Extraordinary 13C enrichment of diether lipids at the Lost ...

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Geochimica et Cosmochimica Acta 73 (2009) 102–118

Extraordinary 13C enrichment of diether lipids at the LostCity Hydrothermal Field indicates a carbon-limited ecosystem

Alexander S. Bradley a,*, John M. Hayes b, Roger E. Summons a

a Department of Earth, Atmospheric and Planetary Sciences, Massachusetts Institute of Technology, Cambridge, MA 02139, USAb Department of Geology and Geophysics, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA

Received 3 March 2008; accepted in revised form 1 October 2008; available online 17 October 2008

Abstract

Active and inactive carbonate chimneys from the Lost City Hydrothermal Field contain up to 0.6% organic carbon withdiverse lipid assemblages. The d13C values of total organic carbon range from �21.5& vs. VPDB at an extinct carbonatechimney to �2.8& at a 70 �C, actively venting carbonate chimney. Samples collected at locations with total organic carbonwith d13C > �15& also contained high abundances of isoprenoidal and nonisoprenoidal diether lipids. Samples with TOCmore depleted in 13C lacked or contained lower amounts of these diethers.

Isoprenoidal diethers, including sn-2 hydroxyarchaeol, sn-3 hydroxyarchaeol, and putative dihydroxyarchaeol, are likely toderive from methanogenic archaea. These compounds have d13C values ranging from �2.9 to +6.7& vs. VPDB. Noniso-prenoidal diethers and monoethers are presumably derived from bacteria, and have structures similar to those producedby sulfate-reducing bacteria in culture and at cold seeps. In samples that also contained abundant hydroxyarchaeols, thesediethers have d13C values between �11.8 and +3.6&. In samples without abundant hydroxyarchaeols, the nonisoprenoidaldiethers were typically more depleted in 13C, with d13C as low as �28.7& in chimneys and �45& in fissures.

The diethers at Lost City are probably derived from hydrogen-consuming methanogens and bacteria. High hydrogen con-centrations favor methanogenesis over methanotrophy and allow the concurrent growth of methanogens and sulfate-reducingbacteria. The unusual enrichment of 13C in lipids can be attributed to nearly complete consumption of bioavailable carbon invent fluids. Under carbon-limited conditions, the isotope effects that usually lead to 13C-depletion in organic material cannotbe expressed. Consequently, metabolic products such as lipids and methane have d13C values typical of abiotic carbon.� 2008 Elsevier Ltd. All rights reserved.

1. INTRODUCTION

The discovery of the Lost City Hydrothermal Field(LCHF) in 2000 (Kelley et al., 2001, 2005) marked the firstrecognition of a warm ocean-floor hydrothermal ecosystem,hosted by peridotite, with chemistry controlled by the ser-pentinization of ultramafic rock. Ultramafic rocks are a sig-nificant component of the oceanic lithosphere (Fruh-Greenet al., 2004), may have been widespread on early Earth

0016-7037/$ - see front matter � 2008 Elsevier Ltd. All rights reserved.

doi:10.1016/j.gca.2008.10.005

* Corresponding author. Present address: Department of Organ-ismic and Evolutionary Biology, Harvard University, Cambridge,MA 02138, USA.

E-mail address: [email protected] (A.S. Bradley).

(Shock and Schulte, 1998). They can be expected on otherterrestrial planets containing water (Sleep et al., 2004), suchas Europa (McCollom, 1999). Because of the alkalinity andreducing power produced by serpentinization, the alter-ation of ultramafic rocks by water has important geochem-ical and biological consequences. Study of theseenvironments may bear on prebiotic chemistry, early Earthevolution, and the potential for life elsewhere.

Reducing power, in the form of hydrogen and methane,is abundant in Lost City fluids. Hydrogen is produced di-rectly during serpentinization (Schroeder et al., 2002) andcan be coupled to reduction of CO2 to produce methane.Formation of methane is thermodynamically favored inenvironments where chemistry is controlled by serpentiniza-tion (Sleep et al., 2004), and may be biologically or abiotically

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13C-enriched organic material at Lost City Hydrothermal Field 103

catalyzed. Both the abiotic geochemical reaction(Proskurowski et al., 2008) and production of methane bymethanogenic archaea (Schrenk et al., 2004; Brazeltonet al., 2006) are likely to be important processes at LostCity.

Determining the relative contributions of abiotic andbiological methane to Lost City vent fluids is a challengethat holds significance for our understanding of the evolu-tion of the biosphere. Methane is an important trace gasin the modern atmosphere and was probably abundant inthe atmosphere during the first two billion years of Earthhistory (Catling et al., 2001; Kasting and Ono, 2006), whenit may have played an important role in regulating globaltemperature (Pavlov et al., 2001; Lowe and Tice, 2004).The role of methanogens in moderating the hydrogen levelin the atmosphere is a critical component of Precambrianclimate models (Kasting, 2005) and the abundance ofhydrogen in the Precambrian atmosphere is hotly debated(Tian et al., 2005, 2006; Catling, 2006). Understandingthe potential for contributions to atmospheric methane bysites like Lost City, and the importance of biology in mak-ing that contribution, will influence these models. Methanehas also been detected in the Martian atmosphere (Formi-sano et al., 2004). Its source is unknown but derivationfrom peridotite-hosted hydrothermal sites is notinconceivable.

Biological methanogenesis requires no direct or indirectbyproducts of oxygenic photosynthesis and may be one ofthe more ancient metabolic strategies on Earth. There isevidence for methanogenesis as far back in Earth historyas the Archean (Ueno et al., 2006), although some of thisevidence has been subsequently challenged (Sherwood Lol-lar and McCollom, 2006), and some biologists dispute theearly evolution of methanogenic archaea (Cavalier-Smith,2002). Hydrogen as an electron donor can also be coupledto other electron acceptors such as sulfate and, when theseelectron acceptors are present, organisms performing thesereactions generally outcompete methanogens (Kristjanssonet al., 1982; Hoehler et al., 1998).

The LCHF is located on the peridotitic Atlantis massif15 km west of the axis of the mid-Atlantic ridge at a depthof 750–900 m. Fluids are alkaline, reducing, and cool rela-tive to those at magma-driven hydrothermal systems on themid-ocean ridge axes. Cooling of the underlying massif isthe main driver of hydrothermal circulation at Lost City(Allen and Seyfried, 2004), with additional contributionsfrom exothermic serpentinization reactions (Kelley et al.,2001, 2005). Fluid temperatures range from <40 to 90 �Cwith pH between 9 and 11, Ca2+ concentrations up to30 mmol/kg, CH4 concentrations up to almost 2 mmol/kg,and hydrogen concentrations up to nearly 15 mmol/kg.Recently, Proskurowski et al. (2008) have reported theabundances of 13C, 14C, and 2H in volatile hydrocarbonsfrom the Lost City hydrothermal fluids. Their results showthat the methane derives entirely from mantle carbon butcannot quantitatively resolve microbial and abioticcontributions.

The most spectacular features of the LCHF are massivecalcium carbonate and brucite chimneys, which grow up to60 m tall from the peridotite seafloor. Radiocarbon mea-

surements indicate that the carbonate has been precipitat-ing at its present location for at least 30 ky (Fruh-Greenet al., 2003). The carbonate has d13C between +1.4 and+2.9& and d18O between +1.5& and +5.2& relative toVPDB, while 87Sr/86Sr values are between 0.70760 and0.70908. Calcium carbonate precipitates as vent fluids comeinto contact with seawater. The Ca2+ source is dominantlyhydrothermal, while the CO3

2� derives predominantly fromseawater (Fruh-Green et al., 2003; Ludwig et al., 2006).Measurements of Sr isotopes and wt% Mg in carbonatechimneys have shown that mixing ratios of seawater andvent fluids vary between chimneys (Ludwig et al., 2006).

The carbonate towers host a rich microbial ecosystem.Analyses of 16S rRNA gene sequences and cell counts byfluorescence in situ hybridization (FISH) show that assem-blages in active vent structures are dominated by archaea(Kelley et al., 2005; Brazelton et al., 2006), with Methanos-arcinales prevalent in active, high-temperature vent struc-tures. ANME-1 group methanotrophs are present in twosamples from less active carbonate veins and at one activevent (Schrenk et al., 2004; Kelley et al., 2005; Brazeltonet al., 2006). Studies of bacterial diversity indicate that Fir-micutes, along with methane-oxidizing and sulfur-oxidizingbacteria, are present in fluids at higher-temperature, activevents (Kelley et al., 2005; Brazelton et al., 2006). The coex-istence of obligate aerobes and obligate anaerobes in thesame samples suggests that different microbial communitiesinhabit various environments around the vents, possibly ananaerobic community inhabiting reducing anaerobic hydro-thermal fluid within carbonate structures and aerobic mi-crobes inhabiting zones on carbonate surfaces wherehydrothermal fluid mixes with oxygenated seawater.

Here we report on the chemical and isotopic composi-tion of organic matter in the carbonate chimneys at theLCHF. These data shed light on the nature of the Lost CityMethanosarcinales and on the biological production ofmethane.

2. ANALYTICAL METHODS

Carbonate samples were collected during Atlantis cruiseAT-7-41 using the submersible Alvin and stored in tefloncontainers at �20 �C until processing. Marker locations re-fer to specific sites within the hydrothermal field and havebeen standardized across multiple expeditions and reports(Kelley et al., 2005). The first four digits of each samplenumber specify the Alvin dive number. The last four specifythe time of collection (1640 = 2:40 PM). These numbers arealso standardized across all reports.

Total organic carbon contents of carbonates weredetermined by weighing freeze-dried and finely crushedcarbonate samples (20–40 mg) in triplicate into clean silvercapsules. The silver capsules were placed in an evacuatedchamber for seven days with vapor in equilibrium withconcentrated HCl and several grams of P2O5, whichserved as a dessicant. Complete removal of carbonatewas verified by addition of 50–100 ll of concentratedHCl directly to the silver capsules at 60 �C. Sampleswere dried at 60 �C overnight, and residual CaCl2 was al-lowed to remain with the samples. Silver capsules were

104 A.S. Bradley et al. / Geochimica et Cosmochimica Acta 73 (2009) 102–118

combusted in a Fisons Elemental Analyzer at 1030 �Ccoupled to a ThermoFinnigan Deltaplus XL isotope ratiomonitoring mass spectrometer. Inspection of the resultingCO2 traces with ThermoFinnigan IsoDat software con-firmed the absence of any residual carbonate in the sam-ples. Total organic carbon contents were calculated byintegration of peak areas and comparison with an externalstandard with known carbon content. Stable carbon iso-tope ratios were determined using an external CO2

standard calibrated to international reference materialsNBS-22 oil, CH-6 sucrose, and an internal lab standard(acetanilide). They are reported relative to the ViennaPee Dee Belemnite (VPDB) isotopic standard.

Subsamples of carbonate were freeze-dried and crushedto a fine powder, then ultrasonically extracted three times(ca. 30 min) in a mixture of dichloromethane (DCM):methanol (3:1, v/v), and all three extracts were combined.Extracts were centrifuged at 2000 rpm for 15 min to re-move residual carbonate particles and then the bulk ofsolvent was evaporated at 35 �C under a stream of drynitrogen. Elemental sulfur was removed from the extractsby passing them over a column of activated copper, fol-lowed by filtration of the extract through a 40-lm com-busted glass Buchner funnel. Total lipid extracts wereweighed and are reported as lg of lipid per gram of dryrock extracted.

Aliquots of lipid extracts were analyzed as their tri-methylsilyl ethers and esters by reacting with N,O-bis(tri-methylsilyl)trifluoro-acetamide (BSTFA + 1% TMCS) inpyridine at 60 �C for 30 min. The remainder of the totallipid extract was separated over silica gel into five frac-tions using an elution scheme of solvents of increasingpolarity: aliphatic hydrocarbons 1% dead column volume(DV, measured by slow addition of the first eluent withcalibrated syringe) hexane, aromatic hydrocarbons 2 DV4:1 hexane:DCM, ketones 2 DV DCM, alcohols 2 DV4:1 DCM:ethyl acetate, fatty acids and diols 2 DV 7:3DCM:methanol. Individual lipids were identified using aHP 6890 gas chromatograph fitted with a PTV injectoroperated in splitless mode and equipped with a VarianCP-Sil-5 (60-m length, 0.32-mm inner diameter, and

δ13C

TOC (‰

)

TOC (%)0.0 0.1 0.2 0.3 0.4 0.5

0

-5

-10

-15

-20

-25

Fig. 1. (a) A cross-plot of total organic carbon versus d13CTOC for all csamples. (b) A cross-plot of diether content versus d13CTOC for samples wi13C contain higher amounts of isoprenoidal and nonisoprenoidal diether

0.25-lm film thickness) fused silica capillary column andcoupled to an Agilent 5973 mass-selective detector. Lipidswere identified by comparisons of mass spectra and reten-tion times with authentic standards or samples where thesecompounds have previously been characterized. Alcoholsand fatty acids were analyzed as TMS derivatives. Dietherlipids were identified by comparison to similar authenticstandards, and we report their masses without attemptingto solve their detailed structures. Lipids were quantifiedrelative to coinjected standards.

Carbon-isotopic compositions of individual lipids weredetermined using a TraceGC gas chromatograph fittedwith a PTV injector and equipped with a Varian DB-1(60-m length, 0.32-mm inner diameter, and 0.25-lm filmthickness) fused-silica capillary column and coupled to aThermoFinnigan Deltaplus XL isotope-ratio-monitoringmass spectrometer via a combustion interface at 850 �C.Column temperatures were programmed from 60 �C at10 �C/min to 100 �C, to 320 �C at 4 �C/min, and then heldisothermal for 20 min. Carbon isotope ratios were deter-mined relative to an external CO2 standard that was reg-ularly calibrated relative to a reference mixture of n-alkanes (Mixture B) provided by Arndt Schimmelmann(Indiana University). All lipid isotopic compositions werecorrected by mass balance for the carbon present in theTMS group(s).

3. RESULTS

3.1. Total organic carbon

Thirty-seven carbonate samples had total organic car-bon contents averaging about 0.2% and varying betweenapproximately 0.05% and 0.6% TOC (Fig. 1a). The 13Ccontent of the organic carbon was widely variable withd13C from �27.7& to �2.8&. TOC and d13C were uncor-related (Fig. 1a). Samples taken from various parts of thesame carbonate tower had values of d13CTOC varying byup to 14.4&, but replicate measurements for a single sam-ple yielded variations <0.5&. Accordingly, larger varia-tions reflect natural heterogeneity within a tower.

δ13C

TOC (‰

)

0 5 10 15 20 25

0

-5

-10

-15

-20

-25

diether concentration (μg/g)

ActiveInactiveFissure

arbonate chimney samples, grouped as active, inactive, and fissureth lipid analyses, showing that samples with TOC highly enriched inlipids.


3.2. Lipid analyses

3.2.1. Isoprenoidal ether lipids and alkanes

Archaeol was detected in approximately one-half of thesamples (Table 1). Concentrations of archaeol ranged up to600 ng of archaeol per gram of dry carbonate. In most casesarchaeol was enriched in 13C relative to marine phytoplank-tonic products, which typically have d13C near �20& at 30�north (Goericke and Fry, 1994). Archaeol had d13C valuesup to +6.0&, and the average value of d13Carchaeol in car-bonate chimneys was +1.5&. Only one sample containedarchaeol with d13C < �4.2&. That sample, withd13Carchaeol = �77&, was an inactive carbonate collectedon the eastern fringe of the vent field (Marker X2, 30 m eastof Marker H; sample 3880-1557).

With the single exception of sample 3880-1557, all sam-ples containing archaeol also contained sn-2 hydroxyar-chaeol, sn-3 hydroxyarchaeol, and a putativedihydroxyarchaeol. Both sn-2 hydroxyarchaeol and dihydr-oxyarchaeol were more abundant than archaeol. Concen-trations of archaeol were strongly covariant with those ofsn-2 hydroxyarchaeol and dihydroxyarchaeol. The d13Cvalues of archaeol covaried with those of sn-2 hydroxyar-chaeol (Fig. 2a), but not with those of dihydroxyarchaeol(Fig. 2b). The concentration of sn-2 hydroxyarchaeol aver-aged 2.6 times that of dihydroxyarchaeol, and both of thesecompounds were more abundant than sn-3 hydroxyarchae-ol (Fig. 3c).

Two unidentified compounds with mass spectra similarto those of hydroxyarchaeols were common at LostCity—one eluting after sn-3 hydroxyarchaeol and the otherjust after dihydroxyarchaeol (Fig. 3a and b). The mass spec-tra of these compounds showed prominent ions at m/z 143,237, and 341, and a smaller ion at m/z 515. This pattern issimilar to that seen in putative dihydroxyarchaeol. A frag-ment at m/z 278 in the spectrum shown in Fig. 3a may indi-cate that a nonhydroxylated phytanyl moiety is bound toglycerol by an ether linkage (Hinrichs et al., 2000a). To-gether with the retention times, these spectra suggest thatthese unknown compounds are structurally related to thehydroxyarchaeols.

The hydrocarbon 2,6,10,16,19-pentamethylicosane(PMI; +7 6 d13C 6 +10&; Table 1) was present in twosamples and had d13C values that were the highest of anycompound detected.

3.2.2. Nonisoprenoidal ether lipids

Nonisoprenoidal ether lipids were present in nearly allsamples analyzed (Table 1). Diethers were most abundantand ranged in mass from 556 Da (C31-TMS) to 664 Da(C39:2-TMS), with saturated and unsaturated side chainsranging in length from 13 to 18 carbons. The range ofether–lipid structures at Lost City is similar to that de-scribed in cold-seep carbonate crusts associated with Med-iterranean mud volcanoes (Pancost et al., 2001a;Bouloubassi et al., 2006), including the Series I, II, andIII components. Similar structures have been describedfrom other AOM environments (Hinrichs et al., 2000a; El-vert et al., 2005), and we detect a wide range of structures,including several additional series of diethers that do not

fall into the categories described in these previous reports.Retention indices and mass spectra were consistent withthe presence of unbranched alkyl chains along with iso-,anteiso-, and x7 methylation patterns. Hydrogen deficitswere commonly due to alicyclic rings (cyclopropyl, cyclo-hexyl) rather than to double bonds. Several samples alsocontained glycerol monoethers, with C16:1 and C18 noni-soprenoidal side chains, with unknown double bond posi-tions, being most abundant.

The nonisoprenoidal ether lipids, like the isoprenoidaldiethers, were frequently enriched in 13C. The most extremeenrichments in these compounds are associated with sam-ples in which hydroxyarchaeols are also abundant. In sam-ples containing both sn-2 hydroxyarchaeol anddihydroxyarchaeol, the d13Cvalues of nonisoprenoidal die-thers ranged from �11.8& to +3.6& (Table 1 and Fig. 4)and those of monoethers ranged from �19.2& to �3.9&.Nonisoprenoidal diethers were typically 2–10& more de-pleted in 13C than hydroxyarchaeols, while nonisoprenoidalmonoethers were the most 13C depleted of these three com-pound classes.

In carbonate tower samples where hydroxyarchaeolswere not abundant, nonisoprenoidal diethers had d13Crang-ing from �14.3 to �28.7&. Monoether d13C values rangedfrom �19.6 to �26.7&. There was one exception: a samplefrom Marker 3 (3881-1408) in which archaeal lipids wereabsent had nonisoprenoidal diethers with d13C averagingnear �6&.

3.2.3. Fatty acids

Free fatty acids, potentially derived from both eukary-otes and bacteria, were detected in nearly all samples. Con-centrations ranged to over 1500 ng per gram of dry rock.Abundances and d13C values of fatty acids are listed inTable 1. Fatty acids had highly variable contents of 13C,spanning a range from approximately �1& to �27&.The most 13C-enriched fatty acids were found in samplescontaining abundant hydroxyarchaeols. In many of these,the 13C content of fatty acids showed a strong pattern inwhich saturated fatty acids were more enriched in 13C thanmonounsaturated fatty acids (Table 1). The most 13C-de-pleted fatty acids at Lost City were found in the carbonateveins, where all detected lipids had d13C < �15&.

3.2.4. Polycyclic triterpenoids

Sterols were detected in all samples analyzed. Choles-terol, commonly derived from animals but also from somealgae such as eustigmatophytes, was the most frequently de-tected sterol and occurred in concentrations up to 580 ngper gram of dry carbonate. Stigmasterol and b-sitosterol,phytosterols often derived from marine algae, were alsocommonly detected. Ergosterol, a common fungal sterol,was detected in trace amounts in a few samples. Cycloarte-nol was commonly detected in concentrations approaching500 ng per gram of dry rock. Its identity was confirmed byits mass spectrum and by coinjection with an authenticstandard.

Sterols were not in any case as exceptionally 13C-en-riched as diethers. Cholesterol ranged in d13C from approx-imately �28& to �23.2&. Phytosterols had a slightly wider

Table 1Individual lipid abundances and d13C values of selected samples. Sample number and Marker (Kelley et al., 2005) are noted and correspond to locations and samples noted in other reports (Kelleyet al., 2005; Brazelton et al., 2006; Ludwig et al., 2006; Proskurowski et al., 2006). d13CTOC and information about vent activity (A = active, I = inactive, or F = fissure Ludwig et al., 2006) andtemperature is supplied where that information is known. All lipid concentrations are in lg lipid per gram of dry rock. Blank entries indicate that that lipid was not detected in a particular sample.

Marker 2 2 3 near 7 7 7 7 B C C H H X1 X2

Sample number 3864-1524 3864-1537 3862-1219 3865-1322 3867-1225 3867-1228 3879-1605 3876-1436 3869-1404 3869-1446 3881-1325 3881-1228 3876-1133 3880-1557

Temperature 64 53.5 59 — 34 — 52 90 70 70 46 — — —

Vent activity A A A F A I A A A A I A F F

TOC (%) 0.11 0.12 0.15 0.10 0.25 0.36 0.44 0.07 0.20 0.34 0.10 0.09 0.15 0.11

d13Croc (%0, VS. VPDB) �15.5 �8.8 �12.2 �15.1 �5.2 �7.5 �6.5 �16.2 �7.8 �4.7 �18.8 �16.9 �16.3 �20.0

Total lipid (lg/g) 304 253 92 35 235 217 364 6 53 91 3 36 76 105

Concentration

(lg/g)

d13 C

ð&ÞConcentration

(lg/g)

d13 C

ð&ÞConcentration

(lg/g)

d13 C

ð&ÞConcentration

(lg/g)

d13 C

ð&ÞConcentration

(lg/g)

d13 C

ð&ÞConcentration

(lg/g)

d13 C

ð&ÞConcentration

(lg/g)

d13 C

ð&ÞConcentration

(lg/g)

d13 C

ð&ÞConcentration

(lg/g)

d13 C

ð&ÞConcentration

(lg/g)

d13 C

ð&ÞConcentration

(lg/g)

d13 C

ð&Þconcentration

(lg/g)

d13 C

ð&Þconcentration

(lg/g)

d13 C

ð&Þconcentration

(lg/g)

d13 C

ð&Þ

archaeal lipids Archaeol 0.02 �0.4 0.12 �0.8 0.01 �2.5 0.61 0.7 0.42 6.0 0.16 1.3 0.03 6.7 0.10 5.7 0.01 �4.2 0.01 �77.0

sn-2 hydroxyarchaeol 0.15 2.4 0.21 0.2 0.07 1.3 5.94 1.3 6.66 4.2 1.80 0.7 0.39 5.1 0.97 4.8 0.01 �2.2 0.18 0.6

sn-3 hydroxyarchaeol 0.03 1.9 — 0.01 0.2 1.25 �2.1 0.67 �1.5 0.39 0.5 0.03 0.8 0.12 6.0 0.08 4.8

Dihydroxyarchaeol 0.05 3.8 0.09 1.1 0.02 1.2 3.47 �0.4 1.97 3.8 1.09 0.5 0.11 6.6 0.40 5.3 0.09 �2.9

PMI 0.12 7.1 0.22 10.0

fatty acids C16:1 fatty acid 0.02 �12.9 0.11 �13.5 0.26 �28.3 0.17 �13.2 0.28 �25.4 0.06 �19.5 0.10 �20.7 0.02 �27.3 0.08 �17.0 0.00 0.24 �14.7 0.39 �21.5

C16 fatty acid 0.06 �7.3 1.62 �6.8 0.75 �8.8 0.15 �13.4 1.56 �7.5 0.09 �8.6 0.73 �9.1 0.01 �31.4 0.04 �25.7 0.52 �4.3 0.01 0.33 �12.0 0.12 �27.4

C18:1 fatty acid 0.06 �13.3 0.47 �13.4 0.15 �17.6 0.08 �20.1 0.41 �22.5 0.03 �26.5 0.18 �16.8 0.04 �25.4 0.14 �20.3 0.01 0.85 �12.0 0.16 �27.5

C18 fatty acid 0.02 �8.7 0.53 �8.9 0.10 �5.4 0.01 �20.4 0.51 �1.1 0.10 �2.2 0.21 �8.7 0.01 �30.2 0.02 �15.3 0.21 �8.0 0.01 0.11 �12.4 0.02 �25.0

polycyclic triterpenoids Cholesterol 0.04 �27.7 0.35 0.25 �28.5 0.10 �24.7 0.29 �26.1 0.10 �26.1 0.17 �27.8 0.01 �27.7 0.06 �26.3 0.49 �27.5 0.06 �23.3 0.01 �26.4 0.58 �27.0 0.09 �28.6

Brassicasterol (C28:2) 0.05 0.03 �20.1 0.09 �25.9 0.02 �21.7 0.12 �28.2 0.01 �28.1

Stigmasterol (C29:2) 0.03 �20.3 0.13 �24.9 0.04 �27.0 0.20 �24.6 0.01 �25.5 �27.7

Stigmastanol (C29) 0.10 �25.8 0.09 0.02 0.15 �22.3 0.01 �23.4 0.09 �28.6 0.05 �26.4

Cycloartenol 0.10 �15.4 0.49 �15.9 0.15 �17.6 0.04 �25.1 � 0.27 �27.5 0.02 �29.8

Diploptene 0.08 0.04 �19.2 0.03 �21.4 0.14 0.03 0.03

bacterial ether

lipidsa

Tetrahymanol 0.03 �4.5 0.15 �3.5 0.04 �15.8 Trace 0.04 0.04 0.08 0.01 �15.7 0.14 �4.6 0.06 �16.9 0.04 �26.5

Diplopterol 0.01 �3.2 0.74 �18.5 Trace 0.07 0.03 0.04 0.23 0.02 �19.3 0.10 �20.4 0.7 �18.2 0.12 �22.0

C19:1 GME 0.01 �6.0 0.15 �6.0 0.01 �21.4 0.03 �9.3 0.01 �13.5 0.003 �6.1 0.04 �6.7 0.00 0.04 �14.0 0.06 �26.4 0.02 �23.9

C21 GME 0.02 �10.6 0.14 �8.1 0.02 �19.6 0.03 �11.5 0.07 �19.2 0.01 �12.9 0.004 �10.6 0.06 �3.9 0.00 0.13 �9.7 0.08 �23.3 0.03 �27.7

C33:1 GDE 0.14 �4.3 0.42 �4.9 0.33 �5.0 2.37 �2.0 0.26 �5.6 0.06 �1.1 0.47 2.6 0.02 �30.3

C33 GDE 0.16 �6.0 0.25 �6.4 0.12 �5.5 0.08 �6.1 0.05 0.8 0.14 �2.2 0.00 �18.2 0.01 �6.8 0.04 �36.2

C35:2 GDE 0.03 �4.6 0.17 �2.9 0.07 �19.8 0.93 �6.0 3.50 �3.5 0.05 �5.3 1.03 �1.4 0.02 �25.6 0.19 �22.1 0.20 �41.3

C36:1 GDE 0.34 �4.6 0.01 �45.0

C37:2 GDE 0.03 �6.4 0.28 �2.5 0.12 �18.9 0.24 �7.7 0.03 �36.9

a Representative structures are shown in the Appendix. GME and GDE refer to glycerol mono- and diethers, respectively. In CX:Y, X refers to the sum of glycerol and substituent (but notderivative) carbon atoms and Y indicates the number of rings or double bonds which must be present.

106A

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-4.0

-2.0

0.0

2.0

4.0

6.0

8.0

-6.0 -4.0 -2.0 0.0 2.0 4.0 6.0 8.0-4.0

-2.0

0.0

2.0

4.0

6.0

8.0

-6.0 -4.0 -2.0 0.0 2.0 4.0 6.0 8.0

13Csn-2 = 0.43* 13Carch + 1.7R2 = 0.77 R2 = 0.27

δ13C

sn-2

hydr

oxya

rcha

eol (

‰)

a b

δ13Carchaeol (‰)

δ13C

dihy

drox

yarc

haeo

l (‰)

δ13Carchaeol (‰)

δ δ

Fig. 2. (a) Correlations of d13C values of archaeol vs. sn-2 hydroxyarchaeol, (b) correlations of d13C values of archaeol vs. dihydroxyarchaeol.Black lines are 1:1 lines.

50 100 150 200 250 300 350 400 450 500 550 600 650 700

143

57

237103 341

175441278

721556515367 426

165

73

123

50 100 150 200 250 300 350 400 450 500 550 600 650 700

83

143273

217

161

121

237

97

69

55 x10

300 400 500 600 700

341

445 515425401 715531

337

sn-2

sn-3 diOH

a b

65 66 67 68 69 70 71 72 73time (min)

c

rela

tive

inte

nsity

rela

tive

inte

nsity

m/z

m/z

rela

tive

inte

nsity

a

b

Fig. 3. Mass spectra of unknown compounds detected eluting after sn-3 hydroxyarchaeol (a) and after dihydroxyarchaeol (b).(c) Chromatogram showing relative elution times and peak heights of compounds a,b, sn-2 hydroxyarchaeol (sn-2), sn-3 hydroxyarchaeol(sn-3), and dihydroxyarchaeol (diOH).


range of d13C, from approximately �20& to �29&.The 13C contents of cycloartenol were more variable,with the lowest d13C value near �30& in the carbonatevein X2, while cycloartenol in carbonate chimneys con-taining abundant archaeol lipids had d13C as high as�15.4&.

The C30 hopanoids diploptene and diplopterol, whichare derived from bacteria, were also detected in a numberof the vent chimneys. Concentrations of these hopanoidproducts ranged up to 740 ng per gram of dry carbonate.Their d13C values ranged from �26& to �2&. The polycy-clic triterpenoid tetrahymanol, often derived from marine

Archaeols

Glycerol diethers

Fatty acids

Glycerol monoethers

Cycloartenol

Other sterols

CH4 DIC

Sites X1, X2

ArchaealLipidsAbundant

ArchaealLipidsRare

Tetrahymanol

-80 -70 -60 -50 -40 -30 -20 -10 0 10δ13C (‰)

Fig. 4. Ranges of carbon isotopic compositions for different compound classes at the LCHF. Ranges for methane and DIC are measuredfrom fluid samples over the entire field (Kelley et al., 2005). Ranges for lipids are reported by grouping carbonate samples into those withabundant archaeal lipids (3862-1219, 3864,1524, 3864-1537, 3867-1225, 3867-1228, 3869-1404, 3869-1443, 3869-1446, 3879-1605), rarearchaeal lipids (3865-1322, 3876-1133, 3876-1219, 3876-1436, 3881-1132, 3881-1325, 3881-1408), and the ANME-dominated samples(Brazelton et al., 2006) at markers X1 and X2 (3880-1557).


ciliates, was detected in concentrations up to 150 ng pergram of dry carbonate. The 13C content of tetrahymanolranged from �3.5 to �26.5&, similar to the range in bacte-rial lipids.

4. DISCUSSION

Organic material in Lost City carbonates must derivefrom a combination of sources: from the microbial commu-nities inhabiting the carbonate chimney interiors, from or-ganic detritus in the marine water column which istrapped in carbonate chimneys as they precipitate, frommicrobial and animal communities on the chimney exteri-ors, and potentially from the subsurface from which fluidsare derived. Examination of the structures and isotopiccompositions of biomarker lipids allows us to begin to dis-entangle these sources and to improve our understanding ofthe microbial communities and carbon cycling in Lost Cityvents.

4.1. Total organic carbon

Exceptional enrichment in the 13C content of total or-ganic carbon is most prevalent in actively venting structures(Fig. 1a). Of samples that are characterized as active orinactive, with one exception (3867-1228) only active struc-tures contained total organic carbon with d13C > �10&,and inactive structures (except 3867-1228) all containedTOC with d13C < �15&. This strongly implies that themicrobial community within active vents is the source of13C-enriched organic carbon.

Examination of the biomarker content of the samplescontaining 13C-enriched organic carbon revealed anotherpattern: 13C-enrichment was detected only in those samplesin which diether lipids are abundant. In Fig. 1b, the d13C oftotal organic carbon is plotted versus the total content ofdiether lipids (isoprenoidal and nonisoprenoidal) in a rangeof samples. It is evident that total organic carbon hasd13C > �10& only in those samples in which concentra-tions of diethers exceed ca. 1 microgram per gram of rock.This suggests that biomass derived from diether-producingorganisms is the source of 13C-enriched organic material, asdiscussed below.

4.2. Archaeal Methanogenesis at Lost City

In environmental samples, hydroxyarchaeols, particu-larly sn-2 hydroxyarchaeol, are usually related to the pres-ence of the archaeal orders Methanosarcinales orMethanococcales, including the ANME-2 group of archa-eal methanotrophs (Hinrichs et al., 2000b; Pancost et al.,2001b; Blumenberg et al., 2004). There are a few exceptionsto this rule, however. Within the Methanobacteriales,Methanosphaera species have been reported to synthesizesn-2 hydroxyarchaeol (Koga et al., 1998; Sprott et al.,1999), as has Methanobrevibacter (Sprott et al., 1999).One halophilic archaeon, Natronobacterium, is also re-ported to synthesize the compound (Upasani et al., 1994).The compound sn-3 hydroxyarchaeol is presumed to derivepredominantly from methanogens and has been reportedfrom lipid extracts of Methanothermococcus thermolithotro-

phicus (Summons et al., 2006b).


In many Lost City carbonates, sn-2 hydroxyarchaeolwas the single most abundant lipid detected. Its concentra-tion was strongly covariant with those of archaeol anddihydroxyarchaeol, suggesting that perhaps all of thesecompounds are synthesized by a single archaeal strain, orat least by organisms that commonly co-occur. Carbon iso-topic patterns confirmed the close association of archaeoland sn-2 hydroxyarchaeol, although they failed to demon-strate a similar relationship between archaeol and dihydr-oxyarchaeol (Fig. 2), suggesting an additional source forone of these compounds.

Unknown compounds with mass spectra indicating astructural relationship to hydroxyarchaeols were also de-tected at Lost City (Fig. 3). Compounds with similar reten-tion times and spectra have been detected in sediments atthe Haakon Mosby Mud Volcano (Elvert and Niemann,2008), which contains ANME-3 methanotrophs. TheANME-3 group is very closely related to the methanogensfound at Lost City (Losekann et al., 2007). These com-pounds may be specific to this clade.

Microbiological evidence from Lost City carbonates isconsistent with the derivation of hydroxyarchaeols fromMethanosarcinales. Archaeal diversity is low in Lost Citycarbonates (Schrenk et al., 2004; Brazelton et al., 2006),which are dominated by a phylotype denoted as LCMS(Lost City Methanosarcinales). It is the sole archaeal phyl-otype in most carbonate chimneys. Exceptions are samplesfrom Marker C, which contain 16S rRNA gene sequencescorresponding to both LCMS and ANME-1, and two sam-ples from carbonate veins hosted in serpentinite, which con-tain ANME-1 only (X2) or ANME-1 together with a verysmall amount of LCMS (X1). At high temperatures, Meth-anosarcinales are generally outcompeted by methanogensthat conserve energy without the involvement of cyto-

methane

MethanotrophsAOM communities

HMMV HR ERB NW B

Location

-140

-120

-100

-80

-60

-40

-20

0

+20

δ13C

(‰)

Fig. 5. Ranges of the d13C values of archaeal diethers and of methan(Niemann et al., 2006; Losekann et al., 2007); HR: Hydrate Ridge (Elvert2005); ERB: Eel River Basin (Hinrichs et al., 1999); NW BS: Dniepr Castudy) and methanogen cultures grown on several substrates (Summons

chromes (Thauer et al., 2008). However, Methanosarcinalesare the group of methanogens that can achieve the highestgrowth yields on hydrogen and CO2 (Thauer et al., 2008),and the very high concentrations of hydrogen may helpto explain their success at Lost City.

In marine sediments and cold seeps, hydroxyarchaeolsfrom methanotrophic archaea commonly are extremely de-pleted in 13C, with d13C values ranging from about �80 to�130& (Hinrichs et al., 1999, 2000b; Boetius et al., 2000;Pancost et al., 2000). In sharp contrast, the hydroxyarchae-ols at Lost City are highly enriched in 13C, with an averaged13C value near +2& (Table 1). PMI, also likely derivedfrom Methanosarcinales, has d13C = +10&, the most 13C-enriched lipid yet reported to date from a naturalenvironment.

The striking enrichment must hold clues to the nature ofthe Methanosarcinales at Lost City. The LCMS phylotypehas, in some cases, been included in the newly definedANME-3 group, apparently on the basis of the close corre-spondence of its 16S rRNA gene sequence to sequencesfrom environments where methane is being oxidized anaer-obically, such as the Haakon Mosby Mud Volcano (Knittelet al., 2005). In such cases, products of the ANME-3 group,notably archaeol and sn-2 hydroxyarchaeol, are highly de-pleted in 13C (Niemann et al., 2006). Later reports have de-fined the ANME-3 clade more narrowly, and excludeLCMS (Losekann et al., 2007). The isotopic enrichment ob-served here supports this view, particularly indicating thatthe LCMS phylotype is not methanotrophic. Three conver-gent lines of evidence support this deduction.

First, values of d13C for methane at Lost City rangefrom �13.6 to �9.4& (Kelley et al., 2005). The archaeal lip-ids are significantly enriched in 13C relative to methane(Fig. 5), reversing the relationship observed in archaeal

archaeal diethers

Lost City Methanogens

H2/CO2 Acetate TMAS

Substrate

cultures

e in AOM communities (HMMV: Haakon Mosby Mud Volcanoet al., 1999; Boetius et al., 2000; Heeschen et al., 2005; Teichert et al.,nyon, Northwest Black Sea (Michaelis et al., 2002), Lost City (thiset al., 1998; Londry et al., 2008).


methanotrophs (Elvert et al., 1999; Hinrichs et al., 1999).Second, DIC is lower in abundance than methane but moreenriched in 13C. This pattern would not occur if largeamounts of methane were being oxidized to DIC, althoughsmall amounts of methane oxidation could occur withoutdisrupting this pattern. Third, concentrations of hydrogenwithin the vent fluids approach 15 mM, providing a strongthermodynamic drive for methanogenesis (Amend andShock, 2001).

Accordingly, the LCMS phylotype is likely methanogen-ic. Methanosarcinales are physiologically diverse and capa-ble of producing methane from a variety of substratesincluding CO2, acetate, methanol, and trimethylamine(Thauer, 1998). The extraordinary abundance of hydrogenin Lost City vent fluids suggests that it may be the most via-ble electron donor for methanogenesis. It follows thatmethanogens are likely autotrophic, although heterotrophicmethanogenesis using substrates such as acetate, formate,or methanol cannot be absolutely excluded. Formate ormethanol might form by reduction of CO formed fromCO2 under some hydrothermal conditions, but the veryhigh pH at Lost City limits CO2 availability and these sub-strates are unlikely to be available in abundance (Seewaldet al., 2006).

Naturally occurring lipids with 13C-enrichments on theorder of those detected at Lost City are unusual. Lipidswith d13C = +4& have been detected in a geothermal sin-ter in New Zealand (Pancost et al., 2006). At that site,13C-enrichment occurs in fatty acids and ether lipidslikely derived from Aquificales, as well as in archaeol de-rived from archaea (Pancost et al., 2006). Although sim-ilar lipids are detected at Lost City, Aquificales are notdetected in surveys of microbial diversity (Brazeltonet al., 2006).

Unusual enrichment of 13C in natural samples may de-rive from the presence of unusually enriched substrates.For example, the 13C-enrichment of archaeols in NewZealand sinters (Pancost et al., 2006) is postulated topotentially derive from 13C-enriched DIC. Another possi-bility is that 13C-enriched lipids are derived from organ-isms with carbon assimilation pathways that do notstrongly discriminate against the heavier isotope of car-bon. Most photosynthetic organisms fix CO2 via the Cal-vin cycle, which discriminates against the heavier isotopeof carbon by 22–30& (Hayes, 2001). However, withinBacteria both the reductive tricarboxylic acid (rTCA) cycleand the 3-hydroxypropionate pathways are carbon-fixa-tion pathways that do not strongly discriminate againstthe 13C compared to the enzymes of the Calvin cycle(Hayes, 2001).

Neither assimilation of 13C-enriched substrates, nor aunique carbon-fixation pathway is likely to be responsiblefor the enrichment in 13C in lipids of the organisms atLost City. Delta values of inorganic carbon in Lost Cityvent fluids range between �8& and +3& (Kelley et al.,2005) and thus coincide with those of the archaeal lipids.To date there has been no reported evidence at Lost Cityfor carbon substrates more enriched in 13C than DIC, soextreme enrichment in substrate is not the most attractiveexplanation for the enrichment in lipid. Small discrimina-

tions by the carbon fixation pathway can also be excluded,at least for the archaeal lipids, which are the most 13C-en-riched lipids detected. These lipids are likely derived frommethanogenic Methanosarcinales, in which the carbonassimilation pathway is well known. The biochemicalpathway of methanogenesis is well studied and in all casesthe final production of methane is catalyzed by methyl-coenzyme M reductase (Thauer, 1998). DNA sequencescorresponding to this gene have been amplified from LostCity carbonates (Brazelton et al., 2006), confirming thatMethanosarcinales are cycling methane. Autotrophicmethanogenesis is understood to proceed via a pathwaysimilar to the acetyl-CoA pathway, and carbon isotopefractionations by methanogens have been well studied(Games et al., 1978; Botz et al., 1996; Hayes, 2001; Houseet al., 2003; Valentine et al., 2004; Conrad, 2005; Penninget al., 2005, 2006; Londry et al., 2008). Taken together,these lines of evidence strongly suggest that no alternatecarbon-fixation pathway is responsible for the 13C enrich-ment seen in archaeal lipids. Diminished isotopic fraction-ation between CO2 and biological methane has beenreported as a result of very high temperatures and pres-sures (Takai et al., 2008), but occurs well beyond theranges relevant at Lost City.

The absence of isotopic fractionation can be explainedby carbon limitation. If available inorganic carbon is con-sumed nearly completely, isotopic discrimination will beminimized. Indeed, at the marker-7 and marker-C vents(the locations at which archaeal lipids were particularlyabundant), concentrations of dissolved inorganic carbonin the vent fluids were 4.0 and 0.1 micromolar, respec-tively (Proskurowski et al., 2008). Moreover, at pH 11and 70 �C more than 99% of DIC is carbonate and con-version of carbonate to CO2 is not enzymaticallycatalyzed.

Although the carbonate in the towers derives from sea-water (Fruh-Green et al., 2003), methane carbon containsno 14C and must primarily derive from the mantle(Proskurowski et al., 2008). Two situations are thus possi-ble: (1) a significant portion of the methane is biogenicand the Lost City Methanosarcinales are scavenging resid-ual mantle carbon from the fluids ascending in the ventchannels. (2) The methanogens producing the biomasssampled here are utilizing seawater DIC and the resultingCH4 is diffusing from the exterior walls of the carbonatetowers rather than mixing into the methane-rich fluids inthe axial channels.

The interpretations offered by Proskurowski et al.(2008) do not exclude microbial contributions to themethane in the Lost City hydrothermal fluids. The ab-sence of radiocarbon in the CH4 pertains to the sourceof the carbon, not the source of the methane. It excludesproduction of methane from seawater DIC but does notrequire an exclusively abiotic synthesis. Abundances ofabiotic hydrocarbons produced by Fischer–Tropsch reac-tions are predicted to conform to the Anderson–Schulz–Flory (ASF) distribution: a log-linear relationship show-ing decreasing hydrocarbon concentration with increasingchain length (McCollom and Seewald, 2007). When com-pared to concentrations of other volatile hydrocarbons,


the concentration of methane at Lost City is elevated byat least two orders of magnitude relative to that predictedby an ASF distribution (Proskurowski et al., 2008). Thed13C values of volatile hydrocarbons ethane, propane,and butane are lower than that of methane: between�13& and �16&. This is consistent with abiotic forma-tion, perhaps through Fischer–Tropsch type reactions(Proskurowski et al., 2008). Lost City methane has d2Hbetween �99& and �147& vs. SMOW (Proskurowskiet al., 2006, 2008). Ethane and propane have similar 2Hcontents (Proskurowski et al., 2008). Microbial methaneis typically depleted by 150&–275& vs. SMOW (Whiti-car, 1999; Valentine et al., 2004), so the methane at LostCity is more enriched in 2H than would be expected froma purely biological source. However, the activity of meth-anogens suggests that biological methane must be pres-ent. Lost City methane is likely a mixture, consisting ofbiological and abiotic inputs. The d2H content of mea-sured methane would be a function of the mixing propor-tions and d values of each endmember. Constraining thed values of these endmembers is difficult. Unusual condi-tions produce unusual carbon isotope ratios in biologi-cally produced methane (Takai et al., 2008). Theassumption that hydrogen isotope ratios in biologicallyproduced methane from Lost City fall within the usualranges might not be valid.

Organic compounds produced abiotically by hydrother-mal synthesis can be depleted in 13C relative to their sourcematerial (McCollom and Seewald, 2006). Organic carbonor methane in hydrothermal deposits cannot be distin-guished as biotic or abiotic based solely on a d13C valueof less than �25& (McCollom and Seewald, 2006; Sher-wood Lollar and McCollom, 2006). The isotope results atLost City extend this concept to the full range of 13C con-tents found in nature. If ultramafic ecosystems rich inhydrogen were important on the early Earth, preserved or-ganic compounds of biological origin detected in those sys-tems may not have the characteristic 13C depletion that iscommonly associated with life.

4.3. Bacterial lipid distribution and 13C content

The co-occurrence of isoprenoidal and nonisoprenoidalether lipids, with the former slightly more depleted in 13C,is common at sites where methane is being consumedanaerobically. At such locations, a combination of geo-chemical and microbiological techniques has confirmedthat the nonisoprenoidal ether lipids are produced by sul-fate-reducing bacteria that are the syntrophic partners ofarchaea that are consuming methane anaerobically (Hin-richs et al., 2000b; Orphan et al., 2001; Orphan et al.,2002; Blumenberg et al., 2004). The archaeal members ofthese consortia that produce isoprenoidal diethers typi-cally belong to the ANME-1 phylogenetic cluster. Bacteriaassociated with ANME-2 archaea tend to produce noni-soprenoidal glycerol ether lipids. The bacterial membersin either case belong to the sulfate-reducing Desulfosarci-

na/Desulfococcus group.At Lost City the archaea are methanogenic and micro-

biological studies fail to detect d-proteobacterial sulfate-

reducers (Brazelton et al., 2006). Instead, the dominantsulfate-reducing bacteria in these environments are Firmi-cutes related to the hydrogen-utilizing sulfate reducerDesulfotomaculum (Brazelton et al., 2006). Accordingly,Clostridia are candidates for producers of the nonisopre-noidal diethers. Clostridia commonly produce ether–lipidplasmalogens and incorporate them in their cellular mem-branes (Goldfine, 1997). However, studies of Desulfotoma-

culum have not detected diethers (Pikuta et al., 2000;Londry et al., 2004). At least one Clostridium, Ammoni-

fex, does produce diethers (Huber et al., 1996), and theFirmicute lipid biosynthesis pathway shares many charac-teristics with archaea (Skophammer et al., 2007). At manyhydrothermal settings, diether lipids are produced by Aqu-ificales (Jahnke et al., 2001), but rRNA gene sequences re-lated to this group were undetected at Lost City(Brazelton et al., 2006).

The coexistence of sulfate-reducing bacteria and metha-nogens is unusual. In marine sediments, sulfate-reducingbacteria outcompete methanogens for hydrogen due tothe higher energetic yield of sulfate reduction comparedto CO2 reduction (Hoehler et al., 1998) and the greater sub-strate affinity of the enzymes of sulfate-reducing bacteriafor hydrogen (Kristjansson et al., 1982). However, undervery high hydrogen concentrations, methanogens and sul-fate-reducing bacteria can coexist. Enzymatic affinities forhydrogen are in the micromolar range for both sulfate-reducers and methanogens (Kristjansson et al., 1982); themillimolar hydrogen concentrations at Lost City exceed thisthreshold and allow coexistence.

Bacterial diethers are slightly depleted in 13C relative toarchaeal diethers in most Lost City carbonates (Fig. 4). Thefact that d13C values are still highly positive relative to typ-ical marine organic material suggests that carbon-limitationmay be playing a key role in bacterial ecology at Lost City,just as it does with the archaea.

Another possibility is that bacteria are acquiring car-bon via an alternative pathway that discriminates against13C to a different degree than archaea. Bacteria are unlikemethanogens in that they have a wide range of plausiblecarbon-acquisition pathways. It is conceivable that majorbacterial community members are fixing carbon autotro-phically via a pathway that does not discriminate stronglyagainst 13C. For example, the Aquificales species Thermoc-

rinus fixes CO2 via the rTCA acid cycle, and when grownautotrophically its lipids are depleted in 13C by only 2&

relative to CO2 (Jahnke et al., 2001). The small discrimina-tion conferred by this pathway has been invoked (alongwith 13C-enriched DIC) to explain the 13C-enriched lipidswith d13C up to +4& likely to be derived from Aquificalesin New Zealand hot springs (Pancost et al., 2006) and avariety of 13C-enriched lipids with d13C up to +2& fromthe Turtle Pits hydrothermal field at the mid-AtlanticRidge (Blumenberg et al., 2007). Aquificales species pro-duce diether lipids that are similar to the nonisoprenoidaldiethers detected at Lost City, but microbial diversitystudies fail to detect these taxa at Lost City (Brazeltonet al., 2006).

A third possibility is that bacteria and archaea areobtaining carbon from different substrates. The main


sulfate-reducing bacteria at Lost City, Desulfotomaculum

(Brazelton et al., 2006), is capable of growth on a varietyof carbon substrates, including acetate, formate, ethanol,lactate, and pyruvate. Bacteria may be able to outcompetemethanogens for competitive substrates like acetate(Kristjansson and Schonheit, 1983). Information regardingthe concentrations of these species in Lost City vent fluids islacking, however.

One relationship that may cast light on these isotopicvalues is shown in Fig. 4, which shows that bacterial die-thers are slightly depleted relative to archaeal diethers insamples in which archaeal diethers are abundant, but moredepleted in 13C relative to archaeal diethers in samples inwhich archaea-derived material is rare. In samples withabundant archaeal lipids, bacterial diethers have a rangeof d13C values between about +4& and �12&, while inchimneys with rare archaeal lipids the range of d13C extendsdown to about �29&. Excluding the two samples 3881-1408 and 3876-1436, each of which contains only low con-centrations of nonisoprenoidal diethers and no detectedarchaeal lipids, the maximum d13C for nonisoprenoidal die-thers in samples with rare archaeal biomarkers is near�14&—lower than the minimum d13C for samples thatcontain abundant archaeal biomarkers. Although archaeallipids were undetected in sample 3881-1408, this samplecontained 16S rRNA sequences corresponding to Lost CityMethanosarcinales (Brazelton et al., 2006). The implicationis that the presence of this methanogenic archaeon, partic-ularly at high abundance, is responsible for 13C enrichmentfor all organisms in the vent fluids—perhaps through draw-down to limitation of a limiting substrate, most likely inor-ganic carbon.

Other bacterial biomarkers in Lost City carbonate,such as fatty acids and hopanoids, are less taxonomicallyspecific than diether lipids and have a greater range ofd13C values. These compounds are probably derived froma greater range of sources. The hopanoids diploptene anddiplopterol have d13C values that range from �15& to�22&. The d13C values of hopanoids do not correlatewith the abundances of archaeal lipids, as those of thenonisoprenoidal diethers do. A likely interpretation is thathopanoids are dominantly derived from aerobes living atthe interface of reduced vent fluids and oxidized seawater.Most hopanoids are derived from aerobic bacteria,although a number of recent studies have demonstratedthat some anaerobes are also capable of producing them(Sinninghe Damste et al., 2004; Fischer et al., 2005; Hart-ner et al., 2005; Blumenberg et al., 2006). Organisms incontact with aerobic seawater could be expected to have13C contents typical of marine organic material, whilethose in the anaerobic vent fluids would have 13C contentsreflecting the conditions, in many cases carbon-limited, inthe vent fluids.

The isotopic compositions of fatty acids may also beconsistent with this model. While samples with rare archa-eal lipids contain fatty acids with d13C values between�20& and �30&, the d13C values of fatty acids in sampleswith abundant archaeal lipids extend over a much widerrange, with d13C values up to �1&. However, there is apattern to these results, with saturated fatty acids tending

to be more 13C-enriched than unsaturated fatty acids. Thisis interpreted to mean that saturated fatty acids in thesesamples are predominantly derived, along with the etherlipids, from the carbon-limited anaerobic environment in-side the carbonate chimneys. Mono-unsaturated fatty acidsprobably originate both from this environment and fromorganisms inhabiting the chimney exterior and overlyingwater column. This pattern may have to do with the regu-lation of membrane fluidity, which is increased by eitherincreasing degree of unsaturation or increasing tempera-ture. Organisms inhabiting lower temperature environ-ments can be expected to have a higher degree ofunsaturation.

4.4. Anaerobic methanotrophy at Lost City

Microbial community analysis suggests that methane-oxidizing ANME-1 archaea dominate the archaeal commu-nity at two sites, X1 and X2, and were present at Marker C(Brazelton et al., 2006). Sample 3880-1557 from site X2contained an archaeol with a d13C value unlike those de-tected from the rest of the Lost City field (Fig. 4). Thelow value of d13Carchaeol implies that archaea at this siteconsume methane. No hydroxyarchaeols were detected inthis sample. Archaeal lipids from environments containingabundant ANME-1 methanotrophic consortia are charac-terized by a low ratio of sn-2 hydroxyarchaeol to archaeol(Blumenberg et al., 2004), so the lack of detected sn-2hydroxyarchaeol is not inconsistent with ANME-1 domi-nating this sample.

The origin of methane at this site is unknown. Archae-ol at site X2 has d13C = �77&. In several studies fromAOM environments that report the 13C content of bothmethane and lipids, archaeol is depleted in 13C relativeto methane by 7& to 55& (Hinrichs et al., 1999; Michae-lis et al., 2002; Teske et al., 2002; Reitner et al., 2005). Thed13C of Lost City methane at the main area of venting is�13.6& to �9.4& (Kelley et al., 2005), but was not mea-sured at X2. It is possible that methane at site X2 derivesfrom a different pool, with a d13C value distinct frommethane at the main area of venting. Based on the d13Cvalue of archaeol, we hypothesize that methane at siteX2 should fall in approximately the range �27& to�70&.

The d13C values of bacterial diethers found in samples atX1 and X2 span a range of nearly 30&. This could indicatethat these compounds derive from a mixture of bacteriawith different carbon acquisition physiologies or differentcarbon sources, potentially including carbon derived frommethane. At site X2, diethers with the most positive valuesof d13C are enriched relative to archaeol by slightly lessthan 50&. While not diagnostic, this difference is withinthe range found between archaeal and bacterial diethersat AOM sites (Hinrichs et al., 2000b). However, no bacte-rial syntrophic partners or sulfate-reducing Firmicutes havebeen detected in the carbonate vein sites (Brazelton et al.,2006), so the source of the nonisoprenoidal ether lipids re-mains uncertain. Conceivably, the ANME-1 organisms atthese sites are operating without a syntrophic partner(Orphan et al., 2002).


4.5. Eukaryotic lipid distribution and 13C content

Cholesterol is the most frequently detected sterol at LostCity and is probably derived from animals inhabiting thesurfaces of the carbonate chimneys (Kelley et al., 2005;DeChaine et al., 2006) and from detritus trapped fromthe water column. C28 and C29 sterols and stanols, also fre-quently detected in carbonates, are likely derived from phy-toplankton or other eukaryotic inputs. Ergosterol wasdetected in a few samples and is likely to be a product ofone of the two fungal lineages detected at Lost City(Lopez-Garcia et al., 2007).

In several samples the most abundant sterol was cycloar-tenol. Cycloartenol, the immediate product of the cycliza-tion of oxidosqualene in plants and algae as well as inseveral groups of protists, is a ‘protosterol’ (Summonset al., 2006a). In most organisms cycloartenol is a biosyn-thetic intermediate that is modified to yield a demethylatedsterol. To date it has been reported as the accumulating fi-nal product in only one organism, the myxobacteriumStigmatella aurantiaca Sg a15 (Bode et al., 2003). In otherorganisms concentrations of cycloartenol detected in cellu-lar extracts are typically minor, reflecting its role as a bio-synthetic intermediate. Its accumulation can be enhancedby metabolic inhibitors (Hata et al., 1987; Haughan et al.,1988) or by genetic mutation of the downstream sterolmodification pathway.

The high abundance of cycloartenol in Lost City car-bonates implies that an organism inhabiting this environ-ment is accumulating cycloartenol as a final product.Given that the animal protosterol is always lanosterol andthat C28 and C29 phytosterols are dominant in marine algae,animals and algae can probably be excluded as the source.Myxobacteria are not detected in 16S rRNA gene surveysat Lost City (Brazelton et al., 2006), and can probably alsobe excluded. The most probable source of abundant cyclo-artenol is one or more of the protists inhabiting the carbon-ate chimneys.

A diverse population of protists has been detected inLost City carbonates. Ciliates are the most dominant,and other alveolates, fungi, heterokonts, radiolaria andother cercozoa, and heterolobosea have also been detected(Lopez-Garcia et al., 2007). Detailed information aboutsterol biosynthetic pathways within these groups of pro-tists is sparse. Among protists in which the sterol syntheticpathway is known, most make cycloartenol as their pro-tosterol (Summons et al., 2006a). The alternate path-way—the lanosterol pathway—is ubiquitous amongopisthokonts and also occurs among kinetoplastids anddinoflagellates (Summons et al., 2006a). These groupscan be ruled out as possible sources for cycloartenol,but large protistan diversity remains (Lopez-Garciaet al., 2007). Given the abundance of cycloartenol in somecarbonates, its source is likely to be an important part ofthe microbial ecology.

Synthesis of cycloartenol requires only one molecule ofO2 (Summons et al., 2006a), but subsequent modificationto a sterol that is fully demethylated on its a-face, such asstigmasterol or cholesterol, requires an additional ten mol-ecules of O2. For this reason we speculate that cycloartenol

might be a favored sterol from an amphiaerobic eukaryoteinhabiting vent fluids with only sporadic access to oxygen-ated seawater.

The triterpenoid tetrahymanol (Ten Haven et al., 1989;Harvey and McManus, 1991), commonly attributed to cil-iates, was also detected in several Lost City vent carbon-ates. Tetrahymanol has a structure similar to sterols, butone respect in which it differs is that oxygen is notrequired for tetrahymanol biosynthesis. Gene sequencesrepresentative of ciliates have been found in Lost Citycarbonates (Lopez-Garcia et al., 2007), and ciliates arethe most plausible source for tetrahymanol. Consumptionof bacteria by ciliates in AOM environments at the Kazanmud volcano (Werne et al., 2002), and consumptionof methanogens by thermophilic anaerobic cilates(Baumgartner et al., 2002) have also been reported. Theco-occurrence of cycloartenol and tetrahymanol is likelynot fortuitous.

Variability in triterpenoid d13C is greater than would beexpected if all sterol inputs were marine. Cycloartenol hasd13C values as high as �15& (Fig. 4), consistent with der-ivation from protists feeding in part on 13C-enriched ar-chaea and bacteria in the carbonate chimneys. In samplesin which its d13C values is as high as �3.5&, tetrahymanolshows very strong evidence of being derived from an organ-ism obtaining its carbon from 13C-enriched vent material(Fig. 4). However there is no strong correlation betweend13C of tetrahymanol and total organic carbon. To a lesserextent, C28 and C29 sterols are also enriched in some sam-ples with d13C up to a maximum of near �20& at MarkerC, suggesting that their sources may not entirely derivefrom the water column.

In some samples, tetrahymanol with high d13C valuesoccurs together with 13C-enriched archaeal lipids, suggest-ing a close relationship between methanogens and ciliates.Some anaerobic ciliates possess hydrogenosomes—cellularorganelles related to mitochondria that generate hydrogen.Hydrogenosomes often host symbiotic methanogens,which serve to keep intracellular hydrogen pressures low(Hackstein and Vogels, 1997; van Hoek et al., 2000).However, the millimolar concentrations of hydrogen inLost City vent fluids may be too high for methanogensto consume at a rate high enough for hydrogenosomesto function. Strong covariation between the d13C of tetra-hymanol and sn-2 hydroxyarchaeol is lacking, suggestingthat the relationship between these organisms is not ubiq-uitous. The relationship between the organisms may bepredator and prey, similar to that of Trimyema minutum

and M. thermolithotrophicus (Baumgartner et al., 2002),but the 13C depletion of tetrahymanol relative to archaeallipids in the samples implies that methanogens are not thesole food source for ciliates.

4.6. Implications for Earth history and astrobiology

The hydrogen-rich, anaerobic environment within thecarbonate towers at Lost City is likely to be similar tosome early Earth environments. Autotrophic methano-genesis using CO2 or bicarbonate as an electron acceptorproceeds exergonically without need for any products of


oxygenic photosynthesis. Thus, the dominant primaryproducer in this ecosystem may be independent of solarenergy. This is in contrast to the organisms in manyother anaerobic ecosystems that usually require indirectbyproducts of photosynthesis such as sulfate or nitrate.Ecosystems independent of the sun, and certainly inde-pendent of the byproducts of oxygenic photosynthesis,would have been important on the early Earth. Bacterialcomponents of the ecosystem at Lost City partly rely onsulfate derived from seawater, but sulfate may also havebeen present on early Earth, and was present in ecosys-tems before the origin of oxygenic photosynthesis(Shen and Buick, 2004). The microbial community atLost City therefore may offer an analogue for early Earthecosystems.

Genomic studies offer further evidence that the micro-bial community at Lost City might be relevant to under-standing ecosystems on the early Earth. Recent attemptsto root the universal tree of life based on analyses of inser-tions and deletions within sets of paralogous genes havesupported the view that the widely accepted root betweenarchaea and bacteria may be an artifact of long branchattraction (Skophammer et al., 2007). Instead, several alter-native sites for the root have been proposed within theGram Positive Bacteria, including a root on the branchleading to the Firmicutes plus Archaea (Skophammeret al., 2007). While this placement of the root is untradi-tional, it argues for a mesophilic cenancestor that may haveinhabited a site like Lost City (Skophammer et al., 2007).Furthermore, Firmicutes and Archaea play major roles inthe Lost City ecosystem (Brazelton et al., 2006). Furtherwork on microbial and genomic evolution will have to beundertaken to determine if this correspondence iscoincidental.

The chemistry of the Lost City vents suggests that simi-lar ancient systems may have been important not only fortheir ecological role, but also as a reactor in which somecritical aspects of life may have emerged. The alkaline con-ditions at Lost City are favorable for some aspects of pre-biotic chemistry, such as formation of RNA-bearingvesicles (Russell, 2003). The warm, reducing, and alkalineconditions are appropriate for the emergence of biochemi-cal pathways such as methanogenesis and acetogenesis. Ithas been suggested that these biochemistries originated insuch an environment (Martin and Russell, 2007). The ther-modynamic drive for methanogenesis at Lost City is under-scored by the fact that the reaction occurs both with andwithout biological catalysis.

Quantifying the relative flux of biologically derivedmethane versus abiotic methane at Lost City is a majorchallenge for future study. Solving this problem will beimportant for constraining the composition of the earlyatmosphere. Methane is often invoked to solve the faintyoung sun problem (Kasting, 2005), but models applyingthis to the Hadean and Archean Earth require an accurateunderstanding of the potential roles for archaeal methano-gens, including the environments in which they live, the tim-ing of their evolution, and the amount of methane that can

be produced in their absence. The possible production ofabundant biological methane that is 13C-enriched compli-cates our understanding of the timing of the evolution ofbiological methanogenesis, which is based, in part, on theappearance of 13C depleted organic carbon in the geologicrecord (Hayes, 1994).

5. CONCLUSIONS

The hydrogen-rich chemistry of Lost City vent fluidsand the isotopic compositions of the archaeal and bacte-rial diether lipids lead us to conclude that the Lost CityMethanosarcinales phylotype (LCMS) is a methano-gen. Similar 13C enrichments in nonisoprenoidal diethersdemonstrate that the conditions causing unusual d13Cvalues are not restricted to archaea, but are most preva-lent where archaea are dominant. The most plausibleexplanation for these data is that the archaea-rich ventcommunities are carbon-limited and the full extent offractionation between DIC and biomass is not beingexpressed.

Conditions of CO2 limitation are rare on Earth. As a re-sult, depletion in 13C is usually necessary (but not sufficient)evidence that preserved organic carbon is biological in ori-gin. The results from Lost City suggest that even this biasmay not be appropriate for all environments. Ultramaficecosystems may have been important on early Earth, andmay be present elsewhere in the solar system. The resultspresented here suggest that biological organic carbon insuch systems can have a wide range of 13C contents. Manyinvestigations have emphasized that 13C-depleted organiccarbon is not necessarily a signal of life. Similarly, our re-sults show that enrichment of 13C is not a reliable indicatorof abiotic origin.

ACKNOWLEDGMENTS

The authors thank Drs. Deborah Kelly, Jeff Karson, andmembers of the Lost City team for the opportunity to partici-pate in the 2003 cruise and for assistance in collecting samples.At WHOI, Sean P. Sylva assisted with TOC analyses and pro-vided invaluable assistance with carbon isotopic measurement.Critical reviews from Rich Pancost, Martin Blumenberg, andMarcus Elvert, and the editorial handling of Jaap SinningheDamste substantially improved this manuscript. We gratefullyacknowledge the NASA Graduate Student Researchers Program(GSRP) for the award of a fellowship to ASB and especially toLinda Jahnke and Tori Hoehler (NASA Ames Research Center)for their interest and support of this project. JMH acknowl-edges support from NSF OCE Cooperative Agreement0228996. RES was supported by grants from the National Sci-ence Foundation OCE 0452410 and Biogeosciences 0525453.R.E.S. and J.M.H. thank the Hanse-Wissenschaftskolleg, Del-menhorst, Germany for their generous support during revisionof this manuscript.

APPENDIX A

Structures of compounds mentioned in the text.


OH

OH

diploptene diplopterol tetrahymanol

C33:1 GDEO

OH

O

O

OH

OOH

O

OH

OOH

O

OH

OOH

OH

OH

O

O

OH

O

O

OH

O

O

OH

O

O

OH

O

O

archaeol

sn-2 hydroxyarchaeol

sn-3 hydroxyarchaeol

dihydroxyarchaeol

2,6,10,15,19 pentamethylicosane

C33 GDE

C35:2 GDE

C36:1 GDE

C37:2 GDE

cholesterol brassicasterol stigmasterol cycloartenol

HO HOHO HO

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