y. Phycol. 29,69-78(1993)

THE BIOCnp:MlCAL COMPOSITION OF MARINE MICROALGAEFROM THE CLASS EUSTIGMATOPHYCEAE'

Joh7i K. Volkman^

CSIRO Division of Oceanography, Marine Laboratories, GPO Box 1538, Hobart 7001, Tasmania, Australia

Malcolm R. Brown

CSIRO Division of Fishorics, GPO Box 1538, Hobart 7001, Tasmania, Australia

Graeme A. Dunstan

CSrRO Division of Oceanography, GPO Box 1538, Hobart 7001, Tasmania, Australia

and

S. W.JeffreyCSIRO Division of Fisheries, GPO Box 1538, Hobart 7001, lasniaiiia, .Australia

ABSTRACT

The biochemical composition of four strains ofmicroal-gnc from the class luistigmatojihyccdi' iras dt'tcrmincd toassess their usefulness us live feeds for mariculture andto establish characteristic features for use in chemolaxo-nomir studies. We studied Naniioc hloropsis salina (strainCS-l90)Jrom Scotland, txi'o strains o/ Nannocliloropsisoculata (CS-179 and CS-216) from Japan, and an un-named eustij^matophyte (CS-2-f6) isolated from Queens-land icater^ that appears to he closely related to N. ocu-lata. Gross compositio)ial features were similar: totalcarbohydrate ranged from 5.2% (N. oculata CS-179) to8.9Vc (N. salina) of cell dry ireight. Polysaccharide com-prised /•/7f (N. oculata CS-179) to SS'i {CS-246)ofthistotal. Glucose was the priixipal polysaccharide sugar{45.2-66.2% of total sugars). Other sugars included fu-cose, galactose, mantiose, rhamnose, ribose, and xylose(2.0—14.0%). Arahi)i()se xt<as a minor ronstitue)tt in allspecies (0.6-l.77(). Protein x'aried from /".iSTf (N. sa-linaj to 22. lVc(N. oculata CS-2l6)ofthe cell dry weight.The major amino acids were arginine, glutamate, andaspartate (7.2-10.4% of total amino acids), irith methi-onine, cystine, histidine, tryptophan, hydroxy-proline, or-nithine, and y-aminobutyric acid much less abundant(0.03-2.6%). Lipid content ranged from 8.2% (N. ocu-lata CS-216) to 16.9% (N. salina) of cell dry weight, thelatter x'alur reflecting enhanced concentrations oj triacyt-glycerols in N. salina. The major fatty acids were palmiticacid (16:0), palmitoleic acid j 16:l(n-7)], and eicosapen-taenoic acid / 20:5(n-3)/ irith lesser a mounts of la u ric acid(14:0), linoleic acid j lS:2(n-6)], and others. The sterolsco)isisted almost entirely of cholesterol, which is an essentialcon.slitucnt oj crustacean diets. Chlorophyll A ranged from0.6%, (N. oculata CS-216) to 1.77c (N. oculata CS-179and N. salina) if cell dry weight. Chlorophylls b and cwere not delected. All strai)is contained a characteristic

|' Adtlrcss lor !('(|ii<'Ms

pattern of carotenoid pigments, which included violaxan-thin, ^-carotene, zeaxanthin, and a pigment tentatixriyidentified as I'aucheriaxanthin-ester. The distinctii'e pig-ment anil lipid compositional data can be used as che-motaxonomic markers for Nannochloropsis and for as-signing inicroalgac to the class Eustigmatophyceae.Naiinochloropsis oculata is -widely used as an algal feedin mariculture, and based on the .•iimilarity of the bio-chemical data, both N. salina and the unnamed tropicalspecies should «/.w prove to be nutritionally valuable livealgal feedstocks. Feeding trials will be needed to confirmthis.

Key index uvrds: amino acids; chemota.xonomy; Eustig-matophyceae; mariculture; Nannochloropsis; nutrition;pigments: polyunsaturated fatty acids; sugars

The class F.ustiginatt)plnceae was proposed byIlibbetdand Leedale (1970, 1971. 1972) to accom-modate certain species of yellow-green microalgaethat had pre\ iously been assigned to the class Xati-thophyceae. These two algal classes are thought tohave diverged from the brown algal line (Chro-mophyta) more recently than the dinoflagellates andraphidophytes, but before the emergence of pryni-nesiophytes and diatoms ("Taylor 1987). Xan)iochlo-ropsis oculata was originally named Xannochloris oeii-lata Droop, atid \annochloropsis salina was formerlyknown as ,Monallantus salina Bourrelly (Berland etal. 1970, Antia et al. 1975, Hibberd 1981). Fustig-tnatophytes can be either unicellular or filamentous,and the coccoid forms may bear a superficial resem-blance to some green algae, in both color and cellmorphology, when examined tnuler light micros-copy. For this reason, strain CS-179 was previouslyand incorrectly referred to as "marine" Chlorella(Chlorojih)ceae), but Maruyania et al. (1986) showedfrom its cell ultrastructure and fatty acid and pig-ment coni]>ositioiis that this alga is idetitical lo thepreviously described Xaniiochloropsis oculata.

Nannochloropsis oculata has been used as a live algal

70 JOHN K. VOLKMAN El" AL.

feedstock in mariculturc for many years (e.g. Wa-tanabe et al. 1983), and it grows well under mas.sculture. It contains vc;ry high concentrations of the[jolyunsaturated fatty acid 20:5(n-3), which is con-sidered essential for good growth and survival oflarval and juvenile stages of many marine animals(e.g. Enright et al. 1986). As part of a study of thenutritional quality of microalgae used for maricul-ture (Jeffrey and Garland 1987, Volkman et al. 1089,Brown and Jeffrey 1992), we undertook a detailedexamination of the gross biochemical compositionof c)ther coccoid algae from the F.ustigmatophyceaeincluding Nannorhlorojjsis salina and an Australianisolate thought to be clcjsely related to A', oculata.Amino acids, sugars, fatty acids, and pigments wereanalyzed to determine which features of the bio-chemical composition might prove to be character-istic of this unusual group of microalgae and to assesstheir potential as feedstocks for mariculture and bio-technology.

MATERIALS AND METHODS

Cultures were- obtained from the CSIRO Algal Culture Col-letlion (JelFrey 1980). S'nniiiirhldroliMs salmn (strain CS-190) wasobtained originally Irom Scotland (SMllA 201). Snitnnrhlornfni'iiKulnIn (strain CS-2U)) and a mitroalKa, previously referred to as"marine CA/orW/rt" (strain CS-179), but tiow ktiown to be a strainof ,V. uculnia (Maruyama et al. 1986), were obtained Irom ]a|jan.A tropical c<Mcoi<l yellow-green alga (strain CS-246) was isolatedfrom phytoplankton from Queensland waters by Mr. J. Marshall(Southern Kishe-ries Research ("eiitre. Deception Bay). DitiialiellriteTlwlnln (C.S-I75) was obtained from the Culture Collc-ction ofMarine Phytoplankton, Bigelow Institute for Ocean Sciences,Maine. This green nlga was chosen as a reprcsentalive chloro-phylc in the pigment studies.

Culture conditions. F.:uh alga was cultured under conditionsknown from previous studies tr) give rise to gocxl growth ratesand longevity in culture. Nnnnorhlorujnis \iilitin (C.S-190)t \'. ocu-latii (C.S-2I6), and I), tfrtinlecin (CS-Pf)) were grown at 20" C(±0.5° C) in 1.0 L of medium tV. ([effrey 1980) on glass shelvesilluminated from fx-neath with 70-80 nV.m ' s ' white lluores-cent light (Philif)s Daylight tuf>es) on 12:12 h LD cycles. Nan-niiihloropsn orutata (C.S-179) and the untiamed coccoid eustig-matophyte (CS-246) were grown in I .("> L of 1/2 medium (Guillardand Ryther 1962) in 2-L F.rlenmeyer flasks at 2O''and 27''C( + 0.r)°C), res(X"c(ivcly. under the same light (onditions. Medium IK issimilar lo medium I »)f Guillarrl and Ryther (19r)2) except thatrthylenediatninetctraacetate has Ix-en added to se(|uester tracemetals; medium f/2 is medium f diluted by W)7( so that nutrientconcentrations are hall those in fK. Irradiance was measured witha Biospherical Optics light meter. A repliiate tulture of CS-240was also grown 3 months later and analyzed lor lipid classes andfatty acid com|>osition to assess the repriwlucibility of these data.The flasks were swirled twite daily to keep tells in suspensionand to increase CO, availability, but the cultures were not aerated.Cultures were harvested after 7 days in mid to late log phase,and cell counts were determined at the time of harvesting usinga Nrubaurr hemt«ytometer. Cell volumes were estimated ac-cording tcj .Smayda (1978). Aliquots were collected for determi-nation of dry weight (2 x 100 mL), chltirophyll ii (2 x 5 mL),carotent)ids (10 niL), and lipitls (200 mL). The remainder of theculture was harvested by centrilugation (.5000 x p for 10 mill),washed with 100 mL of 0S> M ammonium formate (to removeresidual salts lrt)m the seawater medium), and tentriluged again.The NU()crnatant was discarded. The resultant cell pellet was

free7e-tlrie<l and stored at —20° C until analy/etl within 2 wc-eksfor gross composition, amino ac ids, and sugars.

Drytri'iphi ilrlrrmiunlion. Duplitate port ions (100 inL) from eachculture were filteretl through prewfighetl, prtcombustetl (450°C, 2 h), glass-liber fillers (VVhatnian (".FK, 17 mm. nominal pore.size 0.7 ^m). The filters were washed with 0.5 M ammoniumformate (30 tnL), dried at 100° C for 4 h to volatilize amtnoniutuformate and water, and then weighed to determine the thy weightof cells in each 100 ml. of culture.

Chlorophyll a ilclermntation. Duplicate alit|uots (.'j mL) Ironi thecustigmato|)hyte cultures were tentriliiged ai 2000 x f^ for b min.The supernatant was tliscardt tl, and dimethyl sulloxide (0.2 niL)was atltled. The cell pellet was sonicated for 5 min in a transonicS2 sonicator bath. Att-tone (9O'/f, 2.8 ml.) was addetl. and thesuspension wasst>nicated fora further 5 min. Ihe solvent mixturewas then left in the dark for 30 min to ttimplete the extraction.The extracts were (enlrilugtd (2000 x g for r> min). antl chlo-rophyll II in the supernatant was tletermined spt ctrophotoinetri-cally with a Shimadzu spectrophotonu ter, using the appropriateequations of JelTrey aiul Humphrey (197.5) for chlorophyll n.

Determiniitioii of nirotniouls. Duplicate ali(|uots (10 ml.) Itomeach culture were filteretl thttuigh glass-fiber filters (WhattnanGFF, 25 mm), which were storetl at -20° C |)rior to analysis.Filters were ground in 9O'/{ acettjiie (3 mL), and the extract wasanalyzed by reverse-phase high-[)erformance lit|uid chrtimatog-raphy (lll'LC)usiiig the metliotl of Wright et al. (1991). Pigmetitswere tietectetl by monitoring at 436 nm antl identified by com-parison of absorption spectra and retention intlites with those ofauthentic standards (Wright et al. 1991).

Protein unit (imiiio nriit romJHisitioii. Portitiiis (4 mg) of the frcczc-dried algae were hydrt)lyzed at 1 10° C with 2.0 ml. t)f 4 M nieth-anesulft)nic acid (containing 0.2% tryptamine and 0.2 ^iM iior-leuciiieasan internal stantlard) in vacuum-se.iIt tl hytlrolysis tubes.Aliqutjts (0.4 ml.) were removed at 24 h antl tlilutetl tt) 8.0 tnL.Samples were |)urifietl, as describetl by Lazarus (1973), by apply-itig the sample tt) l-niL vt>lutnes t)f calion-exchange resin (AC!50\V-X8, 100-200 mesh, H" form; Bio-Rad Lal)oratt>ries) con-tained in 0.8-x-4-cm polypropylene columns (Bio-RatI). Lipidsand neutral sugars were removetl by successive washc's with 0.01M Jl(;l (5 mL) and water (1 tnL). The amino acids were eliitedfrom the resin with 2 M ammonium hytlroxitle (10 ml.). Theeluants were f rc'cze-drietl. Kadi sattiple was divitled itilo two: otie-half was reat tetl with phenyl isothit>cyanate (Pierce C^hemical Co.,Rocklord, 11.) tti liirm phenylthiocarbamyl (PTC) amino acitis(liidlingmeyer et al. 1984): tbe remaining half was reat tetl with9-fluorenylmethyl t hlortilormate (KMOC: Alth it h Chemical Ct).,Milwaukee, Wl) tt) lorm FMOC amino acids (Biiittm 198()). TheP rC amint) acids were separated by revt rse-phase I IPI.C (Vangantl Sepulveda 198.5) using a .Spheristirb ODS II column (3 >ini,150 X 4.6 mm ID) and a Varian Model 5000 I IPI.C. This systetnwas the primary tjiiantifitatioti tnetht)d because it separated allamino acid tierivatives ext c pt ftir trypttiphan and t)rnithine. 1 helatter were t|uantifietl as their I'\H){; derivatives, which wereclearly rest)lved Irom all other FMOC iimitio acids by reverse-phase IIPI.C: (liruton 1986) also using a Spherisorb ODS II ct)l-umn. P I (', amino acids wetc detected with a IJV-DO tnulti|)le-wavflengtli detetttir (Varian) set at 254 nm. For KMOC aminoat ids. the s.inic tic-tettor was set at 264 nm. Peak areas (or allaii.ilyses were c|uantilied with a Varian Vista 402 combined in-tegrator and plotter. Total protein content was calculatetl bysumtning the anhydto amino acid residues.

DrIrrmiiKilioii "/ l<iliil lijnrh h\ ii'cii^lif. Iree/e-dried algae (10-20mg) were placetl in Mitii-vials (5 ml.; Pierce Chemical C'o.) andextractc'd as outlitied by Whyte (1987). This method providedthree fractions: a lipid fraction, a fraction enriched in mont)- andoligosaccharides, antl a residue ct>ntaining polysaccliarides. Di-methyl sulft)xitle (0.2 ml.) was added lo each vial, and the sus-pension was sonicated for 5 tniti. A 2-tnL portion of chlorofortn-methanol-water (2:4:1, v/v/v) was addetl to each vial. The sus-

EUSTIGMATOPHYTF. BIOCHEMISTRY 71

TABIK 1. Cnnlnits of loin I rnrhohvlrnles. prolrhts. Itpids, and (hlori>l>hylt a in ihr four cultxim o/̂ Nannochloropsis. The ax'fragf cofffirifnls oft'liridliiin bnsrd on pm'ious work (liroirn li al. 19^1) were ns foUou's: cells-ml. '; ±9^ ; dry u7, ±2.5*^.' C(irboh\drnle pg-ccU', ±9.6'~c: prolein pp'cell ', ±9.6%; lipid pg-rell~', ±9.9'7c: chlorojiliyll a pj;(ell '. ±9.2'^. These rnleulatioiis liike itilo nccotinl the error in cell counts.

iV. satinaCS-190

(re)

.V. omlatnCS-216

(fF.)

X, oculataCS-179

Unnamrdsp. CS-246

(r/2)l counl^ and toll diy weights

Cfllsnil. ' al harvest (x 10")Dry weight (|>j;ccll ')

VVfiglu ofconstiluciu (pg-ccll')Total carbohydrate

Mono- and oligosacc haridesHolysa<( haride

Total proteinI'otal li|<ids

I'olar lipidsTriacylglyciTolsStcrols

Chlorophyll a

5.38.3

0.730.130.601.51.40.760.600.040.14

9.59.3

0.600.0830.522.10.760.660.060.040.056

14.73.1

0.160.0270.130.620.340.320.0060.0190.051

8.05.3

O.SI0.040.271.10.700.650.0050.0260.077

pension was mixed and centrifuged. The siipernatanl was re-moved and transferred to another test-tnbe. The cell pellet wasextra<tc<l liirther by adding additional 2-nil. portions of chlo-rolornt-tnethanol-water (2;4;1, v/v/v) utitil theextrait was freeol color. The resiiltatit su|)ernatants were c<)inbine<l and sepa-rated into (lilorolorm and aqueous niethanol layers by the atl-dition ol 5 tnl. ol water and 5 tnl. of chlorolbrni. The chlorolorinlayer was washed with water (5 ml.), concentrated under vacuum,and weighed to <lct<Tniine the total lipiil lor cotilirmation ol thedata obtained by thin-layer chromatography-Hame ionizationde-tection (I l.C-KI D).

Cdrhiilndriileedmpdsilioii. The aciiieous tnethanol layer from thetotal lipids procedure describetl above was concentrated to halfits volume and diluted to 10 ml, with water. This fraction, whichcotitained mono- ati<l oligosaccharides, was assayed ((uatititativelyfor carbohydrate by the phenol-sulfuric acid tiiethod of Dubois<'t al. (1956). The polysaccharide-cotitaitiing residue retiiaitiingalter the extrat tioti of tile algal satn]>les was (lrie<l under vacuumand Indroly/ed witli 4 ml. of 0.5 M 11,SO, at 100° C lor 4 h.Ali(|uots of the hy<lrolysates were assayed (|uatititatively for car-bohydrate using the coloriinetric method of Dubois et al. (1056).The remainder of the hydrolysates was analy/ed (]ualitatively lor

< oniponeni sugars as out lined below. These methods provide dataon readily hydroly/ablecarbohydtates rather thati structtnal car-bohydrates siu h as cellulose.

Suffiir nnnposilion o/ pol\s(icflinride. The hydrolysate ol the poly-sacTharide-<ontaitntig tesidue was neutrali/etl by adding an ex-cess of solid Ha("()|,. Thesatnple was filtered through a glass-fiberfilter (Whatman CIK/F, 25 mm), and the filttate was Iree/e-dried.The constitu<'nt neutral sugars were converted to alditol acetatederivatives as described by Ul.ikeney et al. (1983). These wereseparated on an aliimiiuim-clad tapillary-column (25 m x 0.53mm 11), lM'-225 Itum S.C..F.. l'ty. Ltd.! Melbourne, .Vustralia)fitted to a Hewlett-Packard 5890 gas chromatograph equippedwith a llame ioni/ation detector.

Oelerminiilion oj lipiil cliisuw h\ IIX.-I'II). ("ells wete harvestedby lilteiing 200 nil. of culture medium through a 47-mm-<liain-eter Whatman C.F/K glass-fiber filter. The filter was exttactedaceording to the HIigli and Dy<'r (1959) procedure with chloto-lorm-methatiol-water (1:2:0.8, v/v/v, 5 x 5 ml-) with the ad-dition of sonication to enhatue lipid recoveries. The modifiedliligh and Dyer method is superior to the use of hexaiie/isopro-paiiolor tosoxhlet extraction with methylene chloride/methanolfor the exttactioti o( lipids from Chlorella (C'.nckert et al. 1988).Iti one r«-plicate o( (;S-246, HCl was added to the extractionsolvents to examine previous suggestions (Dubinsky and .\aron-

son 1979) that this tesulted in higher lipid yields. Chloroformatid purified water (Milli-Q* system) were added to the combinedexttacts to bring the chloroform-methanol-watcr ratio to 1:1:0.9 (v/v/v), and the solvent layers were allowed to separate.Lipids were tecovered in the lower chloroform phase, and theaqueous phase containing salts and other water-soluble materialwas discarded. The solvents were removed under vacuum. Chlcvroform was added, and the total lipid extract (TLK) was storedunder nitrogen at —20° C until it was analy/ed a few days later.

The identities and concentrations of the major lipid classeswere determined by analy/ing a portion of the TLK with anIatro.scan Mk III Tli-10 TLC-FlDanaly/er (latron Laboratories,Japan), as described by \'olkman et al. (1986), except that silicaS-III chromarods were used. 'The solvent system was hexane—diethyl ether-acetic acid (60:17:0.2, v/v/v), which resolves hy-drocarbons, triacylglycerols, free fatty acids, sterols. and polarlipids. Itidividnal polar lipids were not idetuified.

.•\nother aliquot of the TLF. was reduced to dryness utlder N,and reacted with methanol-hydrochloric acid-chloroform (10:1:1, v/v/v, 3 ml.) for 2 h ,it 80° C to transesterify triacylglycerolsand more polar esters. This method also esterifies any free fattyacids if present. Distilled water (3 niL) was then added, and theleaction protlucts were extracted with chloroform-hexane (1:4,v/v, 3 x 3 ml.).

Fatty acid methyl esters were analysed with a Shimad/u GC-9A gas chromatograph equipped with an FID and cool OCI-3on-column injector (S.G.F'.. Pty. Ltd., Melbourne, .Vustralia)..Analyses were carried out on both a nonpolar niethyl siliconefused-silica capillary column (50 x 0.32 mm 11): Hewlett-Packard)and a i«ilar Supelcowax 10 capillaty column (60 m x 0.32 mm1D: SufU'lco). Peak areas were <)uatitified with a Shimadzu C-R3Acombined computing integrator and plotter. Details are given byVolkman et al. (1989, 1992). Gas chromatography-mass sjiec-trometiy analyses were carried out with a Hewlett-Packard 5890GC and 5790 Mass Selective Detector (MSD) fitted with a directcapillary inlet. Helium was used as the carrier gas. F.lectron impactmass spectra were ;u-i)uired and processed with a 1 lewlett-Packard59970/\ computer workstation. Typical MSD opetating condi-tions were as lollows: electron multiplier, 2000 volts; transferline, 310° C, electron impact energy of 70 eV, 0.8 scans/s; massrange, 40-600 daltons.

RESULTS

Cultures. All cultures were harvested at mid to latelogarithmic phase of growth at cell densities per

JOHN K. VOLKMAN ET AL.

Total Protein

Total Llpid

Total Cartxjhydrata

Chlorophyll a.

25

20

15

10

25

20 ' ̂ 1

• ^ ^ 1 § 10 ' ^ ^ 1

Nannochloropsissalina

CS-190

Nannochloropsisoculala

CS-216

20

15

10

2S

NannochloropsisoculataCS-179

un-namedCS-Z46

Fic. I. Proximate toin[K)sitioti (proteins, hydtoly/ablc car-Ixihyclratcs, lipi(l», and chlorophyll) of <-ustigniut(>phylcs as a per-centage of dry wcighl (not corrected for ash content).

millilitcr ratiging from 5.3 x 10« (̂ V. snlina) to 14.7X 10« {N. oculntn CS-179) (Table 1). Tbt-sc cc-ll den-sities arc typical of nf)iiaeratcfl cultures, but they are5—10 times less than can be achieved when culturesarc aerated with COj-eririched air. The average dryweights per cell lor strains grown in f K, medium werehigher than those grown in f/2 medium (e.g. 3.1pg-cell~' cf. 9.3 pg-cell"' for strains CS-179 and CS-216, respectively, of A', orulatn), possibly reflectingthe influence of differences in culture conditions(Table 1). Cells of A', salina were oblate ellipsoid inshape with a typical average length of 3.3 and widthof 1.9 ^m, whereas cells of A', orulaln and CS-246were spherical with a typical diameter of 3.0 fim.Average cell volumes in starter cultures ranged from11 nm^ (A', uiliiin and CS-24()) to 15 /im' (A', onilalaCS-179) to 18 /im' (A', orulala CS-216). Cells up to48 ^tm' and as small as 4 ^m' were infrequentlydetected.

Gross biochemical composition. Under the growthconditions used in our study, protein was the majororganic constituent in all strains, followed by lijiidand then carbohydrate (Table 1; Fig. 1). Total car-bohydrate was highest in A', salinri (8.8% tiry wt) andlowest in N. oculata CS-179 (5.2%). Folysacchari<leranged from 74% (,V. ociilnia CS-179) to 88% (strainCS-246) of the total carbohydrate. Protein was

TABI.K 2. Sii/^ar rumfmsilion (iivi/^hl Vija/ llii' Iwlyuiriharides isolnlrdJi^otn Oil* jtntr itiicrotil^fn'. Mraii J'ftlut's from dulilmttf ttcrwatized snin'pies. The nvprngr<. of ihr cnfljinrnh o/ x'riririlion were ± ?. J% /or meanvalues >l.0'/( and ±7.6'/i Jor mean vahti's <1.0'/c.

ArabinoseFiifoseGalattoseGlucoseMatuioseR ham noseRiboseXylose

,V, mlinaCS-190

1.28.98.8

60.52.0

11.23.93.4

CS-"2I5

1.78.0

14.045.2

2.712.17.19.1

S. ftnttninCS-179

0.77.06.3

00.63.7

10.46.44.8

Uruumccl»p. CS-^Ki

0.06.94.2

06.25.48.64.53.3

slightly lower in N. .mliria (17.8% dry wt) than ineitber of the two strains of .V. oculata (20.2 and 22.1 %)or CS-246 (21.2%). Conversely, lipid contents in N.oculala were similar (8.2 and 11.1% (by wt), but thevalue was almost twice as high (16.9%) in A', salina(Fig. 1). Chlorophyll a ranged from 1.5 to 1.7% ofdry weight in all but N. oculata CS-216, where it wasonly 0.6% (Fig. 1). This is despile the fact that thelight regime was the same for all ( ultures. Note thatchlorophyll a was extracted with the lijjids, and sothe weights determined for total lipids were adjustedaccordingly.

Sugar comfmsition oj thr Jmlysaccharicles. Each of thefour eustigmatophytes contained the same suite of

TABLF. 3. Amino arid rnmjiitsiiuin (weif^hl 'A) 0/ liydrol\snle\ firefiaredfrom the mirroalpne. Mean value', frdiii dujiluale derivatized samjdes.The ax<eraf^es 0/ the riiel/irienis n/variation were +2.'J'A fur mean values> l.O'/c and ±6,0'/c Jar mean values <t.O'/c. Cnrrerliims Jor losses ofihreonine and sfrine have hern included.

F.ssential amino acid.tAininine1 listidineIsoleucincI.eiK ineI.ysliieMethioninel'heiiylalatiin<-I'rolitii-''I hrconineTryptojihanValiiie

V MlhmiCs-r.io

7.62.14.H7.05.72.06.27.46.31.46.0

Nonessential amino acidsAlanine^-aminolnitytic addAspailate(lysline('•liitamale(ilydnellydroxy-prolincOrtiithine.SerineTyrosine

7.30.52H.30.87

10.45.70.200.195.44.6

' Data frntn Brown (1091).'' Kssentiiil lor inolluM:i> only.

mulfililc:s-7i(i

8.32.64.96.65.62.36.55.70.81.65.9

6.70.557.91.29.05.30.080.405.95.5

/'I hiCS-173

9.62.54.96.45.42.66.47.10.31.75.7

(i.'l0.877.21.29.25.40.03O.'̂ 25.05.3

Ml,

CSiilfi

9.02.45.00.45.32.40.27.66.11.25.7

(•>..'>

0.707.00.92

10.05.30.040.765.75.2

flitrvac"

7.51.74.46.98.21.35.24.45.11.05.4

5.61.3

10.00.72

12.29.10 . 1 •\

0.645.04.5

EUSTIGMATOPHYTE BIOCHEMISTRY 78

IAHLK. 4. Com/wsilions of total fnttt aeids as relative perrenUij^es with concenlrntions per cell and per dry wfif^hl of algae. A = Bli^h and Dyerextraction; B = lilif^li and Dyer extraction with addition »/ acid to the solvents. Subtotals inelude contributions from minor fatty acidi not lifted. Theaverafres of the coefficients of variation were ±2.0''< for mean values > I.O'l and ±3.5'^c for mean x'alues < l.C^c.

Saturated fatty acids14:015:016:018:0Subt<>t:il

Monouiisaluratcd iatty acid.sIO:l(n-O)lO: 1(11-7)ir.:l(ii-ri)l("):l(ii-l:?)^l7:l(n-8)18:1(11-0)

18:1(11-7)Subtotal

l'olyunsaturatt'd Iatty aiidsl():2(ii-7)l<>:2(ti-l)ir.:;Hn-l)18:2(11-9)18:2(11-0)18:3(11-0)18:;HII-: ' )20::Hn-r))20 : :HI I -^ )

20:4(11-0)20:5(n-3)22:6(n-3)Suljtolal

Otiu-is

CoiuTntratioiispg fatty acid-crll 'nig fatty acid-g ' dry wt

,V. sniiaiiCS-1',)O

(20- C. A)

5.00.5

27.81,0

34.5

04SIM

OLI0;28.S0;S

m:i

0.50.1M0̂ 21.50.40.80.9»r4.0

24.20.5

0.93112

.V. itrulnlnC.S-216

(20" C. A)

3.30.4

17.80.9

22.4

h

20.60.20.30.77.70.9

36.4

0.40.10.10.22.90.30.40.20.17.1

28.4—

40.8

0.9

0.5382

.V. orutainCS-179

(20" C. A)

4.60.5

14.20.0

20.0

0.129.4

0.20.40.86.30.3

37.4

0.80.10.20.S2.0;

0.10.40.18.8

28.8—

42.2

0.8

0.2788

C,S.246(27- C. A-)

5.40.620.10.6S6.8

tr20.9

0.10.50.S4.60.5

26.9

0.70.2t r0.22.70.80.90.10.26.4

mi—

45.7

0.9

0.39—

Unn;imc<l »p.CS-246

(27* C. A')

5.10.3

21.00.5

27.5

—22.1

0.10.80.12 20.0

25.9

0.80.40.2tr2.30.90.10.30.35.9

34.4—

40.0

0.9

0.4483

CS-246(27- C. B)

5.50.4

27.00.8

33.7

—23.3

0.10.90.1 .2.30.7

27.5

OJ<r.f

• 0.1

m

O.t04mS.I

28.0—

38.2

0.9

0.3401

' Rrplicatc tulturfs gn)wn 3 months apart.'' Indicates tli.ii tin- fatty acid was not dcti-ttod (<0.05% of total fatty acids).

sugars (Table 2). Ghicosc was the principal polysac-charidc sugar (()0.5-G(i.29( except lor strain CS-216,where it represented 45.2% of the total sugars). Fu-cose, galactose, mannose, rhamnose, ribose, and xy-lose were tiuuh less abutulant (2.0-14.0% of totalsugars), atid arabinose was tiot abtindant in any spe-eies (0.6-1.7%).

Amitio arid composition. The amitio acid coiiiposi-tioris of ilu- fotir etistigtnatophytes were also re-markably similar (Table 3). Aspartate, glutamate,and argiiiine were generally present in highest con-cetitratiotis (7.2-10.4% of total aniino acids), where-as tnethionitie, cystine, histiditie, try|)tophati, hy-droxy-proliiie, ornitliine, aiul 7-ainitiobutyric acidwere found in lowest concentrations (0.03-2.6%).Nnnnochlorofms salina contained more alanine butless tyrositie, arginitie, histidine, and methionine thaneither straiti ol'A'. orulttlti or the Qui't-tislatul isolateCS-246.

Lifiifl class coinliosition. In contrast to the results foramino acids and sugars, the lipid compositions

showed significant differences (Table 1; Fig. 1). P olar lipids greatly predominated in the two strains ofA', oculata and in strain CS-246 (87-94%), but in N.saliva they comprised just 54% of the total lipids dueto the very high content of triacylglycerols (43% cf.1-8% in the t)tlier strains; 'Table 1). Sterols w-erepresent in very low concentrations (20-40 fg cell"')atid consisted maitily of cholesterol (see Volkman etal. 1992). Previous studies (Wilkman et al. 1992)have shown that acid hydrolysis of the polar lipidsyields significant amounts of unusual Cjo-C-jj straight-chain 1,15-alkyl diols atid unsaturated alcohols inaddition to the fatty acids.

/•V///V aciil compositions. 'The four algae contaitiedthe satne limited suite of fatty acids, but there were.some differetices in relative amounts (Table 4). Therewas excellent agreement between the total fatty aciddistributiotis in the two satiiples of CS-246 cultured3 motitlis apart (Table 4). Palmitic (16:0), palmitole-ic 116:1 (n-7)], and eicosapentaenoic [20:5(n-3)] eachcomprised 14-35% of the total fatty acids atid col-

74 JOHN K. VOLKMAN El AL.

0 OS -1 m) Ntnnochlomptlt oaOUM CS-t79

5 to ' 5

Raunllon Urn* (mln)

20

(436 nm)

0 -"r

b) Dunalieila terttolttcta CS-175 9 10

10

Rvtsnrron lim» {

FIG. 2. HP[,C; chronialo^rams ol chlorophyll and carotc-iioidpigmt-iits ill a) \iinnorht<>rnl/\n onitnin strain CS-17'.) fompar<-(l loihow in b) a common green alga. Diinnlietla terliolerl/i. Peak iden-tifications: 1. chlorophyllide n (chlorophyll breakdown product);2. <ra;n-neoxanthin; 3. 9'-ai-nfoxan(hiri; 4, violaxanthin: !>.vaucheriaxanthin-ester; 6. antheraxanthin: 7, lurcin; 8. /ea-xanthin: 9. chlorophyll tn 10. chlorophyll <r. 11, chlorophyll aepimcr; 12. o-carotene: 13./J-carolene.

lectivcly comprised over 75*% of the total fatty acids.In each alga, saturated fatty acids consisted mainlyof 16:0. with smaller amounts of 14:0 and very mi-nor amounts of 15:0 and 18:0 (Table 4). Traceamounts of Cjo-C,o saturated fatty acids were de-tected in two ofthe algae. The main monounsaturat-ed fatty acids in each strain were palmitoleic acid116:l(n-7)l and oleic acid | I8:l(n-9)|. Several minor16:1 double-bond isomers were identified as 16:l(n-9), 16:l(n-5). and the /rrt«.v-monounsaturated fattyacid I6:l(n-13)/. The latter was presetu in very lowabundance (< 1.0%) compared to most other classesof algae, particularly those frorn the Chlorophyta(Volkman et al. 1989). At least 14 polyunsaturatedfatty acids were identified, but only 3 were presentin concentrations above 19̂ of total fatty acids. 'These-were 18:2(n-6) (1.5-2.9%). 20:4(ti-6) (4.0-8.8%), and20:5(n-3) (16.1-34.4%). Note that the highest con-centration of 20:5(n-3) occurred in the unnamedeustigmatophyte (34.4%). whert-as in iV. salinn, whichcontained abundant triacylglycerols. it was only16.1%. No Cjj polyunsaturated fatty acids were de-tected in any of the sami)les.

Contrary to an earlier study (Dubinsky and Aaron-son 1979). we did not observe any increase in lipidyield when acid was incorporated intcj the solventsused to extract strain CS-246 (extractic^n method B;Table 4). Indeed, the total amount of fatty acids

decreased by about 25%, and the concentrations ofpolyunsaturated fatty acids were lower, presumablyreflecting some degradation of the nic)re labile lijiidconstituents. A possible explanation for this differ-ence is that we used sonicaticjn to facilitate the sol-vent extraction, which is ktiown to be very effectiveat liberating lipid constituents from cells, whereasDubinsky and Aaroti.son used soxhlet extraction,which is less efficient (Guckert et al. 1988).

Pigment composition.'The four eust igmatophytes hadremarkably similar |)igiTieiit [)rofiles. A rejiresenta-tive UPLC cbromatogiani showing the distributicjiiof chlorophyll and carotenoid pigments in N. oculata(CS-1 79) and a typical green alga, Dunalieila tertiolec-ta, is shown in Figure 2. 'The cinly chloro|5liyll de-tected in the eustigmatopjhytes was chlorophyll a,whereas chlorophyll b was a major comj)on(-nt in thegreen alga. 'The eustigmatophytes containc-d viola-xanthin, j8-carotene, and /.eaxanthin plus several mi-nor carcjtenoids that were not characterized. Bycomparison, the greeti alga contained these samecarotenoids plus a high concetitration of lutein andsmall amounts of tv-carotene, anthetaxanthin. and9'-r!.v-neoxanthiti, which were not found in the eu-stigmatophytes. The HPLC chromatograms fromthe eustigmatophytes also contained a pignu-iu (peak5) that eluted bc-tween c hl()ro|)liyll a and violaxan-thin, whcxse ultraviolet-visible spectrum and reten-tion index were consistent with its identification asvaucheriaxanthin-ester. 'This ])igmenl is consideredto be charactc-ristic of species from the T.ustigma-tophyceae (NorgArd et al. 1974, Atitia and Clieng1982), and it is not present in species from the Pra-sinophyceae or Chlorophyceae (Fig. lb and BrownandJefTrey 1992). . .

DISCUSStON

Gross biochemical composition. 'The gross biochemi-cal compositions of the eustigmatophytes were re-markably sitnilar whc-n expressed oti a dry weightbasis (Fig. 1), but scjme diflcrences became apparentwhen the data were expressed per cell (Table I).The cells of the two strains grown in 1/2 mediutn(iV. oculata CS-179 and the tropical sttain CS-24())weighed less, and the.se strains contained less car-bohydrate, protein, and lipid thati the other twospecies (Fig. 1). In fact, the concetitrations of mostbiochemical classc-s pc-r cell in CS-179 were- muchlower than found iti the other species, in large- partreflecting the low weight of the cells iti this culture.Nannochloropsis oculata CS-216 grown in IT. mediumcontained nujie carbohydrate and |)rotein pc-r cellthan the satnplc-s growti iti f/2 tnedium. Nannoehlo-ropsis salina, which was also grown in fF. medium,contained less protein but more lipid and carbohy-drate than strain CS-2H), jjossibly reflecting geneticdifrc-reiues betwc-c-n these- two species. The cc-11masses of 9.3 atid 3.1 pg-cell ' for the two strainsofN. oculata (Table 1) are only just outside the ratigeof 4-9 pg-cell ' reported by Hodgson et al. (1991)

EUSTICMATOPHYTE BIOCHEMISTRY 75

for yV. nnihila fjjrown in S88 medium. However, lipidcoiueius ol 0.76 and 0.34 pgcell ' were much lowerthan values of 1.5-4 p g c e l l ' reported by these au-thors for actively growing cells.

Any comparison ol biochemical data for relatedstrains inust take into account their physiologicalstate when harvested, and this in turn will dependon a great variety of environmental factors such aslight, temjieralure, nutrient concentrations, etc. (e.g.Shilrin and Chisholm 1981, Sukenik and Cartneli1990, Hodgson et al. 1991). Even when culturedunder presumed identical conditions, some strainswill grow better than others, and so biochetnicaldifferences can be apjjai'ent between cultures har-vested at the same time but at different growth stages.In our study, we used two slightly different culturemedia that we had shown |)reviously gave goodgrowth for tin- algae being studied. All cultures wereharvested alter 7 days when the algae were at asimilar growth stage (i.e. mid to late log). The resultsobtained are broadly similar, but differences in theproi^ortions ol the major biochetnical fractions mayreflect some difference between the compositiotis ofthe two culture media used. Bulk nutrients such asnitrate and phosphate were still abmulant wheti thealgae were harvested, but theconcetitration ol somemicronutrient may have becotiie litniting. The like-lihood of genetic differences between strains is sug-gested by their variable growth responses and thesefactors might also have itiduenced the gross com-position. For example, there was a considerablyhigher triacylglycerol content in \'. saliiia (at theexpense of total protein) than iti other species, in-cluditig strain CS-216, which was grown under thesame conditions.

I'igmenl composition. The pigment distributiotiswere remarkably similar and tjuite chai-acteristicwheti cotnpared to other algal classes such as thegreen algae. Distinctive features include the lack ofchlorophylls }i and c, abundance of violaxanthiti, andpresence of vaucheriaxanthin-ester. Clearly, the useof HPLC to identify the pignuMits pn^vides a verycharacteristic fingerprint for assigning species to thisclass. Possible roles of violaxatuhin and vaucheria-xanthin-ester as antennae pigments in iV. sniina arediscussetl by Brown (1 987). The pigment fingerprintof the unnamed eus(igmatoj)liyte CS-24() fromQiu-ensland wati-rs confirmed that it is a eustigma-tophyte, but further ultrastructural study isre(juiredto establish its taxotiomic position. Based oti its cellsi/e and shape and lipid composition it appears tobe more closely related to X. oculata than to A\ sn-liuci.

Siijrnr.i; and nmino adds. We are not aware of anyother reports of the sugars iti eustigmatophytes tocompare to our data. The high proportioti of glu-cose in the |iolysactharides is also found in manyother microalgae, and iti some species it can com-prise up to 909c of the total sugars (Clui et al. 1982,Whyte 1987, Brown 1991). Such large amounts of

glucose could signify the presence of carbohydratereserves of the leucosin type (a /3-1,3-glucan) pre-viously known to be present in chrysophytes (Sze1986).

The amino acid compositions of the eustigmato-phytes are similar to the cotnpositions of other mi-croalgal species grown under comparable cultureconditions, although the proportion of proline iselevated compared to diatoms, prymnesiophytes, andprasinophytes (Brown 1991, Brown and Jeffrey1992). Jatneset al. (1989) also found a similar aminoacid composition atid a high proportioti of proline(10.59() iti ati utispecified species of Xnnuorhloropsh,even though the culture conditions used in that studywere quite differetit (e.g. continuous light, pH 6.5).It appears frotn these data and other studies thatamino acid cotnpositions are little affected by dif-feretices in culture conditions, even in those caseswhere the amount of protein is significantly differ-etit.

Lipids and fatty acids. The high abundance of polarlipids and low proportion of triacylglycerols foundin the two straitis of .V. oculata atid in strain CS-246is typical of microalgae grown under nutrient-suf-ficietit cotiditions (e.g. V'olkman et al. 1989). Highcotuentratiotis of triacylglycerols are commotilyfound in species grown to statiotiary phase when thenitrogen (tiitiate) content of the medium is depleted(Hodgson et al. 1991 and references theteiti). Thehigher abutidance of triacylglycerols iti .V. salina isthus unusual because the cells were still activelygrowing wheti harvested.

Hodgson et al. (1991) showed that the abundanceol 20:5(ti-3) iti .V. oculata declined as the culture ageincreased, such that its relative proportion in sta-tiotiary phase was otily half that during exponentialgrowth (15.4"^ cf. 26.4-28.29f). This was accom-patiied by a buildup of triacylglycerols, which theauthots ptesutiied were deficietit in 20:5(n-3), al-though paradoxically their data show that the con-tent of 20:5(n-3) in triacylglycerols during exponen-tial growth was actualh' tjuite high (34.9-37.59?;Hodgson et al. 1991). In a similar way, the lowerabundance of 20:5(n-3) iti .V. salina (Table 4) mayreflect the much higher contetit of triacylglycerols]iresetit compared to the other eustigmatophytes,but triacylglycerols wete tiot isolated to etiable usto test this. As a getietal rule, the fatty acid com-])ositioti of itidividual lipid classes tends to be morecotistatit atid, hetice, of tnore value as a biochemicalmarker, but differences between cla.sses can be con-siderable.

The tnajor fatty acids iti the eustigmatophytes were14:0, 16:0, 16:l(n-7), and 20:5(n-3). Similar fattyacid distributions have beeti teported previously for;V. oculata (Matuyama et al. 1986, Hodgson et al.1991), ,V. salina (F.mdadi and Berland 1989), andsome unidetitified Nannochloropsis strains (Suen etal. 1987, Suketiik et al. 1989). A similar pattern ofabimdance is also fbutid in most diatoms, except that

76 JOHN K. VOLKMAN V.T AL.

most diatoms contain some 22:6(n-3), which was ab-sent from the eustigmatophytcs, and a much highercontent of C,,; polyuiisaturatcd falty acids. Both al-gal groups lack significant amounts of Cjg polyun-saturated fatty acids, whereas in green algae thesefatty acids are commonly major constituents. Theclose similarity between fatty acid biosynthetic path-ways in diatoms and eustigmatophytes is further ev-ident in the positions of the double bonds. In bothclas.ses of algae, the 16:2 and 16:3 isomers have ter-minal double bonds at positions n-4 and n-7, whereasin greeti algae these <louble bonds occur at positionsn-3 and n-6 (e.g. Volkman et al. 1989, Dunstan etal. 1992). Thus, despite superficial physical similar-ities between eustigmatophytes and some coccoidgreen algae, the two algal classes can be readily dis-tinguished from their fatty acid compositions as wellas from their pigment profiles.

Our previous studies of the neutral lipids in thesesame four algal samples showed that the major sterolwas cholesterol (Volkman et al. 1992). These algaelack C,8 sterols and contain both E and Z isomers ofthe 24(28) double bond in Q,, A"*«'"-diutisaturatedsterols, which is quite unusual (Volkman 1080). Suenet al. (1987) also identified cholesterol as the onlymajor sterol present in another species of Nannochlo-ropsis (strain QII). 'I"he four eustigmatf)phytes alsocontain a monoutisaturated Cjj 1,15-alkyl diol andCjo^Cjj unsaturated n-alcohols and 1,15-alkyl diols,which are thought to be present as part of an un-identified polar lipid (Volkman et al. 1992). I he.sealkyl diols ar<' abutidant in many maritie sediments,but until our study there had been no reports ofthese unusual compounds in laboratory cultures ofmicroalgae. Further studies are required to sub-stantiate that they are unique to this algal class.

In summary, the characteristic pigment, fatty acid,sterol, alcohol, and alkyl diol compositions distin-guish eustigmatophytes from all other algal groups.These data not only provide strong support for theoriginal classification of these algae as a separateclass within the Chromophyta, but they also providea chemf)taxonomic signature for the group.

Relei'niire to maricnllureand hinlcrhiiology. 'I he halo-tolerant alga Xannoflilorf>l)sis saliiia grows well in out-door ponds, and it has been extensively investigatedas a source of biomass and lipids with growth ratesup to 24.5 g m ' day ' (Boussiba et al. 1987). Lipidcontents up to 74% of ash-free dry weight (as Mnn-allanlus salinn: Shif rin and Chisholni 1981) have beetiachieved, \annorhlnrnp.sis ontlatn is widely used as amariculture food species, and it also grows well un-der conditions of mass culture. Its small size, as wellas its high concentrations of the essetitial polyunsat-urated fatty acid 20:5(n-3), has made this alga par-ticularly suited for feeding to the rotifer Brarhinnuspliralilis and the brine shrimp Artcmia satina (for fishlarviculture; W'atatiabe et al. 1983, Sato 1991) andas food for larvae of the oyster Crasso.slren x<ir(rinirnGmelin (Uupuy 1975). The complete absence of C22

polyunsaturated fatty acids does not appear to limitthe nutritional value of this species. This alga is apossible source of Oils rich in the 20:5(n-3) fatty acidfor use as a food supplement (e.g. Borowitzka andBorowit/ka 1988,Gladue 1991). The lack of 22:6(n-3) would be an advantage in studies designed toinvestigate the recjuirement for specific polyunsat-urated fatty acids in the diet of marine animals.

The |)re.sence of cholesterol, which is an essentialconstituent of prawn diets (Teshima et al. 1983),rather than thecom|)lex suite of C-24 alkylated phy-tosterols cotnmoii to most microalgae (Volkman1986), may be an advantageous feature for larvalcrustacean feeding. Also, all of these species contaitihigh contents of vitamin ("(Brown and Miller 1992).

One purpose in this study was to compare thebiochemical composition of eustigmatophyte CS-246and N. snlinn to thoseof JV. onilata, which has provento be very successful as a maricultute feedstock. Inview of the small differences observed in the grossbiochemical compositions, it seems likely that thesetwo eustigmatophytes could also be used successfullyas live inicroalgal feeds. The slightly higher contentof carbohydrate in N. \aliiiti may be of nutritionalsignificatice for the feeding of molluscs (Whyte etal. 1989). Although N. salinn contains proportion-ally only half as much 20:5(n-3) fatty acid as theother species, the amount per cell is actually higherand would clearly meet the dietary requirements ofmost marine animals.

Differences in the sugar composition of polysac-charides from mirroalgae tnay be of tiutritiotial sig-nificance, because the efficieticy with which marineanimals digest polysaccharide is dejx'tident on thetype [)resent (Kristenseti 1972, Onishi et al. 198.')).I he glucose-rich polysaccharides found in eustig-matf)j)hytes should be effectively broken df)wn bythe digestive enzymes of molluscs and crustaceans(Kristensen 1972). The total carbohydrate concen-trations are tiot high, but Thomas et al. (1984) dem-onstrated that the carbohydrate yield iti tiitrogeti-deficient cultures of M salina (as Monallantus salina)can be doubled at the expense of protein sytuhesiswith no change in lipid content. Mowever, this resultdiffers from the findings of Shif rin and C'liisholm(1981) atid needs to be tested by further study.

Algal protein is considered (o be of high nutri-tional value if its esseTitial amino acid compositionis similar to that of the feeding animal (Webb andChu 1983). 'I"he amino acids essential for fish andcrustaceans include valine, leucine, isoleucine, ly-sine, histiditie, argitiine, try{)t()|)hati, phenylalanine,threonine, atid tnethionine (Harrison 1975, Kana-zawa and Teshima 1981, Cowey and lacon 1983),whereas molluscs also require proline (Harrison1975). The concentrations of the essential atiiitioacids in the eustigmatophytes were, in most in-stances, either ec|uivalent to or greater thati the lev-els iti larvae of the oyster Cra.s\oslrm gif^as Thunberg(Table 3), suggesting that they should provide a high-

EUSTIGMATOPHYTE BIOCHEMISTRY

quality protein for larval molluscs. The lysine con-criitrations arc low, but ihis is also true of tiianyotluT species of marine microalgae that have beenused successfully as feedstocks (Brown 1991, Brownand Jeffrey 1992).

These biochemical data provide a useful guide asto the likely value of eustigmatophytes as live algalfeeds for mariculture, but thiscati only be confirmedby feeding trials with appropriate target animal spe-cies.

Conclusions. The gross biochemical compositionsof two strains of the marine eustigmatophyte AV;«-nochlorop.sis oculata (CS-216 atid CS-179), Nannochlo-ropsis .wlina (CS-190), and an unnamed tropical coc-coid eustigmatt)phyte from tropical Australian waterswere similar. Small cjuantitative differences werenoted, most of which are likely to have resulted fromdifferences in culture conditions. The distributionsof individual lipid classes also matched closely, apartfrom an elevated content ol triacylglycerols in N.salina. The compositional data for fatty acids, aminoacids, sugars, and pigments combitied with the pres-ence o( cholesterol, utiusual C5o-C,2 1.15-alkyl diols,and C50-C52 mono- and diiuisaturated n-alcohols aresufficiently distinctive to be used as chemotaxonom-ic markers for this class of algae. High concentra-tions of the essetitial fatty acid 20:r)(n-;^) combinedwith g<)od-(juality protein and carbohydrate andpresence of cholesterol and vitamin C make theseeasily cultured microalgae very useful live diets formarine animals in mariculture.

\V<- tliank Ms. Jcaiiiiic-Marir I.rroi lor lulluriii^ ihc alj;ac andlor |)rovi{liiiji(oll (ounls.aiul M>. Stephanie liarrcll lor assistancewitli lipid analyses. Dr. Peter Niihols. Dr. Kilward Butler, andMr. David Niihiils contritjutcd many iiselul inipiovemenis to ihetnaniiscript. The maiuisiripl bctu'lited from the lonslriietive< oiiniients o( two anoiiyniotis reviewers. 1 his work was (iitulediti part by Kishitig Industry Research Trust .Account (FIR T.A)Krant 1'.180/81. Fishing Itidustry Research and Development('oinuil (FIRDC;) ^rant l ' .)88/69 atid Australian Research Couti-til (ARC) ^rant A 188:51830.

Antia. N. J., Bisalputra. I . . Cheng, J. Y. & Kalley, J. V. 1975.l'i^ll\etlt anti cytologital evidetiee lor ret lassifu ation of A'fiH-uorlilnris unihiln atid Mniuillaiiliis MIIIIIII in the Fustigmalo-phyceae./. t'hyml. i\.yM)-l^.

Atitia. N. J. X: Chi-ni-. J. Y. I<)8L'. The ketu-carotenoids of twottiaritiecoecoid tnenihersol the I'usti^atnatophyi eae. lir. l'h\-rol.J. \7:V.)-M).

BtrlatuI, B. R., Bonin, D. J., Daumas. R. A . l.aborde. P. 1,. X:Maestritii, S. Y. 1970. Variatiotis du cotnportenietit phy-.siologicjue de I'alj^ue .\Uiiiiilliiitlii> Miliiut (xanthophycoe) enctdture. Mm. llwl. (lirrl.) 7:82-92.

Bidlingnieyer. B. A., Coheti. S. A. i>t Farviti. T. L. 1984. Rapidanalysis ol amitio acids usitlg prc-column dcrivalization. /CliKiiiKildf;!. :13O:9;»-1O4.

Blaketiey, A. B., Harris. P. |.. Iletiry. R. J. & Stone, B. A. 1983.A sitnple atid tapid pre|)arati<>n olalditol acetates lor iiiotio-saiiharideatialysis. Carlwln,!. Hrs. ll.S:291-9.

Bligh. V. C f^ Dyer. W..|. 1959. A rapid method ol total lipidextra<ti()n and purification. Cnn.J. liiiKlirm. rhyuol. 37:912-7.

Borowit/ka. M. A. S,- Borowit/ka, I.. ,|.. Fds. 1988. .MirronlpitlBiolrchnology. Cambridge LIniversity Press, { .̂amhridm', -177pp.

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