 PERGAMON Phytochemistry 40 "0888# 58Ð62 9920!8311:88:, ! see front matter Þ 0888 Elsevier Science Ltd[ All rights reserved[ PII]S9920!8311"87#99583!2 Highly branched C 14 isoprenoids in axenic cultures of Haslea ostrearia Emma J[ Wraige a \ Lesley Johns a \ Simon T[ Belt a\ \ Guillaume Masse  b \ Jean!Michel Robert b \ Steven Rowland a\  a Petroleum and Environmental Geochemistry Group\ Department of Environmental Sciences\ University of Plymouth\ Drake Circus\ Plymouth PL3 7AA\ UK b ISOMer\ Faculte  des Sciences et des Techniques\ Universite  de Nantes\ 1 rue de la Houssinie re\ 33961 Nantes Cedex 2\ France Received 7 October 0887 Abstract The hydrocarbon compositions of axenic cultures of the diatom Haslea ostrearia grown in the presence of penicillin\ streptomycin and kanamycin were examined at lag\ exponential and stationary growth phases[ The production of highly branched isoprenoid "HBI# C 14 trienes to pentaenes with the 1\5\09\03!tetramethyl!6!"2!methylpentyl#pentadecane carbon skeleton was demonstrated at all three phases "1299Ð6999 fg cell −0 #[ Of the ten HBI trienes to hexaenes reported previously from non!axenic cultures of H[ ostrearia\ four were present in the axenic samples[ In addition\ two novel trienes and a pentaene were found[ The most abundant of the new trienes was isolated from a larger\ non!axenic batch culture and identi_ed from 02 C! and 0 H!NMR data as 1\5\09\03! tetramethyl!6!"2!methylpent!3!enyl#pentadec!4\8!diene[ Interesting di}erences in HBI isomer distributions were observed among the three growth phases[ For example\ the newly identi_ed\ non!methylenic triene above\ only occurred in the exponential growth phase[ As a primary producer of these alkenes\ several of which have demonstrated cytostatic activity\ Haslea ostrearia\ and perhaps related Haslea species\ is clearly worthy of further study[ Þ 0888 Elsevier Science Ltd[ All rights reserved[ Keywords] Haslea ostrearia^ Bacillariophyceae^ Microalgae^ Diatoms^ Isoprenoid alkenes^ Highly branched isoprenoids^ C 14 alkenes^ Axenic cultures 0[ Introduction Recently\ a family of highly branched isoprenoid "HBI# C 14 polyenes was reported in a non!axenic culture of the diatom\ Haslea ostrearia "Gaillon# Simonsen "Volkman\ Barrett\ + Dunstan\ 0883#[ Hence a primary source was suggested for some of the HBI hydrocarbons which have been found in contemporary marine environments world! wide "reviewed in Robson + Rowland\ 0875^ Rowland + Robson\ 0889#\ but for which no source was known previously[ The compounds are also important in a number of other respects[ The highly branched structures have led to the suggestion that the phosphate esters of such compounds may have been important in the for! mation of vesicles during the early evolution of life on Earth "Ourisson + Nakatani\ 0883# and it has recently been demonstrated that polyunsaturated HBI com! pounds can be produced in the presence of unactivated  Corresponding authors[ S[ T[ Belt] Tel[] ¦33 0641 122 931^ fax] ¦33 0641 122 924^ e!mail] sbeltÝplymouth[ac[uk[ S[ Rowland] Tel[] ¦33 0641 122 902^ fax] ¦33 0641 122 924^ e!mail] srowlandÝ plymouth[ac[uk[ clay by abiotic dimerisation of geraniol and farnesol "Nagano + Nakanishi\ 0887#[ Isolation of individual HBI alkenes from non!axenic\ large scale batch cultures of H[ ostrearia and charac! terisation by NMR and mass spectral methods has con! _rmed the highly branched structures and revealed the positions and stereochemistry of the double bonds "Belt\ Cooke\ Robert\ + Rowland\ 0885#[ Cytostatic tests on some of these pure alkenes have shown that some are active in slowing the growth of lung cancer cell lines in vitro "Patent GB!8697823[5#[ Clearly\ given the non!axenic nature of the algal cul! tures studied to date and the possibility of HBI formation in sediments by abiotic processes\ it is important that the role of H[ ostrearia as a primary producer be con_rmed\ particularly if the bioactivity of the compounds is to be investigated further "cf[ Gerwick + Roberts\ 0883^ Borowitzka\ 0884#[ This makes the demonstration of HBI biosynthesis from an axenic algal culture imperative[ In the present study we report the production of HBI trienes to pentaenes in the three principal growth stages of H[ ostrearia grown axenically in the presence of three anti! biotics[ These growth phases "lag\ exponential\ station!