Phytochemistry\ Vol[ 38\ No[ 4\ pp[ 0116Ð0128\ 0887Þ 0887 Elsevier Science Ltd[ All rights reserved\ Pergamon Printed in Great Britain

9920Ð8311:87:,*see front matterPII] S9920Ð8311"86#99832Ð5

EFFECT OF HIGH IRRADIANCE AND IRON ON VOLATILE ODOURCOMPOUNDS IN THE CYANOBACTERIUM MICROCYSTIS

AERUGINOSA

KATHRYN WALSH\$ GARY J[ JONES\% and R[ HUGH DUNSTAN�$

$ Department of Biological Sciences\ The University of Newcastle\ NSW 1297\ Australia^ % CSIRO Division of WaterResources\ PMB 2\ Gri.th\ NSW 1579\ Australia

"Received 03 Au`ust 0886#

Key Word Index*Microcystis aeru`inosa^ cyanobacterium^ heptadecane^ iron^ irradiance^photoxidation^ terpenoids^ volatile odour compounds[

Abstract*The cyanobacterium\ Microcystis aeru`inosa was exposed to direct sunlight for 2\ 5 or 8 h in mediacontaining either low or high concentrations of iron\ in order to determine any e}ects on the composition ofvolatile odour compounds "VOCs# released under photooxidative conditions[ The most abundant VOCsdetected included aliphatic hydrocarbons "C04ÐC10#\ naphthalene and the terpenoid compounds\ b!cyclocitral\and b!ionone[ Exposure to sunlight and low iron concentrations resulted in a decrease in b!cyclocitral\ b!ionone\ heptadecane and the total VOCs concentration after 8 h with respect to the control cultures[ Six VOCsdetected in the low iron cells were not detected in any of the high iron cells[ However\ those VOCs present inthe high iron cells\ in general\ occurred at higher concentrations than the equivalent low iron cells after exposureto the sunlight conditions[ Consequently\ it was concluded that exposure to both high irradiance and high ironconcentrations in~uenced the VOCs composition in cyanobacteria and this was interpreted to represent acellular change during the photooxidation!promoting conditions[ Þ 0887 Elsevier Science Ltd[ All rightsreserved

INTRODUCTION

The presence of cyanobacteria in Australian waters isoften implicated in the production of unpleasant tastesand odours\ whether through excretion of volatileodour compounds "VOCs#\ such as geosmin or 1!methylisoborneol "MIB#\ or as a result of bacterialdecay of surface scums ð0Ð2Ł[ VOCs have been associ!ated with many di}erent types of phytoplankton\including cyanobacteria\ algae and diatoms\ whenthese species periodically reach bloom proportions inthe freshwater environment ð3Ð6Ł[ Consequently\ thepresence of VOCs in recreational or drinking water inAustralia often leads to public concern\ which hasstimulated research investigating the chemical natureof these compounds[

Volatile products of cyanobacteria can be classi_edas nor!carotenoids "degradation products of car!otenoids or ketones and alcohols with irregular ter!pene structures#\ fatty acid degradation products\ suchas aldehydes\ that may be liberated during lipox!ygenase action or lipid peroxidation\ metabolitesreleased during growth\ terpenoids\ sulfur

� Author to whom correspondence should be addressed[

0116

compounds\ aromatic compounds and amines ð7Ð00Ł[The terpenoids are secondary metabolites of higherplants and microorganisms and\ like the sterols andcarotenoids\ are synthesised from isoprenoid units bythe mevalonate pathway ð01Ł[ The function of ter!penoids varies widely from their use in plants as aninsect attractant during pollination\ a natural insectrepellent and as an inhibitor of seed germination andplant growth ð02Ł[ There is also evidence indicatingthat the production of volatile and terpenoid com!pounds\ such as geranyl acetone\ provide a com!petitive advantage of some phytoplankton species asallelopathic agents\ by inhibiting the growth of sur!rounding phytoplankton ð02\ 03Ł[ Additionally\ vol!atile compounds have been implicated as chemicalmessengers in the aquatic environment\ particularlyas attractants during fertilisation of marine Phae!ophyceae ova ð3\ 04Ł[ These products include octad!iene\ ectocarpene "5!"cis!0!butenyl#cyclohepta!0\3!diene#\ dictyopterene A "trans!0!"trans!0!hexenyl#!1!vinylcyclopropane# and dictyopterene C? "5!butyl!cyclohepta!0\3!diene#\ and these have also been iso!lated from algal phytoplankton samples ð3\ 04Ł[

The most abundant VOCs detected in Microcystisspecies taken from the _eld are b!cyclocitral\ b!ionone\some terpenoid!derived compounds and aliphatic

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hydrocarbons ð0\ 2\ 00Ł[ However\ there is little lab!oratory!based research investigating the e}ects of par!ameters\ such as irradiance and nutrient availability\on the production and:or content of VOCs from cul!tured strains[ Presumably\ since nor!carotenoids andfatty acid derivatives are degradation products ofmembrane components\ exposure of cyanobacteria toprolonged irradiance could lead to their increased pro!duction[ In addition\ carotene!oxygenase activity\responsible for speci_c carotenoid cleavage\ is stimu!lated in the presence of ferric ions ð09\ 05Ł[ Therefore\the presence of pro!oxidant forms of iron "both Fe1¦

and Fe2¦# ð06Ł may also be responsible for alterationsin the production of VOCs[ Iron concentrations varywidely within natural surface waters of Australianwaterways ð07Ł[ Therefore\ the aims of the presentstudy were to investigate the production of VOCsfrom a laboratory strain of M[ aeru`inosa exposed toprolonged periods of sunlight\ in the presence of lowand high concentrations of iron[

RESULTS

Composition of volatiles

The lipophilic volatile fraction from M[ aeru`inosaconsisted mainly of either cyclic components "such asb!cyclocitral#\ terpenoids or hydrocarbon!based com!pounds "Fig[ 0#[ A series of long!chain aliphatichydrocarbons were detected ranging from penta!decane to henicosane[ Heptadecane "Peak 11^ Fig[ 0#was the most abundant aliphatic hydrocarbon fromall irradiance and iron treatments\ ranging from 03 to69) of the total volatile concentration "Tables 0 and1#[

There were nine compounds designated as degra!dation products of carotenoids or fatty acids "Tables0 and 1#[ These included b!cyclocitral and b!ionone\and several short!chain compounds\ such as 2!penten!1!one\ 4!hexadecyne and 1\3!nonedienal[ The terpeno!ids\ cedrol "2R!octahydro!2\5\7\7!tetramethyl!0H!2a\6!methanoazulen!5!ol# "Peak 06^ mass spectrumFig[ 1A# and thujopsene "0\0a\3\3a\4\5\6\7!octahydro!1\3a\7\7!tetramethylcyclopropaðdŁnaphthalene# "Peak00^ mass spectrum Fig[ 1B#\ were detected in all lowand high iron cells exposed to the sunlight regime\ranging from 0[3Ð2[8) and 4[7Ð01[8)\ respectively[Five naphthalene derivatives "Figs 1D\ E and F# weredetected in all the low iron cells and the high iron cellsexposed to 2 and 5 h standard and sunlight irradiance[Several volatile compounds produced were not ident!i_ed but had similar mass spectra to cedrol "Fig[ 1A#and\ hence\ are referred to numerically as unknownterpenoids 0Ð3[ Diethyl phthalate and bis"1!methyl!propyl#phthalate "0# and "1# were detected in all theiron and irradiance treatments[

Sunli`ht exposure with low iron

The concentrations of volatile components from M[aeru`inosa grown in the low iron medium in response

to the sunlight or standard irradiance are summarisedin Table 0[ The nor!carotenoid\ b!cyclocitral "Peak2#\ was signi_cantly lower in the low iron cells afterexposure to 2\ 5 and 8 h sunlight\ compared with thecorresponding cells exposed to the standard irradiance"Fig[ 2A#[ b!Ionone "Peak 7# was also lower in thecells exposed to sunlight for the 2 and 5 h periods\compared with the equivalent standard irradiancecells "Fig[ 2B#[ b!Cyclocitral and b!ionone from thestandard irradiance cells increased by a factor of 4between the 5 and 8 h exposure periods[ However\there were no similar alterations observed in b!cyclo!citral and b!ionone concentrations in the cor!responding cells exposed to the sunlight regime[

The two naphthalene!derived compounds\0\1\2\3\4\5\6\7!octahydro!0\3\8\8!tetramethyl!3\6!methanoazulene "Peak 8^ mass spectrum Fig[ 1C#\0\1\2!trimethyl!3!propenylnaphthalene "0# "Peak 19#\and an unknown terpenoid "3# "Peak 05# were sig!ni_cantly lower in M[ aeru`inosa exposed to sunlightafter 2 h\ compared with the standard irradiance cells[The concentration of pentadecane was signi_cantlylower in the low iron cells after 5 h "but not 8 h#sunlight exposure\ compared with the standardirradiance cells[

Many of the naphthalene!derived compounds werenot signi_cantly di}erent between the sunlight andstandard treatments[ However\ 0\1\2\4\5\7a!hex!ahydro!3\6!dimethyl!0!"0!methylethyl# naphthalene"Peak 03# was negatively correlated with time duringthe standard irradiance exposure "P ³ 9[94#[ 0\1\2!trimethyl!3!propenyl naphthalene "Peak 19# was posi!tively correlated during the sunlight exposure in atime!dependent manner "P ³ 9[94# "Table 0#[ Pro!duction of heptadecane "Fig[ 2C# and octadecaneincreased linearly with time "P ³ 9[94# in both thestandard and sunlight regimes\ whereas nonadecaneand eicosane increased with time only during the stan!dard irradiance regime "Table 0#[ Pentadecanedecreased linearly with time in the low iron cells fromboth the standard and sunlight irradiance treatments"P ³ 9[94#[ The unknown terpenoids 0Ð3 were allnegatively correlated with exposure time in the stan!dard irradiance cells but were not correlated withexposure time in the sunlight treated cells "Table 0#[

Although the amounts of VOCs produced by cellsin the sunlight irradiance were always lower than thoseproduced in the standard irradiance cells\ the totalconcentrations of volatile components did not di}ersigni_cantly between the sunlight and standardirradiance regimes after the 2\ 5 and 8 h exposureperiods[ The production of the total volatile com!ponents in the cells exposed to the standard and sun!light irradiance treatments was positively correlatedwith time "P ³ 9[94# "Table 0\ Fig[ 2D#[

Sunli`ht exposure with hi`h iron

The changes in the volatile components of M[ aeru!`inosa grown in the high iron medium in response to

Irradiance and cyanobacterial volatile compounds 0118

Fig[ 0[ Chromatogram of volatile components extracted after 5 h standard irradiance exposure from high iron M[ aeru`inosa[All volatile compounds that were assessed are listed below[ Compounds that occurred at low abundances and are not

presented on the chromatogram are numerically represented in elution order by T0Ð09 � trace[

0 1\2!Dimethyl!0\3!hexadiene1 0!Methanolcyclohexene2 b!Cyclocitral3 Propanoic acid\ 1!methyl!2!hydroxy!1\3\3!trimethyl!

pentyl ester4 d2!Geosmin "internal standard#5 4!Hexadecyne6 1\5!bis"0\0!dimethylethyl#!3!methylphenol7 b!Ionone8 0\1\2\3\4\5\6\7!Octahydro!0\3\8\8!tetramethyl!3\6!

methanoazulene09 Diethyl phthalate00 Thujopsene01 Unknown terpenoid 002 Unknown terpenoid 103 0\1\2\4\5\7a!Hexahydro!3\6!dimethyl!0!"0!

methylethyl#naphthalene04 Unknown terpenoid 205 Unknown terpenoid 306 Cedrol07 0\1\2\3\3a\4\5\7a!Octahydro!6!methyl!3!methylene!

0!"0!methylethyl#naphthalene"0#

08 0\1\2\3\3a\4\5\7a!Octahydro!6!methyl!3!methylene!0!"0!methylethyl#naphthalene"1#

19 0\1\2!Trimethyl!3!propenylnaphthalene"0#

10 0\1\2!Trimethyl!3!propenylnaphthalene"1#

11 Heptadecane12 2!Penten!1!one13 Cyclohexen!0!one relative14 Octadecane15 bis"1!Methylpropyl#phthalate"0#

16 Nonadecane17 bis"1!Methlpropyl#phthalate"1#

T0 NaphthaleneT1 1\3!NonedienalT2 0\3!Diene!1\5!bis"0\0!dimethylethyl#!1\4!

cyclohexadieneT3 PentadecaneT4 1!Methyl!\ 0!"0\0!dimethylethyl#propanoic acidT5 HexadecaneT6 Benzoic acid\ pentyl esterT7 EicosaneT8 HenicosaneT09 Propanetricarboxylic acid[

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Table 0[ Concentration of volatile components "mg g−0 geosmin equivalents:dry wt# from the low iron M[ aeru`inosa exposed to the sunlight and standard irradiance

2 h Sunlight 2 h Standard 5 h Sunlight 5 h Standard 8 h Sunlight 8 h Standard Correlationsn � 3 n � 3 n � 3 n � 3 n � 4 n � 4 P ³ 9[94

Peak no[ Volatile odour compounds Mean S[E[ Mean S[E[ Mean S[E[ Mean S[E[ Mean S[E[ Mean S[E[ Sun Standard

Breakdown products0 1\2!Dimethyl!0\3!Hexadiene * * * * * * * * 9[46 9[24 9[09 9[092 b!Cyclocitral ��9[96 9[96 1[97 9[24 ���9[91 9[91 1[99 9[16 ��9[32 9[17 00[19 1[89 9[575 4!Hexadecyne * * * * * * * * 9[10 9[10 9[10 9[107 b!Ionone ��9[97 9[97 9[77 9[19 ���9[94 9[92 9[77 9[91 9[43 9[23 4[05 1[19

12 2!Penten!1!one 9[43 9[94 9[52 9[24 9[91 9[91 9[92 9[92 0[72 0[36 1[06 0[57 9[5413 Cyclohexen!0!one relative * * 9[26 9[05 * * * * 9[62 9[62 * *T1 1\3!Nonedienal * * 9[96 9[96 * * * * * * 9[07 9[07T2 0\3!Diene!1\5!bis"0\0! * * 9[95 9[95 9[98 9[95 9[06 9[98 * * 9[14 9[14

dimethylethyl#!1\4!cyclohexadieneT09 Propanetricarboxylic acid * * * * * * * * 0[11 0[11 * *

Naphthalene:cyclic compounds1 0!Methanolcyclohexene * * * * * * * * 9[13 9[13 * *3 Propanoic acid\ 1!methyl!\ 2! 9[47 9[05 9[57 9[29 9[13 9[96 9[31 9[01 9[85 9[69 9[57 9[46

hydroxy!1\3\3!trimethylpentylester

6 1\5!bis"0\0!Dimethylethyl#!3! 0[34 9[38 9[87 9[21 9[95 9[93 9[02 9[91 9[20 9[19 0[02 9[72 −9[46methylphenol

8 0\1\2\3\4\5\6\7!Octahydro! �9[16 9[00 9[62 9[04 9[94 9[92 9[97 9[95 0[21 0[90 9[38 9[260\3\8\8!tetramethyl!3\6!methanoazulene

00 Thujopsene 1[84 9[30 4[58 0[30 9[32 9[04 9[79 9[23 00[60 5[06 5[81 2[3401 Unknown terpenoid"0# 9[11 9[02 9[62 9[18 9[91 9[91 9[93 9[91 9[89 9[53 * * −9[5702 Unknown terpenoid"1# 9[22 9[10 9[80 9[12 9[96 9[93 9[04 9[96 0[45 0[90 * * −9[7903 0\1\2\4\5\7a!hexahydro!3\6! 0[48 9[56 0[66 9[44 9[08 9[97 9[03 9[93 1[37 0[90 9[24 9[24 −9[46

dimethyl!0!"0!methylethyl#naphthalene

04 Unknown terpenoid"2# 9[10 9[01 9[66 9[12 9[93 9[92 9[12 9[01 9[86 9[86 * * −9[6605 Unknown terpenoid"3# ���* * 9[26 9[94 9[92 9[92 9[94 9[92 9[78 9[77 * * −9[7606 Cedrol 9[79 9[06 0[53 9[26 9[04 9[94 9[11 9[00 1[19 1[19 * *07 0\1\2\3\3a\4\5\7a!Octahydro!6! 1[21 9[28 2[87 9[68 9[23 9[04 9[52 9[29 8[21 3[17 2[99 0[03

methyl!3!methylene!0!"0!methylethyl#naphthalene"0#

Irradianceand

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08 0\1\2\3\3a\4\5\7a!Octahydro!6! 9[66 9[00 0[42 9[25 9[02 9[94 9[06 9[98 2[38 0[53 2[07 1[25methyl!3!methylene!0!"0!methylethyl#naphthalene"1#

19 0\1\2!Trimethyl!3!propenyl 9[26 9[04 9[63 9[04 9[01 9[94 9[12 9[98 1[21 9[65 1[35 0[03 9[59naphthalene"0#

10 0\1\2!Trimethyl!3!propenyl �9[17 9[00 9[73 9[06 9[09 9[93 9[11 9[95 0[21 9[58 0[15 9[36naphthalene"1#

T0 Naphthalene * * * * * * * * 9[42 9[21 9[96 9[96T6 Benzoic acid\ pentyl ester * * * * * * * * * * 9[76 9[67T4 1!Methyl!\ 0!"0\0!dimethylethyl# * * * * * * * * * * 3[63 2[24

propanoic acid

Phthalates09 Diethyl phthalate 9[63 9[13 0[49 9[63 9[98 9[95 9[09 9[95 3[56 2[55 6[89 4[1815 bis"1!Methylpropyl#phthalate"0# 1[71 9[40 3[49 0[97 9[43 9[15 9[83 9[23 7[61 2[23 00[99 5[2217 bis"1!Methylpropyl#phthalate"1# 2[11 9[55 4[00 0[02 9[55 9[23 0[11 9[30 7[39 2[43 6[39 0[12

Aliphatic hydrocarbonsT3 Pentadecane 1[48 9[87 2[30 0[95 �9[43 9[03 1[55 9[50 * * * * −9[61 −9[65T5 Hexadecane 9[69 9[04 9[81 9[27 �9[00 9[93 9[35 9[98 9[71 9[42 1[90 9[6911 Heptadecane 8[31 9[82 01[84 3[69 2[07 9[45 8[25 1[18 45[20 00[39 000[09 18[70 9[62 9[6214 Octadecane 9[84 9[20 0[97 9[25 9[02 9[91 9[45 9[10 02[37 2[90 12[06 4[21 9[63 9[6316 Nonadecane 9[22 9[04 9[32 9[04 9[93 9[91 9[93 9[91 9[69 9[33 2[16 0[94 9[51T7 Eicosane * * * * * * * * 9[08 9[08 0[81 9[80 9[45T8 Henicosane * * * * * * * * * * 0[38 0[00

Total volatile components 22[59 6[49 44[25 05[03 6[33 1[36 10[80 5[25 028[21 42[34 102[51 63[13 9[50 9[69

Signi_cant di}erences between the standard and sunlight treatments for each time period are represented by �P ³ 9[94\ ��P ³ 9[90 and ���P ³ 9[990[ Signi_cant correlations "r values\ P ³ 9[94#between VOCs and time are shown for the sunlight and standard irradiance treatments[ Peak numbers correspond to those in Fig[ 0[

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Table 1[ Concentration of volatile components "mg g−0 geosmin equivalents:dry wt# from the high iron M[ aeru`inosa exposed to the sunlight and standard irradiance[

2 h Sunlight 2 h Standard 5 h Sunlight 5 h Standard 8 h Sunlight 8 h Standardn � 1 n � 2 n � 4 n � 4 n � 2 n � 2

Peak no[ Volatile odour compounds Mean Mean S[E[ Mean S[E[ Mean S[E[ Mean S[E[ Mean S[E[

Breakdown products0 1\2!Dimethyl!0\3!Hexadiene * * * * * 9[25 9[25 * * 9[28 9[282 b!Cyclocitral 9[66 4[07 1[62 * * 0[81 0[12 * * 8[85 0[505 4!Hexadecyne * * * 9[24 9[24 9[58 9[32 * * * *7 b!Ionone * 1[26 1[26 9[34 9[34 9[73 9[49 * * * *

12 2!Penten!1!one * * * 2[27 1[96 1[06 0[08 2[59 2[59 2[79 2[7913 Cyclohexen!0!one relative 0[78 * * 0[18 0[09 9[78 9[52 * * 0[35 0[98T1 1\3!Nonedienal * * * * * * * * * * *T2 0\3!Diene!1\5!bis"0\0!dimethylethyl#!1\4!cyclohexadiene 2[51 0[43 0[43 * * * * * * * *T09 Propanetricarboxylic acid 0[30 2[08 2[08 2[30 1[36 4[36 2[46 0[38 0[38 * *

Naphthalene:cyclic compounds1 0!Methanolcyclohexene * * * * * 9[55 9[34 * * 9[35 9[353 Propanoic acid\ 1!methyl!\ 2!hydroxy!1\3\3!trimethylpentyl * * * * * 9[58 9[58 * * * *

ester6 1\5!bis"0\0!Dimethylethyl#!3!methylphenol 1[28 4[80 0[56 �0[29 9[60 9[34 9[26 * * * *8 0\1\2\3\4\5\6\7!Octahydro!0\3\8\8!tetramethyl!3\6! 3[83 0[10 0[10 3[95 0[89 2[51 1[09 1[50 1[50 0[63 0[33

methanoazulene00 Thujopsene 27[53 01[08 5[47 28[07 07[20 15[31 02[76 28[70 24[56 19[02 03[0701 Unknown terpenoid"0# 2[34 9[58 9[58 2[13 0[43 1[51 0[32 2[12 2[12 0[14 0[1402 Unknown terpenoid"1# 6[03 0[18 0[18 4[55 1[79 3[25 1[15 6[13 4[72 1[99 1[9903 0\1\2\4\5\7a!hexahydro!3\6!dimethyl!0!"0! 6[62 9[58 9[58 5[81 2[72 4[26 1[46 * * 3[82 2[28

methylethyl#naphthalene04 Unknown terpenoid"2# 2[97 * * 3[05 1[03 2[61 1[97 * * * *

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05 Unknown terpenoid"3# * * * * * 1[08 0[40 * * 0[68 9[5406 Cedrol 8[38 0[72 0[72 00[75 5[00 6[60 4[38 6[76 6[76 4[48 3[2007 0\1\2\3\3a\4\5\7a!Octahydro!6!methyl!3!methylene!0!"0! 20[13 7[80 4[62 22[04 02[49 06[73 8[85 12[47 19[36 00[53 7[03

methylethyl#naphthalene"0#

08 0\1\2\3\3a\4\5\7a!Octahydro!6!methyl!3!methylene!0!"0! 7[67 1[35 0[59 00[13 3[87 6[48 2[81 3[56 3[56 2[91 1[33methylethyl#naphthalene"1#

19 0\1\2!Trimethyl!3!propenyl naphthalene"0# 09[33 1[99 0[09 7[98 3[49 3[67 0[86 3[34 1[87 0[86 0[0410 0\1\2!Trimethyl!3!propenyl naphthalene"1# 3[69 0[83 0[96 2[85 1[01 2[02 0[24 * * 0[10 9[70T0 Naphthalene * * * * * * * * * * *T6 Benzoic acid\ pentyl ester * * * * * * * * * * *T4 1!Methyl!\0!"0\0!dimethylethyl#propanoic acid 0[10 1[51 0[48 2[09 0[84 * * * * * *

Phthalates09 Diethyl phthalate 09[04 0[83 0[83 09[63 2[52 3[78 1[76 5[76 5[76 4[18 3[9715 bis"1!Methylpropyl#phthalate"0# 14[26 7[54 4[02 36[59 11[90 29[34 7[38 03[62 01[69 7[02 4[3317 bis"1!Methylpropyl#phthalate"1# 13[99 8[74 4[08 41[46 17[63 32[77 06[03 00[25 8[56 6[06 3[74

Aliphatic hydrocarbonsT3 Pentadecane * * * * * * * * * * *T5 Hexadecane �4[61 1[99 0[90 * * * * * * * *11 Heptadecane 013[56 57[18 10[89 31[98 06[21 68[17 32[24 �278[31 053[80 61[40 04[9714 Octadecane 00[31 5[04 1[41 4[94 1[04 8[79 5[91 �22[01 00[69 3[43 9[6916 Nonadecane * * * 0[35 0[35 9[60 9[33 * * * *T7 Eicosane * * * * * * * * * * *T8 Henicosane * * * * * * * * * * *

Total volatile components 231[13 049[75 61[48 293[29 035[08 161[40 025[11 443[94 183[17 057[86 66[15

Signi_cant di}erences between the standard and sunlight treatments for each time period are represented by �P ³ 9[94\ ��P ³ 9[90 and ���P ³ 9[990[ Peak numbers correspond to those in Fig[0[ There were no signi_cant correlations between VOCs and time for the sunlight and standard irradiance treatments[

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Fig[ 1[ Mass spectra of terpenoids and naphthalene derivatives detected in the volatile fraction of M[ aeru`inosa\ A[ Cedrol "Peak 06#\ B[ Thujopsene "Peak 00#\ C[ 0\1\2\3\4\5\6\7!Octahydro!0\3\8\8!tetramethyl!3\6!methanoazulene "Peak 8#\ D[ 0\1\2\4\5\7a!Hexahydro!3\6!dimethyl!0!"0!methylethyl#naphthalene "Peak 03#\ E[ 0\1\2!Trimethyl!3!propenylnaphthalene "Peak 19 and 10#\

F[ 0\1\2\3\3a\4\5\7a!Octahydro!6!methyl!3!methylene!0!"0!methylethyl#naphthalene "Peak 07 and 08#[

Irradiance and cyanobacterial volatile compounds 0124

Fig[ 2[ Concentrations of "A# b!cyclocitral "Peak 2#\ "B# b!ionone "Peak 7#\ "C# heptadecane "Peak 11# and "D# the totalvolatile components extracted from M[ aeru`inosa grown in low and high iron media and exposed to either standard "Ž# or

sunlight "ž# conditions[

the sunlight and standard irradiance are summarisedin Table 1[ Six of the volatile compounds from thelow iron cells were not detected in either the standardor sunlight irradiance cells after incubation in the highiron medium[ However\ those volatile componentsthat were present in the high iron cells\ generallyoccurred at greater abundances than the equivalentlow iron cells[ The high iron cells had a di}erent VOCpro_le compared with the low iron cells "Tables 0 and1#[ The total volatile concentration was greater in thehigh iron cells exposed to 8 h sunlight than the lowiron cells exposed to 2 and 5 h standard and sunlightirradiance "P ³ 9[94# "Fig[ 2D#[ Furthermore\ thetotal volatile concentration from all the high iron cul!tures was signi_cantly higher than all the low ironcultures "P ³ 9[9990\ n � 36#[

b!Cyclocitral decreased in cells exposed to both thesunlight and standard irradiance after the 5 hirradiance periods "Fig[ 2A#[ However\ after 8 hexposure to the standard irradiance\ the productionof this carotenoid degradation product increased sig!ni_cantly\ compared with those cells from the sunlightregime[ b!Ionone was detected in the 2 and 5 h stan!dard exposures and the 5 h sunlight exposure\ but wasnot detected in the other high iron samples "Fig[ 2B#[

Heptadecane and octadecane were the only ali!phatic hydrocarbons detected in the high iron cellsafter 8 h standard or sunlight irradiance exposure"Table 1#[ Both of these compounds occurred in sig!ni_cantly greater concentrations after 8 h sunlightirradiance\ compared with the cells exposed to the

standard irradiance[ The high iron cells exposed to 8h sunlight had a signi_cantly greater concentration ofheptadecane than all the other irradiance treatmentsin both the low and high iron cells "P ³ 9[94# "Fig[2C#[ By comparison\ pentadecane\ eicosane and hen!icosane were not detected in any of the high ironcells\ whereas hexadecane and nonadecane were onlydetected at low concentrations in the 2 h and 5 hirradiance treatments\ respectively[

1\5!bis"0\0!Dimethylethyl#!3!methylphenol "Peak6# decreased with time in the high iron cells exposedto the sunlight irradiance "P � 9[995\ r � −9[66\n � 09#[ Additionally\ after 5 h sunlight irradiance\the concentration of 1\5!bis"0\0!dimethylethyl#!3!methylphenol "Peak 6# was signi_cantly greater\ com!pared with the equivalent standard irradiance\ highiron cells "Table 1#[

DISCUSSION

The production of a large range of volatile com!ponents from phytoplankton species has been welldocumented ð6\ 08Ð12Ł[ Several VOCs detected in M[aeru`inosa in the present study\ such as b!cyclocitral\b!ionone\ 2!pentene!1!one and the aliphatic hydro!carbons "Tables 0 and 1#\ were consistent with volatilecomponents previously reported for Microcystis spec!ies ð2\ 00Ł[ However\ 5!methylhept!4!en!1!one and ger!anyl acetone were not detected in samples from thisstudy[ It may be possible that the production of theseVOCs were reduced after successive generations in

K[ WALSH et al[0125

culture[ The production of the numerous naph!thalene!derived or terpenoid compounds detected inour study has only been previously reported in _eldsamples of M[ aeru`inosa ð19\ 10Ł and not in lab!oratory cultures[ Comparison of the mass spectra ofthese naphthalene and terpenoid compounds\ andtheir common fragmentation ions "such as m:z � 008\050 and 193\ Fig[ 1#\ illustrate the structural similarityof these VOCs in M[ aeru`inosa[ Furthermore\ VOCcomposition and total volatile concentration alteredwith both high iron and high irradiance conditions\suggesting that photooxidative conditions may pro!mote the production of nuisance VOCs from cyano!bacterial sources within the natural environment[

Although phthalate derivatives can be indicativeof sample contamination with plastics ð13Ł\ diisoocytlphthalate\ diethyl phthalate\ diisobutyl phthalate anddibutyl phthalate have been detected in cyanobacterialextracts from a freshwater lake in Canada ð19Ł anddimethyl phthalate is a metabolite of the fungus\ Gib!berella fujikuroi ð14Ł[ All care was taken to avoid theuse of plastics during sample preparation[ Addition!ally\ the abundances of the phthalate componentswere observed to ~uctuate in a similar manner tomany of the remaining VOCs "with respect to theinternal standard# during the irradiance and irontreatments[ Consequently\ the diethyl and bis"1!methylpropyl# phthalates detected in the cyano!bacterial extracts were considered to be metabolicproducts of M[ aeru`inosa[

Terpenoids\ such as cedrol "Peak 06#\0\1\2\3\4\5\6\7!octahydro!0\3\8\8!tetramethyl!3\6!methanoazulene "Peak 8# and thujopsene "Peak 00#\were observed in the volatile fraction of M[ aeru`inosafrom our study "Fig[ 0#[ These terpenoids have pre!viously been characterised from plant volatile extractsincluding cedar\ cypress and the Japanese Hiba tree\respectively ð14Ł[ Cedrol\ a C04 sesquiterpenoid\ is alsoreferred to as cypress or cedar camphor ð14Ð16Ł[ Someterpenoids are thought to be allelopathic agents[Exposure to volatile terpenes can lead to the accumu!lation of lipid globules in the cytoplasm\ reduction inorganelles\ including mitochondria\ and disruption ofmembranes surrounding mitochondria and nucleið02Ł[ The terpenoids observed in M[ aeru`inosa maybe phytotoxic to other phytoplankton and:or zoo!plankton grazer species or may be released as a resultof cellular stress initiated by photooxidation\ but theexact function of these compounds still remainsunclear[

Heptadecane was the major volatile componentdetected within the cells after exposure to the iron andirradiance treatments[ The prominent appearance ofheptadecane is a characteristic marker of most cyano!bacteria ð10Ł and may be an important constituentof their membrane[ Production of long!chain alkanesoccurs in higher plants\ fungi\ bacteria\ algae\ insectsand animals\ often as components of waxes "for abarrier against water!exchange# or as constituents inthe myelin sheaths of peripheral nerves ð17Ð29Ł[ The

formation of long!chain alkanes were originallythought to occur via decarboxylation of fatty acidsð17Ł[ However\ although the reaction mechanism hasnot been fully elucidated\ there is evidence that theproduction of long\ odd!chain hydrocarbons\ such asheptadecane\ are derived during the enzymic decar!bonylation of aldehydes generated from an even!chain\ precursor fatty acid ð18Ł[ In addition\ metalchelators\ such as EDTA\ were found to stronglyinhibit the production of these alkanes\ suggestingthat the decarbonylase reaction is catalysed by a met!alloenzyme ð18\ 29Ł[ Consequently\ the observedincrease in the production and:or release of hep!tadecane after prolonged sunlight exposure in the highiron cells from our study\ may be due to the stimu!lation of a metalloenzymic decarbonylase within thecyanobacteria[

The production of heptadecane from fatty acids vialipid peroxidation processes is unlikely to occur in M[aeru`inosa[ This is because during decarboxylation\the parent unsaturated fatty acid would be requiredto have a chain length equal to C19ÐC11\ with a doublebond close to the carboxyl group\ to yield a hydro!carbon with a chain length of C06[ Since these types offatty acids were not detected in abundance in theextracts from M[ aeru`inosa ð20Ł\ the formation of thehigh concentrations of heptadecane observed in thepresent work are unlikely to have originated directlyfrom photooxidation and lipid peroxidation[ Furtherconsideration as to the biochemical role and the mech!anism of production of heptadecane in M[ aeru`inosais therefore warranted[

Release of heptadecane into the surrounding watermay act as an allelopathic agent as the general bioac!cumulation of aliphatic hydrocarbons are well knownto be toxic ð13\ 21\ 22Ł[ For example\ unsaturatedhydrocarbons\ such as myrcene\ limonene and a!phel!landrene\ have been reported to cause a 49)reduction in seed germination at concentrations of 1mM ð02Ł[ a!Phellandrene has a structure resemblingthat of 0\3!diene!1\5!bis"0\0!dimethylethyl#!1\4!cyclohexadiene "T2\ Table 0 and 1# and may thereforehave an allelopathic function in cyanobacteria againstcompeting phytoplankton species[ The volatile com!pound\ 2!penten!1!one "Peak 12# may have a similarfunction as 3!methyl!2!penten!1!one "mesityloxide#\which is produced and excreted in large quantities bythe cyanobacterium\ Anabaena cylindrica ð00Ł[ Mes!ityloxide is thought to be a metabolite originatingfrom fatty acid biosynthesis in cyanobacteria ð00Ł[Growth rates of cyanobacteria "Synechococcus# arenot a}ected by concentrations up to 099 mg l−0 ofmesityloxide ð00Ł[ Consequently\ this volatile com!pound may be either a degradation product of fattyacids and:or be used as a defence against other species[

Alterations in the production of volatile com!ponents were observed during exposure to the sunlightirradiance in the high iron cells[ The increase in theconcentration of the total volatile components afterexposure to both the sunlight and standard irradiance

Irradiance and cyanobacterial volatile compounds 0126

in M[ aeru`inosa was predominantly due to theincrease in heptadecane "Peak 11#\ 0\1\2\3\3a\4\5\7a!octahydro!6!methyl!3!methylene!0!"0!methylethyl#naphthalene "0# and "1# "Peaks 07 and 08#\ and thu!jopsene "Peak 00#[ The increase in the total volatilecomponents with time in the low iron cells during thesunlight exposure and the high iron cells during boththe irradiance regimes\ suggests that the length ofirradiance exposure is important in the productionand release of VOCs "Tables 0 and 1#[ Furthermore\alterations in the concentration of numerous indi!vidual volatile components were observed to be time!dependent\ particularly in the low iron cells[ Thesetime!dependent alterations in the volatile compositionof M[ aeru`inosa were concluded to occur primarilyin response to sunlight irradiance in the low iron cells[In addition\ several VOCs were not detected in thehigh iron cells after either the standard or sunlightirradiance\ demonstrating that incubation in the highiron media promoted a di}erent VOCs pro_le\ com!pared with the low iron cells[

Cleavage of b!carotene by b!carotene 6\7 "6?\7?#!oxygenase stoichiometrically yields two molecules ofb!cyclocitral and one molecule of crocetindial ð09\ 05Ł[However\ the oxidative cleavage of b!carotene duringincreased oxygen concentrations may also yield nor!carotenoid products including the b!apo!carotenalseries ð23Ł and b!ionone ð03Ł[ Nor!carotenoids have ademonstrated antimicrobial\ growth modifyingactivity against photoautotropic organisms\ bacteriaand fungi ð03Ł[ If these nor!carotenoids play an activeallelopathic role for M[ aeru`inosa\ then their pro!duction would presumably be under tight regulationand not rely entirely on photooxidation of caro!tenoids[ b!Cyclocitral and b!ionone may be formedby a carotene!oxygenase or by photooxidative degra!dation of b!carotene\ in addition to other cleavageproducts of the b!apo!carotenal series[ It was not poss!ible from the data obtained to determine the mech!anism of production of these nor!carotenoids but theydid indicate that the concentrations of b!cyclocitraland b!ionone were related to the concentration of b!carotene in the low iron cells "Table 0 and ð24Ł#[

The concentrations of b!cyclocitral and b!ionone"Figs 2A and B# from the low and high iron cellsexposed to the sunlight irradiance were lower thanthe respective standard treatments for 2\ 5 and 8 hexposure time[ In contrast\ b!cyclocitral and b!iononehave been detected in natural Australian waters inassociation with speci_c blooms of cyanobacteria ð2Ł[The presence of b!ionone has been shown to activatethe biosynthesis of b!carotene ð03Ł[ Alterations in theconcentrations of nor!carotenoids from M[ aeru`inosawere concluded to result from time!dependentexposure to sunlight irradiance\ which was furtherenhanced in the presence of high iron concentrations[

The detection of degradation products\ such asalkenes\ alkynes\ ketones and alkadienals "Tables 0and 1# is evidence for the peroxidation of unsaturatedfatty acids in M[ aeru`inosa exposed to pho!

tooxidative conditions ð25\ 26Ł[ Saturated and unsatu!rated\ short!chained alkanes\ alkenes\ ketones andreactive aldehydes "such as alkenals\ 1!alkenals and1\3!alkadienals#\ are recurrent oxidation products offatty acid chain cleavage ð8\ 26\ 27Ł[ The increase inthe alkyne and alkadiene components\ 1\2!trimethyl!0\3!hexadiene "Peak 0#^ 4!hexadecyne "Peak 11# and0\3!diene!1\5!bis"0\0!dimethylethyl#!1\4!cyclo!hexadiene "T2#\ as well as the ketone\ 2!penten!1!one"Peak 12#\ with time\ in the low iron cells may bedue to prolonged irradiance exposure[ However\ lipidperoxidation radical products are known to be short!lived "−09−8 s# ð06Ł[ Many of the degradation prod!ucts detected were absent from the high iron cellsexposed to 8 h irradiance\ suggesting that under theseconditions\ formation and consequently breakdownof these degradation compounds may have occurredmore rapidly[ In addition\ the formation of morestable peroxidation by!products\ such as malon!dialdehyde\ traditionally detected using non!volatileextracts\ can reach a maximum concentration afteronly 0 h "in rat liver microsomes# ð28\ 06Ł\ comparedwith the 8 h exposure periods in our study[ Conse!quently\ the detection of lipid degradation productswas proposed to be reliant on the length of irradianceexposure and the concentration of pro!oxidantcations\ such as iron[

In conclusion\ alterations in the production ofVOCs were observed in the cells exposed to sunlightand:or incubated in the high iron media compared tothose cells from the standard conditions[ Further!more\ the concentration of many volatile componentsincreased with sunlight exposure over time in the lowand high iron cells[ Microcystis aeru`inosa exposed tothe high iron concentrations produced signi_cantlyhigher concentrations of the total VOCs than the equi!valent low iron cells after 8 h exposure time[ There!fore\ exposure to high solar irradiance and high ironconcentrations altered the volatile composition of thecyanobacteria[

An increase in the concentration of VOCs fromcyanobacterial sources as a result of natural pho!tooxidative conditions prevalent during the Aus!tralian summer months\ could lead to problematictaste and odour problems in water supplies[ Fur!thermore\ there is potential to use VOCs as sensitivebiomarkers for some species of cyanobacteria ð2Ł[Consequently\ further examination of the environ!mental and biological factors which in~uence the pro!duction of volatile compounds from cyanobacteriamay serve to further enhance water!treatment facili!ties[

EXPERIMENTAL

Culturin` and irradiance exposure

Stock cultures of M[ aeru`inosa "strain MASH90!CSIRO Culture Collection\ Hobart\ Australia# weregrown and maintained in 499 ml conical ~asks con!

K[ WALSH et al[0127

taining 299 ml of either standard ASM medium ð39Łmodi_ed by substitution of metal stocks with MBLtrace metals "0 mM FeCl2# ð30Ł or iron!enriched ASMmedium containing an additional 29 mM iron as ferriccitrate[ Cultures were grown at 14> under 01]01 cyclicillumination using cool white ~uorescent lamps andan incident photon ~ux density of 24Ð34 mmol m−1

s−0[ Cultures grown in low and high iron media weredirectly transferred from conical ~asks to open PyrexPetri dishes "49×199 mm# immediately prior toirradiance exposure at the same iron concn[ Cells inthe original low and high iron media were exposedto either standard arti_cial irradiance "as for stockcultures# or sunlight regimes for either 2\ 5 or 8 hexposure time during the summer months of Octoberto mid!March "0883Ð0884#[ Temperature andirradiance were recorded regularly throughout thesunlight exposure periods[ Attempts to minimisetemp[ ~uctuations during sunlight exposure were car!ried out by placing each Petri dish inside a larger\water!_lled Petri dish lid[ All cyanobacterial sampleswere maintained at −19> after irradiance exposure insealed glass Schott bottles "49 ml# containing 1 dropsof 0 mM HgCl1 to prevent bacterial growth priorto extraction[ Cultures exposed under the standardculture conditions "standard irradiance and low iron#\served as a ref[ for comparison with the highirradiance "sunlight# and high iron treatments[ Theterms {{high|| and {{low|| concn are used only in arelative sense[ The low iron concn corresponds to thatpresent in ASM culture medium "0 mM FeCl2#\whereas the high iron treatment contained anadditional 29 mM ferric citrate in ASM medium "_naliron concn of 20 mM#[ Consequently the iron concnsemployed were within a range comparable to concnsmeasured in Australian inland waterways ð07Ł[ Sun!light exposure in the low iron expt had incident radi!ation ranging from 0099Ð0799 mmol m−1 s−0\ with anaverage irradiance of 0329 mmol m−1 s−0[ During the8 h exposure in low iron\ the temp[ of the culturesincreased from 14> to 33>\ with an average temp[ of21>[ Sunlight exposures in the high iron expt had inci!dent radiation ranging from 799Ð0899 mmol m−1 s−0\with an average irradiance of 0409 mmol m−1 s−0[ Thetemp[ of the high iron cultures increased from 14> toa maximum of 31>\ with an average temp[ of 28>[Whilst these temp[ changes were not ideal from acontrolled experiment perspective\ they do re~ect con!ditions experienced by a surface scum on a hot Aus!tralian summers| day[

Extraction of volatile odour compounds

VOCs from M[ aeru`inosa exposed to sunlight orstandard irradiance and grown in either the low orhigh iron media were extracted within 37 h after har!vest via closed loop stripping and analysed by GC!MS ð31Ł[ This involved the release of VOCs duringsample aeration and concn onto an activated carbon_lter[ Brie~y\ ca 49 ml sample was dild to 899 ml with

Milli Q H1O with subsequent addition of dry NaCl"89 g# and the int[ standard\ deuterated geosmin "8ml\ 30[5 ng mg−0#[ Prior to extraction\ contaminatingorganic components present in the head space of theapparatus were removed onto an axillary carbon _lterby commencing stripping for 09 s[ VOCs were col!lected during stripping via sparging with air onto asecond\ clean\ activated carbon _lter maintained at ca39>[ Cyanobacterial samples were maintained at 14>during the closed loop stripping process "89 min#[After closed loop stripping\ the activated carbon _lterwas washed ×2 with CS1 to concentrate the VOCsand the sample was then stored prior to analysis"−39>#[ The sample was manually injected "0 ml# with!out derivatisation[

GC!MS

VOCs were separated according to the method ofref[ ð31Ł[ The GC was _tted with a fused silica capillarycolumn "14 m\ 9[19 mm int[ diameter\ coated with9[29 mm cross!linked Me silicone# and split:splitlessinjector[ The GC!MS was run with the following set!tings^ inj[ temp[ 159> and a 0[4 min hold at 14> withthe splitter o} for 0 min[ The splitter was then turnedon and the column heated from 14Ð79> at 09> min−0\and then 4> min−0 to 149>\ with a 4 min hold at 149>[

Volatile components were identi_ed by comparisonof their MS with ref[ library spectra and the RIs ofavailable standards "b!cyclocitral\ b!ionone and thealiphatic hydrocarbons#[ Concentrations of volatilecompounds were calculated with respect to the concnof the int[ standard "d2!geosmin#[

Acknowled`ements*We gratefully thank Cheryl Orrfor assistance with volatile odour compound analysisand Bruce Petersen and Bruce Cole for helpful dis!cussions[ This research was supported by a grant fromHunter Water\ Newcastle\ Australia[

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