Lipid composition of a Recent Polynesian microbial mat sequence

Advances in Organic Geochemistry 1985 Org. Geochem. Vol. IO. pp. 705-709. 1986

0146-6X30/86

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Lipid composition of a Recent Polynesian microbial mat sequence J. P. BOUDOU’, J. TRICHET~, N. ROBINSON~ and S. C. BRASSELL) ‘Universite de Paris VI, CNRS L.A. 196, Laboratoire de GCochimie et Mttallogenie, 4, Place de Jussieu, 75230 Paris Cedex 05, France ‘Universitt d’orlians, CNRS UA 724, Laboratoire de Geologique Appliqule, 45046 Orleans, France ‘Organic Geochemistry Unit, University of Bristol, School of Chemistry, Canto&s Close, BS8 ITS, U.K. (Received

5 November

1985; accepred

I8 March

1986)

Abstract-The extractable lipid composition of four layers of a microbial mat from Hao, French Polynesia, shows differences that reflect both the imprint of their microbial populations and the selective diagenetic transformations of specific microbial compounds. The uppermost layers, principally composed of cyanobacteria and other bacteria, contain lipids typical of such microbes, namely n-heptadecane, heptadecene, hexadecanoic acid, and various sterols. With increasing depth the selective degradation of lower n-alkane homologues occurs; n-alkenes also show enhanced degradation. The predominant sterols in the bottom horizon of the mat are C,, and C,, components, including dinosterol and other 4-methylsterols presumably derived from dinoflagellates. In addition, there is an increase in the proportion of stanols with depth, perhaps arising from preferential degradation of

As-stenols. Key

words:

microbial mat, Hao, French Polynesia, lipids, microorganisms, cyanobacteria, diagenesis,

sterols

INTRODUCllON

eral descriptionof thesemats and their hydrocarbon characteristicsis given elsewhere(Boudou et al., in Microbial mats are simple ecosystemsof especial interest to environmental biogeochemists.Their in- press).In brief, thesemicrobialmatsarepopulatedby dividual layers tend to be populated by specific stratified communitiesof aerobiccyanobacteriaand pigmentedphotosynthetic purple bacteria, underlain organisms(e.g. cyanobacteria,purple photosynthetic bacteria, sulphate-reducingbacteria) so that differ- by anaerobicbacteria in the deepersediments. Herein, we evaluate the extractable lipid comencesin the organic matter of various mat horizons position of the mat horizonsand assess the microbial can be assessed in terms of the contributions from, transformations affecting these compounds. and effectsof, thesedifferent microorganisms.Studies of microbial mats also bear on the nature of PreEXPERIMENTAL PROCEDURES cambrian environments,since they are present-day analoguesof ancient stromatolites. Sample details Many previous studieshave been performed on The collection and description of the four sediment layers various contemporary microbial mat systems,no- is described elsewhere (Boudou et al., in press). In summary, tably from Baja California, Western Australia, Abu the four sections comprise: the uppermost green layer of cyanobacteria (layer G; &I .Ocm); a pink-violet layer Dhabi, Texaslagoonsand Gavish Sabkha(documen- living of Diamented nhotosvnthetic bacteria (layer V; I .0-l .2 cm); ted in Boudou et al., in press).They have shownthat the‘ ipper and lowe; sections of underlying red sediment lipids from modem microbial mats present some (layers RI and R2; 1.2-4.5 and 4%l2cm, respectively). similaritiesand can be correlatedto a limited extent Lipid extractions, separations and analyses with those from Precambrianstromatolites (Philp, Freeze-dried samples were extracted (CHCl,/MeOH, 2:3) 1980).More recently, Boon et al. (1983)have made by ultrasonication, yielding crude lipid extracts which were a detailedstudy of the laminatedmicrobial mat from separated on an alumina column to give fractions containthe hypersaline,mesothermalSolar Lake, Sinai. They ing hydrocarbons (hexane), wax esters (hexane-diethylether, soughtto evaluate the altered molecular“signature” 9: 1) and .oolars (methanol). The polar fractions were separateb into neutrals and acids by partitioning between aqueof the original cyanobacterial ecosystemand found ous KOH and hexane-diethylether. The acid fractions obthat many molecularmarker compoundsof the top tained were methylated (BFjMeOH) prior to GC analysis. mat living community are quickly transformed or The neutral fractions were separated into ketones and alcohols by preparative TLC (silica gel G, hexanecompletely degradedduring burial. 2: I) and the latter were derivatised with In this paper we report the extractable lipid com- diethylether BSTFA to form TMS ethers prior to GC analysis. positionof four layersof a particular non-hypersaline All fractions were analysed by gas chromatography (GC) microbial mat from Hao, French Polynesia.A gen- and Computerised-GC-MS (C-GC-MS). GC analysis, 705

706

J. P. BOUDOU et al. Table

I. Abundance

of the extractable Mation from GC) Abundances”

G V RI R2 “Obtained

(pg gg’ dry, extracted Wax esters t carboxylic acids

Hydrocarbons

Seclion

lipid fraclions and isolated of Hao microbial mat

2600 6900 2500 820

690 1500 970 800 by summing

concentrations

using on-column injection was carried out on an Erba Science 4160 instrument fitted with an open tubular flexsil column (25 m x 0.3 mm i.d.), wall-coated with OV-I. A typical run wasprogrammed from 60 to 280°C at 4°C mm-’ and held isothermally at 280°C. H, was used as carrier gas. CCX-MS analyses were performed on a Finnigan 4000 mass spectrometer coupled to a Finnigan 9610 gas chromatograph with on-line INCOS 2300 data system (Brassell er al., 1980). GC conditions were similar to those described above, with the column leading into the ion source of the mass spectrometer.

Compound

iden@cation

and quanritation

Individual lipid components were identified and quantified according to previous criteria (Boudou er al., in press).

of individual

classes

(quan-

sediment) Ketones

Alcohols

II I2 I7 15

780 I600 I200 860

components.

alkanes. Among the n-alkanes, the higher oddcarbon-number homologues(i.e. n -Cr,, n-C,, and n-C,,), presumably derived from vegetation surrounding the pond, become progressively more significantrelative to lower homologues,sothat n-C,, is the dominant alkane in the deeper Rl and R2 layers. The C,, and Cg methyl mid-chain branched alkanesare thought to originate from cyanobacteria (Han et al., 1968;Boudouet al., in press)and appear to persistwith depth relative to their linearanalogues, indicating their preferential survival. Desmethylsterols

Unlike the majority of prokaryotes, cyanobacteria biosynthesise significantamountsof sterols,producRESULTS AND DISCUSSION ing desmethylsterols,often 24-methyl and 24-ethyl In this communication we concentrate on the substituted,with double bondsin any of the 5, 7 or overall lipid concentrations and the distributions of 22 positions(Nes and McKean, 1977).Thesesterols, hydrocarbons and steroidal alcohols and ketones, however, are not specificto cyanobacteriaand their plus other componentswhere appropriate. occurrencein the mat layers may also reflect contributions from other aquatic microorganismsand Total lipid profile from higher plants. Despitetheir relative abundance The extractable lipids of the samplesvary both in in cyanobacteria,A’,‘-sterolswerenot detectedin the their total amountsand in the relative quantities of mat, except for a trace of the Cr7compoundin the G the individual lipid fractions (Table 1). The highest layer. This observationisconsistentwith the reported concentration is observedin the pigmentedpurple rapid removal of suchlabile sterolsin other aquatic photosynthetic bacterial layer (V) where wax esters environments (Robinson et al., 1984a).Diagenetic and carboxylic acids dominate. In the lower layers transformationsof sterolswere observedto proceed this lipid fraction decreases most markedly, in terms with depth in the Hao microbial mat. Thus, A’-sterols of their absoluteand relative abundance,presumably dominate the distribution in the cyanobacterial mat due to diagenetic alteration and/or incorporation layer (G), whereasin the bottom horizons (RI and into kerogen. Thus, the concentration profiles pro- R2) AS-sterolsand 5cr(H)-stanolsare equally promivide a crude assessment of the extent of reworking/ nent (Fig. 1; Table 2). The similarities in their incorporation of organic matter in the mat layers. distributions and the increasein the proportion of A more detailed appraisal can, however, be made stanolswith depth provides supportive evidencefor from the changesin the distributions of individual microbially-mediatedsteno1to stanol conversionin compounds. the upper aerobic mat layers (cf. Gaskell and Eglinton, 1976).The sterolsof this mat appearto be Hydrocarbons affected by selectivediageneticprocesses sincethere Detailed descriptionsof the hydrocarbon profiles is a shift towards a greater proportion of C19and C,, in the Hao microbial mat are given elsewhere compoundsat the expenseof Cr7and CZssterolswith (Boudou et al., in press). In summary, the hydro- increasingdepth (see Fig. 1). The occurrence of carbon compositions show marked changeswith A*-sterenesdominated by C27 components in the depth, despitethe similarity in the total amountsof lower mat layer suggeststhat sterol dehydration hydrocarbons in the four mat levels (Table 1). In might preferentially affect Cr7 homologues, a the uppermost, cyanobacterial layer, the aliphatic phenomenonthat has also been observed in Solar hydrocarbonsare dominated by componentstypical Lake microbial mat (Boon et al., 1983), but not, of such organisms,namely n-C,, and C,,:,. With as yet, in open marine environments(Brasselland increasingdepth the dominanceof theseconstituents Eglinton, 1983). Perhaps the reduction of stenols disannears: the alkenes more ranidlv than the enhancesthe pool of stanolsavailable for dehydra.

I

Lipids of a Polynesian microbial mat

707

Table 2. IderMies Peak (Fig. I)

JL

OLi

1

C27

5

C*7

15 I6 I7

so0

z

2 3 4

8 9 IO II I2 13 I4

75-

i

G,

6 7

IOO-

I8 I9

25-

20

o-

l-l

21

RI

75

I 50

22 23 24 25

C,,

C*7 CU C,, C,, C*, C28 C*, G8 C**

Cl8 C29 C*9 CD -C G9 C*9 CZ9 { C29 CM

CM C29 C,,

of Hao

microbial

mat

Assignment SB(H)-Cholesr-7-en-38-o1 SB(H)-Cholestan-38-01 Unknown Cholesla-5,22-dien-3p-ol Cholest-5-en-3fi-ol 51r(H)-Cholestan-38-01 ZCMethylene-Sa(H)-choleslan-38-01 Unknown 5a(H)-s~m-3fi-ol Cholesla-5.7-dien-38-01 Unknown 5a(H)-slan-3p-ol 24-Methylcholesta-5.22-dien-3P-ol 5a(H)-Cholest-7-en-3P-ol 24-Methylenecholest-5-en-3/J-ol 24-Methylcholesc-5-en-3b-ol 24-Methyl-Sa(H)-cholestan-38-01 23,24-Dimethylcholesta-5.22-dien-3p-ol [email protected] 4cr,24-Dimethyl-Scl(H)-cholest-22-en-3/3-ol 24-Ethyl-501 (H)-cholest-22-en-3/3-ol 24-Ethylcholest-5-en-3/3-ol 24-Ethyl-5a(H)-cholestan-3j-ol 4a,24-Dime~hyl-5a(H)-cholestan-3~-ol 4u.23.24-Trimelhylcholesla-5.22-dien-3p-al’ 4u,23.24-Trimelhyl-5a(H)-cholesl-22-en-3/3-ol” Unknown 24-Elhyl-5ol(H)-cholest-7-en-3/3-o1 Unknown 4-methylslerol

“A’-Dinosterol. ‘Dinosterol.

25 01

50

C No.

I

of swols

L

J

1

III

I

Llll R2

I

c

might be symbiontsof higherorganisms.As with the desmethylsterols,a preferential lossof A5-dinosterol (peak 21 in Fig. 1; Table 2) relative to dinosterol (peak 22 in Fig. 1; Table 2) is observed. Steroidul

25

ketones

tion to stereneswith C2, sterolsbeing reducedat a faster rate.

Steroidal ketonesare presentin the microbial mat in small quantities, but increasewith depth (Fig. 2; Table 3). In all layers, steroidal ketonedistributions are dominated by 5a (H)-cholestan-3-one.The levels of Cz9steroidal ketones, however, increasesignificantly in the lower horizons of the mat. Suchdistributionsmay resultfrom a preferentialdegradationof lower sterol homologuesin the upper mat layers(cf. Edmundset al., 1980).

4a-Methylsterols

Other components

4a-Methylsterols are presentin low abundancein the upper greenlayer, but becomemajor compounds in the bottom mat (Table 2; Fig. 1); thus,they appear to be relatively resistant to degradation compared with other sterol components(cf. Robinson ef al., 1986).The most likely sourceis from dinoflagellate input (Boon et al., 1979;Robinsonet nl., 1984b).A similar distribution of 4a-methylsterolshave, however, been found in Solar Lake microbial mats, although dinoflagellatesare not observedamongthe presentphytoplankton assemblage (Edmunds,1982). Similarly, in Hao ponds dinoflagellateshave never beenreported, nor in other French Polynesianponds. As the ecology of the Hao microbial mat is very variable (Defarge, 1983),the Dinophyceae might have beenpresentin the pondsduring someperiodswhich have not yet been investigated. Alternatively, they

The presenceof higher plants close to the Hao microbialmat isevidencedby the detectionthereinof derivatives of oxytriterpenoids, including amyrins, amyrenones,friedelan-3-oneand various other pen-

0i

II

IL

5

111111111

IO

15

20

I

I

I

25

Fig. 1. Histogram of sterol distributions in the four layers of Hao microbial mat. Compound assignments are given in Table 2.

tacyclic

non-hopanoid

triterpenoids

in low

abun-

dance. The n-C,0 alkanol becomesdominant in the bottom mat, due either to an increasedhigher plant contribution in the past, or to preferentialmicrobial degradation of lower homologues,especiallythe nC,, alkanol. The extendedhopanoid 178(H),2 1p(H)bishomohopan-32-01, which is probably herederived from microbial degradation of the polyhydroxybacteriohopanes of cyanobacteria,plusother aerobic or facultative anaerobicbacteria,is presentin minute concentrationsin the top mat, but becomesa major compoundin the bottom mat. A similarobservation has been made for Solar Lake (Boon el al., 1983).

708

J. P. Bouwu

et al. Table

G 4-

Peak (Fig. 2)

3-

I 2 3 4

2-

5

3. Identities

G,

O-

8

-

9 IO II I2 I3 I4 I5 I6 I7 I8

ketones

C No.

6 7 I-

of steroidal

C27 C, C, C, CL GO C29 L1

of Hao

microbial

mat

Assignment SB(H)-Cholestan-3-one Sa(H)-Cholestan-3-one Unknown Stan-3-one 24-Methyl-S/J(H)-cholestan-3-one 24-Methyl-k(H)-cholestan-3-one 24-Ethyl-5,9(H)-cholestan-3-one 24-Ethyl-SK(H)-cholestan-3-one 24-Methyl-5a(H)-cholest-22-en-3-one Cholesta-3,5-dien-7-one 4a-Methyl-Sa(H)-cholestan-3-one 4a,24-Dimethyl-Sa(H)-cholestan-3-one Cholest-Cen-3-one 23,24-Dimethyl-SE(H)-cholestan-3-one 24-Ethyl-Sa(l&zholest-22-en-3-one 23,24-Dimethyl-Sa(H)-cholest-22-en-3-one 24-Ethvlcholesta-3.5-dien-7-one 4a,23,i4-Trimethyi-Sa(H)-cholestan-3-one 24-Ethylcholest-7-en-3-one

characteristics in terms of the occurrence and abundance of specific lipid types. Such variation suggests that there are fundamental differences between the contributions of particular organisms to each series of microbial mat. Acknow1edgements-C-GC-MMS data were’obtained using the NERCfunded facility (Research Grants GR3/2951 and GR3/3758 to Professor G. Ealinton) in Bristol. We thank the SERC for a studentship (%R). REFERENCES

Fig. 2. Histogram of steroidal ketone distributions in the four layers of Hao microbial mat. Compound assignments are given in Table 3.

Boon J. J., Rijpstra W. I. C., De Lange F., De Leeuw J. W., Yoshida M. and Shimizu Y. (1979) The Black Sea sterol-a molecular fossil for dinoflagellate blooms. Nature 277, 125-127. Boon J. J., Hines H., Burlingame A. L., Klok J., Rijpstra W. I. C., De Leeuw J. W., Edmunds K. E. and Eglinton G. (1983) Organic geochemical studies of Solar Lake laminated cyanobacterial mats. In Advances in Organic Geochemistry 1981 (Edited by Bioray M. et al.), pp. __ 207-227. Wiley, Chichester. - - Boudou J. P.. Trichet J.. Robinson N. and Brassell S. C. (1986) Profile of aliphatic hydrocarbons in a recent Polynesian microbial mat. lnf. J. Environ. Anal. Chem. In press.

Brassell S. C. and Eglinton G. (1983) Steroids and triterpenoids in deep sea sediments as environmental and diagenetic indicators. In Advances in Organic Geochemistry 1981 (Edited by Bjoroy M. et al.), pp. 684-697. Wiley, Chichester. CONCLUSIONS Brassell S. C., Gowar A. P. and Eglinton G. (1980) Computerised gas chromatography-mass spectrometry in The lipid compositions of layers of the microbial analyses of sediments from the Deep Sea Drilling Project. mat from Hao show two major features: (i) the In Advances in Organic Geochemistry 1979 (Edited by selective degradation of particular components, Douglas A. G. and Maxwell J. R.), pp. 421-426. notably lower n-alkanes, alkenes and AS-stenols, and Pergamon Press, Oxford. Defarge C. (1983) Contribution a l’ttude giochimique et (ii) the selective preservation of specific components, petrologique des formations protostromatolitiques de such as long-chain n-alkanes, mid-chain branched Polynbie. Application a la connaissance des mecanismes alkanes and higher-carbon-number sterols. Overall, de la precipitation des carbonates de calcium au sein de the lipid distributions show several similarities to matieres organiques stdimentaires. Ph.D. Thesis, Universite d’orltans, France. other microbial mat sequences, such as Solar Lake Edmunds K. L. H. (1982) Organic geochemistry of lipids (Boon et al., 1983). Yet, the organic geochemical and carotenoids in the Solar Lake microbial mat investigations performed to date of such environsequence. Ph.D. Thesis, University of Bristol, U.K. ments,from diversegeographicalareas,revealseach Edmunds K. L. H., Brassell S. C. and Eglinton G. (1980) mat to exhibit, to a greater or lesserextent, unique The short-term diagenetic fate of Sa (H)-cholestan-3/I-ol:

Lipids of a Polynesian microbial mat in sifu radiolabelled incubation in algal mats. In Advances in Organic Geochemisrry 1979 (Edited by Douglas A. G. and Maxwell J. R.), pp. 427-434. Pergamon Press, Oxford. Gaskell S. J. and Eglinton G. (1976) Sterols of a contemporary lacustrine sediment. Geochim. Cosmochim. Acta 40, 1221-1228. Han J., McCarthy E. D., Van Hoeven W., Calvin M. and Bradley W. H. (1968) Organic geochemical studies, II. Preliminary report on the distribution of aliphatic hydrocarbons in algae, in bacteria and in a Recent lake sediment. Proc. Nor. Acad. Sci. U.S.A. 59, 23-33. Nes W. R. and McKean M. L. (1977) Biochemistry of Steroids and Other Isopentenoidr, 690 pp. University Park Press, Baltimore. Philp R. P. (1980) Comparative geochemical studies of recent algal mats and sediments of algal origin. In Biogeochemitry of Ancient and Modern Erbironments (Edited by Trudinger P. A., Walter M. R. and Ralph B. J.), pp. 173-185. Aust. Acad. Sci., Canberra.

709

Robinson N., Cranwell P. A., Finlay B. J. and Eglinton G. (1984a) Lipids of aquatic organisms as potential contributors to lacustrine sediments. In Advunces in Organic Geochemistry I983 (Edited by Schenck P. A., De Leeuw J. W. and Lijmbach G. W. M.). Org. Geochem. 6, 143-152. Pergamon Press, Oxford. Robinson N., Eglinton G., Brassell S. C. and Cranwell P. A. (1984b) Dinoflagellate origin for sedimentary C-methylsteroids and Sa (H)-stanols. Nature 308, 439-441. Robinson N., Cranwell P. A., Eglinton G., Brassell S. C., Sham C. L.. Gonhen M. and Pollingher U. (1986) Lipid geochemistry of Lake Kinneret. in Advances ii Organic Geochemistry 1985(Edited by Leythaeuser D. and Rullkiitter J.), pp. 733-742. Pergamon Journals, Oxford. Trichet J. (1970) Etude des premiers stades d’ivolution de la matitre organique dans des mares en milieu recifal (Polynesie francaise). In Advances in Organic Geochemistry 1966 (Edited by Hobson G. D. and Spcers G. C.), pp. 265-284. Pergamon Press, Oxford.