Org. Geochem. Vol. 18, No. 1, pp. 131-138, 1992 0146-6380/92 $5.00 + 0.00 Printed in Great Britain. All rights reserved Copyright © 1992 Pergamon Press plc C30--C32 alkyl diois and unsaturated alcohols in microalgae of the class Eustigmatophyceae JOHN K. VOLKMAN, l STEPHANIE M. BARRETT, 1GRAEME A. DUNSTAN I and S. W. JEFFREY2 tCSIRO Division of Oceanography, Marine Laboratories, G.P.O. Box 1538, Hobart 7001, Australia 2CSIRO Division of Fisheries, Marine Laboratories, G.P.O. Box 1538, Hobart 7001, Australia (Received 5November 1990;returned for revision 2January 1991;accepted in revised form 13February 1991) Abstract--A study of the neutral lipids of the marine microalgae Nannochloropsis oculata Droop (2 strains), Nannochloropsis salina Hibberd and an unnamed species, all from the little-studied yellow-green algal class Eustigmatophyceae (Chromophyta), has shown that these microalgae contain unusual C30-C32 1,15-alkyl diols and a monounsaturated C32 1,15-diol. Alkane diols are abundant in many marine sediments, but this is the first report of their occurrence in cultured microalgae. This result argues against the suggestion, based on the identification of the same diols in a natural bloom of the cyanobacterium Aphanizomenonflos-aquae from the Baltic Sea, that these compounds are specificcyanobacterial markers. C32homologues were more abundant than C30homologues in the eustigmatophytes, as in the field sample of A. flos-aquae, whereas in most sediments the reverse is found. This suggests that the biological origin for these compounds in sediments may well be microalgae other than those studied here. The diols do not occur as free lipids in the eustigraatophytes, at least under the culture conditions used, but they could be liberated from more polar lipids by acid hydrolysis. In contrast, the major form of the alkyl diols in sediments appears to be as free lipids and not esters. Acid hydrolysis also liberated C30-C32mono and diunsaturated straight-chain alcohols. The carbon number distributions of the alcohols and diols are identical, and the relative proportions of homologues are similar, suggestingthat both compound classes are formed by the same biosynthetic pathway. The sterol compositions are also unusual with cholesterol comprising about 75% of the total sterols. C2s sterols were not detected, but small amounts of the C29 sterols 24-ethylcholesta-5,24(28)E-dien-3fl-ol and 24-ethylcholesta-5,24(28)Z-dien-3fl-ol were found in all algae. N. salina also contained 24-ethylcholesterol. Key words--alkyl diols, n-alkenols, biomarkers, sterols, microalgae, cyanobacteria, Eustigmatophyceae, Nannochloropsis, chemotaxonomy INTRODUCTION Long chain 1,15-alkyl diols and alkan-15-one-l-ols are a common feature of the extractable lipid distri- butions in most Recent marine sediments (reviewed by Morris and Brassell, 1988). In all sediments analysed thus far, the C30 homologue predominates with much smaller amounts of C2s, C3t and C32 compounds. The mid-chain hydroxy group occurs at position 15 except for homologues having 30 or fewer carbon atoms, where 1,13- and 1,15-isomers both occur. Homologues with odd carbon numbers are usually very minor components. This restricted car- bon number distribution and occurrence of a few specific positional isomers implies a common biologi- cal source from an organism, or group of organisms, that is ubiquitous in marine ecosystems. Long-chain alkyl diols and keto-ols were first identified in Recent Black Sea Unit 1 and Unit 2 sediments where they are particularly abundant (50/ag/g for the C30 diol; de Leeuw et al., 1981). Since then they have been recognized in marine sediments from the Miocene to the Quaternary and in a few lacustrine sediments (Smith et al., 1983; Morris and Brassell, 1988 and references therein). Most studies have been restricted to analyses of the solvent extractable lipids, but ten Haven et al. (1987) carried out a detailed study of free and bound lipids in late Quaternary Mediterranean sapropels. C30 diols and keto-ols were abundant in the soxhlet extractable lipids, but hydrolysis of the sediment residue also liberated shorter chain C24 and C26 alkyl diols with the mid-chain hydroxy group at positions 12, 13 and 14. The biological source of alkyl diols has been the subject of considerable speculation. Some of the suggestions have included coccolithophorids (de Leeuw et al., 1981; Nichols and Johns, 1986), mycolic acid type lipids of bacterial origin (de Leeuw et al., 1981), and methanogenic bacteria (Shaw and Johns, 1986). The matter seemed to be settled by the identifi- cation of C304232 alkyl diols in a natural bloom of the cyanobacterium Aphanizomenon flos-aquae (Morris and Brassell, 1988), but we demonstrate in this paper that certain species of microalgae may be a more likely source. EXPERIMENTAL Microalgae Cultures of Nannochloropsis oculata (strain CS- 216) and N. salina (strain CS-190; original source 131 OG 18/I--I