7) with small amounts of 16 : 1 (n-5) and no 16 : l(n-9) (Table IV). In contrast, the green algae contained low concentrations of 16: 1 and the main isomer was 16 : 1 (n-9) with small amounts of 16 : 1 (n-7) and no 16 : l(n-5) (Table V). Hexadecanoic acids were also minor constituents in Chroo~~on~s supine; the main isomer was 16 : 1 (n-7) with small amounts of the (n-9) isomer and no (n-5) isomer. Both pr~nesiophytes contained mainly the 16 : 1 (n-7) isomer (Table IV). The major 18 : 1 fatty acid isomer in all but one species is oleic acid but each contains a significant amount of vaccenic acid [ 18 : 1 (n-7)]. Only one culture was not axenic, so these data show that vaccenic acid is a genuine component of the fatty acids of many microalgae. In Chroomonas salina, it is z 1.5 times as abundant as oleic acid (Table V). It is likely that the 18 : 1 (n-7) isomer is produced by chain-elongation of 16 : 1 (n-7) as occurs in bacteria.

CONCLUSIONS

From the data presented here, a number of observations can be made that are relevant to the use of microalgae as feed stocks in mariculture.

(i) The 10 species of micro~gae grow well in hatcheries world wide but several studies have shown that their nutritional quality varies. Much of this variation can be explained by major differences in fatty acid composition, particularly with respect to the proportions of long-chain polyunsaturated fatty acids (Tables IV, V).

(ii) The three green algae, N. atomus, D. tertiolecta and T. suecica, do not contain high concentrations of polyunsaturated C,, and C,, fatty acids which are considered essen-

FATTY ACID AND LIPID COMPOSITION IN MICROALGAE 231

tial dietary constituents for many animals. Fish larvae fed on rotifers raised on D. tertio-

lecta showed stunted growth and a high mortality rate (Scott & Middleton, 1979). For similar reasons, freshwater C~I#rell~ spp. are often unsuitable as food although some marine Chlorella spp. containing high concentrations of 20 : 5(n-3) can be used as food for many fish (Watanabe et al., 1983) including barramundi (Lates calcarifer, L. Rodgers, pers. comm.).

(iii) Nutritional deli&en&es in a diet can be avoided by the use of mixed algal diets. For example, green algae can be used to provide a high c~bohydrate content (reviewed by Brown et al., 1989) and their lack of C,,- and C,,-polyunsaturated fatty acids can be met by including another species of the same size range which is rich in these compounds. Growth rates in molluscs have been improved using this strategy (e.g., Enright et al., 1986b; Laing & Millican, 1987; Helm & Laing, 1987).

(iv) A high ratio of (n-3) to (n-6) polyunsaturated fatty acids is often cited as an index of high nutritional value for many animals (e.g., Watanabe et al., 1983). However, this index is of limited value without a consideration of which fatty acids are actually present. D. tertiolectu has a high proportion of (n-3) fatty acids (Fig. 3c) but these are mainly C,, fatty acids which are less nutritionally valuable than the C,, and Czz fatty acids. Polyunsaturated C,,fatty acids with double bonds at (n-l), (n-4) and (n-7), as occurring in diatoms, are not included in this index.

(v) Many diatom species are very satisfactory foods. Most contain a high concen- tration of total fatty acids (4.2-27.8 y0 dry wt; Orcutt & Patterson, 1975) and a signiti- cant proportion of 20 : 5(n-3). Waldock & Nascimento (1979) showed that C. calcitrans was superior to Fyramimonas virginica or isoc~rys~sga~bu~a as food for larval Crassostrea virginica. Similar results were obtained by Helm & Laing (1987). In both cases, the important nutritional factor was probably the content of 20 : 5(n-3) which is abundant in C. calcitruns (Fig. 3b) but only a minor compound in the other algae. All four of the diatoms we studied were shown by Enright et al. (1986a) to be superior to Isochrysis sp. clone T.ISO when fed singly to juvenile Ostrea edulis, probably due to the higher content of 20 : .5(n-3), and significant amounts of 22 : 6(n-3), in the diatoms.

(vi) P. lutheri which is widely used in mariculture as a food for rotifers fed to larval fish has, like C. salina, a high proportion of polyunsaturated fatty acids (Fig. 3b). Both species are useful in temperate hatcheries but neither grows well at tropical temperatures (S. W. Jeffrey, unpubl. data). It is likely that taxonomically related strains will have similar fatty acid compositions and work is in progress to identify suitable strains for use in tropical m~i~ulture operations.

(vii) Laboratory studies have shown that major changes in the fatty acid composition of microalgae can result from modification of the culture conditions (e.g., Orcutt & Patterson, 1974; Ballantine et al., 1979; Borowitzka, 1988) but little attention has been given to this in hatcheries. These variations can be exploited to maximise the nutritional quality of the algae. For example, Enright et al. (1986) were able to alter the growth rates of Ostrea edulis by manipulating the nitrate and silicate levels in the cultures of C. gracifis fed to the oysters. Highest growth rates were obtained with culture conditions that

238 J. K. VOLKMAN ET AL.

produced highest concentrations of 22 : 6(n-3) in the algae. Our data provide a reference for compositions that can be expected under defined conditions. By changing these conditions, the nutritional value of some of these algae can undoubtedly be improved.

(viii) The dietary significance of many, or even m&t, of the fatty acids quantified in Tables IV and V is unclear or unknown. Most studies have focussed on a few fatty acids such as 20 : 4(n-6) 20 : 5(n-3) and 22 : 6(n-3). More studies are required to identify the role of other polyunsaturated fatty acids in microalgal and animal biology.

ACKNOWLEDGEMENTS

We thank M. R. Brown for helpful discussions, P. Deprez and D. Holdsworth for laboratory assistance and J.M. Leroi for culturing the algae and for measuring cell counts and volumes. This work was funded by FIRTA Grant 86/81 (Department of Primary Industry, Canberra) to S. W. Jeffrey and CD. Garland and MST Grant 84/1852 (Department of Science, Canberra) to J. K. Volkman and H. J. Bavor.

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Antia, N. J., R. F. Lee, J. C. Nevenzel & J. Y. Cheng, 1974. Wax ester production by the marine cryptomonad Chroomonas salina grown photoheterotrophically on glycerol. J. Protozool., Vol. 21, pp. 768-771.

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growth stage. Phytochemistry, Vol. 18, pp. 1459-1466. Beach, D. H., G. W. Harrington & G.G. Holz, Jr., 1970. The polyunsaturated fatty acids of marine and

freshwater cryptomonads. J. Protozool., Vol. 17, pp. 501-510.

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