Lipid biomarkers in ooids from different locations and ... · Lipid biomarkers in ooids from...

Lipid biomarkers in ooids from different locations andages: evidence for a common bacterial floraR. E. SUMMONS,1 L . R . BIRD,1 , 2 A . L . GILLESPIE,1 S . B . PRUSS, 3 M. ROBERTS4 AND

A. L . SESSIONS5

1Department of Earth, Atmospheric and Planetary Sciences, Massachusetts Institute of Technology, Cambridge, MA, USA2Department of Geosciences and Penn State Astrobiology Research Center, The Pennsylvania State University, University Park,

PA, USA3Department of Geosciences, Smith College, Northampton, MA, USA4NOSAMS, Woods Hole Oceanographic Institution, Woods Hole, MA, USA5Division of Geological and Planetary Sciences, California Institute of Technology, Pasadena, CA, USA

ABSTRACT

Ooids are one of the common constituents of ancient carbonate rocks, yet the role that microbial com-

munities may or may not play in their formation remains unresolved. To search for evidence of microbial

activity in modern and Holocene ooids, samples collected from intertidal waters, beaches and outcrops in

the Bahamas and in Shark Bay in Western Australia were examined for their contents of lipid biomarkers.

Modern samples from Cat and Andros islands in the Bahamas and from Carbla Beach in Hamelin Pool,

Western Australia, showed abundant and notably similar distributions of hydrocarbons, fatty acids (FAs)

and alcohols. A large fraction of these lipids were bound into the carbonate matrix and only released on

acid dissolution, which suggests that these lipids were being incorporated continuously during ooid

growth. The distributions of hydrocarbons, and their disparate carbon isotopic signatures, were consistent

with mixed input from cyanobacteria together with small and variable amounts of vascular plant leaf

wax [C27–C35; d13C �25 to �32&Vienna Pee Dee Belemnite (VPDB)]. The FAs comprised a complex

mixture of C12–C18 normal and branched short-chain compounds with the predominant straight-chain

components attributable to bacteria and/or cyanobacteria. Branched FA, especially 10-MeC16 and

10-MeC17, together with the prevalence of elemental sulfur in the extracts, indicate an origin from

sulfate-reducing bacteria. The iso- and anteiso-FA were quite variable in their 13C contents suggesting

that they come from organisms with diverse physiologies. Hydrogen isotopic compositions provide further

insight into this issue. FAs in each sample show disparate dD values consistent with inputs from auto-

trophs and heterotrophs. The most enigmatic lipid assemblage is an homologous series of long-chain

(C24–C32) FA with pronounced even carbon number preference. Typically, such long-chain FA are

thought to come from land plant leaf wax, but in this case, their 13C-enriched isotopic signatures com-

pared to co-occurring n-alkanes (e.g., Hamelin Pool TLE FA C24–C32; d13C �20 to �24.2& VPDB; TLE

n-alkanes d13C �24.1 to �26.2 �&VPDB) indicate a microbial origin, possibly sulfate-reducing bacteria.

Lastly, we identified homohopanoic acid and bishomohopanol as the primary degradation products of

bacterial hopanoids. The distributions of lipids isolated from Holocene oolites from the Rice Bay Forma-

tion of Cat Island, Bahamas were very similar to the beach ooids described above and, in total, these

modern and fossil biomarker data lead us to hypothesize that ooids are colonized by a defined microbial

community and that these microbes possibly mediate calcification.

Received 5 January 2013; accepted 24 May 2013

Corresponding author: R. E. Summons. Tel.: 617 452 2791; fax: 617 253 8630; e-mail: rsummons@mit.

edu

© 2013 John Wiley & Sons Ltd 1

Geobiology (2013) DOI: 10.1111/gbi.12047

INTRODUCTION

Ooids, small spherical to ellipsoidal coated grains character-

ized by concentric layers of calcium carbonate, are among

the most prevalent components of carbonate sediments

known from the geological record (e.g., Rodgers, 1954;

Fl€ugel, 1982; Richter, 1983) and are thought to form in

agitated settings, such as shallow, carbonate environments

like those in the Bahamas today. Differences concerning

the definition of these grains (e.g., Peryt, 1983) led to one

of the most fundamentally debated questions of their ori-

gin: Are ooids biogenically produced grains? Nonetheless,

some consensus surrounds the classification of ooids, which

is based largely on microfabric and mineralogy (Tucker &

Wright, 1990), and they are distinguished from oncoids by

the regular, even nature of laminae that surround the

nucleus. Ooids are also generally 2 mm in size or smaller,

although ‘giant’ ooids (pisoids) are reported from the Pro-

terozoic, and rarely in the Phanerozoic (see Trower &

Grotzinger, 2010 for a review). These descriptive charac-

teristics of ooids do not require a common mode of forma-

tion, so these grains are generally thought to represent a

polygenetic grouping that can form from biogenic and/or

chemical precipitation, with the relative importance of each

process being unclear. However, despite the long-recog-

nized association of ooids and organic matter (e.g., (Bre-

hm et al., 2006; Davies et al., 1978; Folk & Lynch, 2001;

Reitner et al., 1997; Pl�ee et al., 2008), recent work has

called into question the role of microbes in the accretion

of ooid laminae (Duguid et al., 2010). Endolithic borings,

common features of modern ooids, suggest a prominent

effect from endolithic micro-organisms that can lead to

micritization of ooid cortices and cement precipitation in

these borings (Harris, 1977; Duguid et al., 2010); what

remains debated is whether micro-organisms, locally and

directly, foster the precipitation of carbonate layers around

the cortex.

Despite the pervasive nature of ooids as grains in carbon-

ate strata throughout geological time, they occur in only a

handful of places in the modern ocean. Two of these

regions were selected for analysis in this work: the Bahamas

and Western Australia (Fig. 1). These two areas provide a

suitable comparison of the microbial constituents of mod-

ern ooids from contrasting depositional environments.

Ooids are common components of carbonate sand in

several areas of the Bahamian Archipelago, including the

northwestern part of Cat Island, for example, (Kindler,

1992; Kindler & Hearty, 1996; Mylroie et al., 2006;

Glumac et al., 2011) where ooids comprise beach sand

and make up much of the exposed Holocene eolianite,

both of which were sampled here. Similarly, at Joulter’s

Cays to the northwest of Cat Island (see Fig. 1), ooid

dunes line tidal channels, which can be as large 200 m

wide (Harris, 1979; Shinn et al., 1993).

Hamelin Pool in Western Australia (see Fig. 1) is a hy-

persaline basin that is well known for its microbial mats

and stromatolites colonized by cyanobacteria, coccoid and

filamentous algae, and diatoms (Burne & Moore, 1987).

At Carbla beach, on the eastern shore of Hamelin Pool

and in intimate association with the well-documented stro-

matolites, ooids are also abundant. Unlike most occur-

rences in the Bahamas, these particular ooids are forming

in hypersaline waters thereby providing an important envi-

ronmental variant for the present work.

Lipid biomarker studies of sedimentary organic matter

are one means to access clues about processes involved

in formation and diagenesis of sedimentary rocks and

their component minerals. Carbonates are particularly

suitable for this approach as they are shown to trap lip-

ids characteristic of the microbes present when they

formed. Methane seep limestones, for example, bear the

distinctive biomarker and isotopic signatures of both

aerobic and anaerobic methane-oxidizing bacteria and

archaea (Michaelis et al., 2002; Birgel et al., 2006, 2008;

Birgel & Peckmann, 2008). Massive carbonate towers at

the active vents of Lost City Hydrothermal Field bear

molecular evidence, in the way of DNA and biomarker

lipids, for both methanogens and sulfate-reducing bacte-

ria (Bradley et al., 2009). However, to date, there are

relatively few detailed studies of lipids associated with

ooids or oolites. Reitner et al. (1997) investigated the

organic matrices of Great Salt Lake ooids. Jenkins et al.

(2008) detected lipids from methanotrophic archaea in

ooid-like coated grains found in a Cretaceous methane

seep deposit in Japan. Edgcomb et al. (2013) investi-

gated the nature and diversity of the bacterial community

colonizing Highborne Cay ooids by comparing the pat-

terns of solvent-extractable lipids with clone libraries of

small subunit ribosomal RNA gene fragments.

Here, we report an analysis of the distributions of

FAs, hydrocarbons, and triterpenoids present in modern

and Holocene ooids from different localities. The FA

data for each sample, including their stable isotopic com-

positions, show remarkable parallels between localities

that indicate that the ooids have been colonized by a

common microbial community throughout their forma-

tion and further suggests that this community contrib-

uted, at least in part, to calcification of the ooid cortex.

Our results are interpreted in the light of the report by

Edgcomb et al. (2013) whose 16S ribosomal RNA clone

library data for ooids from Highborne Cay showed that

cyanobacteria were the most diverse taxonomic group

detected, followed by Alphaproteobacteria, Gammaprote-

obacteria, Planctomyces, Deltaproteobacteria, and several

other groups. These authors also observed that the over-

all bacterial diversity within ooids is comparable to that

found within thrombolites and stromatolites at the same

locality.

© 2013 John Wiley & Sons Ltd

2 R. E. SUMMONS et al.

SAMPLES AND METHODS

Sampling locations are illustrated in Fig. 1. Modern ooid

samples were obtained from the intertidal zones of beaches

in the Bahamas and from Shark Bay in Western Australia.

Bahamian samples were collected from Alligator Point on

the leeward side of the Exuma Sound coast of northern

Cat Island (intertidal) and from Joulters Cays (subtidal),

Andros Island in the Bahamas (Harris, 1977). A third sam-

ple of modern ooids was obtained from 1 m deep subtidal

waters at the north end of Carbla Beach in Hamelin Pool,

Shark Bay, Western Australia. These samples were kept

frozen, or refrigerated at <4°C, prior to analysis.

Holocene oolites were collected from outcrops of the

North Point and Hanna Bay members (1–4 and 5 ka,

respectively; Carew & Mylroie, 1985; Boardman et al.,

1987, 1989) of the Rice Bay Formation on Cat Island

(Mylroie et al., 2006; Glumac et al., 2011). Eolian oolites

comprise the Holocene North Point and overlying Hanna

Bay members on northern Cat Island; both units consist

of spherical to elliptical, fine to medium sand-size ooids

(Glumac et al., 2011). Bulk samples were collected and

stored in the dark at room temperature until processing.

The outer edges of the North Point and Hanna Bay

oolite samples were pigmented dark green and thus

inferred to be colonized by viable micro-organisms. All

Miami

TheBahamas

20 mi50 KM

AndrosTown

NichollsTown Nassau

Mangrove CaySettlement

SettlementLittle CreekSettlement

Greencastle

Governer’sHarbour

LowerBogue

ArthursTown

FreetownSettlement

McQueen’s Settlement

Victoria HillSettlement

100 mi200 km

Perth

Bunbury

Albany

PortDenison

Geraldton

Western Australia

Cat Island

San Salvador Island

Alligator Point

North PointHanna Bay

Hamelin Pool (Carbla)

Joulters Cays

Andros Island

Fig. 1 Modern and Holocene ooid sampling localities in the Bahamas and Western Australia.


Lipid biomarkers in ooids and oolites 3

pigmented surfaces were removed using a rock saw and the

inner white sediment crushed with a mortar and pestle.

The Cat Island beach ooid sample was extracted intact

while the subtidal Carbla Point and Joulters Cays ooid

samples were first powdered in a puck mill. The Joulters

Cays, Cat Island, and Carbla Point samples were also ana-

lyzed petrographically; they were embedded in epoxy and

thin sectioned to examine their internal fabrics.

Small-scale extraction by sonication

Approximately 15 g of each sample was divided into three

60-mL centrifuge tubes. A mixture (40 mL) of 9:1 dichlo-

romethane (DCM)/methanol was added to each centrifuge

tube followed by sonication for 15 min and centrifugation

at 2200 rpm for 40 min or until the finest particulates had

settled completely. After repeating the procedure, the clari-

fied solvent extracts were transferred to glass vials, gently

evaporated under a stream of dry nitrogen, and weighed.

All extracts, denoted TLE, were faintly colored.

Large-scale extraction by accelerated solvent extraction

To obtain some additional lipid for stable isotope analysis,

extra sample was subjected to Accelerated Solvent Extrac-

tion (Dionex ASE 350). Each sample (~100 g) was

weighed and placed in a large-capacity stainless steel cell

with a layer of baked sand above the sediment. They were

extracted with 9:1 DCM/methanol at 100°C, with the sys-

tem pressure set to 1000 psi. Clean sand blanks were pre-

pared and run at the beginning and end of the ASE cycle.

These extracts were clear and treated as above. The Joul-

ters Cays ooids and North Point extracts from the ASE

were both faintly colored.

Carbonate-bound lipids

The solid carbonate residue from the 15 g sonication

extraction was placed in a 1000 mL beaker and dissolved

by gradual addition of hydrochloric acid (2N). A viscous

light brown foam formed during sample digestion and this

was captured with minimal loss. After dissolution was

complete, the entire residue was diluted with HPLC-pure

water and extracted five times with DCM. As before, these

extracts were concentrated under a stream of dry nitrogen,

filtered through a sodium sulfate column to remove any

residual water, and then dried and weighed. Lipids released

by acid dissolution are denoted TLE2. Extract weights are

given in Table 1.

Removal of elemental sulfur

Joulters Cays ooids contained a substantial amount of ele-

mental sulfur evident as crystals forming when the satu-

rated hydrocarbon fractions were taken to dryness. Other

ooid TLE and TLE2 samples contained sulfur in trace

amounts. Elemental sulfur was removed by standing the

extracts, dissolved in DCM, in contact with freshly acti-

vated copper shot followed by filtration.

Bulk d13C

Samples for analysis of bulk organic carbon isotopes were

decalcified using 6N HCl until no further reaction was

observed. The residues were ground to a fine powder and

weighed in triplicate into tin cups. The d13C values were

determined using a Fisons (Carlo Erba) NA 1500 elemen-

tal analyzer fitted with a Costech Zero Blank Autosampler,

coupled to a Thermo Finnigan Delta Plus XP isotope ratio

mass spectrometer. The data were calibrated against a CO2

working standard and normalized to acetanilide, sucrose,

and urea isotopic standards.

Derivatization

Internal standards (2 lg each of epiandrosterone,

5a-androstane, and 3-methyltricosane) were added to aliquots

of each sample prior to derivatization. These aliquots were

first treated with 200 mL dry methanolic HCl (prepared

by adding acetyl chloride to cool, dry methanol) with heat-

ing 60°C for 24 h in order to convert glycerides into fatty

acid methyl esters (FAME). The samples were taken to

Table 1 Gravimetric yields of total lipids and fatty acid methyl esters from modern and Holocene ooid samples by two extraction methods

North Carbla Joulters Cays Cat Island North Point Hanna Bay

TOC (LO I) % 3.1 2.1 1.9 1.6 1.8

d13Corg �15.8 � 1.3 �17.7 � 1.6 �17.4 � 0.2 �20.7 � 2.0 �15.5 � 2.5

Lipid % TOC

ASE TLE 0.68 0.05 0.05 0.22 0.09

Sonication TLE 3.70 0.47 0.28 1.03 4.30

Sonication TLE2 1.09 0.66 0.42 0.74 2.13

Sonication TLE FAME 0.005 0.002 0.002 0.012 0.023

Sonication TLE2 FAME 0.032 0.004 0.018 0.011 0.015

FAME, fatty acid methyl esters.



dryness, transferred to gas chromatography-mass spectrom-

etry (GC-MS) vials and then further derivatized by adding

50 lL BSTFA and 50 lL pyridine with further heating to

60°C for 30 min prior to analysis.

Gas chromatography-mass spectrometry analysis

Samples were analyzed using an Agilent 7890A GC

equipped with a Gerstel programmable temperature vapor-

izing (PTV) injector and interfaced to an Agilent 5975

Mass Selective Detector. The GC was fitted with a J&W

DB5-HT (30 m length, 0.25 mm inner diameter, 0.25-lmfilm thickness) capillary column using helium as the carrier

gas. After a pulsed splitless injection with the oven held at

60°C for 5 min, the column ramped to 340°C at 20°C per

min and then held for 20 min. For quantification of

FAME, the analyses were repeated using an Agilent 7890A

GC fitted with a flame ionization detector and operated as

above. FAME were identified by a combination of their

mass spectra and GC retention times relative to authentic

normal and branched FAME mixtures obtained from Supe-

lco Analytical.

Gas chromatography-isotope ratio mass spectrometry

(GC-IRMS) analysis

Prior to isotope analysis, large aliquots of the TLE and

TLE2, constituting 50% of the total were methylated with

acidic methanol, as above, and subject to liquid chroma-

tography over ca. 10 cm columns of silica gel in a Pasteur

pipette. Five fractions were obtained using an elution

sequence of solvents of increasing polarity. Aliphatic hydro-

carbons were eluted in the first fraction with hexane [1⅜column dead volume (DV) determined empirically for each

silica bed] followed by aromatic hydrocarbons in 2 DV 4:1

hexane/DCM, ketones, and FAME in 2DV DCM, alco-

hols in 2 DV 4:1 DCM/ethyl acetate and diols 2 in DV

7:3 DCM/methanol. Stable carbon isotope analysis on the

hydrocarbon and FAME fractions was performed using a

Thermo Trace GC fitted with a PTV injector and equipped

with a J&W DB-1 (60 m length, 0.32 mm inner diameter,

0.25 lm film thickness)-fused silica capillary column and

coupled to a ThermoFinnigan Deltaplus XL isotope ratio

mass spectrometer via a combustion interface operated at

850°C. Column temperatures were programmed from

60°C at a constant flow of 2.5 mL per min and a tempera-

ture gradient of 10°C per min to 100°C, followed by a

temperature gradient of 4°C per min to 320°C then

isothermal for 20 min. Stable carbon isotope ratios were

determined relative to an external CO2 standard that was

cross-calibrated relative to a reference mixture of n-alkane

(Mixture B) provided by Arndt Schimmelmann (Indiana

University). Data are presented in the conventional d13Cnotation as part-per-thousand (&) deviations from VPDB.

No corrections were made for the carbon or hydrogen

added by methylation. Analytical uncertainties under these

conditions are conservatively estimated as �0.4&.

Due to limited abundance, hydrogen isotopic analyses

were only feasible for the FAME fraction, and then only

for the most abundant compounds. These analyses were

performed at Caltech on a Thermo Trace GC coupled to a

Delta+ XP IRMS via a GC/TC pyrolysis interface operated

at 1440°C, as described by Zhang et al. (2009). GC con-

ditions were similar to those for d13C analyses. D/H ratios

of analytes were measured relative to CH4 reference gas

added to the GC effluent via an external loop and are

presented in the conventional dD notation as part-per-

thousand (&) deviations from Vienna Standard Mean

Ocean Water (VSMOW). No further normalization to the

SMOW/SLAP scale was attempted. FAME dD values were

not corrected for the hydrogen added by the methyl ester

derivatives. This correction typically adjusts all dD values in

the same direction by <10&. Squalene was added to every

sample as an internal standard and yielded a dD value

of �169.2 � 1.4& as compared to its value determined

by offline combustion/reduction of �168.9 � 1.9&(A. Schimmelmann, Indiana University). Analytical uncer-

tainties for hydrogen isotopic analyses are conservatively

estimated as �5&.

The H-isotopic composition of Hamelin Pool water was

determined using a spectroscopic Water Isotope Analyser

(Los Gatos Research, Inc., Mountain View, CA, USA).

The sole sample was analyzed five times, with six replicate

injections for each analysis, against two separate water stan-

dards with dD values near 0&. The resulting standard

error (r/√n) for the pooled data was 0.6&.

Radiocarbon analysis

Four samples of TLE were submitted to NOSAMS for

AMS radiocarbon analyses. These were combusted in evac-

uated quartz tubes with 100 mg CuO and the resultant

carbon dioxide reacted with Fe catalyst to form graphite.

The graphite was then analyzed for radiocarbon content

on NOSAMS’s 500 kilovolt AMS system using NBS oxalic

acid I (NIST-SRM-4990) as the normalizing standard. As

per convention, data were corrected to a d13CVPDB value

of �25& using 13C/12C ratios measured concurrently on

the AMS system and are reported using the D14C nomen-

clature.

Bulk composition by loss on ignition

Bulk weight percent organic matter, weight percent

carbonate, and weight percent ash of each sample were

estimated using loss on ignition (LOI) techniques.

300–400 mg of rock powder was weighed into a small

ceramic crucible and combusted in a Barnstead/Thermolyne



30 400 furnace at 550°C for 4 h. Once cool, the powders

were weighed, and mass loss at this step taken as the

organic matter content. The powders were then recombu-

sted at 950°C for 2 h, and the further mass loss taken as

weight percent carbonate. Material remaining after the final

combustion (primarily silicate, oxide, and sulfide minerals)

was considered ash.

RESULTS

Petrography

Ooids from Joulter’s Cays and Cat Island, Bahamas, and

North Carbla, Hamelin Pool, Australia were examined in

thin section in order to evaluate their relative preservation

states. The Joulters Cays sample contained ooids with vari-

able preservation; some are heavily micritized from endo-

lithic borings (Fig. 2A,B) while others preserve relatively

undisrupted cortical layers (Fig. 2A). The nuclei of ooids

at these localities are typically peloidal and only rarely skel-

etal. The outer layers of many ooids are well preserved, but

the inner part of the cortex is commonly bored and/or

micritized. Cat Island ooids show a similar preservational

style to those analyzed from Joulter’s Cays (Fig. 2C,D).

They are also occasionally cemented together to form

grapestone (Fig. 2D), and skeletal grains are rare. Hamelin

Pool ooids preserve exceptional outer cortical fabrics and

show little evidence for endolithic boring in their outer-

most laminae (Fig. 2E,F). These thin sections demonstrate

that other grains are generally rare in the ooid populations

selected for biomarker analysis, presumably as a result of

sorting in the depositional environment (Glumac et al.,

2011), and demonstrate that there is variability in the pres-

ervation of ooid cortices between localities.

Bulk compositions

The total organic carbon contents, as determined by loss

on ignition, were between 3.1 and 1.6% (Table 1). The

three modern samples all had slightly more organic carbon

than the Holocene oolites. Bulk d13Corg values, determined

after decalcification, were variable from �15.5 to �20.7&VPDB. The decalcified residues themselves, which con-

tained a significant proportion of inorganic material, were

isotopically heterogeneous based on a dispersion in the EA

data.

200 μm 200 μm

200 μm 200 μm

1

2

A B

C D

250 μm 250 μm

E F

Fig. 2 Photomicrograph of ooids embedded

in epoxy from Joulter’s Cays and Cat Island,

Bahamas, and Carbla Beach, Shark Bay,

Australia. (A) Joulter’s Cays ooids. Arrow 1

shows ooids with well-preserved outer cortex

and Arrow 2 shows micritized ooid;

(B) Joulter’s Cays ooids, one with round

endolithic boring (arrow); (C) Cat Island ooids,

with well-preserved cortical fabrics (arrows);

(D) Cat Island ooids, two of which show

micritic envelope around ooids (arrow)

resulting in grapestone formation; (E and F)

Carbla ooids showing well-preserved cortical

fabrics and little endolithic boring and/or

micritization of laminae.



Total lipid extracts

As shown in Table 1, and relative to the weights of ooids

extracted, the extraction by sonication appeared to afford

significantly higher total lipid extract yields (by a factor of

~5) than was produced by the ASE extraction. In contrast

to the total lipid extracts, the FA yields from the ASE

extraction were broadly similar to those obtained by soni-

cation despite there being minor differences in relative

abundance. Given the compositional similarities, we focus

the remainder of the results and discussion on the compo-

sition of the lipids extracted by the sonication procedure.

One point of note is that the yields of alcohols, including

sterols, which were a very minor fraction of the total TLE,

were slightly higher in the ASE extracts. The reason for

the large weight discrepancy between TLE obtained by the

sonication protocol vs. that obtained by ASE remains

unknown. However, it is possible that there was some very

fine-grained inorganic material in the ASE extracts that was

not removed by simple filtration.

Fatty acid methyl esters

Fatty acids were abundant and diverse in structure as

evident in the total ion chromatograms of the FAME frac-

tion of the TLE from Carbla ooid sample (Fig. 3). Individ-

ual compounds were initially identified using mass

chromatograms of the characteristic 74 Da McLafferty

rearrangement ion of the FAME. Identifications were con-

firmed, where possible, by comparison of the mass spectra

and retention times of the FAME with those of authentic

standards. The relative abundances of FAME in the

Holocene and Modern samples, as quantified from the

GC-FID chromatograms, are presented in the form of his-

tograms in Figs 4 and 5. Saturated, normal (straight-chain)

FAME with chain lengths from C10 to C32 were accompa-

nied by branched FAME with chain lengths of C12 to C18.

Monounsaturated C16 and C18 FAME were minor or trace

compounds in most of the samples, and they were more

often identified in the TLE than in TLE2 (Fig. 3). Traces

of a C16:2 FAME were detected in the Carbla and Cat

Island ooids, but the loci of unsaturation were not deter-

mined due to the low abundances of these components.

With two exceptions, n-C16 and n-C18 were the most

abundant FA in all samples, and their concentrations were

in the ranges 3–11 lg g�1 TOC and 2.5–46 lg g�1 TOC,

respectively. The exceptions were iso-C16 FA (85 lg g�1

TOC) being dominant in the Hanna Bay TLE and a C18:2

FA (6.7 lg g�1TOC) in the Cat Island sample. The C16:0

and C18:0 FAME showed similar patterns in the TLE and

TLE2 samples with the C16:0 FAs generally being more

abundant. The one exception was the Hanna Bay TLE2

sample that shows a slightly higher C18:0 abundance. For

Fig. 3 Total ion chromatograms from the gas

chromatography-mass spectrometry analyses

of fatty acid methyl esters in (A) TLE and (B)

TLE2 from the North Carbla ooid sample.



FA larger than C18, there was an overt even-over-odd car-

bon number preference, and their overall abundances

decreased progressively with chain length. These long-

chain fatty acids (LCFA) ranged from C22 to C36, generally

with a maximum at C22 or C24, and were accompanied by

very low amounts of iso, anteiso, and mid-chain methyl

analogs as can be seen in the chromatogram of the

North Carbla TLE FAME (Fig. 3A). The concentration of

n-C24:0 FAME ranged from 0.8 to 3.9 lg g�1 TOC in the

TLE to 2–22 lg g�1 TOC in TLE2. Of all the samples,

the ooids from Carbla Beach had the highest concentra-

tions of LCFA.

A common feature of all the samples analyzed was a rela-

tively high abundance of branched FAME, with C14–C18

iso- and anteiso FAME the most commonly identified.

The iso-C16:0 FAME was present in all the samples

(0.8–85 lg g�1 TOC in TLE and 1.0–20 lg g�1 TOC in

TLE2) and, as mentioned above, the most abundant FA in

the carbonate-bound fraction of the Holocene oolite from

Hanna Bay (85 mg g�1). Iso- and anteiso-C15:0 FAME

were also present in all samples. Another common feature

was the presence of a mid-chain monomethyl FAME with

17 carbon atoms. Comparison with a mixture of authentic

standards confirmed that the predominant isomer was

10-methylhexadecanoic acid (10-methyl-C16:0). In fact, for

the North Point sample, the 10-methyl-C16:0 and n-C17:0

FAME were present in almost equal concentrations. Some

samples also contained trace amounts of a FAME that, on

the basis of relative retention time, was probably 10-methyl

C17:0.

In addition to the FAME that were formed by transeste-

rification of complex ester-linked lipids, we also identified

low abundances of TMS FA derivatives in most samples.

These were likely derived from free FAs that were incom-

pletely methylated in the first step of our procedure. All

the TLE samples contain TMS C16:0 and C18:0 as well as

traces of other FA. We also observed that, compared to

TLE, the TLE2 samples contain more TMS VLCFA with

an even-over odd carbon number preference.

Other lipids

Trace amounts of n-alcohols, identified as their trimethylsi-

lyl derivatives, and n-alkanes are present in both Modern

and Holocene ooids. Their low abundances, however,

precluded a detailed comparison across the sample set. The

0

50

100 North Point TLE North Point TLE2

0

50

100 Hanna Bay TLE Hanna Bay TLE2

Fig. 4 Histograms showing relative abundances

of fatty acids in two Holocene ooid grainstones

from Cat Island, Bahamas. The columns labeled

TLE and TLE2 compare the compositions of

freely extractable lipids with those released by

acid dissolution. The data are all for sample that

were extracted using sonication.



Holocene samples contained saturated alcohols from n-C10

to n-C32 while, in the modern ones, we could only detect

these compounds as low as n-C14, and the abundances of

alcohols were greater in the TLE compared to the TLE2.

Samples that contained longer chain length alcohols exhib-

ited a distinct even-chain length preference as was the case

with the FA. This could be seen most clearly in the TLE

samples and the Joulters Cays ASE-extracted sample (data

not shown). The fossil oolite samples from North Point

and Hanna Bay had larger amounts of C16 and C18,

whereas the Modern samples were more enriched in C24

and C26. Many of the samples contained more than one

isomer for the C15–17 alcohols as shown by the presence of

multiple peaks with identical mass spectra but slightly

different retention times. The low abundances of alcohols

in the extracts precluded formal identification of these

isomers.

Steroids were detected in low abundance in most of the

samples. In contrast to the FAs, the distributions of sterols

differed markedly across the sample set. Cholesterol

(~900 ng g�1 TOC) and cholestanol (~600 ng g�1 TOC)

were the most prominent sterols in the Joulters Cays TLE,

0

50

100 Cat Island TLE

Cat Island TLE2

0

50

100 Joulters Cay TLE Joulters Cay TLE2

0

50

100 Carbla TLE Carbla TLE2

Fig. 5 Histograms showing relative abundances

of fatty acid methyl esters derived from TLE

and TLE2 for three samples of Modern ooids

from Joulters Cays and Cat Island in the

Bahamas and from North Carbla in Hamelin

Pool, Western Australia.



and the same compounds were present in TLE2. In the

North Point sample sitosterol, stigmasterol, and campester-

ol were identified in trace amounts. Sitosterol, stigmasterol,

and cholesterol along with stigmasta-3,5-diene were identi-

fied as trace components in the North Carbla sample.

Apart from the detection of traces of cholestanol in all the

samples, there did not appear to be any clear trend in the

sterol contents of the ooids.

In contrast to the variable patterns of sterols, several

hopanoids were consistently present in the samples. The

most abundant were methyl esters of the C31–C33 hopa-

noic acids and the TMS ether of bishomohopanol. Of

these compounds, bishomohopanoic acid methyl ester was

the most abundant. The methyl ester of bishomohopanoic

acid has a prominent molecular ion at 484 Da and is

accompanied by fragment ions at 469 (M-15), 369 (penta-

cyclic ring system), 263 (base peak; ring D cleavage with

side chain), and 191 Da. In the particular case of the

North Carbla TLE and TLE2, bishomohopanoic acid

methyl ester was visible in the total ion chromatograms

(Fig. 3) and was estimated to be present at a level of

~0.4 lg g�1 TOC based on a comparison of peak areas

with those of the FAME. Further, three isomers were pres-

ent with the predominant one identified as having the bio-

logical 17b, 21b-22R configuration based on its position

as the last eluting isomer and the relative intensities of the

191 (A+B ring fragment) and 263 (D+E+ side chain) Da

fragment ions. Smaller amounts of the 17a, 21b and 17b,21a isomers were also identified on the basis of elution

order and the relative intensities of the 191 and 263 Da

fragment ions (Peters et al., 2005).

Carbon isotopic compositions of FAME

The C-isotopic compositions of ooid FAs vary widely

although some consistent trends can be discerned (Table

5). In particular, the FAME from the freely extractable

lipid (TLE) were distinct from those in the carbonate-

bound fraction (TLE2). In almost all cases, the same com-

pound in the bound fraction was more enriched in 13C

than the one in the freely extractable fraction. FAME from

the North Carbla sample were more enriched in 13C com-

pared to those from the Bahamian Archipelago and, as

well, they varied over a narrower range than those of the

other samples. The C-isotopic compositions of the

branched FAs show greatest disparity with, for example,

the iso-C16 FAME having a d13C value as low as �39.1&VPDB in the Hanna Bay TLE and as high as �15.5& in

the North Carbla TLE2. Four measurements were possible

for the 10-methyl-C16 FAME and they too varied widely

from �36.9 to �20.0&. The most abundant n-C16:0 and

n-C18:0 FAME varied within a narrower range of values

from �19.2 to �24.5& (C16:0) and �18.8 to �26.5&(C18:0). The long-chain C20:0–C32:0 FAME followed a sim-

ilar trend of narrow isotopic dispersion within each sample

of TLE and TLE2.

Hydrogen Isotopic compositions of FAME

Hydrogen isotopic measurements were possible for a few

compounds in each sample with the North Carbla TLE

sample providing greatest coverage of FAME (Table 2).

Here, the prominent n-C16:0 and n-C18:0 FAME had dDvalues of �129.6 and �121.6& VSMOW, respectively.

The even carbon number long-chain FAME varied over a

similar range while the C23, and above, odd carbon num-

ber counterparts were slightly different and mostly a little

enriched in D. As with carbon isotopes, the North Carbla

FAME were more D-enriched than those of the Hanna

Bay and North Point samples. The greatest contrast was in

the H-isotopic compositions of the short-chain branched

FAME that were as heavy as +4.8& VSMOW in the case

of the iso-C15 from North Carbla TLE. The anteiso-C15:0

and iso-C15:0 were also significantly more enriched than

co-occurring n-C16:0 and n-C18:0 FAME. Short-chain

branched FAME in the North Point and Hanna Bay

samples were 60–100& more D-enriched than n-C16:0 and

n-C18:0 FAME, echoing the trend in the North Carbla

ooid sample. The hydrogen isotopic composition of the

Table 2 Hydrogen isotopic compositions of fatty acid methyl esters present

in the free and carbonate-bound lipid extracts from three samples of ooids

Carbon # Isomer

Hanna Bay North PointNorth Carbla

TLE TLE2 TLE TLE2

14 n �160.5

15 Iso 4.8

Anteiso �63.4

n �113.6

16 Iso �123.8 �112.0 �46.9

n �197.1 �171.0 �129.6 �111.1

17 10-Me �79.6

n �101.7

18 Iso �134.0

n �203.6 �198.3 �121.6 �114.4

19 n �116.1

20 n �120.8 �96.9

21 n �135.2

22 n �116.2 �99.6

23 n �111.3

24 n �125.3 �94.1

25 n �116.6

26 n �114.8 �71.2

27 n �107.3

28 n �128.2

29 n �109.2

30 n �123.7

The North Point and Hanna Bay samples are Holocene oolites while the

North Carbla sample is from contemporary beach ooids. The increased

sample demands of H-isotopic measurement precluded analysis of more

compounds.



water at the North Carbla ooid collection site was +14.2&VSMOW.

Carbon isotopic compositions of hydrocarbons

Normal alkanes are only a trace component of the organic

matter that was freely extractable from the ooids. On the

other hand, they are easily isolated in a clean fraction by

liquid chromatography that renders them amenable to iso-

topic analysis (Table 3).

Radiocarbon

Radiocarbon analyses of the North Carbla total lipid

extracts (Table 4) revealed that the carbon was roughly

contemporaneous with modern seawater. The carbonate-

bound lipids were, however, significantly older. A similar

comparison was attempted for the supratidal beach ooids

from Cat Island where TLE was measured to be

5000 year �35 BP. The TLE2 from this sample was lost

during sample preparation.

DISCUSSION

The samples studied here comprise unconsolidated ooids

from subtidal environments of Shark Bay and the Baha-

mas, beach ooids from Cat Island in the Bahamas, and

lithified Holocene oolites from the North Point and

Hanna Bay members of the Rice Bay Formation on Cat

Island. Thus, they represent a range of ages, diagenetic

histories, and preservation states. Petrographic examination

confirms this with variable extent of borings, micritization,

and cortex preservation. Within any single sample, there

are populations with different states of preservation, and

these differences are magnified across localities. Because

ooids grow over long periods in environments that typi-

cally experience near constant wave activity and occasional

major storm events (Duguid et al., 2010), it is to be

expected that individual grains in any sample will have

spent variable proportions of their existence in, above, and

beneath seawater. Thus, it seems quite remarkable that the

distributions of FAs recovered from the three samples of

contemporary ooids are quite similar and so distinctive. As

such, they provide insights into the nature of the biologi-

cal communities that have colonized them over 1000 year

timescales. The close similarity of the FAME distribution

patterns of freely extractable lipids in TLE and carbonate-

bound lipids in TLE2 indicates that a biosignature for the

microbial communities that have colonized the ooids dur-

ing their existence are recorded in the carbonate matrix.

While the prominent n-C16:0 and n-C18:0 FA are ubiqui-

tous in bacteria and eukaryotes and do not allow us to

identify specific sources, the short-chain, odd carbon num-

bered, and branched FA can be confidently attributed to

bacteria (Kaneda, 1991) and suggest that specific kinds of

bacteria have colonized the ooids during and/or after their

formation.

The nature of the short-chain branched FAME identi-

fied here in TLE and TLE2 specifically suggest an associa-

tion between sulfate-reducing bacteria and ooids. Sulfate-

reducing bacteria have been shown to possess diverse

cellular FAs that range from C12 to C19 (Taylor & Parkes,

1983; Dowling et al., 1986, 1988; Kuever et al., 2001)

including branched FAs, present in their phospholipids,

that are quite distinctive. The 13-methyl C15:0 (iso-C15:0)

and 12-methyl C15:0 (anteiso-C15:0) FAs identified in these

ooid samples have been previously identified in Desulfobul-

bus and Desulfobacter sp. (Taylor & Parkes, 1983), for

example. Even more diagnostic is the 10-methyl C16:0 FA

that is identifiable in all the samples and that is commonly

found in members of the Deltaproteobacteria. It has been

found in cultures of Desulfobacter sp. (Taylor & Parkes,

1983; Dowling et al., 1986, 1988) and in closely related

genera Desulfobacula and Desulfotignum (Kuever et al.,

2001). The 10-methyl C16:0 FA is also prevalent in envi-

ronmental samples where sulfate-reducing bacteria are

prominent (Hinrichs et al., 2000; Labrenz et al., 2000;

Elvert et al., 2003). 10-Methyl C16:0 has also been identi-

fied in Geobacter metallireducens (Lovley et al., 1993), but

unlike the aforementioned sulfate-reducing species, and

the ooid FAME, it is accompanied by significant amounts

of unsaturated C16 and C18 acids. Some samples also

contained trace amounts of 10-methyl C17:0 which, along

Table 3 Carbon isotopic compositions of hydrocarbons isolated from the

freely extractable total lipids for four samples of ooids

n-alkane Hanna Bay North Point Cat Island North Carbla

Carbon # TLE TLE TLE TLE

17 �14.1 �26.6 �21.7

18 �24.1 �19.3

19 �20.3 �28.1 �22.8

20 �22.0

21 �18.8 �22.5

22 �25.3 �26.6 �21.9

23 �22.2 �27.9 �21.9

24 �23.5 �29.7 �21.6

25 �22.3 �28.5 �24.4 �22.0

26 �24.8 �31.2 �22.3

27 �25.4 �28.9 �28.8 �24.1

28 �27.0 �30.4 �23.2

29 �27.8 �30.3 �30.7 �24.9

30 �27.5 �31.5 �28.8

31 �28.2 �32.3 �29.5 �25.8

32 �28.3 �31.8 �23.7

33 �27.7 �29.7 �26.2

34 �26.8

35 �27.6

36 �27.7

37 �26.3



with 10-methyl C15:0 and 10-methyl C18:0, has also been

reported in Desulfobacter spp. (Dowling et al., 1986).

Based on these data, we suggest the short-chain branched

FAs identified here are primarily sourced from sulfate-

reducing bacteria.

Very long-chain FA with a pronounced even-over-odd

carbon number preference are often attributed to an origin

from vascular plants due to their widespread occurrence in

leaf waxes and prevalence in sediments with significant

terrigenous plant inputs (Eglinton & Hamilton, 1967;�Rezanka, 1989; Prasad et al., 1990; Rielley et al., 1991).

However, the tropical shallow marine environments where

these ooids were collected do not favor vascular plants

being an important source of sedimentary organic matter.

While eolian processes can deliver plant organic matter, an

absence of significant riverine drainage to Hamelin Pool

(Logan & Cebulski, 1970) and these Bahamian environ-

ments places a limit on the deposition of terrigenous

organic matter. Moreover, in the present sample set,

another major leaf wax component, long-chain hydrocar-

bons with an odd-over-even preference are only present in

trace amounts and are isotopically distinct, in both C and

H, from the co-occurring FA. Thus, the very long-chain FA

and long-chain n-alkanes appear to have distinctly different

sources. Besides the ubiquitous vascular plants, very long-

chain FA have been detected broadly across the eukaryote

domain (�Rezanka, 1989; �Rezanka & Sigler, 2009).

Comparable observations of abundant medium- to long-

chain FAs have been made in other marine settings, most

recently in studies of the lipids preserved in sediments in

the Santa Monica Basin (Gong & Hollander, 1997;

Pearson et al., 2001) and in the Santa Barbara Basin (Li

et al., 2009). Pearson et al., 2001, in their study of Santa

Monica Basin sediments, observed a series of even-prefer-

ence C20+ FA, with a maximum abundance at C24. Among

other characteristics, Pearson & Eglinton (2000) reported

that the C24 FA had D14C values near to the end-member

DIC values determined for post-bomb and pre-bomb sedi-

ments from which the FA were isolated. Co-occurring odd

carbon numbered n-alkanes had more depleted D14C val-

ues. Where they could be measured in the ooid samples,

d13C values of the n-alkanes were more depleted than the

FA by 4–5& (Tables 3 and 5). These data suggested the

C20–C30 FA sources were distinct from the co-occurring

odd carbon numbered n-alkanes with the latter originating

from a mixed fossil carbon and contemporary terrigenous

higher plant sources (Pearson & Eglinton, 2000). Their

observations are also consistent with those of an earlier

study by Gong & Hollander (1997) who reported a similar

C24 predominance in the long-chain FAs and d13C values

that could not be reconciled with sources from vascular

plants. Rather, they hypothesized mixed input from plants

and bacteria to explain their data. In the Santa Barbara

Basin study of Li et al. (2009), hydrogen isotopic values

for C22–C26 FAs varied from �49 to �138& VSMOW

and were also distinct from co-occurring leaf wax compo-

nents. The authors tentatively attributed these compounds

to aquatic macrophytes, but they were among the most

D-enriched compounds detected in this study. There was

also significant overlap with the dD values of the odd

carbon number and branched FAs attributed to bacteria.

When all these results are considered, it seems likely that

there can be a primary bacterial source for C22–C36 FAs

with an even carbon number preference in the marine envi-

ronment. Based on their detection in a cultured strain of

spore-forming Desulfotomaculum sp. (�Rezanka et al.,

1990), we hypothesize that sulfate-reducing Fimicute bac-

teria are a candidate source for the long-chain FA that are

prevalent in ooids.

Hopanoic acids and hopanols

Hopanoic acids and hopanols are among the common

diagenetic products of bacteriohopanepolyols (BHP).

All the ooid extracts examined here contained 17b,21b-bishomohopanol, identified in the GC-MS analyses of

the silylated TLE, and attributable to bacteria. Because the

potential sources of BHP are so diverse, no specific state-

ments can be made regarding the origin(s) of these

biomarkers other than to note that their abundance was

much higher than sterols. The most abundant hopanoid in

all samples was 17b, 21b-bishomohopanoic acid that

appears as a late-eluting peak in the GC traces of all FAME

samples. Overall, the distributions of FAs and hopanoids

combined with the low abundances of sterols and their

diverse and inconsistent compositions suggest that the

organic matter preserved in the ooids and oolites is largely

of bacterial origin.

Carbon and hydrogen isotopic compositions of the short-

chain and branched FAME

A key inference that can be drawn from the carbon and

hydrogen isotopic data for ooid TLE and TLE2 FAME is

Table 4 Radiocarbon analysis results for North Carbla lipid extracts

Description NOSAMS Accession # d13C F Modern Fm Error Age Age Error D14C

North Carbla TLE OS-83162 �19.84 0.944731 0.0027 455 25 �62.0

North Carbla TLE2 OS-83163 �15.29 0.906024 0.0027 795 25 �100.4



that environmental conditions have varied during ooid

formation in the different locations represented in this

study. Although the same compounds are present in the

freely extractable and carbonate-bound lipids of all the

samples, their isotopic compositions differ. In the case of

the North Carbla ooid sample, the carbonate-bound lipids

are, on average, about 350 years older based on D14C

values. Further, the d13C and dD values for carbonate-

bound FA are consistently enriched by ~0.5–3.0 and

~10–30&, respectively. Hamelin Pool lies in an arid

region of Western Australia and is an evaporitic basin by

virtue of its connection to the open ocean being

restricted by a shallow sill, the Faure Sill (Logan, 1961;

Playford & Cockbain, 1976). This high salinity seawater

carries distinctive distribution of isotopic signatures for

the water and carbon as shown by 13C and 18O enrich-

ment of the endemic organisms from microbes (Des

Marais et al., 1992) to metazoa (Edmonds et al., 1999).

The C- and H-isotope data for Hamelin Pool ooid lipids

suggest that organic matter entrapped in the carbonate

matrix incorporated C and H, which was isotopically

different compared to the most easily extracted lipids, or

that some fractionation process operated during entrain-

ment of the organics in the carbonate. In the one case

where we could measure D14C values, the carbonate-

bound fraction was, on average, older than the freely

extractable fraction by ca. 350 years. This older 14C age

for the carbonate-bound lipids in the North Carbla sam-

ple suggests the 13C and D differences carry a signal for

earlier paleoenvironmental conditions. Similar patterns are

seen in the C-isotopic compositions of free and carbon-

ate-bound lipids in the Cat Island ooids and the Hanna

Bay and North Point oolites. In all cases, the carbonate-

bound FA are more enriched in 13C by, on average

1–3&. A more detailed isotopic analysis, including 14C

ages of the FAs released during sequential dissolution of

ooid matrix may reveal more about the processes leading

to this offset and the timescales of their emplacement.

Table 5 Carbon isotopic compositions of fatty acid methyl esters present in the free and carbonate-bound lipid extracts from four samples of ooids

Carbon # Isomer

Hanna Bay North Point Cat Island North Carbla

TLE TLE2 TLE TLE2 TLE TLE2 TLE TLE2

13 n �17.2

14 Iso �28.4 �15.6

n �20.5 �18.8 �21.9 �18.7 �25.2 �18.3 �21.4 �18.8

15 Iso �24.3 �18.1 �24.8 �18.8 �19.2 �17.1 �16.3 �14.6

Anteiso �22.2 �17.9 �24.5 �16.4 �15.7

n �23.4 �19.2 �24.6 �18.0 �24.8 �16.6 �17.9 �16.4

16 Iso �39.1 �37.1 �35.8 �25.3 �24.7 �19.0 �16.8 �15.5

n �23.6 �19.8 �24.4 �24.5 �23.7 �21.7 �21.4 �19.2

17 10-Me �24.7 �36.9 �31.9 �20.0

Iso �32.8 �23.8 �24.6 �19.8 �18.9 �15.3 �16.1 �16.6

Anteiso �33.8 �27.2 �25.3 �21.3 �21.8 0.0 �17.5 �15.9

n �26.7 �19.8 �24.1 �17.1 �23.3 �14.4 �18.2 �16.4

18 Iso �39.4 �22.3 �18.2

Anteiso �28.8 �24.6

n �26.5 �21.9 �20.9 �23.6 �23.0 �23.4 �21.8 �18.8

19 n �24.3 �15.2 �25.1 �13.9 �23.1 �12.8 �19.0 �18.1

20 Iso �15.3 �12.7

Anteiso �22.4

n �25.8 �25.2 �25.0 �16.0 �23.4 �14.8 �19.1 �16.3

21 n �15.0 �20.9 �19.9

22 n �26.5 �22.0 �24.5 �16.8 �26.8 �15.7 �20.7 �19.6

23 n �20.8 �17.8 �18.1 �21.2 �20.5

24 n �18.1 �16.5 �28.2 �16.4 �23.3 �16.2 �20.0 �19.0

25 n �20.0 �19.7 �18.4 �21.1 �20.5

26 n �20.1 �15.9 �26.9 �19.5 �23.4 �16.7 �21.1 �22.6

27 n �21.0 �17.9 �21.0 �20.7

28 n �20.2 �18.4 �28.4 �24.0 �33.5 �20.5 �21.1 �20.5

29 n �19.9 �20.7 �23.0 �21.7

30 n �22.7 �18.5 �26.7 �28.5 �23.8 �23.2 �22.8

31 n �24.2 �24.2

32 n �24.3 �25.3

33 n �25.0

34 n �22.0

The North Point and Hanna Bay samples are Holocene oolites while the Cat Island and North Carbla samples are from contemporary beach ooids.



The hydrogen isotopic compositions of FAs can be

informative about the physiology of organisms contribut-

ing these compounds to marine sediments. This is because

the hydrogen isotopic fractionation between lipids and

source water is in part a function of the way NADPH is

formed in different metabolic pathways (Zhang et al.,

2009). Photoautotrophs, for example, typically exhibit a

lipid/water fractionation of 180–200&, while chemoauto-

trophs often show even larger fractionations. In aerobic

heterotrophs, the fractionation is more variable and spans

�150 to +200&. Using these guidelines, we can rational-

ize some of the hydrogen isotopic data for ooid FA and

hydrocarbons. In Hanna Bay and North Point samples,

where ooids formed in open ocean waters, the dD values

of C16 and C18 FAs vary from �171 to �203& VSMOW

suggesting that photoautotrophs were important sources

for these compounds in the Holocene Bahamian ooids. In

comparison, dD values for the same FA from the Carbla

sample were significantly D-enriched at �120 to �130&VSMOW, yet the H- isotopic composition of Hamelin

Pool waters at the ooid collection site was +14.2&VSMOW. The D-enrichment in the TLE2 FA was even

more extreme. These smaller fractionations may reflect a

different balance of autotrophic vs. heterotrophic contribu-

tions to these FA. Alternatively, they may reflect the higher

salinity waters in Hamelin Pool, or a combination of both

factors.

Sample availability restricted measurement of dD values

of branched FA in the North Point sample to iso-C16, iso-

C18, and 10-Me C16 FA. All were D-enriched compared to

the straight-chain compounds with 10-Me C16 FA having a

dD of �79.6& VSMOW, consistent with a source distinct

from photoautotrophs. Branched FAs in the North Carbla

sample are even more enriched and have divergent dD val-

ues suggesting they represent differential inputs from bacte-

ria with heterotrophic physiologies. On the other hand, the

VLCFA in the freely extractable lipid have dD values in the

same range as n-C16 and n-C18 FA in the same sample con-

sistent with photoautotrophic or heterotrophic sources, but

not to the exclusion of other possibilities.

Overall similarities and origins of the ooid geochemical

signatures

Lipids that are readily extracted from ooids and oolites are

very similar in composition to those released after carbon-

ate dissolution. This suggests that the microbial commu-

nity colonizing the ooids has varied little over time during

their formation. The notable characteristics of the lipid dis-

tribution include a predominance of n-C16 and n-C18 satu-

rated FA that, although ubiquitous in nature, likely

represent a predominant source from photoautotrophic

microbes such as cyanobacteria. These are accompanied by

significant amounts of branched and saturated FA that

likely originate from heterotrophic bacteria including

sulfate-reducing bacteria. Very long-chain normal FA, with

a pronounced even-over odd carbon number preference,

together with smaller amounts of iso- and anteiso- coun-

terparts may also originate from bacteria, possibly firmi-

cutes, although we cannot unambiguously ascribe a

particular source at this time. However, a recent molecular

study of Bahamian ooids from Highborne Cay sheds some

light on this. Small subunit (16S) ribosomal DNA data for

a sample of ooids from the intertidal zone, generated from

the living community via analyses of cDNA generated from

RNA, indicate that the overall bacterial diversity was com-

parable to that found within thrombolites and stromato-

lites at the same location (Edgcomb et al., 2013).

Cyanobacteria were the most diverse taxonomic group

detected in the ooids, followed by Alphaproteobacteria,

Gammaproteobacteria, Planctomyces, and Deltaproteobac-

teria including diverse sulfate reducers. Sequences for the

firmicute genus Moorella were also identified. These ooids

had lipid signatures that were very similar to those

observed in the present study. FAs were dominated by

C16:0 and C18:0 each of which was accompanied by lower

abundances of their mono- and diunsaturated analogs.

Odd carbon number and branched C14–C17 FA were pres-

ent, the most abundant of which was 10-Me C16. Also

prominent was the homologous series of even carbon

numbered C22–C30 FA together with the methyl esters of

bishomohopanoic acid and phytanic acid. A diverse assem-

blage of BHP was identified in LC-MS analyses of the ace-

tate derivatives. Bacteriohopanetetrol (BHT) was the most

abundant of these along with the corresponding 2-methyl

and 3-methyl analogs and BHT cyclitol ether. Bacterioho-

paneaminotriol was accompanied by a corresponding 3-

methyl analog. These data, together with IPL precursors of

the FAME are consistent with a diverse and predominantly

bacterial microbial community (Edgcomb et al., 2013). In

particular, the molecular evidence for combinations of pri-

mary producing cyanobacteria and heterotrophs that

include sulfate reducers provides both organic matter and

sources of alkalinity that can drive active carbonate precipi-

tation as observed in other lithifying organosedimentary

biofilms (Dupraz & Visscher, 2005; Dupraz et al., 2009).

CONCLUSIONS

Lipid distributions within the contemporary beach ooids

from Western Australia and from the Bahamas, and Holocene

oolites from the Bahamas, are remarkably similar. They

suggest that a specific community of microbes has colo-

nized these ooids wherever they form and possibly also

contributed to ooid cortex growth. Studies of other, and

more ancient, fossil oolites will be needed to verify this

observation. Despite being limited in the number of mea-

surements, the C- and H-isotopic data set for FAME and



hydrocarbons does firmly establish that the lipids we have

isolated originate from microbes with diverse physiologies

that include photoautotrophs, heterotrophs, and sulfate-

reducing bacteria. By analogy with studies of the microbial

communities forming stromatolites and thrombolites in

similar environments, we hypothesize that colonizing

microbes exist in the form of a bacterial biofilm that allows

these microbes to operate syntrophically. The identification

of sulfate-reducing bacteria biomarkers is particularly sig-

nificant in this regard because they can provide an alkalinity

pump in support of calcification (Riding, 1991; Visscher

et al., 1998; Paterson et al., 2008; Dupraz et al., 2009).

This is an important consideration as it is known that ooid

occurrences are exceptionally rare in environments, and

particularly in the Pacific Ocean and some parts of the

Atlantic, where pH and total alkalinity are significantly

below values that allow facile carbonate precipitation

(Rankey & Reeder, 2009).

The data presented here provide evidence suggestive of a

specific microbiota inhabiting ooids in environments where

they are actively forming. Although our data do not prove

the involvement of microbial biofilms in driving ooid for-

mation, we propose that there is substantial circumstantial

evidence that they do. Specifically, the microbial commu-

nity inhabiting ooids appears very similar to those inhabit-

ing other environments where microbial mats, including

stromatolites and thrombolites (Dupraz & Visscher, 2005)

are undergoing active lithification while a photosyntheti-

cally powered community of biofilm-forming microbes is

observed to play an active role in ooid formation in a

freshwater environment (Pl�ee et al., 2008). Moreover, the

presence and metabolic activity of sulfate-reducing bacteria

in the marine biofilm communities serves to increase local

alkalinity and so provides a plausible mechanism for

enhancing rates of carbonate precipitation. Thus, – in con-

trast to the recent conclusions of Duguid et al. (2010) –

we feel there is substantial evidence that microbes do play

an active role in ooid formation, and that this potential

role deserves further geobiological investigation.

ACKNOWLEDGMENTS

We thank Sara Lincoln and Carolyn Colonero for advice

and assistance in the laboratory. David Fike generously

provided the ooids from Joulter’s Cays. Work at MIT was

supported by grants from the NASA Astrobiology Institute

(NNA08CN84A), the NSF Program on Emerging Trends

in Biogeochemical Cycles (OCE-0849940) and the NSF

Chemical Oceanography Program (OCE-0926372). Fund-

ing for sample collection from Cat Island, The Bahamas,

was made possible by a Smith College Committee on Fac-

ulty Development grant (to SBP). The authors thank two

anonymous reviewers for their critical comments on the

initial submission.

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