Geobiology (2005),

3

, 195–209

© 2005 The Authors Journal compilation © 2005 Blackwell Publishing Ltd

195

Blackwell Publishing Ltd

ORIGINAL ARTICLE

Spatial variations of methanotrophic consortia at cold methane seeps

Spatial variations of methanotrophic consortia at cold methane seeps: implications from a high-resolution molecular and isotopic approach

M. ELVERT,

1,2

E. C. HOPMANS,

3

T. TREUDE,

2

A. BOETIUS

2,4

AND E. SUESS

5

1

Research Center Ocean Margins, University of Bremen, Bremen, Germany

2

Max Planck Institute for Marine Microbiology, Bremen, Germany

3

Royal Netherlands Institute for Sea Research, Den Burg, the Netherlands

4

International University Bremen, Bremen, Germany

5

GEOMAR Research Center for Marine Geosciences, Kiel, Germany

ABSTRACT

Anaerobic methane-oxidizing microbial communities in sediments at cold methane seeps are important factorsin controlling methane emission to the ocean and atmosphere. Here, we investigated the distribution and carbonisotopic signature of specific biomarkers derived from anaerobic methanotrophic archaea (ANME groups) andsulphate-reducing bacteria (SRB) responsible for the anaerobic oxidation of methane (AOM) at different coldseep provinces of Hydrate Ridge, Cascadia margin. The special focus was on their relation to

in situ

cell abun-dances and methane turnover. In general, maxima in biomarker abundances and minima in carbon isotopesignatures correlated with maxima in AOM and sulphate reduction as well as with consortium biomass. Wefound ANME-2a/DSS aggregates associated with high abundances of

sn

-2,3-di-

O

-isoprenoidal glycerol ethers(archaeol,

sn

-2-hydroxyarchaeol) and specific bacterial fatty acids (C

16:1

ω

5c

, cyC

17:0

ω

5,6

) as well as with highmethane fluxes (

Beggiatoa

site). The low to medium flux site (

Calyptogena

field) was dominated by ANME-2c/DSS aggregates and contained less of both compound classes but more of AOM-related glycerol dialkyl glyceroltetraethers (GDGTs). ANME-1 archaea dominated deeper sediment horizons at the

Calyptogena

field where

sn

-1,2-di-

O

-alkyl glycerol ethers (DAGEs), archaeol, methyl-branched fatty acids (

ai

-C

15:0

,

i

-C

16:0

,

ai

-C

17:0

), anddiagnostic GDGTs were prevailing. AOM-specific bacterial and archaeal biomarkers in these sediment stratagenerally revealed very similar

δ

13

C-values of around

−

100‰. In ANME-2-dominated sediment sections,archaeal biomarkers were even more

13

C-depleted (down to

−

120‰), whereas bacterial biomarkers were foundto be likewise

13

C-depleted as in ANME-1-dominated sediment layers (

δ

13

C:

−

100‰). The zero flux site(

Acharax

field), containing only a few numbers of ANME-2/DSS aggregates, however, provided no specificbiomarker pattern. Deeper sediment sections (below 20 cm sediment depth) from

Beggiatoa

covered areaswhich included solid layers of methane gas hydrates contained ANME-2/DSS typical biomarkers showingsubsurface peaks combined with negative shifts in carbon isotopic compositions. The maxima were detectedjust above the hydrate layers, indicating that methane stored in the hydrates may be available for the microbialcommunity. The observed variations in biomarker abundances and

13

C-depletions are indicative of multipleenvironmental and physiological factors selecting for different AOM consortia (ANME-2a/DSS, ANME-2c/DSS,ANME-1) along horizontal and vertical gradients of cold seep settings.

Received 15 June 2005; accepted 25 October 2005

Corresponding author: Marcus Elvert, Tel.: (+49) 42121865706; fax: (+49) 42121865715; e-mail: melvert@uni-bremen.de.

INTRODUCTION

Chemosynthetic microbial communities inhabiting cold seepenvironments provide the base for growth and metabolismof active and rich ecosystems that are decoupled from

photosynthesis (Sibuet & Olu, 1998). The existence of suchecosystems depends on the long-term availability of chemicallyreduced electron donors such as methane or higher hydro-carbons, and sulphide as a product of their turnover (Boetius& Suess, 2004). The dominant process of anaerobic oxidation

196

M. ELVERT

et al

.

© 2005 The Authors Journal compilation © 2005 Blackwell Publishing Ltd

of methane (AOM) is regarded as the major barrier againstmethane efflux from marine sediments into the ocean(Reeburgh, 1996; Hinrichs & Boetius, 2002), especially indiffusion-controlled marine systems where AOM completelyconsumes the upward-diffusing methane (Claypool & Kaplan,1974; Barnes & Goldberg, 1976; Iversen & Jørgensen, 1985).

Since the late 1990s, AOM has been the focus of a varietyof studies highlighting the global environmental importanceof this process (Hinrichs

et al

., 1999; Boetius

et al

., 2000; Val-entine & Reeburgh, 2000). Ribosomal RNA investigationsshowed that AOM is performed by consortia of archaea(ANaerobic MEthanotrophs – ANME-1 and ANME-2 group)related to the order Methanosarcinales and sulphate-reducingbacteria (SRB) of the

Desulfosarcina/Desulfococcus

(DSS) clus-ter (Boetius

et al

., 2000; Orphan

et al

., 2001; Michaelis

et al

.,2002; Knittel

et al

., 2003). Corresponding evidence has beenprovided by biomarker investigations of anoxic marine sedimentsas well as ancient and recent carbonate crusts obtained from coldmethane seep environments (Elvert

et al

., 1999, 2000, 2003;Hinrichs

et al

., 1999, 2000; Thiel

et al

., 1999, 2001a; Pancost

et al

., 2000, 2001b; Zhang

et al

., 2003; Peckmann & Thiel,2004). The significance of certain biomarkers as indicators formethanotrophic consortia has been deduced from the strongdepletion in

13

C, apparently based on the use of methane as themain carbon source. The spatial abundances, distribution andisotopic signature of the biomarkers may change dependingon the amount of living and preserved AOM-consortia biomass,which in turn depends on continuous supply of methaneand sulphate as electron donor and acceptor, respectively.

The main focus of AOM-targeted studies have been areas ofmethane-rich seeping environments at active and passive con-tinental margins. Seep ecosystems have been found in diversegeosphere–biosphere settings (e.g. Black Sea, Hydrate Ridge,Eel River Basin, Mediterranean Sea, Gulf of Mexico). Unfor-tunately, little is known on the environmental factors con-trolling the diversity, structure and biomass distribution ofanaerobic methanotrophs. This investigation here used acombination of high-resolution molecular and isotopic measure-ments of lipid biomarkers from a well-known cold seep envi-ronment (Hydrate Ridge, north-eastern Pacific) to obtain acomprehensive picture of the spatial distribution of anaerobicmethanotrophs under varying environmental conditions (i.e.methane flux, presence of gas hydrates, etc.). The relation oflipid biomarker profiles to microbiological and biogeochemicaldata (cell counts and rate measurements) is presented in detail asdatabase for future molecular ecological and biogeochemicalstudies targeting AOM at recent and ancient methane seeps.

MATERIALS AND METHODS

Geobiological setting

The geophysical and geological setting at southern HydrateRidge (44

°

34

′

N, 125

°

09

′

W; 780 m water depth) and the

biological characteristics of specific seep provinces (

Beggiatoa

sites,

Calyptogena

fields,

Acharax

fields) have been extensivelystudied and described previously (Suess

et al

., 1999; Sahling

et al

., 2002; Torres

et al

., 2002; Tryon

et al

., 2002; Treude

et al

., 2003; Boetius & Suess, 2004). Referring to thesestudies, the differentiation between seep provinces is based onvarious environmental parameters, including the macrofaunalcommunity structure, fluid flow velocity, methane flux andpore water characteristics. Upward fluid flow rates were foundto be highest below

Beggiatoa

mats (10 to 250 cm year

−

1

),medium to low below

Calyptogena

fields with temporarilynegative components (inflow) (2 to

−

10 cm year

−

1

), andwere not detectable at the

Acharax

fields. Methane fluxes outof the sediments range from 30 to 90 mmol m

−

2

d

−

1

at the

Beggiatoa

sites, they are lower than 1 mmol m

−

2

d

−

1

at the

Calyptogena

fields, and are close to zero at the

Acharax

fields.Simultaneously, hydrogen sulphide concentrations in theupper 25 cm of the sediments are highest at the

Beggiatoa

sites(sulphidic sediment surface; sometimes exceeding 26 m

M

inthe deeper parts), lower at the

Calyptogena

fields (sulphidepresent below 3 cm sediment depth; up to 25 m

M

in thedeeper parts), and are only detected below 15 cm sedimentdepth at

Acharax

fields (0.1–0.3 m

M

). Shallow gas hydratesare predominantly found at

Beggiatoa

sites (10–20 cmsediment depth), but are also found deeper below

Calyptogena

and

Acharax

sites.

Sample collection and storage

Sediment samples were obtained during RV SONNE cruisesSO143-1 and SO148-1 in July 1999 and August 2000,respectively, at the crest of southern Hydrate Ridge from thedifferent chemosynthetic provinces (station 19-2:

Beggiatoa

site; station 44:

Calyptogena

field; station 51:

Acharax

field; allSO148-1) near areas of active methane venting using a video-guided multiple corer (TV-MUC). Upon recovery, the TV-MUC sediment cores were sliced into depth intervals of 2 cm(0–10 cm) and 3 cm (10–19 cm). Moreover, a 1.2 m gravitycore, retrieved from an area densely covered with

Beggiatoa

mats (station 55-5: termed ‘gas hydrate’ site; SO143-1), wassampled at distinct sediment horizons across observed gashydrate layers. All sediment sections were transferred intoprecleaned 20 mL glass vials and were kept frozen at

−

20

°

Cuntil lipid extraction.

Lipid biomarker analyses

Lipid extraction, separation into different compound classes,and analyses by gas chromatography (GC), gas chromatography–mass spectrometry (GC–MS), gas chromatography–isotoperatio mass spectrometry (GC–IRMS), and high performanceliquid chromatography–mass spectrometry (HPLC–MS)were conducted according to methods reported previously(Hopmans

et al

., 2000; Elvert

et al

., 2003; Niemann

et al

.,

Spatial variations of methanotrophic consortia at cold methane seeps

197

© 2005 The Authors Journal compilation © 2005 Blackwell Publishing Ltd

2005). The presence of double bonds in hydrocarbonshas been determined by the hydrogenation of selectedhydrocarbon fractions (Elvert

et al

., 2000). Double-bondpositions in fatty acid methyl esters (FAMEs) as well astrimethylsilyl (TMS)-derivatives of AOM-specific short-chain

n

-alcohols and

sn

-1-mono-

O

-alkyl glycerol ethers (MAGEs)have been verified by the preparation of dimethyl disulphide(DMDS) adducts (Elvert

et al

., 2003); specific ring positionsin FAMEs have been determined by the preparation of 4,4-dimethyloxazoline (DMOX) derivatives (Elvert

et al

., 2003).Carbon isotope ratios are reported in the

δ

-notation as per mil(‰) deviation from the Vienna Pee Dee Belemnite standard(VPDB).

δ

13

C values have an analytical error of less than

±

1.0‰ and are corrected for the introduction of additionalcarbon atoms during derivatization. Portions of AOM-derivedcarbon in discrete fatty acids (FAs) were calculated using isotopicmass balance corrections according to Elvert

et al

. (2003).Where the relative abundance of the specific FA cyC

17:0

ω

5,6

was too low to determine precise

δ

13

C-values (

Acharax

field),a mean

δ

13

C-value of −98.4‰ – obtained from the Beggiatoasite and the Calyptogena field – has been used for all sedimenthorizons.

TOC measurements

Contents of total organic carbon (TOC) were determinedfrom the carbonate-free, dried and homogenized sedimentmaterial using a Fisons NA-1200 Elemental Analyser accordingto Elvert et al. (2000). The δ13CTOC was measured byelemental analysis-isotope ratio mass spectrometry (EA-IRMS)using a EuroVector EA 3000 Elemental Analyser connectedvia a ConFlo™ II interface to a Finnigan Delta Plus. Analyticalreproducibility for duplicate runs was better than ± 0.2‰ VPDB.

Aggregate counts and rate measurements

Data on ANME and SRB counts as well as AOM and sulphatereduction (SR) rates from the same (19-2, 51; Beggiatoa siteand Acharax field, respectively) or close by stations (38;Calyptogena field) have been obtained from parallel studies(Knittel et al., 2003, 2005; Treude et al., 2003; T. Treude,unpublished data) that include a detailed description of thecorresponding methods.

RESULTS

Beggiatoa site

Sediment samples from the Beggiatoa site were dominated bylipid biomarkers indicative of anaerobic methane-oxidizingconsortia composed of methanotrophic archaea (ANME) andsulphate-reducing bacteria (SRB) (Fig. 1). Concentrations ofthe most dominant biomarkers were up to 13.59 µg g−1 dw(dry weight) for sn-2-hydroxyarchaeol dominantly derived

from ANME-2 archaea (Blumenberg et al., 2004) and17.31 µg g−1 dw for the FA C16:1ω5c assigned to SRB of theDesulfosarcina/Desulfococcus (DSS) cluster (Elvert et al., 2003)within the δ-proteobacteria (Table 1). Both compoundsshowed very low δ13C-values in the range from −115.9 to−123.5‰ and from −75.4 to −96.3‰, revealing an offset of20–40‰ between archaea and SRB, respectively. Other specificbiomarkers for methanotrophic archaea include crocetane(2,6,11,15-tetramethylhexadecane, Cr), unsaturated 2,6,11,15,19-pentamethylicosenes with 3 and 4 double bonds (PMI:3 andPMI:4), archaeol and sn-3-hydroxyarchaeol (Elvert et al., 1999;Hinrichs et al., 1999; Pancost et al., 2000). Concentrationsand carbon isotope ratios ranged from 0.06 to 4.51 µg g−1 dwwith δ13C signatures of −88.9 to −129.3‰, respectively.

Specific biomarkers indicative for SRB are the FAs C16:1ω5c,ai-C15:0, cyC17:0ω5,6, C18:1ω7c, the n-alcohol C16:1ω5c, the sn-1-mono-O-alkyl glycerol ether (MAGE) C16:1ω5c, and thesn-1,2-di-O-alkyl glycerol ethers (DAGEs) C32:2a (coelutingwith C32:1a) and C32:2b (coeluting with C32:1b) (Hinrichs et al.,2000; Elvert et al., 2003). Concentrations of the SRB biomar-kers ranged from 0.07 to 4.36 µg g−1 dw. The most negativeδ13C-value for bacterial biomarkers of −103.1‰ was obtainedfor the unusual FA cyC17:0ω5,6 in the 4–6 cm sediment hori-zon, closely associated with the maximum in mean SR rate of0.46 µmol cm−3 d−1 and AOM rate of 0.36 µmol cm−3 d−1

(Treude et al., 2003; T. Treude, unpublished data) (Fig. 1).Highest δ13C-values of up to −34.1‰ were detected forthe FA C18:1ω7c in the upper sediment section which prob-ably indicates strong contributions from sulphide-oxidizingBeggiatoa at this site (Elvert et al., 2003).

Downcore concentration profiles of the most dominant ar-chaeal and bacterial biomarkers (alcohols sn-2-hydroxyarchaeoland archaeol for the archaea; FAs C16:1ω5c and cyC17:0ω5,6 forthe SRB) exhibited two distinct maxima at intervals 2–4 and8–10 cm sediment depth (Fig. 2A). The peak in 2–4 cmsediment depth was accompanied by a maximum in counts ofANME-2/DSS consortia (up to 12.7 × 107 cm−3; Fig. 2A)and an increase in 13C-depletion. The maximum in microbialbiomass was also visible in a higher TOC concentration of upto 2.58%, coinciding with a low δ13C-value of −34.7‰.Moreover, highly 13C-depleted carbon isotope ratios of allother archaeal biomarkers such as crocetane, PMI:3, PMI:4,and sn-3-hydroxyarchaeol were detected here (see Table 1).In contrast, the abundance of AOM-related glycerol dialkylglycerol tetraethers (GDGTs), containing cyclopentane rings intheir molecular structure (GDGT-1 and -2) (Pancost et al.,2001b), was low throughout the core (Fig. 3A). There wasalso no remarkable change in the relative abundance ofGDGTs over depth. However, a maximum in GDGT abun-dance at the Beggiatoa site was observed at 4–6 cm sedimentdepth in GDGT-0 (caldarchaeol) and crenarchaeol (contain-ing 4 cyclopentane rings and 1 cyclohexane ring) with theformer being attributed to archaea in general (Schouten et al.,2000), whereas the latter is more specifically found in

198 M

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he Authors

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2005 Blackw

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Table 1 Concentrations and carbon isotopic compositions of archaeal and bacterial lipid biomarkers at the Beggiatoa site

Depth(cm)

TOC(%)

Hydrocarbons (µg g−1 dw) Alcohols (µg g−1 dw) Fatty acids (µg g−1 dw)*

Cr PMI:3 PMI:4 n-C24:1 C16:1ω5c

MAGE C16:1ω5c

DAGE C31:1a

DAGE C32:2a (+C321a)

DAGE C32:2b (+C32:1b) Ar sn2-OH-Ar sn3-OH-Ar

sn2-OH-Ar/Ar ratio ai-C15:0 C16:1ω5c cyC17:0ω5,6 C18:1ω7c

0–2 1.83 0.54 0.44 0.57 n.d. 1.27 0.57 n.d. 1.43 0.48 2.15 5.33 0.19 2.5 1.52 8.05 1.47 1.532–4 2.58 1.33 0.87 1.02 n.d. 3.79 1.41 n.d. 4.36 1.06 4.51 13.59 0.27 3.0 3.40 17.31 4.10 1.244–6 1.79 0.62 0.42 0.52 n.d. 1.04 1.76 n.d. 1.04 0.48 2.15 6.07 0.17 2.8 1.36 9.10 2.32 0.986–8 1.76 0.48 0.33 0.43 n.d. 0.65 0.40 0.11 0.71 0.41 1.42 3.52 0.13 2.5 0.82 5.43 1.19 0.868–10 1.61 0.83 0.46 0.61 n.d. 1.17 0.44 0.12 1.13 0.48 2.25 5.94 0.23 2.6 1.43 9.37 2.07 1.3210–13 1.72 0.82 0.46 0.60 n.d. 0.86 0.35 0.17 0.89 0.41 1.91 4.77 0.19 2.5 1.04 5.78 1.48 1.1713–16 1.54 0.37 0.19 0.29 n.d. 0.23 0.10 0.07 0.21 0.17 0.67 1.43 0.08 2.1 0.59 2.02 0.63 0.8916–19 1.54 0.32 0.11 0.19 n.d. 0.12 0.07 0.07 0.19 0.18 0.48 0.88 0.06 1.8 0.45 0.96 0.26 0.60

Depth(cm)

TOC(‰ VPDB)

Hydrocarbons (‰ VPDB) Alcohols (‰ VPDB) Fatty acids (‰ VPDB)

Cr PMI:3 PMI:4 n-C24:1 C16:1ω5c

MAGEC16:1ω5c

DAGEC31:1a

DAGE C32:2a (+C32:1a)

DAGE C32:2b (+C32:1b) Ar sn2-OH-Ar sn3-OH-Ar ai-C15:0 C16:1ω5c cyC17:0ω5,6 C18:1ω7c

0–2 −29.5 −112.6 −124.7 −122.5 n.det. −96.6 −90.0 n.d. −87.6 −74.8 −111.6 −119.0 −112.2 −62.7 −75.4 −94.0 −36.92–4 −34.7 −119.3 −129.3 −125.7 n.det. −101.9 −93.9 n.d. −98.8 −91.0 −120.4 −123.5 −121.6 −73.8 −80.4 −101.0 −34.14–6 −29.3 −112.5 −122.1 −119.3 n.det. −99.9 −95.0 n.d. −87.4 −75.9 −114.7 −121.1 −111.6 −67.4 −92.5 −103.1 −45.76–8 −28.3 −106.9 −118.3 −115.3 n.det. −97.3 −92.3 −37.3 −76.8 −63.7 −106.5 −119.5 −110.4 −62.6 −90.8 −100.1 −48.48–10 −29.8 −115.8 −120.4 −119.0 n.det. −102.5 −93.8 −45.2 −92.6 −77.6 −111.0 −119.7 −115.8 −75.6 −96.3 −100.5 −56.910–13 −29.4 −113.7 −120.2 −117.8 n.det. −100.2 −93.0 −49.0 −87.3 −75.8 −109.5 −119.5 −113.7 −73.1 −95.8 −101.1 −59.513–16 −27.3 −100.9 −120.0 −118.7 n.det. −94.0 −88.1 −50.9 −57.9 −62.1 −97.9 −116.9 −107.1 −79.4 −95.3 −99.2 −73.616–19 −26.9 −100.0 −121.0 −117.2 n.det. −94.0 −87.5 −48.1 −54.2 −60.0 −88.9 −115.9 −110.2 −87.2 −96.2 −99.1 −80.0

n.d.: not detected; n.det.: not determined; *: after isotopic mass balance correction (Elvert et al., 2003).

Spatial variations of methanotrophic consortia at cold methane seeps 199

© 2005 The Authors Journal compilation © 2005 Blackwell Publishing Ltd

Fig. 1 Gas chromatograms obtained from Hydrate Ridge sediments. Upper panels: Beggiatoa site: (A) alcohol fraction of the 2–4 cm sediment horizon (this study),(B) fatty acid fraction of the 4–6 cm sediment horizon (adapted from Elvert et al., 2003; Fig. 1); Lower panels: Calyptogena field: (C) alcohol fraction and (D) fattyacid fraction from deeper sediment layers (16–19 cm).

200 M. ELVERT et al.

© 2005 The Authors Journal compilation © 2005 Blackwell Publishing Ltd

Fig.

2D

epth

pro

files

of

pore

wat

er s

ulph

ate,

AN

ME-

2/D

SS a

ggre

gate

num

ber,

conc

entr

atio

n an

d ca

rbon

isot

opes

of

spec

ific

lipid

bio

mar

kers

(ar

chae

ol, s

n-2-

hydr

oxya

rcha

eol,

FAs

C16

:1ω

5c a

nd c

yC17

:0ω

5,6)

, and

AO

Man

d SR

rate

s at

the

thre

e ch

emos

ynth

etic

pro

vinc

es o

f Hyd

rate

Rid

ge. A

ggre

gate

cou

nts

wer

e ob

tain

ed fr

om K

nitt

el e

t al

. (20

03);

por

e w

ater

sul

phat

e, a

s w

ell a

s A

OM

and

SR

rate

s, w

ere

take

n fr

om T

reud

e et

al.

(200

3)an

d T.

Tre

ude

(unp

ublis

hed

data

). N

ote

the

diff

eren

t sc

ales

use

d fo

r th

e pr

esen

tatio

n of

lipi

d bi

omar

ker

conc

entr

atio

ns a

s w

ell a

s of

AO

M a

nd S

R r

ates

.

Spatial variations of methanotrophic consortia at cold methane seeps 201

© 2005 The Authors Journal compilation © 2005 Blackwell Publishing Ltd

planktonic Crenarchaeota (Sinninghe Damsté et al., 2002).Independently generated 16S rDNA clone libraries from thesame station showed a dominance of euryarchaeota, whereasonly a few sequences of benthic crenarcheota were retrieved(Knittel et al., 2005).

Concentrations of all biomarkers described above generallydecreased further downcore and carbon isotope values stayedroughly constant. Exceptions are archaeol, and the DAGEsC32:2a and C32:2b, which carbon isotope values stepwise becamemore 13C-enriched, whereas δ13C-values of the FAs ai-C15:0

and C18:1ω7c gradually became more 13C-depleted (Table 1).

Calyptogena field

Concentrations of AOM-specific biomarkers at the Calyptogenafield were significantly lower than at the Beggiatoa site(Fig. 2B, Table 2). The two most dominant GC-amenablebiomarkers, sn-2-hydroxyarchaeol and the FA C16:1ω5c, wereup to 5.6 and 8.8 times decreased in concentration relativeto the Beggiatoa site, respectively (maximum concentration of2.36 µg g−1 dw detected for sn-2-hydroxyarchaeol at 4–6 cmsediment depth). Despite this difference in concentration,stable carbon isotopic compositions of all specific biomarkerswere similar to the Beggiatoa site, showing negative δ13C-values of −93.4 to −134.7‰ for the methanotrophic archaeaand of down to −102.1‰ for the SRB. Similarly to theBeggiatoa site, more 13C-enriched values have been obtainedfor the FA C18:1ω7c in the upper sediment sections.

Downcore profiles show a maximum in concentrationaround 5 cm sediment depth, which is also closely related tothe highest AOM and SR rates (Fig. 2B). A similar subsurfacepeak in AOM-consortia abundance as indicated by biomarkerand rate measurements was observed previously (RV SONNESO143-2, 1999) at another Calyptogena field (see Fig. 4A,Station 185 in Knittel et al., 2003). However, the enrichmentof AOM consortia in this sediment region was not so evidentfrom aggregate counts of ANME-2/DSS consortia sampledduring RV SONNE cruise SO148-1 in 2000. This findingmay result from biomarker sampling of a TV-MUC, which was

not retrieved from the same Calyptogena field, potentiallyintroducing small-scale variability in AOM biomass. Never-theless, maximum concentrations of AOM-specific biomarkers(sn-2-hydroxyarchaeol, archaeol, FAs C16:1ω5c and cyC17:0ω5,6)at the Calyptogena field were accompanied by minima in δ13C-values. The highest abundance of AOM-related GDGT-1 and-2 with concentrations of up to 6.2 and 7.0 µg g−1 dw, respec-tively, was observed at 4–6 cm sediment depth (Fig. 3B).In contrast, the concentration of crenarchaeol, a biological markerfor pelagic input, was continuously decreasing with depth.

A second concentration maximum was observed at 13–16 cm sediment depth (Fig. 2B). A feature of this maximumwas the abundance of a highly 13C-depleted hydrocarboncompound (δ13C-values: −112.5 to −114.3‰), containing24 carbon atoms and one double bond in its structure (n-tetracosene – C24:1; molecular mass = 336). This compound,eluting slightly after the cluster of PMIs and ahead of then-alkane C24 (Kovats retention index (HP-5 MS): 2386), wasidentified after hydrogenation of the respective hydrocarbonfraction. Its finding is accompanied by smaller amounts ofunsaturated tricosenes (δ13C-values: ∼−115‰), previouslyreported to be abundant in a Black Sea microbial mat(Thiel et al., 2001b). In the upper sediment horizons, onlytrace amounts of n-C24:1 were found at this location whereasit was fully absent at the Beggiatoa site (Table 1). The presenceof n-C24:1 was accompanied by highly abundant DAGEsC31:1a and C32:2b (up to 0.71 µg g−1 dw) which showedhighest 13C-depletion in these sediment horizons. Wealso observed a relative increase of sn-3-hydroxyarchaeol andthe FA ai-C15:0, with the latter compound being the mostdominant FA in the lower sediment horizons at the Calyptogenafield. Both compounds, similar to the DAGEs C31:1a andC32:2b, are relatively more 13C-depleted here than in sedimenthorizons above.

Acharax field

Concentrations of AOM-specific biomarkers at the Acharaxfield were between one and two orders of magnitude lower

Fig. 3 Depth profiles of GDGTs at (A) theBeggiatoa site and (B) the Calyptogena field(selective samples analysed).

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Table 2 Concentrations and carbon isotopic compositions of archaeal and bacterial lipid biomarkers at the Calyptogena field

Depth(cm)

TOC(%)

Hydrocarbons (µg g−1 dw) Alcohols (µg g−1 dw) Fatty acids (µg g−1 dw)*

Cr PMI:3 PMI:4 n-C24:1 C16:1ω5c

MAGE C16:1ω5c

DAGE C31:1a

DAGE C32:2a (+C32:1a)

DAGE C32:2b (+C32:1b) Ar sn2-OH-Ar sn3-OH-Ar

sn2-OH-Ar/Ar ratio ai-C15:0 C16:1ω5c cyC17:0ω5,6 C18:1ω7c

0–2 2.14 0.32 0.15 0.12 trace 0.22 0.06 0.09 0.36 0.28 0.59 1.20 0.08 2.0 0.68 1.61 0.18 1.442–4 1.94 0.51 0.24 0.23 0.02 0.33 0.14 0.13 0.50 0.30 1.07 2.12 0.13 2.0 1.04 1.58 0.38 1.514–6 1.84 0.55 0.37 0.63 0.04 0.41 0.18 0.33 0.66 0.50 1.36 2.36 0.22 1.7 1.81 1.95 0.68 2.416–8 1.84 0.52 0.32 0.60 0.04 0.36 0.29 0.23 0.51 0.42 1.23 2.15 0.18 1.8 1.70 1.68 0.75 2.058–10 1.74 0.56 0.20 0.43 0.04 0.25 0.19 0.20 0.42 0.37 1.03 1.53 0.18 1.5 1.21 1.01 0.46 1.5710–13 1.73 0.48 0.15 0.21 0.04 0.06 0.05 0.08 0.14 0.12 0.32 0.46 0.06 1.4 0.59 0.57 0.19 0.9213–16 1.81 0.57 0.17 0.37 0.24 0.19 0.08 0.71 0.25 0.68 1.03 0.79 0.28 0.8 1.21 0.64 0.20 0.9116–19 1.61 0.28 0.08 0.19 0.14 0.09 0.04 0.71 0.16 0.67 0.58 0.33 0.21 0.6 0.68 0.30 0.10 0.26

Depth (cm)

TOC(‰ VPDB)

Hydrocarbons (‰ VPDB) Alcohols (‰ VPDB) Fatty acids (‰ VPDB)

Cr PMI:3 PMI:4 n-C24:1 C16:1ω5c DAGE C16:1ω5c

DAGEC31:1a

DAGEC32:2a (+C32:1a)

MAGEC32:2b (+C32:1b) Ar sn2-OH-Ar sn3-OH-Ar ai-C15:0 C16:1ω5c cyC17:0ω5,6 C18:1ω7c

0–2 −28.7 −100.3 −120.6 −109.9 n.det. −79.5 −55.9 −51.8 −61.9 −55.8 −108.4 −118.4 −98.9 −48.7 −64.7 −92.4 −40.62–4 −29.3 −114.6 −122.3 −112.9 n.det. −90.4 −74.5 −51.1 −68.3 −65.7 −105.7 −121.0 −106.2 −62.9 −72.4 −99.6 −47.34–6 −30.1 −112.6 −122.7 −120.7 n.det. −92.9 −86.4 −54.0 −73.2 −74.8 −103.4 −120.3 −112.4 −83.9 −85.1 −100.0 −68.36–8 −31.5 −109.6 −124.2 −121.2 n.det. −92.4 −90.6 −59.9 −77.7 −79.3 −106.7 −123.0 −116.1 −89.2 −89.2 −102.1 −75.58–10 −29.5 −113.7 −119.3 −117.9 n.det. −90.5 −86.1 −60.7 −68.6 −73.8 −102.9 −118.9 −106.1 −85.6 −85.8 −98.4 −75.710–13 −27.6 −112.0 −116.2 −113.2 n.det. −84.1 −82.9 −50.7 −58.2 −65.6 −93.4 −114.1 −96.0 −65.1 −76.6 −90.3 −64.613–16 −31.1 −114.1 −125.0 −120.3 −112.5 −97.0 −88.2 −95.2 −65.9 −95.6 −96.5 −112.6 −109.9 −93.2 −87.6 −95.1 −78.116–19 −32.4 −111.1 −134.7 −127.9 −114.3 −98.3 −96.8 −100.5 −65.9 −101.8 −101.6 −113.8 −119.5 −102.5 −94.1 −98.1 −83.8

n.det.: not determined; *: after isotopic mass balance correction (Elvert et al., 2003).

Spatial variations of methanotrophic consortia at cold methane seeps 203

© 2005 The Authors Journal compilation © 2005 Blackwell Publishing Ltd

than at the Beggiatoa site and the Calyptogena field (Fig. 2C),as reflected in ANME-2/DSS aggregate counts and ratemeasurements. Constant pore water sulphate concentrationsof approximately 28 mM indicate low sulphate reduction ratesin the upper 12 cm of this chemosynthetic province (Treudeet al., 2003). Nevertheless, biomarkers with 13C-depletedcarbon isotopic ratios have been found within all measuredfractions, exhibiting a minimum δ13C-value of −119.7‰for sn-2-hydroxyarchaeol measured at 10–13 cm sedimentdepth. Generally, there was a slight trend to higher con-centrations of AOM-specific biomarkers with increasingsediment depth (full data set not shown), which wasaccompanied by lowered δ13C-values.

Subsurface gas hydrate site

Figure 4 presents depth profiles and stable carbon isotopevalues of AOM-specific biomarkers as well as pore watersulphide and chloride from the 1.2 m gravity core, containingthick layers of methane gas hydrates at 55 cm, 80 cm andbelow 103 cm sediment depth. Biomarker concentrations inthese subsurface sediments of Hydrate Ridge were maximallya third of those in surface sediments with 4.58 µg g−1 dw forsn-2-hydroxyarchaeol as a marker for ANME-2 archaea and upto 1.80 µg g−1 dw for the FA C16:1ω5c specific for SRB of theDSS-cluster. 13C-depletion of both compounds were similar toall other investigated sites ranging from −113.2 to −120.7‰and from −67.9 to −90.7‰, for sn-2-hydroxyarchaeol and FAC16:1ω5c, respectively. Other prominent AOM-specific bio-markers in the deeper sediments include crocetane, PMI:3,PMI:4, and archaeol indicative of methanotrophic archaeaand n-alcohol C16:1ω5c, MAGE C16:1ω5c, DAGE C32:2a aswell as the FAs ai-C15:0, cyC17:0ω5,6, and C18:1ω7c diagnostic forSRB (Table 3). The concentrations of specific biomarkersincreased just above methane hydrate layers and alsorevealed stronger 13C-depletion here. In contrast, only traceamounts have been found for DAGEs C31:1a and C32:2b as wellas sn-3-hydroxyarchaeol.

DISCUSSION

Spatial variability of methanotrophic consortia at cold methane seeps

Biomarker profiling and microbial diversityANME-2/DSS aggregates at Hydrate Ridge are associatedwith the FAs C16:1ω5c and cyC17:0ω5,6 as indicator for SRBof the DSS group (δ13C-values down to −103‰) (Elvert et al.,2003). Moreover, ANME-2-derived major abundant isoprenoidalglycerol ethers have been detected (δ13C-values down to−123‰) (Boetius et al., 2000; Elvert et al., 2001), exhibitinga strong dominance of sn-2-hydroxyarchaeol over archaeol(Blumenberg et al., 2004) (Table 1). Archaeal glycerol ethersare specifically accompanied by prominent amounts ofFi

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Table 3 Concentrations and carbon isotopic compositions of archaeal and bacterial lipid biomarkers at the gas hydrate site

Depth(cm)

TOC(%)

Hydrocarbons (µg g−1 dw) Alcohols (µg g−1 dw) Fatty acids (µg g−1 dw)*

Cr PMI:3 PMI:4 C16:1ω5c

MAGE C16:1ω5c

DAGE C31:1a

DAGE C32:2a (+C32:1a)

DAGE C32:2b (+C32:1b) Ar sn2-OH-Ar sn3-OH-Ar

sn2-OH-Ar/Ar/ratio ai-C15:0 C16:1ω5c CyC17:0ω5,6 C18:1ω7c

23–25 1.65 0.15 0.08 0.11 0.37 0.27 n.d. 1.28 Trace 1.97 4.18 Trace 2.1 0.40 1.67 0.56 0.3928–30 1.53 0.20 0.16 0.19 0.48 0.32 n.d. 1.78 Trace 2.19 4.85 Trace 2.2 0.60 1.80 0.77 0.6038–40 1.35 0.15 0.08 0.14 0.25 0.22 n.d. 0.99 Trace 1.61 2.89 Trace 1.8 0.50 1.01 0.53 0.3348–50 1.39 0.17 0.12 0.20 0.37 0.18 n.d. 1.14 Trace 2.09 4.33 Trace 2.1 0.80 1.23 0.74 0.6658–60 1.26 0.25 0.02 0.03 0.11 0.04 n.d. 0.69 Trace 1.31 1.46 Trace 1.1 0.34 0.78 0.37 0.2868–70 1.32 0.28 0.06 0.10 0.61 0.28 n.d. 0.90 Trace 1.57 2.53 Trace 1.6 0.29 0.83 0.31 0.2578–83 1.26 0.25 0.02 0.02 0.10 0.08 n.d. 0.68 Trace 1.33 1.45 Trace 1.1 0.10 0.25 0.13 0.0488–90 1.27 0.17 0.01 0.01 0.07 0.04 n.d. 0.55 Trace 1.10 0.94 Trace 0.9 0.06 0.05 0.03 0.02106–108 1.34 0.10 0.01 0.01 0.07 0.05 n.d. 0.66 Trace 1.00 0.73 Trace 0.7 0.04 0.05 0.03 0.01

Depth (cm)

TOC (‰ VPDB)

Hydrocarbons (‰ VPDB) Alcohols (‰ VPDB) Fatty acids (‰ VPDB)

Cr PMI:3 PMI:4 C16:1ω5c

MAGEC16:1ω5c

DAGEC31:1a

DAGEC32:2a (+C32:1a)

DAGEC32:2b (+C32:1b) Ar sn2-OH-Ar sn3-OH-Ar ai-C15:0 C16:1ω5c cyC17:0ω5,6 C18:1ω7c

23–25 −26.9 −108.1 −120.1 −115.2 −90.1 −78.8 n.det. −76.4 n.det. −100.7 −119.2 n.det. −66.3 −87.0 −101.8 −45.728–30 −28.5 −109.3 −122.3 −117.5 −94.5 −84.7 n.det. −88.6 n.det. −103.3 −120.7 n.det. −80.3 −86.2 −100.8 −51.838–40 −27.5 −105.4 −125.6 −115.8 −96.8 −83.9 n.det. −76.6 n.det. −96.9 −118.4 n.det. −87.6 −86.0 −100.0 −43.148–50 −28.4 −105.7 −126.3 −118.7 −95.9 −79.4 n.det. −79.8 n.det. −102.4 −120.6 n.det. −94.4 −90.7 −101.7 −68.858–60 −25.2 −109.6 n.det. n.det. −92.5 n.det. n.det. −63.2 n.det. −82.0 −113.2 n.det. −85.2 −88.4 −99.3 −71.468–70 −25.5 −109.9 n.det. −110.7 −96.2 −81.7 n.det. −73.1 n.det. −95.8 −119.5 n.det. −86.2 −89.1 −98.6 −64.578–83 −25.0 −109.5 n.det. n.det. −88.6 n.det. n.det. −52.1 n.det. −84.2 −113.5 n.det. −68.1 −80.7 −85.4 −45.288–90 −24.8 −107.6 n.det. n.det. −83.1 n.det. n.det. −46.4 n.det. −78.4 −114.3 n.det. −61.0 −67.9 −79.6 −43.1106–108 −23.8 −79.6 n.det. n.det. n.det. n.det. n.det. −34.4 n.det. −71.2 −114.7 n.det. −54.6 −71.3 −85.5 −35.8

n.d.: not detected; n.det.: not determined: *: after isotopic mass balance correction (Elvert et al., 2003).

Spatial variations of methanotrophic consortia at cold methane seeps 205

© 2005 The Authors Journal compilation © 2005 Blackwell Publishing Ltd

irregular isoprenoids such as Cr and pentamethylicosenes(Elvert et al., 1999, 2001) as well as short-chain n-alcoholsand MAGEs, with both showing a very similar chain-lengthdistribution and unsaturation pattern as that found for the FAs(Hinrichs et al., 2000) (Fig. 1; Tables 1 and 2).

The variation in the biomarker inventory of the chemosyn-thetic provinces observed here may be derived from differentarchaeal ANME-2 subgroups present in the particular sedi-ment column (Knittel et al., 2005). At the Beggiatoa site,which is dominated by the subgroup ANME-2a, we foundhigh abundances of specific GC-amenable biomarker com-pounds but no obvious maximum or increase in GDGT-1 and-2 abundance (Fig. 3A), both of which have been attributed tohigh AOM activity in mud volcanoes from the MediterraneanRidge (Pancost et al., 2001b). In contrast, in the upper sedi-ment layers at the Calyptogena field, which is characterized bythe subgroup ANME-2c, we detected a higher abundance ofAOM-related cyclic GDGT lipids (Fig. 3B). The concentra-tion maximum of all specific biomarker components at theCalyptogena field is found at the same sediment depth horizonwhere the highest AOM and SR rates are detected (Fig. 2B)(Treude et al., 2003). Thus, we suggest that ANME-2c archaea– besides the well-known irregular isoprenoids and glycerolethers – also produce GDGTs as cell wall compartments,whereas ANME-2a archaea do not. These results questionthe possibility to distinguish ANME-1 and ANME-2 archaeasolely on the presence (ANME-1) or absence (ANME-2) ofGDGT lipids (Blumenberg et al., 2004).

Isotopic mass balance calculations indicate that within theAOM hotspot at 2–4 cm sediment depth below the Beggiatoamat, the amount of AOM biomass is as high as 16% TOC (or0.41% dry weight). This finding corresponds to the most nega-tive TOC-carbon isotope values (−34.7‰) obtained, and isalso in accordance with high cell numbers of around 1010 cellscm−3 in this zone (Boetius et al., 2000). The amount of FAsderived from SRB involved in AOM in this horizon is also veryhigh and reached abundances of up to 63% of the total fattyacid content (Elvert et al., 2003). In the upper sediment zone(0–8 cm depth) at the Beggiatoa site, linear correlation coef-ficients between specific archaeal lipid content and aggregatenumber are significantly high (r2 [sn-2-hydroxyarchaeol] =0.91; r2 [archaeol] = 0.87), thus enabling an estimate of theamount of archaeal lipid content per ANME-2 cell. Such anapproach integrates over a range of environmental and physi-ological factors that regulate growth conditions of methano-trophic archaea (Nauhaus et al., 2005), and given the extremelyslow growth rates of these still uncultured archaea (Girguiset al., 2003) seems to be appropriate. Normalizing the amountof sn-2-hydroxyarchaeol to the number of archaeal cells inAOM aggregates, there are between 0.62 and 0.90 × 10−15 gsn-2-hydroxyarchaeol and 0.22–0.30 × 10−15 g archaeol perANME-2 cell. The estimate for SRB-specific lipids derivedfrom DSS species involved in AOM such as C16:1ω5c andcyC17:0ω5,6 is about 0.46–0.58 × 10−15 g and 0.10–0.14 ×

10−15 g per DSS cell, respectively, and has been presentedpreviously by Elvert et al. (2003).

Correlation of AOM aggregates with biomarker abundanceis less obvious at the Calyptogena field (Fig. 2B). Althoughaggregate counts are as high as those from the Beggiatoa site,biomarker concentrations are significantly reduced, resultingin biomarker abundances per ANME-2 cell of 0.10–0.36 ×10−15 g for sn-2-hydroxyarchaeol and of 0.05–0.24 × 10−15 gfor archaeol. This indicates a minor coupling of ANME-2/DSS aggregates and biomarker production. Other AOM-performing consortia may be present at the Calyptogena fieldor the specific biomarker content per microbial cell is different,maybe due to reduced methane flux here relative to theBeggiatoa site. Indeed, Knittel et al. (2005) reported differentsubgroups of ANME-2 archaea dominating the respectivesampling locations. ANME-2a/DSS aggregates (called ‘mixedtypes’) are dominating the Beggiatoa sites (up to 80% relativeto the so-called ‘shell type’ ANME-2c/DSS aggregates),whereas at the Calyptogena field, the majority of AOM-performing consortia contains archaea belonging to theANME-2c subgroup (ANME-2c/DSS aggregate detectionof up to 80% abundance). Moreover, the abundance ofcyclic GDGTs indicative for AOM in cold seep environments(Pancost et al., 2001b) is higher at the Calyptogena field thanat the Beggiatoa site (Fig. 3), suggesting a different lipidbiosynthesis in ANME-2c archaea relative to the ANME-2asubgroup. The lowest amounts of AOM biomarkers weredetected in surface sediments at the lowest flux location, i.e.Acharax field, matching the low microscopic abundance ofAOM aggregates, although a contribution by relict AOMbiomarkers cannot be excluded. Nevertheless, the concentra-tions of AOM-specific lipids showed a slow increase inbiomass further downcore, which corresponds to slightlyincreasing SR rates towards the underlying methane-richersediment depths (Fig. 2C), maybe indicating the appearanceof AOM with depth.

We also found evidence for low but with sediment depthincreasing abundances of ANME-1 archaea at the Calyptogenafield, confirming previous ANME-1 cell counts (Knittel et al.,2005). ANME-1 signatures are abundant GDGT-1 and -2(Fig. 3B; Stadnitskaia et al., 2005) as well as low sn-2-hydroxyarchaeol to archaeol ratios (down to 0.6; Table 2)(Blumenberg et al., 2004). The decrease in sn-2-hydroxyarchaeolto archaeol ratios is accompanied by a relative increase in sn-3-hydroxyarchaeol that has been detected earlier in sedimentsand carbonate crusts from Mediterranean mud volcanoesdominated by ANME-1 archaea (Pancost et al., 2001b; Aloisiet al., 2002). Cultured archaea known so far that produce sn-3-hydroxyarchaeol include methanogens such as Methanosaetaconcilii – formerly referred to as Methanotrix concilii (Ferranteet al., 1988) – and Methanococcus voltae (Sprott et al., 1993).Nevertheless, carbon isotope values for sn-3-hydroxyarchaeolat Hydrate Ridge indicate an origin from a methanotrophicarchaeal source that is very probably ANME-1.

206 M. ELVERT et al.

© 2005 The Authors Journal compilation © 2005 Blackwell Publishing Ltd

The biomarker pattern of deeper sedimentary strata at theCalyptogena field is also characterized by relatively high abun-dances of methyl-branched FAs (ai-C15:0, i-C16:0, and ai-C17:0)as well as a diverse suite of DAGEs (Table 2, Fig. 1C,D). Sidechains within the different DAGEs are partly known fromother studies focusing on AOM (Hinrichs et al., 2000; Pancostet al., 2001a) but are also first reported here at Hydrate Ridge(Table 4). DAGEs seem to be especially prominent in ANME-1 systems whereas they are more or less lacking in ANME-2systems (Teske et al., 2002; Blumenberg et al., 2004). Althoughvarious authors attributed these components to bacterialsources and most likely SRB due to reports of their detectionamong thermophilic, deeply branching SRB such as Ther-modesulfobacterium commune and Thermodesulfobacteriumhveragerdense (Langworthy et al., 1983; Sturt et al., 2004),the exact microbial source of these compounds in AOMenvironments is currently missing. All of the dominatingAOM-related biomarkers (DAGEs C31:1a and C32:2b, archaeol,FA ai-C15:0) from the deeper sediment sections at the Calyp-togena field show correlating δ13C-values (around −95‰ at13–16 cm and approximately −101‰ at 16–19 cm sedimentdepth, Table 2). ANME-1 cells are clearly enriched here (Knittelet al., 2005), whereas SRB abundances decreased rapidly(Knittel et al., 2003). Therefore, the question arises if notANME-1 archaea themselves may be potential candidates forthe production of most of the dominating biomarkers foundhere. The possibility of the presence of bacterial-like biomar-kers, i.e. fatty acids or straight-chain alcohols, in archaea hasalready been demonstrated (Carballeira et al., 1997; Nishiharaet al., 2000). Moreover, ANME-1 has been microscopicallyshown to occur as single cells in cold seep environments (Orphanet al., 2002). Nevertheless, we cannot exclude the presence offree-living syntrophic SRB in the vicinity of ANME-1 cells thatproduce a very different biomarker pattern here relative to thatobserved from DSS species living in close association withANME-2 (Fig. 1B,D).

Relation to methane flux

The abundance of biomarkers at the three investigatedchemosynthetic provinces of Hydrate Ridge in this study wasfound to be related to average methane flux as measuredby Torres et al. (2002). Highest concentrations of AOMbiomarkers were observed at the Beggiatoa site, followed bythe Calyptogena field, and are very much reduced at theAcharax field. The effectiveness of AOM consortia to reducemethane efflux from the sediment is very high at HydrateRidge. At the Beggiatoa site, focused methane efflux is about50% of methane consumption (Boetius & Suess, 2004). Theincreased methane efflux pushes the zone of AOM activity andthus the zone of sulphate depletion upwards to the sediment/water interface (Luff & Wallmann, 2003), which is indicatedby subsurface maxima of AOM-consortia biomass andbiomarker abundance in the 2–4 cm sediment horizon (Fig. 2A).At the Calyptogena field, methane fluxes to the overlying watercolumn are reduced to less than 1 mmol m−2 d−1, i.e. only 2%of the influx (Torres et al., 2002). This may be due to thebehaviour of the clams to dwell with their foot into sulphidepockets, thereby mixing sulphate-rich pore water into theupper 10 cm of sediment.

In general, by using mean values of AOM and SRR ratespresented by Treude et al. (2003), we observe a relationbetween methane turnover, the maxima of biomarker concen-tration and their highest 13C-depletion with AOM and SRrate, especially at the Calyptogena field (Fig. 2B). This maysuggest that biomarker concentrations can be used as indicatorsnot only for the depth position of AOM biomass (Werne et al.,2002) but also for AOM activity which has been alreadyobserved in other environmental settings (Bian et al., 2001).However, the understanding of the microbial carbon cycle incold seep sediments is still limited because carbon isotope sig-natures of important components such as pore water methane,DIC, and different DOC compounds have not yet beenanalysed in full detail.

Relation to methane gas hydrates

Biomarkers of AOM consortia in a gravity core containingsolid gas hydrate layers show a general decrease of AOM-derived biomass with increasing sediment depth (Fig. 4).Nevertheless, the concentration profiles of the specificbiomarkers correlate along the whole gravity core and thusindicate the direct link between the lipid-producing archaeaand bacteria.

Biomarkers are more 13C-depleted in sediment horizonsjust above the methane hydrate layers accompanied by anapparent concentration increase. This finding indicates thatmethane stored in the hydrates is available for the microbialcommunity even though the sediments at Hydrate Ridge are inthe hydrate stability field (water depth 770 m; 4 °C bottomwater temperature) (Suess et al., 1999). The methane available

Table 4 Structures of various detected sn-1,2-di-O-alkyl glycerol ethers(DAGEs) in Hydrate Ridge sediments

Compoundsn-1 alkyl moiety

sn-2 alkyl moiety

Source assignment

C29:0 n-C14:0 ai-C15:0 b (Ic)C30:0 ai-C15:0 ai-C15:0 a; b (If)C31:1a cyC16:0 ai-C15:0 a; b (Ig)C31:1b n-C14:0 cyC17:0 a; b (IIa)C32:1a n-C16:1 n-C16:0 aC32:2a n-C16:1 (ω5c ?) n-C16:1 (ω5c ?) aC32:1b n-C16:0 n-C16:1 aC32:2b n-C16:1 (or cyC16:0 ?) n-C16:1 (or cyC16:0 ?) this studyC33:2 n-C16:1 (or cyC16:0 ?) cyC17:0 this studyC34:2 cyhexC17:0 cyC17:0 a; b (IIb)

(a) Hinrichs et al. (2000).(b) Pancost et al. (2001a); in parentheses abbreviation used by the authors.

Spatial variations of methanotrophic consortia at cold methane seeps 207

© 2005 The Authors Journal compilation © 2005 Blackwell Publishing Ltd

could be supplied by the continuous dissociation of gashydrates due to diffusion caused by the intense concentrationgradient between the gas hydrates and the surroundingsediment (Haeckel et al., 2004). By scavenging the porewater methane the microbial community likely enhances thedissociation of methane hydrates. Alternatively, free gas maybe present which migrates from deeper horizons through per-meable conduits into the zone of hydrate stability (Milkovet al., 2005). Indeed, free gas bubbles have been detected inthe hydrate stability zone by CT investigation under in situconditions (Abegg et al., 2003). Below 70 cm sediment depth,biomarker concentrations drop rapidly, indicating a reducedor even inhibited AOM activity probably due to sulphatedepletion and/or the loss of water caused by the formationof massive gas hydrate layers, with the latter being visiblein positive chloride anomalies (Haeckel et al., 2004) (Fig. 4).

An additional in vitro enrichment study from Hydrate Ridgesediments confirmed that a high sn-2-hydroxyarchaeol toarchaeol ratio is indicative of methanotrophic archaea of theANME-2 cluster (K. Nauhaus, M. Albrecht, M. Elvert, A. Boetius,unpublished data), as previously also observed by Blumenberget al. (2004). The ratio of sn-2-hydroxyarchaeol to archaeolcan thus be used for the verification of the predominance ofANME-1 or ANME-2 archaea. Higher values of up to 5.5 areindicative for ANME-2 archaea, whereas environments dom-inated by ANME-1 archaea are indicated by ratios lower than 1(Niemann, 2005). Ratios obtained from the upper 70 cmof methane gas hydrate-containing sediments vary between1.1 and 2.2 (Table 3) and are in the lower range compared tothe sediment sections dominated by ANME-2 from theBeggiatoa site and the Calyptogena field (Tables 1 and 2).Combined with the high abundance of DSS-specific FAsC16:1ω5c and cyC17:0ω5,6 (Elvert et al., 2003), these ratiosindicate the predominance of ANME-2/DSS aggregates in theupper sediment horizons of the gas hydrate-bearing sediments.

DSS-specific lipid concentrations at the gas hydrate sitedecrease stronger compared to archaeal lipids, especially archaeol.The relative increase of archaeol vs. sn-2-hydroxyarchaeol withdepth as well as the elevated carbon isotope values of up to−71‰ measured for archaeol might indicate the presenceof methanogenic besides methanotrophic archaea in thesulphate-depleted sediment zones. Generally, carbon isotopesignatures measured for archaeol as indicator for metha-nogenic sources obtained from other environmental studieswere ranging between −17 and −34‰ (Bolle et al., 2000; Schoutenet al., 2001) and thus, archaeol detected here may result fromboth methanogenic and methanotrophic archaeal sources.

SUMMARY AND CONCLUSIONS

This study focused on the high-resolution characterization ofspecific biomarkers related to archaeal–bacterial assemblagesinvolved in the anaerobic oxidation of methane (AOM)from a well-known cold seep environment. Specific biomarker

patterns obtained from different chemosynthetic provinces(Beggiatoa site, Calyptogena field, Acharax field) varyboth horizontally and vertically in abundance and stablecarbon isotope composition on scales of centimetre to tensof metres. The abundance of biomarkers mainly reflectsAOM biomass, which seems to be related to the amount ofmethane flux from below.

The diversity in biomarker abundance and their variationin δ13C-depletion indicates the presence of various AOM-mediating assemblages (ANME-2a/DSS, ANME-2c/DSS,ANME-1) at the different cold seep sites: (1) High abundancesof sn-2,3-di-O-isoprenoidal glycerol ethers (archaeol, sn-2-hydroxyarchaeol) and specific fatty acids (C16:1ω5c, cyC17:0ω5,6)are associated with ANME-2a/DSS aggregates and occur athigh methane flux sites. (2) Upper sediment sections of thelow to medium flux site contain reduced amounts of bothcompound classes but higher amounts of AOM-relatedglycerol dialkyl glycerol tetraethers (GDGTs) that probablyindicate ANME-2c/DSS aggregates. (3) Deeper sedimenthorizons of the low to medium flux site, where isotopicallysimilar sn-1,2-di-O-alkyl glycerol ethers (DAGEs C31:1a, C32:2b),archaeol, methyl-branched fatty acids (ai-C15:0, i-C16:0, ai-C17:0), as well as diagnostic GDGTs (GDGT-1 and -2) are pre-vailing, provide indications of the predominance of ANME-1archaea eventually in absence of a bacterial partner. (4) Deepersediment strata containing solid layers of methane gas hydratesare characterized by correlating biomarker concentrations ofANME-2/DSS aggregates accompanied by negative shifts inδ13C-values just above the hydrate layers.

ACKNOWLEDGEMENTS

We thank the officers, crew and shipboard scientific party forexcellent support during RV SONNE cruises SO143-1 andSO148-1. We are especially indebted to Dirk Rickert (GEO-MAR, Kiel, Germany) for compiling pore water data and hishelp with sample handling and transport during and after thecruise, and to Katrin Knittel for providing data on aggregatecounts. Gabriele Klockgether and Swantje Lilienthal areacknowledged for their assistance with bulk organic carbonmeasurements. Kai-Uwe Hinrichs provided fruitful commentson an earlier version of the manuscript. Constructive criticismby three anonymous reviewers was highly appreciated. Thisstudy was part of the programs TECFLUX I and II (TECton-ically induced FLUXes, FN 03G0143A and FN 03G0148A)and MUMM (Mikrobielle UMsatzraten von Methan in gashy-drathaltigen Sedimenten, FN 03G0554A) supported by theBundesministerium für Bildung und Forschung (BMBF,Germany). Further support was provided by the Max-Planck-Gesellschaft (Germany) and the Deutsche Forschungsgemein-schaft (DFG, Germany). This is publication GEOTECH-195of the program GEOTECHNOLOGIEN of the BMBF, andpublication no. 0346 of the Research Center Ocean Marginsat the University of Bremen funded by the DFG.

208 M. ELVERT et al.

© 2005 The Authors Journal compilation © 2005 Blackwell Publishing Ltd

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