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PII S0016-7037(00)00582-2

The chemical structure of Gloeocapsomorpha prisca microfossils:

Implications for their origin

PETERBLOKKER,1,

* PIM VAN BERGEN,2

RICHPANCOST,1,

MARGARET E. COLLINSON,3

JAN W. DELEEUW,1,2

and JAAPS. SINNINGHE DAMSTE1,2

1Department of Marine Biogeochemistry & Toxicology, Netherlands Institute for Sea Research, PO Box 59, 1790 AB Den Burg, Texel,The Netherlands

2Department of Earth Sciences, Utrecht University, PO Box 80021, 3508 TA, Utrecht, The Netherlands3Department of Geology, Royal Holloway University of London, Egham, Surrey, TW20 0EX, UK

(Received May 1, 2000; accepted in revised form July 23, 2000)

AbstractTwo Estonian Kukersites (Ordovician) and two samples from the Guttenberg Member (Ordovi-cian) of the Decorah formation (North America) containing botryoidal aggregates ofGloeocapsomorphaprisca were investigated by RuO4 chemical degradation, FTIR, and flash pyrolysis-GC/MS to obtaininformation about the polymeric structure of these microfossils. The products formed upon oxidation by RuO4were analysed by GC/MS and revealed the presence of a wide range of carboxyl and/or carbonyl moietycontaining compounds with carbon skeletons ranging from C5 to C20. The Estonian Kukersites reveal thepresence of a characteristic set of mono-, di-, and tricarboxylic acids. These compounds suggest that theEstonian Kukersites are composed of a polymer consisting of mainly C21 and C23 n-alkenyl resorcinolbuilding blocks. Similarly, although the tricarboxylic acids are not present, the RuO4 degradation productmixtures of the Guttenberg Member samples, suggest a poly(n-alkyl resorcinol) structure. The higher thermalmaturity is most likely responsible for the different chemistry and morphology of theG. prisca microfossilsin these samples. Because compounds like n -alkenyl resorcinols are known to polymerise under oxygenatedconditions even in an aqueous environment, it is not per se necessary that these microfossils are composed ofa selectively preserved biopolymeric cell wall. It is also possible thatG. prisca microfossils are composed ofa cell wall or sheath component that polymerised during senescence or diagenesis of the organism. Copyright 2001 Elsevier Science Ltd

1. INTRODUCTION

The origin and chemical composition ofGloeocapsomorpha

prisca has long been a topic of debate in geochemistry andpalynology and many researchers have focussed on this subject

since the recognition of these microfossils in Ordovician sed-

iments (Zalessky, 1917). Zalessky (1917) made the first link

between the morphology of these microfossils and the extant

cyanobacteriaGloeocapsa and thus named this microfossil G.

prisca. Despite numerous investigations the biological affinity

of G. prisca is still uncertain (Fowler, 1992 for review). For

example, Reed et al. (1986) suggested a non-photosynthetic,

prokaryotic, benthonic, mat-forming organism while Hoffmann

et al. (1987) concluded that the organism was phototrophic

planktonic, and possibly eukaryotic. Foster et al. (1989, 1990)

proposed based on investigations of G. prisca in Estonian

samples, that it must have been a mat-forming cyanobacteria

similar to Entophysalis major from an intertidal environment.

They described three morphotypes designated as Type I, II, and

III. Stasiuk and Osadetz (1990) and Stasiuk (1991) suggested

that the microfossils probably originate from a photosynthetic

cyanophyte with a complex three-stage life cycle. Derenne et

al. (1990, 1992) suggested that the G. prisca microfossils are

the selectively preserved algaenan cell walls of a green mi-

croalga like Botryococcus braunii.

To date it is generally accepted that ancient microfossils are

often composed of selectively preserved nonhydrolysable and

insoluble biopolymers. In recent years, it has become evident

that resistant biopolymeric materials are fairly common innature, and especially green micro-algae from the order of the

Chlorococcales comprise several members biosynthesizing

such a diagenetically resistant material (algaenan) (Blokker et

al., 1998; Largeau and de Leeuw, 1995). To date, the mono-

meric components of a number of resistant biopolymers in

green microalgae have been elucidated. For example, B. brau-

niialgaenan is composed of mainly C32,-dialdehydes (Ber-

theas et al., 1999; Gelin et al., 1994; Metzger et al., 1991),

algaenans fromPediastrum, Scenedesmus,Sorastrum,Coelas-

trum, and Tetraedron are composed of even, unsaturated C30-

C34-hydroxy fatty acids (Blokker et al., 1998; Blokker et al.,

2000), and Nannochloropsis algaenan is composed of C28-C36

diols and C30and C32alkenols (Gelin et al., 1997). Because itwas shown that the isolation procedure for resistant materials

can give rise to the formation of artefacts (Allard et al., 1997),

the discovery of resistant biopolymers in cyanobacteria (Cha-

lansonnet et al., 1988) has become questionable. Consequently,

the cell walls of green microalgae seem to be one of the most

important sources of selectively preserved biopolymers espe-

cially in the lacustrine environment.

Due to the presence of a resistant algaenan in the green

microalga B. braunii and some morphological similarities be-

tween this alga andG. prisca,B. braunii, or a related alga was

suggested as a possible source organism for the G. prisca

microfossils (e.g., Traverse, 1955; Glikson et al., 1989;

* Author to whom correspondence should be addressed([email protected]). Present address: Organic Geochemistry Unit, University of Bristol,Cantocks Close, Bristol BS8 1TS, UK.

Pergamon

Geochimica et Cosmochimica Acta, Vol. 65, No. 6, pp. 885900, 2001Copyright 2001 Elsevier Science LtdPrinted in the USA. All rights reserved

0016-7037/01 $20.00 .00

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Derenne et al., 1992). However, Burns et al. (1982) suggested

that such resemblance is only superficial and probably errone-

ous. Derenne et al. (1992) observed that upon increasing salin-

ity of the B. braunii culture medium, a significant phenolic

component was biosynthesized. Because the presence of phe-

nolic compounds like n-alkyl-phenols and n-alkyl-resorcinols

in the pyrolysates of G. prisca is one of the most striking

chemical features of these microfossils (Derenne et al., 1990;1992) these findings are in favour of the B. braunii origin

theory. Although B. braunii was shown to grow under more

saline conditions, this microalgae is principally a freshwater

organism. In contrast, the deposition ofG. priscaoccurred in a

marine environment as indicated by intercalated limestones

containing the remains of typical marine fauna, indicating a

marine origin for this organism (Foster et al., 1989; Bekker,

1921).

The bituminous fraction of G. prisca-rich rocks typically

reveals an exceptional distribution of odd-over-even n -alkanes

ranging between C9and C19in carbon number and n -alkylcy-

clohexanes exposing a similar distribution (Fowler, 1992 and

references cited therein). To date, it is still unknown if thisfraction in some way reflects the chemical structure of the

kerogen. In general details about the polymeric structure ofG.

prisca microfossils are still scarce. Recently, improved tech-

niques have refined the knowledge of polymeric materials in

organisms and kerogen (e.g. Eglinton, 1994; Largeau and de

Leeuw, 1995; Blokker et al., 2000), which may assist in ob-

taining new information about G. prisca microfossils. In this

study RuO4 degradation is used as a tool to obtain additional

chemical information about the polymeric substance mak-

ing-up the G. prisca microfossils. This technique allows a

detailed look at the complex chemistry of these microfossils

and sheds new light on their biological affinity.

2. GEOLOGICAL SETTING

Both Estonian samples are from the Kohtla quarry, northeast

Estonia. The section in this quarry represents the lowermost

part (the Kivioli Member, Viivikonna Fm.) of the Kukruse

Stage. This age assessment is based on the palynology (under-

taken by Dr. Zwier Smeenk) and other known markers. This

section is part of Graptolite zone N. gracilus (Middle Lan-

deiloEarliest Caradoc Epoch; Middle Ordovician). Kukersite

II comes from a real kukersite layer (bed D in this quarry)

denoted as an argillaceous kukersite with sparse and few skel-

etal debris (TOC 2550%). This sample was collected 20 cm

above Kukersite I, which is from a kukersineous limestone

[Total Organic Carbon (TOC) 525%; bed C/D in this quarry]

characterized as a kukersineous fine-grained argillaceous skel-

etal wackestone.

The North American samples are taken from the Guttenberg

member of the Decorah Formation obtained from the Cominco

SS-9 core (205.4 m and 206.6 m), Millbrook Farms, (Jackson

Co., IA, USA). The Guttenberg Member is composed predom-

inantly of carbonate mudstones with occasional skeletal wacke-

stone and packstone lenses interpreted as tempesite beds (Lud-

vigson et al., 1996). In general CaCO3content ranges from ca.

40% to 80% and thin (ca. 1 m) brown-coloured G. prisca-rich

partings are common and associated with a high TOC content.

The TOC values in the Guttenberg member range from 0.5% to

40%. The organic-rich layers are associated with the presence

of G. prisca microfossils (Jacobson et al., 1988). In the Gut-

tenberg member, the organic matter is represented by stroma-

tolitic as well as disseminated A maceral based on the classi-

fication of Stasiuk et al. (1993). No closed (phenol-rich)

morphotype (Derenne et al., 1992) and disseminated B maceral

(Stasiuk et al., 1993) have been reported in these samples

(Pancost et al., 1998).

3. EXPERIMENTAL

3.1. Determination of Hopanoid Epimerisation

The G. prisca-rich rocks were ground with a mortar and subse-quently extracted with DCM/hexane (4, 1 : 1) and hexane (2). Afterevaporation of the solvent, the apolar hydrocarbon fractions wereobtained from the extracts by column chromatography on activatedAlO2 using hexane as an eluent (elution volume 4 column vol-ume). These fractions were analyzed by GC/MS. The 22S/(22S 22R)C31and C3217,21(H)-homohopane ratios were determined from theintegrated peak areas using the total ion cur (TIC) trace.

3.2. Rock-Eval

Rock Eval pyrolysis was performed following standard procedures(Peters, 1986).

3.3. G. prisca Microfossil Preparation

After grinding the sediments the samples were ultrasonically sus-pended in dichloromethane (DCM) and centrifuged for 1 min at 3000rpm. The DCM float consisted of pure G. priscamicrofossils as shownby light microscopy and was used for further analysis. To simulate theeffect of increased temperature on G. prisca, the Kukersite I samplewas heated under an N2 atmosphere at 100C for 10 h.

3.4. Microscopy

Light microscopy examination was performed on an Olympus BH

microscope (New Hyde Park, NY, USA). Specimens for SEM weremounted onto a negative film from a water droplet following themethods described by Moore et al. (1991) for modern pollen. Speci-mens for TEM were incorporated into an agar pellet by centrifugationand the pellet was embedded in Spurr resin. Sections 60-nm thick werecut by using a ReichertJung ultracut microtome, no fixatives and nostaining were used in order to examine the material as close to thechemically analyzed conditions as possible.

3.5. Fourier Transform/Infrared Spectroscopy

Fourier transform infrared spectroscopy was performed on a BrukerIFS28 scanning (Billerica, MA, USA) over a frequency range of400/cm to 4000/cm using KBr pellets containing 2 mg of dry sample.

3.6. Ruthenium Tetroxide Treatment

This treatment was modified from a procedure reported earlier(Blokker et al., 1998). Briefly: 5 mg of microfossils was ultrasoni-cally suspended in a mixture of 1 mL of chloroform, 1 mL of aceto-nitrile, and 2 mL of an aqueous solution of NaIO4 (0.2 M, pH 34).After addition of 6 mg Ru(III)Cl3, the two phase system was allowedto react in an ultrasonic bath. After 4 h, 3 mL of water, and 2 mL ofhexane were added. The organic layer was removed and quenched withmethanol (MeOH) (0.5 mL). The aqueous layer was extracted with 2mL of hexane (1) and 2 mL of dichloromethane (2) and theseorganic layers were transferred to the first organic extract. The blackRu-salts were precipitated from the organic layer by centrifugation. Theremaining supernatant was washed with 0.5 mL of Na2S2O3 solution(5% in H2O). The extract was dried over Na2SO4, evaporated todryness under a nitrogen flow and derivatized with diazomethane priorto analysis.

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3.7. GC/MS, GC/FID and Curie-point py-GC/MS

GC/MS, GC/FID, and py-GC/MS equipment and conditions were aspreviously described (Blokker et al., 1998). For the analysis of theapolar fractions, the GC column temperature was programmed from70C to 130C at 20C/min followed by 4C/min to 320C with a finalisothermal period of 10 min. For the analysis of the RuO4 productmixture the following temperature program was used: 1 min at 40Cand subsequently heated to 320C at 4C/min. The final temperaturewas held for 10 min. Of all the compound series identified here, withthe exception of compounds 8 and 12, a reference mass spectrum wasavailable in the NIST database. The relative retention time and massspectrometric characteristics were used to identify other members ofthese series. Compound series 7, 8, 9 , 11, and 12 were quantified byintegration of the peak areas in the mass-chromatograms ofm/z 58 and88. The obtained peak areas were multiplied by a response factorcompensating for differences in mass spectral responses. This responsefactor was determined from the relative intensity of the ion used formass chromatography in the mass spectrum of a well-resolved peak ofthe homologue series.

4. RESULTS

4.1. Thermal Maturity

The Tmax for both Kukersite samples is 430C, while the

Guttenberg samples reveal a Tmax of 444C for sample I and

442C for Sample II (Jacobson et al., 1988), indicating that the

Kukersites are thermally more immature than the Guttenberg

samples. The thermal maturity of the samples as determined

from the biomarkers, also indicate that the organic matter of the

Kukersites was exposed to less thermal stress than the Gutten-

berg samples. This is expressed by the presence of a C3117,21(H)-homohopane in the Kukersites and 22S/(22S

22R) C31and C3217,21(H)-homohopane ratios of 0.3. The

Guttenberg samples lack the presence of a C31 17,21(H)-

homohopane and display homohopane ratios of 0.6, which

suggests a thermal maturity equal to or greater than the onset of

oil generation (Seifert and Moldowan, 1986).

4.2. Microscopy

Light microscopic examination of the microfossils that were

isolated from the sediment by preparing a DCM float showed

that this material consisted of virtually 100% G. priscaremains

in all samples. The G. prisca microfossils in the Estonian

Kukersites are yellowish to brown. Although the size of the

obtained microfossils is quite variable, they generally range

from 10 to 80 m. SEM (Fig. 1a) shows that the particles

consist of budding colonies with a smooth surface. TEM (Fig.

1b) shows that the colonies are comprised of concentrical

layers of organic matter around what appear to be cell voids.The Guttenberg Member G. prisca microfossils are comprised

of some larger brown aggregates and are more homogenous in

colour. SEM (Fig. 1c) illustrates that only some remnant mor-

phology has been retained. TEM (Fig. 1d) shows that the

spherical bodies composing the colonies are amorphous solid

masses with only minor changes in image density, which are

possibly the remains of the layers observed in the Kukersites.

4.3. FTIR

The FTIR spectra of all four samples are virtually identical

and all display absorption bands at: 3650/cm (small, broad,

shoulder), 3340/cm (strong, very broad), 2927/cm (strong),

2851/cm (strong), 1710/cm (medium), 1610/cm (medium,

broad), 1457/cm (medium, broad), 1375/cm (medium,

broad), 1283/cm (medium, broad), 1200/cm (medium, broad),

1094/cm (medium, broad), 974/cm (medium, broad shoulder),

837/cm (small), 720/cm (small). The samples from the Gutten-

berg Member contain an additional strong absorption at 1025/

cm.

4.4. Flash Pyrolysis

The TIC traces of Estonian Kukersite I and Guttenberg II

pyrolysates are shown in Figure 2. The Kukersite pyrolysates

are dominated by a large unresolved complex mixture (UCM)

(Fig. 2a). Important contributors to the UCM are phenolic

compounds (van Bergen, unpublished results) such as n-alkyl

and n-alkenyl resorcinols (5-n-alkyl-benzene-1,3-diol). n-Al-

kyl and n-alkenyl resorcinols can be revealed by the m/z 124

mass chromatogram (Figs. 2b) but depending on their concen-

tration they also appear as resolved peaks. For Kukersite I this

mass chromatogram reveals some mono-, diunsaturated, and

saturated C21 and C23 n-alkyl resorcinols, which were notfound in any of the other samples but were reported before in

Kukersite pyrolysates by Derenne et al. (1990, 1992). All

samples also display a series ofn -alkenes and n -alkanes rang-

ing from C7to C20. These compounds are more dominant in the

Guttenberg pyrolysates (Fig. 2c). In the TIC traces of the

Guttenberg pyrolysates the UCM is much less pronounced,

although the mass chromatogram of m/z 124 (Fig. 2d) still

reveals the contribution ofn-alkyl resorcinols,

4.5. RuO4

Degradation

Degradation of polymeric substances with RuO4 is based

upon oxidative cleavage of cross-links such as ether-bonds,which are known to give aliphatic biopolymers like algaenans

their structural integrity (Blokker et al., 1998, 1999; Schouten

et al., 1998; Gelin et al., 1997). Upon RuO4 degradation,

aromatic moieties like n-alkyl benzenes are oxidised resulting

in the generation of fatty acids containing one more carbon

atom than their aliphatic chains (e.g. Trifilieff et al., 1992).G.

priscamicrofossils are degraded with RuO4within 1 h and no

final residue was left after the complete reaction time of 4 h.

Figure 3 shows the TIC traces of the RuO4product mixtures

of the fourG. priscamicrofossil samples. Close examination of

these traces shows the presence of a number of homologous

series of sometimes coeluting compounds. Mass chromato-

grams ofm/z 74, 88, 58, characteristic for compounds contain-

ing a carboxyl, a 2-carboxyl and a carbonyl function, respec-

tively, reveal the presence of these compounds in the Estonian

Kukersite I (Fig. 4). The most complex product mixtures are

obtained from the Estonian Kukersites. In general the com-

pounds in these product mixtures can be subdivided into three

major groups on the basis of the number of carboxyl moieties

(Fig. 5). Group I contains the compounds with one carboxyl

moiety, Group II has two carboxyl moieties, and Group III has

three carboxyl moieties. The relative abundances of these com-

ponents in the RuO4 degradation product mixtures of the four

samples are plotted as bar diagrams in Figures 6 and 7. The

compounds displayed in Figures 6a and 7a show a strong

odd-over-even carbon number predominance, especially in the

887The chemical structure ofG. prisca microfossils

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Fig. 1. SEM and TEM ofG. prisca specimens from the Estonian Sample I (a,b) and Guttenberg Sample II (c,d). Scalebars 10 m.


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higher molecular weight region. All compounds in Figures 6b

and 7b except the dicarboxylic acids (6) display an even-over-

odd carbon number predominance, which is particularly evi-

dent for the higher carbon numbers.

4.5.1. Estonian Kukersites

Of the series of compounds displayed in Figure 5 only 1 and

5are not present in the RuO4product mixtures of the Estonian

Kukersites. From group I only the fatty acids (2) display a

broad range of carbon numbers, ranging from C7to C18with an

even-over-odd carbon number predominance in the C15C18area. Although these normal fatty acids are not abundant in the

product mixtures, their counterparts containing a carbonyl

group are present in higher relative concentrations. Both 1-

oxo- and n-oxo-fatty acids (3 and 4, respectively) reveal a

strong even-over-odd predominance in the C15C18 area. Al-

Fig. 2. The py-GC/MS chromatograms of the Estonian Kukersite I and Guttenberg Member II. Mass-chromatograms(m/z, 124) (b) and (d) reveal then-alkyl resorcinols. Numbers denote carbon number. UCM unresolved complex mixture.


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though the carbonyl moiety of series 4 is present at multiple

positions as illustrated by the broad peaks in the m/z 58 mass

chromatogram (Fig. 4), the oxo-fatty acids are dominated by

the 1 and 9-oxo counterparts.

Compounds from Group II (Fig. 5) can all be found in the

RuO4 degradation product mixtures of the Kukersites. The

dicarboxylic acids (6) represents the most dominant series in

these product mixtures and are found in the range of C5to C14with a maximum at C9. In contrast to the other compound

series, the dicarboxylic acids do not show a specific odd or even

carbon number predominance and display a preference for the

lower carbon numbers. The 10-carboxy 1-oxo-fatty acids

(7) and 11-carboxy 9-oxo-fatty acid (8), both contain an

additional carboxyl moiety in comparison to compounds 3 and

4 (Group I). Series 7 displays a restricted carbon range C17

C19, dominated by the C19member. Series8is only represented

by the C19 member. The position of the mid-chain carboxyl

moiety was determined to be at 10 for all members of series

Fig. 3. The GC/MS chromatograms of the RuO4 product mixtures of the G. prisca microfossils.


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7, while8 contains an11 carboxyl moiety, Since no reference

mass spectrum was available for compound 8, this identifica-

tion remains tentative and is only based on its mass spectrum

(Fig. 8a) and relative retention time. The last series within the

dicarboxylic acid group are the 2-methyl dicarboxylic acids ( 9),

which range from C5to C19in Kukersite II and from C5to C14in Kukersite I. Like the former two compound scries the carbon

number distribution of the 2-methyl dicarboxylic acids in Ku-

kersite II maximizes at C17 and C19.

The last group in the Estonian Kukersite RuO4 degradation

product mixtures contains compounds with three carboxyl moi-

eties (Group III in Fig. 5). Series 10, like the previous three

groups, exhibit a strong odd-over-even carbon number predom-

inance and in both Kukersites product mixtures is dominated

Fig. 4. Total ion current and mass chromatograms ofm/z74, 88, and 58, showing fatty acids, 2-methyl carboxyl moieties,and carbonyl containing compounds in the RuO4product mixture of the Estonian Sample I. Numbers denote longest linearchain length including the carbon of eventual terminal carboxyl moieties.


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by the C17 member. The mid-chain carboxyl moiety of this

series is predominantly at the10 position. The same holds for

the series of 2-methyl tricarboxylic acids (11), containing the

mid-chain carboxyl at the 10 position. In contrast to all other

compound series, series 11 shows a carbon number preference

of C18 and C20 in both Kukersites product mixtures, The last

compound eluting in the chromatograms of the Kukersite prod-

uct mixtures is the 8-carboxy-9-oxo-nonadecanedioic acid (12).

Since no reference mass spectrum is available for this com-

pound, this structure is only identified on the basis of its relative

Fig. 5. Compounds generated upon RuO4degradation of theG. priscamicrofossils. The compounds are grouped on thebasis of the number of carboxyl moieties. x and y are variable and the total chain length of the molecules are mentionedin the text and in Figs. 6 and 7.

Fig. 6. Bar diagrams representing the relative concentration of compounds in the RuO 4product mixtures of the EstonianKukersites. Diagram (a) contains the compounds showing an odd-over-even predominance and (b) contains the compoundsshowing even-over-odd predominance. The abundance of the most abundant compound is set at 100%. The height of thebars representing the dicarboxylic acids has been multiplied by a factor of 0.5. The inset shows the relative amount of thecompound groups from Fig. 5 in the product mixtures.


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retention time and interpretation of the mass spectrum (Fig,

8b). The shorter C17and C16homologues of this compound are

present in the Kukersite product mixtures, but only in very low

concentrations.

4.5.2. Guttenberg Members

Compared with Kukersites, the RuO4product mixtures of the

isolated G. prisca microfossils from the Guttenberg Members

do not show such a strong dominance of certain compounds nor

a strong preference for odd or even carbon numbers. Most of

the compounds observed in the product mixtures display a

broad distribution resulting in a series of coeluting compounds

as displayed in Figure 3. Most striking is the fact that no

compounds could be found in the product mixture containing a

mid-chain carboxyl moiety as observed in compound series 7,

8, and 10 to 12 (Group III) in the Kukersite product mixtures.

However, in addition to the series present in the Kukersites, a

series of methyl ketones (1) and 2-methyl fatty acids (5) are

observed. Since the concentration of methyl ketones is low and

because they coelute with some other compounds, they can

only be revealed by the m/z 88 mass chromatogram. They are

found in the product mixtures ranging from C12to C19 exhib-

iting a odd-over-even predominance.

From group I (Fig. 5) the fatty acids are dominant in the

product mixtures, especially in the Gutterberg I sample. In both

Guttenberg samples they are observed in the carbon number

range of C7 up to C18 displaying a slight even-over-odd pre-

dominance. In contrast to the Kukersites, the 1-oxo- and

n-oxo-fatty acids (3,4) occur in a very broad range of carbon

numbers from C9to C18with a much less pronounced, but still

present, even-over-odd predominance. Consistent with the

Kukersite RuO4 product mixtures, the carbonyl moiety of the

n-oxo-fatty acids is present at multiple positions, but is most

prominently present at 9. 2-Methyl fatty acids (5) are like

series 1 unique for the Guttenberg product mixtures. They can

be found with carbon numbers ranging from C8to C20, reveal-ing an even-over-odd predominance above C12.

The dicarboxylic acids (6) are as prominent as the fatty acids

in the Guttenberg G. prisca RuO4 degradation product mix-

tures. They range from C6 to C18, maximising at C10C11. As

in the Estonian Kukersite product mixtures, the dicarboxylic

acids do not display a preference for odd or even carbon

numbers. The only other member from compound group II

present in the RuO4 degradation product mixtures are the

2-methyl dicarboxylic acids (9). These also display a wide

range of carbon numbers, generally from C6to C19. Especially

for the longer chain-length members there is a preference for

odd carbon numbers.

Fig. 7. Bar diagrams representing the relative concentration of compounds in the RuO4 product mixtures of theGuttenberg Members. Diagram (a) contains the compounds showing an odd-over-even predominance and (b) contains thecompounds showing even-over-odd predominance. The abundance of the most abundant compound is set at 100%. Theheight of the bars representing the 2-methyl fatty acids has been multiplied by a factor of 10. The inset shows the relativeamount of the compound groups from Fig. 5 in the product mixtures.


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5. DISCUSSION

5.1. Chemical Structure ofG. prisca Microfossils in

Estonian Kukersites

Studies using flash pyrolysis have revealed that n-alkyl re-

sorcinols (Derenne et al., 1990, 1992), but also alkylated het-

erocyclic sulphur compounds, alkylbenzenes, and alkylcyclo-

hexanes (Douglas et al., 1991), can be important constituents of

G. priscamicrofossils. In the pyrolysates of the Estonian Kuk-

ersites (Fig. 2a,b)n -alkyl resorcinols show a broad distribution

ranging from C7 to C20 maximizing at C14. The broad distri-

bution indicates that these pyrolysis products are probably

derived from larger polymer building blocks that are thermally

degraded. The Kukersite I pyrolysate contains C21, and C23n-alkyl resorcinols (Fig. 2), thus containing an n-alkyl side

chain comprised of 15 and 17 carbon atoms, respectively. This

specific distribution, only displaying the odd homologues,

could indicate that these resorcinols represent fragments that

did undergo only minor structural changes upon thermal crack-

ing during pyrolysis. In addition, the presence of the mono- and

diunsaturated homologues (see also Derenne et al., 1990, 1992)

suggests the presence of intermolecular linkages on the n-alkyl

side chain in the polymer that were thermally cleaved resulting

in the formation of a double bond at the cleavage position.

Consequently, if we consider the C21 and C23 n-alkyl resorci-

nols as important building blocks of the polymer, the shorter

saturated and unsaturated n-alkyl resorcinols are most likely

fragments from a thermal cleavage at a mid-chain position of

the alkyl side chain, possibly due to the presence of a linkage

to another monomer at this position. Since these shorter homo-

logues are dominated by the C14 saturated and unsaturated-n-

alkyl resorcinols, this suggests that such a cross-link is posi-

tioned around the 8 position on the alkyl side-chain.

Upon RuO4degradation of C21and C23n -alkyl resorcinols,

the resorcinol moieties of these compounds will be oxidised to

a carboxyl moiety generating products that contain the C15and

C17n -alkyl chain and the additional carbon from the carboxyl

moiety, thus C16 and C18 fatty acids, respectively. However,

although fatty acids are present in the RuO4product mixtures,

they are only minor components (Fig. 6). The most dominant

compounds in the RuO4 product mixture of the Estonian Ku-

kersites are series 7, 8, and 10 (Fig. 5), which all contain a

mid-chain carboxyl moiety and have a chain-length preference

of C17and C19instead of C16and C18. This shift in distribution

by one carbon number seems the consequence of the additional

mid-chain carboxyl moiety, which is probably a remnant of a

C-C bond between an n-alkyl resorcinol and another moiety.

Such a moiety linked at the mid-chain position of the n-alkyl

chain of the original n-alkyl resorcinol is most probably a

resorcinol unit from another monomer. More specifically, the

Fig. 8. Mass spectra of the compounds tentatively assigned as (a) 2-(2-oxo-decyl)-nonanedioid acid dimethyl ester and(b) 8-methoxycarbonyl-9-oxononanedecanoic acid dimethyl ester.


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compounds of series 7 were probably linked to two othern-alkyl resorcinols via a C-C bond and a C-O-C bond gener-

ating a carboxyl moiety and a carbonyl moiety, respectively

(Fig. 9a). The specific distribution of compound series 10

suggests that these also originate from the oxidative linkage of

ann -alkyl resorcinol linked via a C-O-C and a C-C bond (Fig.

9b). In case of compound 8 the C-O-C bond between the

resorcinol unit and the alkyl chain was positioned at the 10

position and the C-C bond at the 8 position (Fig. 9c). Although

the carboxyl and carbonyl moieties of compound 8 may orig-

inate from two different monomers linked to the alkyl chain, it

is also possible that a chroman-like structure as in Fig. 9c is

responsible for this particular degradation product.

On the basis of the former information and considerations, a

structure for an n-alkyl resorcinol-based polymer can be pro-

posed (Fig. 10a). The10-carboxy1-oxo-fatty acids (7) may

be generated upon RuO4 degradation of unit e and the 11-

carboxy 9-oxo-fatty acid (8) from n-alkyl resorcinol (a).

Tricarboxylic acids (10) are most likely formed upon degrada-

tion of a monomer (n) linked at a mid-chain position to a

carbon of another n -alkyl resorcinol and at the terminal carbon

of the n-alkyl via an ether linkage. Not only are these former

compounds are explained by this model, the formation of all

other compounds in the RuO4 product mixtures can also be

explained by this polymer model (Fig. 10a). Fatty acids (2) can

be formed from RuO4 degradation of terminal n-alkyl resor-

cinols (b, m), providing the long-chain fatty acids with a

predominance at C16and C18. The shorter homologues can, forexample, be formed upon cleavage of a vicinal set of ether-

linkages (n-alkyl resorcinol unit (g). 1 (3) and n-oxo fatty

acids (4) may originate from cleavage of n-alkyl resorcinols

containing a single ether cross-link at the alkyl chain (c,l,o,p),

although they also may originate fromn-alkyl resorcinols units

containing a carbonyl moiety (h). Long-chain dicarboxylic

acids (6) can be formed upon degradation ofn-alkyl resorcinol

(f), whereas the shorter homologues may originate from cleav-

age of a vicinal set of ether-linkages (g). 2-Methyl dicarboxylic

acids (9) are formed upon oxidation of, for example, n-alkyl

resorcinol (i) linked via a C-C bond at the 1 position to

another resorcinol unit. RuO4 degradation can generate

2-methyl tricarboxylic acids (11) from ann-alkyl resorcinol (k)

linked to two resorcinol moieties via C-C linkages at a mid-

chain and 1 position. The 8-carboxy-9-oxononadecanedioic

acid (12) is possibly formed from a unit such as d, which is

linked to three other monomeric units by two ether and one C-C

bond. Considering that the Kukersites were completely de-

graded by RuO4degradation, we can regard all the degradation

products to be representative of the entire polymer, which

suggests that the G. prisca microfossils in the Kukersites are

completely composed ofn -alkyl resorcinols like in Fig. 10a.

FTIR analysis of Estonian Kukersite microfossils also sup-

ports the polymer structure presented above. The FTIR data

obtained in this study are comparable to those of G. prisca

microfossils reported by Derenne et al. (1990, 1994) and Sta-

Fig. 9. Rationale for the formation of compounds series 7 (a), 10, (b) and 8 (c) upon RuO4degradation of the EstonianKukersites.


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siuk et al., (1993). In all FTIR spectra the oxygen functional-

ities are expressed as absorptions at 3340/cm (hydroxyl

groups), 1710/cm (non-conjugated carbonyl groups), 1200/cm

(probably phenolic or alkyl aryl ether moiety), and the area

between 1094/cm and 974/cm consisting of a combination of

peaks and shoulders (possibly alkyl aryl ethers or dialkyl

ethers). The bands at 2927/cm, 2851/cm, 1457/cm and 720/cm

are due to the presence of alkyl CH2. The peak broadening at

2927/cm, 2851/cm be ascribed to the presence of CH3stretch-

ing absorptions together with the absorption at 1375/cm. The

final group of absorptions originates from the C-H of benzene

moieties. Because these absorptions are generally rather weak

and found in areas around 3000/cm, 16501400/cm, and

between 1200 400/cm they overlap with some other broad

signals in these spectra, which makes it difficult to assign

absorption bands to these moieties. Although some of these are

consequently double assigned, the 974/cm, 837/cm, and

720/cm may reflect 1,3,5-substituted benzene together with

those at 1610/cm. However the latter can also result from

some carboxylate moieties, which could provide and alternative

explanation for the 1375/cm absorption.

The 13C-nuclear magnetic resonance data presented by

Derenne et al. (1990) are also in agreement with the polymer

model in Figure 10. Their spectrum clearly shows the dominant

aliphatic signal at 29 ppm and some smaller and broader peaks

at chemical shifts 155, 142, 108, 103, 60, and a shoulder at 15

ppm. The first four of these peaks can be ascribed to an

aromatic resorcinol moiety and the latter is the chemical shift of

the CH3. The chemical shift at 60 ppm most likely corresponds

to the aliphatic carbon of the ether-linkages that form the

cross-links between the monomers.

5.2. Relation Between G. prisca Microfossils in Kukersites

and the Guttenberg Member

The FTIR spectra of the Guttenberg G. prisca microfossils

are virtually identical to those of the Kukersites, indicating that

these are probably also composed of a poly( n-alkyl resorcinol).

Although the Guttenberg samples display an additional absorp-

tion at 1025/cm this can most likely be ascribed to some

inorganic residues in these samples (see also Derenne et al.,

1994). Upon flash pyrolysis of the Guttenberg G. prisca mi-

Fig. 10. Generalised and simplified reconstruction of the proposed n -alkyl resorcinol based polymer composing the G.priscamicrofossils from the Kukersites (a) and from the Guttenberg (b). The numbers refer to compounds generated fromthese polymeric moieties upon RuO4 degradation shown in Fig. 5.


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crofossils a clear n-alkyl resorcinol series is observed next to

the series ofn-alkenes andn -alkanes (Fig. 2c and d). Although

the abundance of n-alkyl resorcinols generated is lower and

some relative differences between the n-alkenyl and n-alkyl

resorcinols exist when compared with the Estonian samples, the

distribution is very similar to that observed in the Kukersite

pyrolysates (Fig. 2a,b). Not only do the FTIR spectra and

pyrolysates of the two different sets of samples display similarfeatures; also upon RuO4 degradation identical compound se-

ries are observed (Fig. 7). Compound series 2, 6, and 9 in the

Guttenberg RuO4 product mixture display virtually the same

broad distribution as those in the Kukersite product mixtures. In

addition series3 and 4 are also present, displaying a preference

for carbon numbers C16and C18similar to their counterparts in

the Kukersite product mixtures. Although compound series2,

6, and 4 are not specific G. prisca RuO4 products and can be

found amongst the degradation products of for example al-

gaenans (though having another chain-length distribution)

(Blokker et al., 1998, 1999), series 3 and 9 have never been

observed in RuO4 degradation products before and might be

highly specific degradation products ofG. prisca microfossils.The presence of these compounds in the Guttenberg G.

prisca RuO4 product mixture, together with the other data

obtained for these samples suggests that the Guttenberg micro-

fossils are composed of a similar polymer as found in the

Kukersites. However, there are some important chemical dif-

ferences between the RuO4product mixtures of the Guttenberg

Member samples and Estonian Kukersites. For example, series

3 and 4 display a broader carbon chain-length distribution in

the RuO4product mixtures of the Guttenberg Member samples.

The most striking difference is the lack of compounds bearing

a mid-chain carboxyl moiety (pie diagrams, Figs. 6 and 7) and

the presence of two additional compound series 1 and 5 in the

Guttenberg product mixture. Considering that the polymer aspresented in Figure 10a forms the basis of also the Guttenberg

microfossils, these differences must be the consequence of

cleaved C-C bonds at a mid-chain position. Such cleavages

could be the consequence of the higher thermal maturity of the

Guttenberg samples as implied by the hopanoid epimerization

ratios, which indicate that these samples have reached the early

stage of oil generation. Simulation experiments with the more

immature Kukersite I sample revealed that upon heating of this

sample for 10 h at 100C under an inert atmosphere, the

pyrolysate changed, resulting in the loss of C21and C23n-alkyl

resorcinols and a decreased abundance of the UCM. These

changes in pyrolysate composition illustrate that upon minimal

thermal stress indeed chemical changes occur that could ulti-

mately result in a material with the same chemical properties as

the Guttenberg G. prisca microfossils. Although this is a sim-

plified representation, Figure 10b illustrates that early catage-

netic cleavage of the thermally labile C-C bonds to the

resorcinol moieties linked at a mid-chain position, will result in

the loss of series7,8,1012in the RuO4degradation mixture.

Furthermore, the moieties generated by this process will be

responsible for the formation of series 1 and 5 upon

RuO4degradation, although the long-chain homologues of

these series are more difficult to explain by this process. An-

other effect of such C-C bond cleavages is that all compounds

generated upon RuO4degradation will show a more broad car-

bon chain length distribution for compound series 3 and 4.

Because thermal maturity apparently has had an important

impact on the chemistry of the Guttenberg Member samples,

this process also should have affected the morphology of G.

prisca microfossils in these samples. Although the Guttenberg

MemberG. priscamorphotypes superficially resemble those of

the Estonian Kukersite G. prisca microfossils (Fig. 1a and c),

some morphological differences are indeed observed. Although

some of the differences could be the effect of variations in thesalinity levels in the paleoenvironment or other differences in

growth conditions (Derenne et al., 1992), the complete lack of

discernible cell voids in the Guttenberg G. priscamicrofossils,

as illustrated by TEM analysis (Fig. 1d), strongly points to-

wards a post-depositional thermal process. This effect has been

simulated under laboratory conditions by Stasiuk and Osadetz

(1993), who showed that G. prisca microfossils display a sort

of melting phenomenon upon heating up to 290C.

Although the G. prisca microfossils in the Kukersites and

Guttenberg Members are both composed of a poly(n-alkyl

resorcinol), the results presented here indicate that both the

chemical and some of the morphological differences can be

explained by post-depositional thermal processes.

5.3. Biological Occurrence of Resorcinols

n-Alkyl resorcinols (5-n-alkyl-benzene-1,3-diol) are quite

common in nature and especially the C21and C23members can

be abundant in some higher plants such as the Ginkgoaceae and

Anacardiaceae (Kozubek and Tyman, 1999, for a review).

Recently, the occurrence of resorcinol lipids has been demon-

strated in an increasing number of tissues and organisms in-

cluding fruits, seeds, bacterial cysts, bacterial vegetative cells

and senescent organs or cells (Kozubek and Tyman, 1999). A

series of unique resorcinolic lipids were found in the green

microalga B. braunii involving C31, C33, and C35 n-alkenylresorcinols linked via a resorcinolic C-O linkage to long-chain

n-alkenes,n -alkadienes and n-alkatrienes forming high molec-

ular weight molecules (Metzger and Largeau, 1994). However,

in most cases the reported alkyl resorcinols occur as C21 and

C23 homologues, generally having different degrees of unsat-

uration (Kozubek and Tyman, 1999 and references therein).

The function of alkyl resorcinols in nature is only partially

known. However, it is clear that this family of compounds

plays an important role in organism-organism interactions (Ko-

zubek and Tyman, 1999 and references therein), Furthermore,

it was shown that longchain 5-n-alk(en)ylresorcinols can pre-

vent membranes from oxidation processes and even polymerise

under oxic conditions (Tymann, 1991; Kozubek and Tyman,

1999).

Some plants like the oriental lacquer trees (e.g. Toxicoden-

dron vernicifluum, Rhus vernicifera) contain types of com-

pounds very similar to n -alkyl resorcinols namely n -alkyl cat-

echols (3-n-alkyl-benzene-1,2-diol) with carbon numbers of

predominantly C21 and C23. These n-alkyl catechols are the

main constituents of the sap that has been used as natural

varnish and adhesive for more than 5000 yr (Du et al., 1984;

Kumanotani, 1998). It was shown that two main mechanisms

are involved in the polymerisation of these compounds: a

coupling of the catecholic units producing diphenyls and diben-

zofurans and a reaction of the catechol unit with the unsatura-

tion on the alkyl chain. The observed dimeric structures as


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proposed by Kumanotani (1995, 1998) clearly illustrate that the

type of linkages found in theG. priscamicrofossils (Figs. 9 and

10) are easily formed upon oxygen-induced polymerization of

such compounds.

Although to date we can only guess what kind of polymeri-

sation mechanism was exactly involved in case ofG. prisca, it

seems possible that some kind of radical mechanism initiated

the polymerisation process in parallel to the peroxidase induced

radical polymerisation of the phenolic units in lignins

(Freudenberg and Neish, 1968) and aerobic oxidative or lac-

case-catalysed polymerisation of urushiol (Kumanotani, 1995).

The polymerisation of both lignin and urushiol starts with theabstraction of an H radical from a phenolic hydroxyl group,

upon which the activated species can react with other moieties.

In the case ofG. prisca probably the resorcinol moiety of the

n-alkyl resorcinols did undergo such H radical abstraction and

reacted with the other monomers, which probably contained

one or more unsaturations on the alkyl chain. Consequently

most of the intermolecular linkages will involve the resorcinol

as illustrated in Figure 11, showing that both carbon-carbon

bonds and ether-linkages can be created this way. Although this

mechanism is tentative, the structures expected from such a

polymerisation are in perfect agreement with the proposed

polymer structure (Fig. 10a).

5.4. Implications for the Origin ofG. prisca

Although the inference that the G. prisca microfossils are

composed of a poly(n-alkyl resorcinol) is an important contri-

bution to the knowledge of these microfossils, it still remains

unclear if the poly(n-alkyl resorcinol) is a biopolymer or a

structure that is formed from free n -alkyl resorcinols after the

organism died, The inner morphology of the Kukersite G.

priscamicrofossils clearly shows the voids of the original cells

(Fig. 1b) embedded in concentrically layers of organic matter.

Derenne et al. (1992) interpreted these layers as the remnants of

the cellular matrix of a B. braunii-like organism, thus com-

posed of the biopolymer algaenan. Because B. braunii also

makes resorcinolic lipids, though having much longer alkyl-

chains of 2529 carbon atoms instead of 1517 as observed in

the G. prisca polymer, a relation between these organisms is

possible (Metzger and Largeau, 1994).

Algaenan-containing algae like Tetraedron minimum, Pedi-

astrum boryanumnormally release their daughter cells from the

algaenan containing mother cell wall via a fissure at a specific

position in the wall (van Hoek et al., 1995) and B. braunii

daughter cells grow out of the mother cell cup (Burns, 1982). In

this way these algaenan-containing algae avoid being trapped

inside their polymeric algaenan cell wall. Although Derenne et

al. (1992) clearly illustrated that the morphology ofB. braunii

becomes moreG. prisca-like under increased salinity, the inner

morphology observed for the Kukersite microfossils, in which

the daughter cells stay within the mother cell, is unlikely for

organisms having an algaenan-containing cell wall, since this

rigid polymeric structure would obviously restrict cell division

and growth. Furthermore, in case ofB. brauniithe resorcinolic

lipids are not the main building-blocks of the algaenan cell

walls, which is composed of linear polyaldehyde (Metzger et

al., 1993; Gelin et al., 1997, Bertheas et al., 1999). In G. prisca

microfossils, a poly(n-alkyl resorcinol) forms the basis of the

polymeric structure and no evidence is found for the presence

of a linear polyaldehyde or other aliphatic polymer structure

from the RuO4 degradation products.Although compounds like unsaturated n-alkyl resorcinols

can be polymerised by enzyme catalysis as illustrated by uru-

shiol (Kumanotani, 1995) and most likely also by the resorcin-

olic lipids in B. braunii, the sensitivity of these compounds

towards polymerisation suggests the possibility that G. prisca

microfossils consist of a polymer formed after the organism

died. It is possibly that G. prisca lived under conditions which

required protection of a sheath containing anti-microbial

agents, anti-oxidants and/or UV-filters. If G. prisca excreted

n-alkyl resorcinols for this purpose the specific chemical prop-

erties of these compounds could have resulted in polymerisa-

tion during senescence or after death. Polymerisation of the

Fig. 11. Proposed catalytic radical polymerisation mechanism of alkyl resorcinols and formed dimeric structures and theirRuO4 oxidation products.


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n-alkyl resorcinols could have been catalysed by oxygen itself,

trace metals and even lytic enzymes, released upon senescence

of the cells or by certain bacteria thriving on its remains.

Based on the sheath morphology of some cyanobacteria G.

prisca was suggested to be closely related to these micro-

organisms (Foster et al. 1989, 1990; Stasiuk and Osadetz, 1990;

Stasiuk et al., 1991). Cyanobacterial sheaths often contain large

amounts of secondary protective compounds like the naturalUV-filter scytonemin, sometimes in concentrations up to 25

mg/g protein (Dillon and Castenholz, 1999). To date not much

is known about the chemical composition of cyanobacterial

sheaths, but it is known that this mucus can be readily pre-

served (Boon and de Leeuw, 1987; de Leeuw and Largeau,

1993) and is even reported to partially polymerise under certain

conditions (Humm and Wicks, 1987). Cyanobacterial sheaths

probably do not have intrinsic resistant properties like algaenan

containing cell walls, but fossilisation obviously does occur due

to the specific preservation conditions (de Leeuw and Largeau,

1993). Because the water-containing sap of oriental lacquer

trees resembles to a certain degree the sheath of a cyanobacteria

(Humm and Wicks, 1987), maybe such sheaths serve a suitablemicroenvironment for the polymerisation of excreted n-alkyl

resorcinols under certain conditions. Although this theory

seems to favour the cyanobacterial origin of G. prisca, it

remains pure speculative because a poly(n-alkyl resorcinol) has

never been observed in such organisms before.

6. CONCLUSIONS

The results obtained from FTIR, flash pyrolysis and most

importantly RuO4degradation presented in this study illustrate

that the G. prisca microfossils from the Kukersites and the

Guttenberg Member are composed of a polymeric structure

based on unsaturated C21 and C23 n-alkyl resorcinols. These

monomeric units are intermolecularly linked via C-C and C-Obonds between resorcinol moieties and the n-alkyl chains. Both

the chemical and some morphological differences between the

microfossils from these different settings are assigned to the

higher thermal maturity of the G. prisca obtained from the

Guttenberg Member, which altered the chemical properties of

the poly(n-alkyl resorcinol) structure by cleavage of specific

linkages within the polymer. This process disrupted the integ-

rity of the polymer allowing some of the morphological differ-

ences between the Guttenberg and the Kukersite microfossils.

Because poly(n-alkyl resorcinols) are unknown from nature

it is not possible to directly link the chemistry to a certain

organism and answer the question if these microfossits repre-

sent a biopolymer or a polymer formed during senescence orafter the cells died, However, the specific chemical, physical,

and biological properties ofn-alkyl resorcinols could indicate

that they served as a sheath component forming a part of the

matrix in which the original cells were embedded. In this case

polymerisation must have resulted in a resistant polymeric

substance, which could have been selectively preserved in the

sediment in such a fashion that the cell morphology was re-

tained in the shape of readily discernible cell voids in the

immature G. prisca microfossils in the Kukersites.

AcknowledgmentsThis study was supported by the Research Councilfor Earth and Life Sciences (ALW) with financial aid from the Neth-erlands Organisation for Scientific Research (NWO). We thank G.

Ludvigson and B. Witzke of the Iowa Geological Survey for supplyingsamples from the SS-9 core and T. Brain of Kings College London forassistance with the TEM. This is NIOZ contribution 3489.

Associate editor: M. C. Kennicutt II

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Blokker Et Al 2001