www.elsevier.com/locate/chemgeo

Chemical Geology 205 (2004) 367–390

Life at cold seeps: a synthesis of biogeochemical and ecological

data from Kazan mud volcano, eastern Mediterranean Sea

Josef P. Wernea,*, Ralf R. Haeseb,1, Tiphaine Zitterc, Giovanni Aloisid,2,Ioanna Bouloubassie, Sander Heijsf, Aline Fiala-Medionig, Richard D. Pancosta,3,

Jaap S. Sinninghe Damstea, Gert de Langed, Larry J. Forneyf,4,Jan C. Gottschalf, Jean-Paul Foucherh, Jean Masclei, John Woodsideb

the MEDINAUT and MEDINETH Shipboard Scientific Parties5

aDepartment of Marine Biogeochemistry and Toxicology, Royal Netherlands Institute for Sea Research (NIOZ), Den Burg, The NetherlandsbDepartment of Geochemistry, Faculty of Earth Sciences, Utrecht University, Utrecht, The Netherlands

cDepartment of Sedimentology and Marine Geology, Faculty of Earth and Life Sciences, Free University Amsterdam,

Amsterdam, The NetherlandsdLaboratoire d’Oceanographie Dynamique et de Climatologie, Universite Pierre et Marie Curie, Paris, France

eLaboratoire de Chimie et Biogeochimie Marines, Universite Pierre et Marie Curie, Paris, FrancefDepartment of Microbiology, Center for Ecological and Evolutionary Studies, University of Groningen, Haren, The Netherlands

gObservatoire Oceanologique, Universite Pierre et Marie Curie, Banyuls-sur-Mer, FrancehDRO/Environments Sedimentaires, IFREMER, Brest, France

iGeosciences Azur, Observatoire Oceanologique de Villefranche, Villefranche-sur-Mer, France

Received 4 October 2002; received in revised form 30 September 2003; accepted 23 December 2003

Abstract

Recent field observations have identified the widespread occurrence of fluid seepage through the eastern Mediterranean Sea

floor in association with mud volcanism or along deep faults. Gas hydrates and methane seeps are frequently found in cold seep

areas and were anticipated targets of the MEDINAUT/MEDINETH initiatives. The study presented herein has utilized a multi-

disciplinary approach incorporating observations and sampling of visually selected sites by the manned submersible Nautile and

by ship-based sediment coring and geophysical surveys. The study focuses on the biogeochemical and ecological processes and

conditions related to methane seepage, especially the anaerobic oxidation of methane (AOM), associated with ascending fluids

on Kazan mud volcano in the eastern Mediterranean. Sampling of adjacent box cores for studies on the microbiology,

biomarkers, pore water and solid phase geochemistry allowed us to integrate different biogeochemical data within a spatially

0009-2541/$ - see front matter D 2004 Elsevier B.V. All rights reserved.

doi:10.1016/j.chemgeo.2003.12.031

* Corresponding author. Current address: Large Lakes Observatory and Department of Chemistry, University of Minnesota Duluth, 10

University Dr. 109 RLB, Duluth, MN 55812, USA. Tel.: +1-218-726-7435; fax: +1-218-726-6979.

E-mail addresses: jwerne@d.umn.edu (J.P. Werne), ralf.haese@ga.gov.au (R.R. Haese).1 Address as of October 2003: Geoscience Australia, Canberra.2 Current address: Department of Marine Environmental Geology, GEOMAR, Kiel, Germany.3 Current address: Organic Geochemistry Unit, School of Chemistry, University of Bristol, Bristol, UK.4 Current address: Department of Biological Sciences, University of Idaho, Moscow, ID, USA.5 S. Asjes, K. Bakker, M. Bakker, J.L. Charlou, J.P. Donval, R. Haanstra, P. Henry, C. Huguen, B. Jelsma, S. de Lint, M. van der Maarel, S.

Muzet, G. Nobbe, H. Pelle, C. Pierre, W. Polman, L. de Senerpont, M. Sibuet.

J.P. Werne et al. / Chemical Geology 205 (2004) 367–390368

highly heterogeneous system. Geophysical results clearly indicate the spatial heterogeneity of mud volcano environments.

Results from pore water geochemistry and modeling efforts indicate that the rate of AOM is f6 mol m�2 year�1, which is

lower than at active seep sites associated with conditions of focused flow, but greater than diffusion-dominated sites.

Furthermore, under the non-focused flow conditions at Kazan mud volcano advective flow velocities are of the order of a few

centimeters per year and gas hydrate formation is predicted to occur at a sediment depth of about 2 m and below. The methane

flux through these sediments supports a large and diverse community of micro- and macrobiota, as demonstrated by carbon

isotopic measurements on bulk organic matter, authigenic carbonates, specific biomarker compounds, and macrofaunal tissues.

Because the AOM community appears to be able to completely oxidize methane at the rate it is seeping through the sediments,

ultimate sinks for methane in this environment are authigenic carbonates and biomass. Significant differences in organic

geochemical data between this site and those of other cold seep environments, even within the eastern Mediterranean mud

volcanoes, indicate that the microbiological communities carrying out AOM varies depending on specific conditions such as

methane flux and salinity.

D 2004 Elsevier B.V. All rights reserved.

Keywords: Anaerobic oxidation of methane; Cold seep fauna; Gas hydrate; Mud volcano; Biogeochemistry; Microbiology

1. Introduction

Methane cold seeps have become environments of

intensive study in recent years for several reasons.

Among these are: (1) the potential for methane release

from hydrates and the subsequent impact on climate

(Dickens et al., 1995), (2) slope destabilization associ-

ated with gas hydrate dissociation (Henriet and Mie-

nert, 1998), (3) energy sources for chemosynthetically

based life (Sassen et al., 1993; Sibuet and Olu, 1998),

and (4) as a potential energy resource (Hovland, 2000).

Furthermore, the anaerobic oxidation of methane

(AOM) has been identified in methane seep environ-

ments world-wide, and is believed to scavenge signif-

icant amounts of methane (Reeburgh et al., 1993)

which could otherwise be released to the atmosphere,

potentially contributing to global warming (e.g., Dick-

ens et al., 1995).

Mud volcanoes, which result from the extrusion of

fluid-rich mudflows (mud breccias), are often associ-

ated with methane seepage, and, given the appropriate

pressure and temperature conditions, with gas hydrates

(Brown, 1990; Milkov, 2000). Mud volcanoes have

been identified in the easternMediterranean (Cita et al.,

1981, 1989; Cronin et al., 1997), and active methane

seepage has been documented on these mud volcanoes

(Emeis et al., 1996; Limonov et al., 1996; Woodside et

al., 1998). In 1998 and 1999, the joint French–Dutch

MEDINAUT and the Dutch MEDINETH expeditions

were made to investigate eastern Mediterranean mud

volcanoes and their associated cold seeps. These mul-

tidisciplinary investigations combined visual observa-

tions from dives aboard the manned submersible R/V

Nautile (launched from the support vessel N/O Nadir)

and geophysical/acoustic surveys with shore-based

sedimentological, (bio)geochemical and (micro)bio-

logical investigations on seafloor sediment samples

taken from the Nautile and via box core from the R/V

Professor Logachev (MEDINAUT/MEDINETH Ship-

board Scientific Parties, 2000).

One of the major objectives of the MEDINAUT/

MEDINETH expeditions was an in depth biogeo-

chemical and (micro)biological investigation of

AOM. It is now generally accepted that a microbial

consortium consisting at least of methane-oxidizing

archaea and sulfate-reducing bacteria consumes sulfate

and methane in a 1:1 molar ratio during AOM,

producing sulfide and DIC according to the general-

ized net reaction

SO2�4 þ CH4 ! HS� þ HCO�

3 þ H2O ð1Þ

as proposed by Reeburgh (1976). While the consor-

tium hypothesis for net AOM has been proven through

a number of studies (Boetius et al., 2000; Hinrichs et

al., 1999; Orphan et al., 2001), the specific pathways

of methane-derived carbon incorporation into micro-

bial biomass remain debated (Valentine and Reeburgh,

2000; Sørensen et al., 2001). Furthermore, the specific

organisms remain unknown and uncultured, though

significant advances have been made in narrowing

down the organisms based in large part on phyloge-

J.P. Werne et al. / Chemical Geology 205 (2004) 367–390 369

netic studies (Hinrichs et al., 1999; Boetius et al.,

2000; Orphan et al., 2001; Aloisi et al., 2002). This

paper is a synthesis of the geophysical, (bio)geochem-

ical and (micro)biological studies carried out by mem-

Fig. 1. (A) Eastern Mediterranean Sea geodynamic map, with location of th

the Mediterranean ridge (triangles)—(1) Prometheus, (2) Pan di Zucchero

bathymetry map of the Anaximander Mountains and location of mud volc

bers of the MEDINAUTandMEDINETH expeditions.

The focus is on studies of methane cold-seeps on

Kazan mud volcano, located in the Anaximander

Mountains area (Fig. 1).

e main features cited in the text and of the mud diapiric fields within

, (3) Prometheus 2, (4) Olimpi, (5) United Nation. (B) Multibeam

anoes (stars).

J.P. Werne et al. / Chemical Geology 205 (2004) 367–390370

2. Geologic setting and regional tectonics:

formation of the cold seep environment

Eastern Mediterranean tectonics record the initial

stages of collision between Africa and Europe, from

incipient stages south of Crete to more advanced stages

south of Cyprus (and eastwards into the Zagros area

where continental collision between the Arabian prom-

ontory and Europe is now occurring). The complex

deformation of the Eastern Mediterranean results from

the interactions of several plates and micro-plates

(McKenzie, 1972; Reilinger et al., 1997; McClusky

et al., 2000) and develops into two cup-shaped arc

systems, the Hellenic and Cyprus Arcs (Fig. 1). These

two arc systems are characterized by different tectonic

regimes, with the Hellenic arc characterized by more

active subduction (Papazachos and Comninakis, 1978;

Spakman et al., 1988; Chaumillon and Mascle, 1997)

and the Cyprus Arc characterized by a quieter tectonic

and seismic activity (Rotstein and Kafka, 1982; Ben-

Avraham et al., 1988) where subduction may be halted

(Robertson, 1998). The submarine Anaximander

Mountains region (along with the Florence Rise and

the Rhodes Basin) is located at the connection between

these two arc systems, and as a result has been the

subject of several recent studies (Woodside et al., 2000,

2002; Zitter et al., 2003; Ten Veen et al., in press).

A series of mud volcano fields exists within the

Mediterranean Ridge accretionary prism (Cita et al.,

1981, 1989, 1996; Cronin et al., 1997; Mascle et al.,

1999). Two mud volcanoes from the Olimpi field

(Napoli mud volcano and Milano mud volcano) were

investigated in detail during the Ocean Drilling Pro-

gram Leg 160 (Robertson et al., 1998). The Anax-

imander Mountains mud volcano field occurs in a

different context of the eastern Mediterranean colli-

sion zone. This mud volcano field was discovered

from multibeam mapping during the ANAXIPROBE

cruise in 1995 based on high backscattering patches

associated with circular relief (Woodside et al., 1997,

1998), and has received much less vigorous study.

The Anaximander Mountains are a complex of three

seamounts (Fig. 1), rising more than 1000 m above the

seafloor, that rifted southwards from Turkey in late

Miocene–early Pliocene times (Woodside et al., 1997).

The post-Miocene formation of this area is demonstrat-

ed by the absence of evaporites (that were deposited

during the Messinian salinity crisis in most parts of the

Mediterranean Sea Ryan et al., 1973) beneath the

Anaximander Mountains. The gravity signature

(Woodside, 1977; Woodside et al., 1997), lithological

facies (Woodside et al., 1997), and surface deformation

(Zitter et al., 2003) vary between the eastern and

western Anaximander Mountains regions, clearly indi-

cating differences in the geology of the two regions.

The western mountains belong to the Eastern Hellenic

fore-arc deformation zone (Woodside et al., 2000; Ten

Veen et al., in press), whereas the eastern mountains

(the Anaxagoras seamount) form the western limb of

the Cyprus Arc and are affected by a large NW–SE

transpressional wrenching system (Woodside et al.,

2002; Zitter et al., 2003; Ten Veen et al., in press). This

area is intensely crosscut by major N150j and N070jfaulting, inferred to be active shearing based on both

the seismic signature (pop-up flower structures) and the

bathymetric expression.

Most of the mud volcanoes discovered in the Anax-

imander Mountains occur within the Anaxagoras sea-

mount (Fig. 1), associated with both the strike-slip

faults and secondary, probably extensional, faults (Zit-

ter, unpublished results). They are generally identified

as conical topographic features on the order of 50 to

100 m high and 1 km wide (Woodside et al., 1998).

Nonetheless, the mud volcanoes and associated mud-

flows show an important variability in morphology,

size and acoustic characteristics. The shapes vary from

conical for Tuzlukush mud volcano, a quite perfectly

circular dome for Kula mud volcano, an irregular dome

for Kazan mud volcano, to the several km-wide ‘‘mud

pie’’ morphology of the exceptionally large Amster-

dam mud volcano.

3. Kazan mud volcano

Kazan mud volcano is a small dome, about 50 m

high and 1000 m wide, with an elliptical summit at

about 1700 m water depth (Fig. 2). It is located in the

southern part of the Anaxagoras seamount (E30j33.5V,N35j26V) at the interplay between the N150j and

N070j faults. From the sidescan sonar record, the

volcano shows high to very high backscatter (Fig. 2).

The high backscatter signature is believed to be caused

by both the seafloor roughness and the volumetric

heterogeneity of the mudflows produced by clasts

within the mud breccia (Volgin and Woodside,

Fig. 2. Sidescan sonar image and subbottom profile of Kazan Mud Volcano. Bathymetric map of Kazan mud volcano with the interpretation of

sidescan sonar, the position of dive MN10 (in black) and MN12 (in grey), as well as the positions of samples (stars), as follows: (1) MN12BT3,

(2) MNLBC18, (3) MNLBC19.

J.P. Werne et al. / Chemical Geology 205 (2004) 367–390 371

1996). High backscatter is then an indication of the

presence of a mudflow, with mud breccia either

cropping out or near the seafloor (i.e. recent mud-

flows). The backscatter level decreases with increasing

thickness of the hemipelagic sediments covering the

mudflows (i.e. old mudflows). Several other parame-

ters, such as carbonate crusts on the seafloor or the

presence of near bottom gas hydrates can influence the

backscatter signature. The tongue-shaped mudflows of

Kazan mud volcano extend radially about 1 km away

from the summit, with recent mudflows located on the

northern and eastern sides of the volcano. In contrast to

most of the mud volcanoes, part of the summit is

characterized by a low backscatter area, which is

probably less active (Fig. 2). This observation was

confirmed by dives of the submersible Nautile (see

Fig. 2 for dive tracks) from which it was possible to

distinguish two active areas of seepage. The first one is

a plateau on the northern part of the summit associated

with a recent mudflow (Fig. 3a); the second is on the

southeastern flank of the volcano and corresponds to a

patch of enhanced backscatter in the sonar imagery

(Fig. 2). No fresh mud breccia was observed exposed

on the seafloor, suggesting that the strong backscatter

came from carbonate crusts and fields of shells ob-

served during the dives (Fig. 3b). In the area surround-

ing Kazan mud volcano, faults (Fig. 3c) trend in many

directions (Fig. 2), probably due to the position of the

volcano at the intersection of major faulting between

the Anaximenes and the Anaxagoras seamounts (Zit-

ter, unpublished results). Active degassing associated

with faulting around Kazan mud volcano is indicated

Fig. 3. Photographs taken during dives of the submersible Nautile to

Kazan mud volcano. (a) Mud breccia showing size variability of

clasts in extrusive flows. (b) Example of abundant authigenic

carbonate crusts and shell fields. (c) Fault scarp demonstrating

recent tectonism on Kazan mud volcano.

J.P. Werne et al. / Chemical Geology 205 (2004) 367–390372

by typical wipeouts in acoustic facies on the subbot-

tom profiler along a scarp about 1 km west of the

volcano (Fig. 2).

4. Methods

4.1. Geophysics

The sidescan sonar and subbottom profiling data

were acquired with a MAK-1 system during the 6th

Training Through Research (TTR6) expedition. The

vehicle (or fish) operating in a Long Range mode at 30-

kHz frequency, is towed at 150 m above the seafloor at

a speed of 1.5 to 2 knots and is able to insonify a swath

2000mwide (1000m on each side). TheMAK-1 fish is

equipped with a sub-bottom profiler (SBP) operating at

a 6-kHz frequency.

4.2. Sampling

Samples were taken from Kazan mud volcano by a

variety of methods. Carbonate crust samples were

taken either by dives of the submersible Nautile during

the MEDINAUT cruise of the R/V Nadir in Novem-

ber–December 1998, or by box cores taken during the

MEDINETH cruise of the R/V Professor Logachev.

Crust sample MNT12BT3 (35j26.007VN, 30j33.53VE,1706 m water depth (Fig. 2) taken from Kazan is the

focus of both organic geochemical and carbonate

geochemical/mineralogical studies, and was sampled

by submersible dives of the Nautile. Two box cores

taken from the summit of Kazan mud volcano during

the 1999 MEDINETH cruise of the R/V Professor

Logachev (MNLBC18 and MNLBC19, 35j25.936VN,30j33.704VE, water depth 1673 m and 35j25.950VN,30j33.679VE, water depth 1673m, respectively, Fig. 2),

were dedicated to geochemical and microbiological

analysis. Pore water geochemistry and molecular iso-

topic geochemistry were studied in core MNLBC19,

and microbiological analyses were performed on both

MNLBC18 and MNLBC19.

It should be noted that the mud breccia sediments

investigated in this study were taken by box core

rather than sampled directly by submersible. There-

fore, the highly precise targeting of active cold seeps

was not possible, and the likelihood of actually

sampling an active seep in this manner is low, given

an estimated radius around the coordinates of f80 m

as a result of a 5% scope (assumed based on known

currents in the Rhodes Gyre area) in the cable lower-

ing the box core, and the extremely variable terrain on

and around Kazan mud volcano (see discussion be-

J.P. Werne et al. / Chemical Geo

low). These samples, therefore, likely represent a mid-

point in the continuum between highly active seep

sites, such as those examined on Hydrate Ridge

(Boetius et al., 2000; Elvert et al., 2001) and those

in areas of ‘‘normal’’ sediment methane levels, such as

the Kattegat (Bian et al., 2001).

4.3. Clay Mineralogy

The matrix clay mineral composition of the mud

breccia from several mud volcanoes, and of a hemi-

pelagic sample as reference, was defined on the basis

of X-ray diffraction (XRD). XRD analyses of the <2

Am-size fraction were carried out at different percen-

tages of relative humidity (0–50–100%) in order to

identify expanding clay minerals in the complex

mixture. Carbonate was removed and an internal

standard was added to prepare samples for analysis.

Semi-quantitative analyses were performed using

the integrated weighted-peak area and the clay

mineral amount was estimated using the samples

measured at 50% humidity. Results from the matrix

of MNLBC19, sampled at 21 cm deep, are presented

here.

4.4. Macrobiology

Preparation for transmission electronic microscopy

(TEM), included fixing small pieces of bivalve gills

(Myrtea sp. andVesicomya sp.) in 3% glutaraldehyde in

0.4 M NaCl, buffering with 0.1 M cacodylate (pH 7.4)

and post-fixing in 1% osmium tetroxide for 1 h in the

same buffer. The specimens were dehydrated through a

graded ethanol series and embedded in Araldite. Semi-

thin sections were stained with toluidine blue. Ultra-

thin sections were contrasted with uranyl acetate and

lead citrate, and observed under a Hitachi H600 trans-

mission electron microscope.

Stable carbon and nitrogen isotope analyses of

the tissues were made on samples frozen and stored

in liquid nitrogen then freeze dried and acidified in

the laboratory. The acidified samples were com-

busted to CO2 and N2 using standard methods

(Bouton, 1991) before being passed through a mass

spectrometer (Finnigan Delta E). Carbon isotopes are

reported relative to PeeDee Belemnite (PDB) and

nitrogen isotopes relative to atmospheric molecular

nitrogen.

4.5. Geochemistry

Pore water analyses are described in detail in Haese

et al. (2003). Carbonate mineralogy and geochemistry

analyses are described in Aloisi et al. (2000). Lipid

concentration and carbon isotopic analyses are de-

scribed in Werne et al. (2002), Pancost et al. (2000,

2001a,b), and Hopmans et al. (2000).

4.6. Microbiology

Three depth intervals spanning the zone of AOM

were investigated (0–6, 6–22, >22 cm) because of the

larger sample size needed relative to geochemical

analyses. Total counts of bacteria as well as aggregates

as described by Boetius et al. (2000) were determined

by epifluorescence microscopy using DAPI (4V,6V-dia-midino-2-phenylindole hydrochloride) and acridin or-

ange dyes. Most probable numbers (MPN) were

determined according to Heijs et al. (1999) using

appropriate culture media for the functional groups of

organisms determined according to the German Col-

lection of Microorganisms and Cell Cultures (www.

dsmz.de). DNA was isolated from sediment samples

following Aloisi et al. (2002), slightly modified with

respect to extraction times and mechanical lysis. DNA

isolates were cleaned by gel-filtration (WIZARD DNA

clean up kit, Promega Benelux, Leiden, The Nether-

lands) and concentrated to a final volume of 120 Al. Thetotal amount of DNAwas measured spectrophotomet-

rically. Archaeal 16S ribosomal RNA genes were

amplified from the total chromosomal DNA pool by

using a group specific archaeal primer set as described

by DeLong (1992). For the analysis of the microbial

communities, terminal-Restriction Fragment Length

Polymorphism (t-RFLP) was applied using FAM and

HEX-labeled primers in Polymerase Chain Reaction

(PCR). After gel purification, 100 ng of PCR product

was cut for 3 h at 37 jC using 2 U of HHA1 digestive

enzyme, and analyzed on a ABI 310 automated

sequencer.

logy 205 (2004) 367–390 373

5. Results and discussion

5.1. Sediment cores

Box cores discussed herein span the upperf30 cm

of sediment. Sediments consist of grey mud breccia

J.P. Werne et al. / Chemical Geology 205 (2004) 367–390374

with millimetric to centimetric mudstone clasts. The

upper few centimeters of sediment are oxidized. In core

MNLBC19, from 22 to 34 cm there is evidence that the

sediment was gas saturated (this interval corresponds to

a temperature of 11.9 jC measured on board).

5.2. Mud matrix clay mineralogy

The clay mineral composition of the mud matrix of

Kazan mud volcano is largely dominated by smectite

(85%), with 10% kaolinite, 3% palygorskite and 2%

illite. In comparison with the composition of Pliocene

sediments in the Mediterranean Sea (Melieres et al.,

1978) and with the hemipelagic core recovered in the

region of the Florence Rise, the matrix of the mud

breccia is considerably enriched in smectite. Such

smectite-rich formation is typically found in the Med-

iterranean Sea in Messinian deposits (Chamley et al.,

1978), which suggest a possible deeper, Messinian

source for these mud breccia matrix sediments. On

the other hand, the high abundance of hydrated clays

seems to indicate the absence of deep burial and

subsequent diagenetic transformation, supporting a

shallow source for the matrix. The smectite in the

Anaximander area could also be derived from the wea-

thering of ophiolitic rocks found on Turkey, Cyprus,

and probably partly beneath the Anaximander Moun-

tains. As discussed below, the predominance of smec-

tite clays has implications for the interpretations of pore

water geochemistry and the occurrence of methane

hydrates.

5.3. Pore water geochemistry

Profiles of measured pore water geochemical con-

stituents are shown in Fig. 4. Since methane is highly

volatile, methane gas bubbles form and may migrate

laterally and upwards during sample retrieval, leading

to highly variable measured methane concentrations,

which may significantly underestimate the in situ con-

centrations (Paull et al., 2000). In order to overcome

this artifact, the in situ methane concentrations were

reconstructed in a recent study on methane dynamics

(Haese et al., 2003) by using the unambiguous sulfate

profile and the advective flow velocity of 4 cm year�1

estimated for site MNLBC19 using best-fit simulations

of measured depth profiles of conservative solutes (Na,

B) (Haese et al., 2003). Additional assumptions re-

quired for reconstruction of a reasonable methane

concentration profile include near-complete methane

oxidation by AOM, an insignificant rate of methano-

genesis below the depth of AOM, and an insignificant

rate of sulfate reduction related to organic matter (OM)

decomposition. These assumptions are justified be-

cause at this site, AOM is restricted to a narrow depth

interval (14–18 cm below sediment surface), as can be

seen from the coinciding discrete depth of the sulfide

maximum, the minimum in the d13C of DIC, and the

depth of sulfate depletion (Fig. 4). Total organic

carbon and biomarker concentrations and carbon iso-

topic composition (Fig. 4 and Table 2) show the

imprint by AOM at approximately the same depth as

the pore water indicators, and the lack of AOM-related

biomarkers in the shallowest sediments justify the

assumption of (near) complete methane oxidation

within a discrete depth interval. Sulfate reduction

related to the mineralization of OM and methano-

genesis should be negligible due to the low amount

(0.2–0.3 wt.%, Fig. 4D) and refractory nature of the

OM present (most of the OM in these mud breccia

sediments is associated with the ascending mud ma-

trix, not with pelagic sedimentation, and is therefore

already largely degraded, Werne et al., 2002). The

most convincing argument for a negligible contribu-

tion of sulfate reduction related to the mineralization of

OM comes from a sulfate/DIC* (=DIC+Ca) molar

ratio of 1 in the depth interval where both variables

show a concentration gradient (Haese et al., 2003).

This clearly reflects the stoichiometery of AOM;

sulfate reduction related to OM mineralization would

result in a ratio of 0.5.

If our assumptions are valid, according to the net-

stoichiometery of AOM proposed by Reeburgh

(1976) (Eq. (1)), the sulfate flux must be exactly

balanced by the methane flux (Borowski et al.,

1996). Once the methane flux is known, the methane

depth distribution can be reconstructed based on our

assumptions with an analytical solution to the well-

established one-dimensional, steady-state transport

equation for solutes accounting for diffusion and

advection (e.g. Berner, 1980). The modeled methane

depth distribution is shown in Fig. 5.

According to the model results by Haese et al.

(2003), a steep increase in the methane concentration

is predicted from the depth of AOM (14–18 cm) down

to a depth of about 1 m before the concentration

Fig. 4. Depth profiles of pore water and sedimentary geochemical constituents. (A) Sulfate (solid) and sulfide (open) concentration. (B)

Dissolved inorganic carbon, DIC, concentration (solid) and carbon isotopic composition (open). (C) Calcium concentration (solid) and methane

concentration (open). (D) Bulk organic carbon weight percent (solid) and carbon isotopic composition (open). (E) Bulk carbonate concentration

(solid) and isotopic composition (open). In A–C, important depth intervals are marked by different shadings and labeled respectively. Sulfate,

DIC, d13C-DIC, Corg, and d13C-Corg profiles were redrawn from Haese et al. (2003), and methane was redrawn from Werne et al. (2002). The

zone of anaerobic oxidation of methane, AOM, can be identified by the concurrent depth of sulfate consumption and sulfide and DIC

production. Carbonate precipitation is predominant where the calcium gradient is steepest.

J.P. Werne et al. / Chemical Geology 205 (2004) 367–390 375

gradient flattens and an asymptotic maximum methane

concentration is reached at approximately 2 m (Fig. 5).

The predicted asymptotic maximum methane concen-

tration is approximately 130 mmol l�1, which matches

the methane saturation concentration with respect to

gas hydrate under the local pressure and temperature

conditions (Zatsepina and Buffet, 1997). Thus, at site

MNLBC19 gas hydrate formation is expected at a

depth of 2 m and below. Using the approach by

Rempel and Buffet (1997), it was further calculated

that 3.8% of the initial pore space is filled with gas

hydrate in 10,000 years (Haese et al., 2003).

From the simulations of the sulfate and the cor-

responding methane concentration profiles, a depth-

integrated rate of AOM of f6.0 mol m�2 year�1 was

derived (Haese et al., 2003). When compared to cold

Fig. 5. Model results showing: (A) the best-fit simulation to the measured sulfate concentration profile using the analytical solution of the one-

dimensional, steady state transport equation accounting for diffusion and advection, (B) the modeled methane concentration profile, (C) the

derived rate of AOM, and (D) the modeled methane concentration profile down to 5-m sediment depth (redrawn from Haese et al., 2003).

Measured values are shown for comparison in open diamonds. Note the different depth scales. The grey shaded interval represents the zone of

bioirrigation. Once the sulfate flux is calculated, the methane profile can be reconstructed with the assumptions discussed in the text. An

asymptotic maximum methane concentration is reached below approximately 2 m and is very close the methane saturation concentration. This

suggests gas hydrate formation below a depth of 2 m. The rate of AOM is calculated from the combined sulfate and methane models. Note in B

the difference between measured and modeled methane concentrations, suggesting migration and outgassing of methane during core recovery.

J.P. Werne et al. / Chemical Geology 205 (2004) 367–390376

seep sites at Hydrate Ridge (Boetius et al., 2000) and

the Aleutian Trench (Wallmann et al., 1997), the rate of

AOM at site MNLBC19 is comparably low. However,

AOM rates of all diffusion-dominated sites, i.e. at Saa-

nich Inlet, Skan Bay, and Skagerrak, are significantly

lower than at site MNLBC19 (Haese et al., 2003).

5.4. Bulk carbon geochemistry

Concentrations and carbon isotopic compositions

of bulk organic carbon and carbonate are shown in

Fig. 4. The negative isotopic excursion in bulk organ-

ic carbon is a result of methane derived biomass from

AOM (Fig. 4). Note however, that the negative

excursion in the d13C is presumably only noticeable

because the indigenous bulk organic carbon is very

low in concentration. Based on a stable isotope mass

balance, it was calculated that the biomass-carbon

derived from AOM only makes up 0.05 wt.% carbon

of the dry sediment (Haese et al., 2003).

AOM leads to the formation of 13C-depleted DIC,

which diffuses upwards along a concentration gradient

and is consumed by carbonate precipitation. The

required calcium is replenished by the downward flux

from the bioirrigated zone (Fig. 4). Despite high rates

of carbonate precipitation as reflected by the steep

calcium concentration gradient, neither enhanced bulk

carbonate concentration nor a discrete minimum in the

d13C of bulk carbonate can be observed (Fig. 5). This

finding opposes observations made on the bulk organ-

ic carbon record and is related to relatively high

background (associated with ascending mud matrix)

carbonate concentrations. This means that the identi-

fication of paleo-seep environments based on the bulk

carbon geochemical record may only be possible in

sediments poor in background and pelagic bulk inor-

ganic or organic carbon. The effect of masking the

AOM imprint on the carbon records may, however, be

compensated by high rates of AOM over long time

periods (Raiswell, 1988). As discussed below, discrete

nodules of authigenic carbonate and individual bio-

markers are the most appropriate indicators for tracing

carbon transformations based on the geologic record.

5.5. Authigenic carbonates

Anaerobic oxidation of methane is often associated

with the precipitation of authigenic carbonates (Ritger

et al., 1987; Stakes et al., 1999; Pierre et al., 2000;

Aloisi et al., 2000, 2003), which can be considered a

terminal sink for methane-derived carbon in marine

sediments. AOM in eastern Mediterranean mud vol-

canoes has resulted in the widespread precipitation of

authigenic carbonates and carbonate crusts (MEDI-

Fig. 6. (a) Photo taken by submersible Nautile of authigenic

carbonate crust MN12BT3 in situ, prior to sampling. (b) Photo of

sample MN12BT3 in the lab after sampling.

J.P. Werne et al. / Chemical Geology 205 (2004) 367–390 377

NAUT/MEDINETH Shipboard Scientific Parties,

2000). The mineralogy and stable carbon and oxygen

isotope compositions of these carbonates have been

discussed in detail by Aloisi et al. (2000), so only data

from carbonates occurring on Kazan mud volcano will

be summarized herein.

Mud volcano-associated authigenic carbonates in

the eastern Mediterranean can be divided into several

categories based on mineralogy and morphology,

including crusts, nodules, and carbonate-cemented

sediments (both lithified pelagites and lithified mud

breccia sediments). Crusts range in thickness from

millimeter scale up to decimeter scale, with variable

morphology ranging from mounds to slabs (Figs. 3b

and 6) (Aloisi et al., 2000). The authigenic carbonate

crusts are composed of dolomite, aragonite, and

calcite in varying proportions, ranging from 70%

aragonite (Type ‘‘A’’ crusts of Aloisi et al., 2000) to

90% calcite (Type ‘‘D’’), and carbonate nodules are

up to 90% dolomite (Aloisi et al., 2000). Both low-

Mg and high-Mg (up to 12 mol% Mg) calcites have

been identified (Aloisi et al., 2000). Many of the

crusts are associated to some extent with macrofauna

such as pogonoforan tube worms and bivalves (Figs.

3b and 6; see discussion of macrofauna below).

The carbon isotopic composition of authigenic

carbonates (d13Ccarb) on Kazan mud volcano ranges

fromf0xto nearly �50x(PDB, Fig. 7; data from

Aloisi et al., 2000). Typically, carbonate crusts are

much more 13C depleted than poorly lithified mud

breccia, which is in turn generally more 13C depleted

than unlithified pelagites, due to the formation of fine-

grained authigenic carbonate minerals (Aloisi et al.,

2000). Controls on cold-seep carbonate d13C are

complex mainly due to uncertainties regarding mixing

ratios of different carbon sources having distinct iso-

topic compositions (bottom water DIC, OM, methane-

derived DIC, DIC in deep fluids) and the extent to

which carbon isotopes fractionate during AOM (Aloisi

et al., 2000). The d13Ccarb at Kazan mud volcano

suggests that methane is the major source of carbonate

carbon for crust formation and that this source is

somewhat diluted, probably by mixing with relatively13C-rich bottom water or rising fluids. OM decompo-

sition could play a minor role in contributing to the

DIC pool (Luff and Wallmann, 2003), however, given

the modeling results of Haese et al. (2003), such an

effect is expected to be minimal.

The oxygen isotopic composition of authigenic

carbonates (d18Ocarb) ranges from values of �1xto

+1x(PDB) in the case of nodules and unlithified

sediments, and from +2xto approximately +4.5xin lithified sediments and crusts (such as sample

MN12BT3) (Fig. 7). The enriched d18Ocarb signal is

interpreted by Aloisi et al. (2000) to indicate carbonate

precipitation from 18O-enriched diagenetic fluids, rath-

er than as a temperature effect. Possible processes

producing 18O-rich fluids include smectite dehydration

and gas hydrate destabilization. Pore water chemistry

and clay mineralogy suggest that the former process

might dominate here. In other areas of brine seepage in

the eastern Mediterranean (such as Nadir Brine Lake

or Napoli mud volcano in the Olimpi field), extreme18O-enrichment of the carbonate crusts is probably due

to precipitation from 18O-enriched fluids sourced from

relic Messinian brines (Aloisi et al., 2000). However,

Fig. 7. Cross plot of d13C vs. d18O of carbonates and unlithified sediments from Kazan mud volcano. 13C depletion and 18O enrichment of

carbonates relative to unlithified sediments and nodules is believed to indicate authigenic production as a byproduct of AOM (or possibly

aerobic oxidation of methane).

J.P. Werne et al. / Chemical Geology 205 (2004) 367–390378

this hypothesis cannot be applied to the 18O-enriched

fluids of Kazan mud volcano, since no brines are found

in the Anaximander Mountains due to the absence of

the Messinian evaporite formations.

5.6. Organic geochemistry

Molecular isotopic indicators of AOM, such as13C depleted compounds typically associated with

methanogens and sulfate reducers, have been identi-

fied in samples from various modern cold-seep

environments (Hinrichs et al., 1999, 2000; Elvert et

al., 2000, 2001; Thiel et al., 2001), including eastern

Mediterranean mud volcanoes (Pancost et al., 2000,

2001a; Werne et al., 2002; Aloisi et al., 2002), as

well as in ancient methane seeps (Thiel et al., 1999)

and non-methane seep environments characterized by

AOM such as the Kattegatt Strait (Bian et al., 2001)

and the water column of the Black Sea (Schouten et

al., 2001).

5.6.1. Organic geochemistry of authigenic carbonates

Authigenic crust sample MNT12BT3 has been

characterized as a lithified pelagite (Aloisi et al.,

2000). The carbonate-carbon is 13C depleted (to

�46.5xVPDB), clearly indicating a methane-de-

rived source (Aloisi et al., 2000). The suite of archaeal

biomarkers identified in this crust includes archaeol (in

highest abundance, Table 1), hydroxyarchaeol, croce-

tane, and pentamethylicosane (PMI, see Appendix A

for structures). Of these compounds, those that were

present in sufficient quantities for carbon-isotopic

analysis are extremely 13C depleted, ranging from

�69x(in the case of PMI) to �112x(archaeol,

Table 1), clearly indicating a methane-oxidation source

for these compounds. Alkyl diethers derived from

sulfate reducing bacteria (SRB) (series I and II of

Pancost et al., 2001b) were also identified in this crust.

Hopanoid compounds, such as bishomohopanol, were

also identified, but the low concentrations precluded

isotopic measurement (Bouloubassi et al., unpublished

Fig. 8. Photo taken by submersible Nautile showing white patches

that were observed on Kazan mud volcano, believed to be

filamentous microbial ‘‘mats’’, perhaps Beggiatoa.

Table 1

Biomarkers and carbon isotopic data from crust MNT12BT3

Compound Relative

abundance

d13C(xVPDB)

Archaeol ++ �112

Hydroxyarchaeol + n.d.

Crocetane + n.d.

PMI:0 + �69

Series I alkyl diethers + to ++ �94 to �99

Series II alkyl diethers + f�70

‘‘Background’’ biomarkers + to +++ �23 to �32

Series I and II alkyl diethers refer to Pancost et al. (2001b).

‘‘Background’’ biomarkers (e.g., hopanoids, fatty acids, steroids,

and n-alkanes derived from bacteria and photoautotrophs) are

probably incorporated into authigenic carbonates from the ascend-

ing mud matrix that was lithified. See Appendix A for compound

structures. ‘‘n.d.’’ indicates no data.

J.P. Werne et al. / Chemical Geology 205 (2004) 367–390 379

results). In other studies, this compound has been

attributed to bacteria oxidizing sulfide (Pancost and

Sinninghe Damste, 2003), which seems reasonable in

this environment as well given the visual observation

of white filamentous Beggiatoa-like bacterial mats by

submersible (Fig. 8; and MEDINAUT/MEDINETH

Shipboard Scientific Parties, 2000) and the identifica-

tion of colorless sulfur bacteria in samples from Kazan

mud volcano sediment (Heijs, unpublished data, see

discussion on microbiology below). Other biomarker

compounds identified in comparable or greater abun-

dance than the AOM consortium biomarkers include

terrestrially derived n-alkanes, sterols, and fatty acids

which are believed to be associated with OM in the

ascending mud breccia based on their typical carbon

isotope compositions (f�24xto �32x, Table 1).

5.7. Organic geochemistry of mud breccia sediments

Themolecular isotopic signature in coreMNLBC19

taken from the mud breccia of Kazan mud volcano

shows similarities to the crust, but also shows some

striking differences. For example, 13C depleted (to

�123.8x) archaeal compounds such as archaeol, hy-

droxyarchaeol, and PMI have been identified in the

sediments (Table 2, some data fromWerne et al., 2002).

We have also identified several non-isoprenoid dialkyl

glycerol diethers attributed to SRB, but only series II

compounds of Pancost et al. (2001b) were identified in

the sediments (compared to dominantly series I in the

crust).

Hopanoid compounds such as diploptene and dip-

lopterol were also identified with ‘‘intermediate’’

degrees of 13C depletion (f�55xto�70x), which

were attributed to aerobic methane-oxidizing bacteria

(Werne et al., 2002) based in part on decreasing down-

core concentration trends. h,h-bishomohopanol was

also identified in surficial sediments (Werne and Sin-

ninghe Damste, unpublished results), as in the crust.

This compound has been attributed to sulfide-oxidizing

bacteria in other studies (Pancost and Sinninghe Dam-

ste, 2003).

A series of glycerol dialkyl glycerol tetraethers

(GDGTs) was also identified in the sediments of Kazan

mud volcano (Werne and Sinninghe Damste, unpub-

lished results). Based on molecular isotopic studies in

other mud volcano cold seep sediments (Pancost et al.,

2001a,b) and in the water column of the Black Sea

(Schouten et al., 2001), it has been proposed that some

of these GDGT compounds are derived at least in part

from anaerobic methane-oxidizing archaea. In Kazan

mud breccia sediments, however, carbon isotope anal-

ysis of C40 isoprenoids released from these GDGTs by

chemical degradation show 13C enriched values, in the

range of �22x, indeed the most 13C enriched com-

pounds in the extractable OM (Werne and Sinninghe

Damste, unpublished results). Such values clearly

indicate a non-methane-related source for these com-

pounds, likely derived primarily from Crenarchaeota

ubiquitously occurring in the pelagic zone of the water

column (Schouten et al., 2000), and are therefore more

Table 2

Concentration (mg/g sediment) and carbon isotopic composition (xVPDB) of selected biomarkers

Depth (cm) Concentration, mg/g sed d13C, xVPDB

1 2.5 11 17 21 28 1 2.5 11 17 21 28

Pristane 0.23 0.36 0.38 0.48 0.36 0.42 �29.1 �28.8 �27.8 �27.9 �28.3 �31.0

n-C17 0.05 0.06 0.06 0.10 0.08 0.07 – �29.7 – �28.5 �27.6 –

n-C19 0.12 0.11 0.16 0.22 0.13 0.13 �29.5 �28.5 �28.4 – �29.5 �29.0

n-C29 0.31 0.23 0.24 0.29 0.17 0.20 �29.7 �29.8 �30.0 �29.4 �30.3 �30.5

n-C31 0.30 0.27 0.30 0.30 0.16 0.21 �31.3 �30.7 �30.9 �30.5 �31.0 �32.7

Hydroxyarchaeol – 0.19 0.20 0.93 0.61 0.29 – �103 �107 �111 �111 �101

Archaeol – 0.07 0.07 0.32 0.16 0.10 – �94.0 �94.9 �102 �101 �80.5

PMI:3c – – 0.03 0.04 0.02 – – – �86.7 �102 �123 �53.4

Diether 1 – – 0.01 0.06 0.06 0.03 – – – �90.9 �88.5 �70.0

Diether 2 – – 0.02 0.08 0.12 0.10 – – – �90.4 �96.7 �82.0

Diether 3 – – 0.04 0.07 0.10 0.03 – – �85.3 �90.8 �73.3 �61.5

Diether 4 – – 0.01 0.06 0.06 0.05 – – – �89.5 �94.5 �73.2

Diplopterol 0.20 0.14 – – – – �55.5 �62.4 – – – –

Diploptene 0.08 0.06 0.02 0.01 – – �54.1 �59.7 – – – –

Bishomohopanol 0.22 0.16 – – – – �45.7 �52.8 – – – –

GDGT-1302 0.82 0.45 0.28 0.12 0.20 0.18 Biphytanes released from GDGTs �21.5xto �35.3x.

GDGT-1300 0.17 0.09 0.06 0.02 0.05 0.05

GDGT-1298 0.17 0.17 0.13 0.08 0.14 0.10

GDGT-1296 0.06 0.07 0.08 0.04 0.07 0.05

GDGT-1294 0.00 0.00 0.06 0.04 0.06 0.04

Pristane and n-alkanes are matrix-associated organic matter; archaeol, hydroxyarchaeol, and PMI:3c (a tri-unsaturated pentamethylicosane) are

derived from anaerobic methane-oxidizing archaea; diethers are derived from syntrophic sulfate reducing bacteria (SRB); hopanoids are from

aerobic bacteria, and glycerol diether glycerol tetraethers (GDGTs) are mostly matrix associated, but with some contribution from anaerobic

oxidation of methane (AOM).

J.P. Werne et al. / Chemical Geology 205 (2004) 367–390380

likely to be associated with the ascending mud matrix

than with in situ microbial processes.

Numerous indicators of the ‘‘background’’ OM (i.e.,

that OM that is associated with the ascending mud

matrix) such as n-alkanes (d13C values of f�31x)

were identified at relatively high abundance in the mud

breccia sediments (Werne et al., 2002). The higher

proportion of non-AOM related OM is to be expected

in mud breccia sediments or lithified pelagite carbo-

nates compared to more active seep vents or pure

carbonate crusts, simply due to dilution.

5.8. Microbiology

Preliminary results of terminal restriction fragment

length polymorphism (t-RFLP) analyses of 16S rDNA

genes indicate that the microbial communities, though

similar, do display definite variability with depth based

on the presence or absence of specific phylotypes in a

given community (Fig. 9). Further experiments are

Fig. 9. t-RFLP chromatograms of bacterial and archaeal communities in se

in microbial community structure at different sediment depths.

currently underway to define these differences more

rigorously by means of the eubacterial and archaeal

16S rDNA genes.

Some further information is available, however,

through total bacterial counts as well as counts of the

aggregate clusters composed of methane-oxidizing

Archaea and sulfate-reducing bacteria (cf., Boetius

et al., 2000). Epifluorescence microscopy utilizing

both the DAPI and Acridin Orange methods con-

firmed the existence of structured consortia, probably

consisting of anaerobic methane-oxidizing archaea

and SRB (Boetius, personal communication) (Fig.

10). Aggregate concentrations in the mud breccia

sediments of Kazan mud volcano range from

1.79�107 to 4.2�108 cells/cm3, however, the counts

obtained by two methods differed by an order of

magnitude (Table 3). It is possible that this simply

reflects a difference in the methods (e.g., DAPI

reflects more ‘‘viable’’ cell counts, and Acridin Or-

ange more ‘‘total’’ counts). It is also possible that

diments from Kazan mud volcano, illustrating qualitative differences

J.P. Werne et al. / Chemical Geology 205 (2004) 367–390 381

Fig. 10. Archaea/SRB aggregates identified using fluorescence in situ hybridization (FISH) and stained with DAPI. Archaea are shown here in

green and SRB are shown in red.

Table 4

Most probable number (MPN) counts

Dilution Water 0–6 cm 6–22 cm >22 cm

WI WII WIII SI SII SIII SI SII SIII SI SII SIII

Sulfate-reducing bacteria

100 � � � / / / / / / / / /

1000 � � � � � � � � � � � �10,000 � � � � � � � � � � � �100,000 � � � � � � � � � � � �

Colorless sulfur bacteria

100 +++ +++ +++ / / / / / / / / /

1000 � � +++ � � � � +++ � � � �10,000 � � +++ � � � � � � � � �100,000 � � � � � � � � � � � �

Methanogens

100 � � � / / / / / / / / /

1000 � � � �x � � � + � � � �10,000 � � � � � + � � �x � � �

J.P. Werne et al. / Chemical Geology 205 (2004) 367–390382

DAPI numbers may be more reliable, due in part to

the very low quantities of DNA that were extractable

from these sediment samples.

Another interesting feature of the microbiological

data is found in the most probable number (MPN)

counts. Data from MPN counts indicate the likely

existence of colorless sulfur bacteria (e.g. sulfide

oxidizers) and methanogens in sediments from Kazan

mud volcano above 22 cm depth, but give no indica-

tion of the existence of SRB or aerobic methane

oxidizers (Table 4). These results suggest a number

of possibilities. First, it is not surprising to see

evidence of colorless sulfur bacteria in the MPN

counts given the visual observation of ‘‘white

patches’’ by submersible dives (Fig. 8) which are

believed to be sulfide oxidizers similar to Beggiatoa.

Second, it is possible that there are no SRB in these

sediments, but this seems very unlikely given the

biomarker and pore water geochemical data discussed.

Alternatively, it is possible that the SRB that are living

syntrophically with the anaerobic methane-oxidizing

archaea are not culturable under the conditions uti-

lized, and therefore do not appear in the MPN counts.

Table 3

Microbial cell and aggregate counts (DAPI and Acridin Orange)

from core MNLBC18/19

Sample

interval

(cm)

DAPI counts

(cells/cm3)

Acridin Orange

counts (cells/cm3)

Aggregate counts

(aggregates/cm3)

0–6 2.14�107 4.2�108 few

6–22 1.79�107 2.6�108 few

>22 2.99�107 1.3�108 F106

aggregates/cm3

As these consortia have proven difficult to culture (cf.

Boetius et al., 2000; Michaelis et al., 2002), the

second hypothesis seems more likely.

100,000 � � � � � � � � � � � �

Methane-oxidizing bacteria

100 � + � / / / / / / / / /

1000 � � � � � � � � � � � �10,000 � � � � � � � � � � � �100,000 � � � � � � � � � � � �Symbols used are: (�) indicates not present; (/) indicates not

analyzed due to pre-dilution of 100 times; (+), (++), and (+++)

indicate the relative level of likely concentration; (x) indicates

sample contaminated by oxygen. WI, WII, WIII and SI, SII, SIII

indicate water and sediment samples used for culture work, run in

triplicate.

l Geology 205 (2004) 367–390 383

5.9. Chemosynthetic macrofauna

Dense communities composed of small bivalves and

vestimentiferan tube worms were discovered on east-

ern Mediterranean mud volcanoes (Fig. 11, cf. Fig. 2b

in MEDINAUT/MEDINETH Shipboard Scientific

Party, 2000). During the 1998 MEDINAUT cruise,

living individuals of several species were sampled by

submersible from the top of Kazan mud volcano

(35j25.983VN, 24j33.594VE, 1707 m water depth;

Fiala-Medioni et al., 2001; Olu-Le Roy et al., in press).

Among the organisms collected were lucinid bivalves:

the most abundant species was identified asMyrtea sp.

(Olu-Le Roy et al., 2001, in press); few samples of

another species were found and described as Lucinoma

kasani (Salas and Woodside, 2002). Other living

bivalves included a small vesicomyid, identified as

J.P. Werne et al. / Chemica

Fig. 11. Photo taken by submersible Nautile showing types of

chemosynthesis-dependent macrofauna observed on Kazan mud

volcano. (a) Vestimentiferan tube worms. (b) Lucinid, vesicomyid,

and mytilid bivalves.

Isorropodon perplexum (Olu-Le Roy et al., 2001, in

press; von Cosel and Salas, 2001) and a small mytilid

attached to carbonate crusts and identified as Idas

modiolaeformis (Olu-Le Roy et al., 2001, in press).

Some tubes of living worms belonging to Lamellibra-

chia sp. (Olu-Le Roy et al., 2001, in press) were also

collected at the site. On top of a strongly reduced

sediment, white-grey bacterial mats (Fig. 8) were

observed as well as many echinidae sea urchins and a

number of unidentified crabs.

TEM studies of the gills of Myrtea sp. and Vesico-

mya sp., as well as trophosomes of vestimentiferan

worms demonstrated that they were composed of

bacteriocytes housing sulfide-oxidizing bacteria of

various morphological types as found in other symbi-

otic organisms from vents or cold seeps (Fiala-Medi-

oni and Felbeck, 1990; Fisher, 1995). In contrast, the

mytilid gill houses both sulfide-oxidizing and meth-

anotrophic symbionts (Fiala-Medioni et al., 2001) as

found in Bathymodiolus puteosrpentis (Cavanaugh et

al., 1992) or B. azoricus (Fiala-Medioni et al., 2002).

Carbon isotopic compositions of the gills of bivalves

also suggested chemosynthesis-based metabolism,

ranging from values �27.7 xto �30.5 xfor M.

sp. and I. perplexum and from �23.6xto �26.6xfor the tube worm Lamellibrachia sp. (Fiala-Medioni

et al., 2001); these values are in the range of those

found for other sulfide-oxidizing symbionts in differ-

ent vent or cold seep sites (Fiala-Medioni and Felbeck,

1990; Kennicutt et al., 1992; Fisher, 1995).

The values measured on the Kazan mud volcano

mytilid I. modiolaeformis are more depleted than those

of the lucinid, ranging from �44.2xto �44.6x(Fiala-Medioni et al., 2001). Such values reflect the

presence of methylotrophic symbionts which may use

methane as an energy source as found for B. childressi,

a cold seep species collected off Louisiana (Nelson and

Fisher, 1995). TEM and isotopic results led to the con-

clusion that these species are mainly relying, through

their symbionts, on reduced sulfur produced in the se-

diment, with the exception of the mytilid which is prob-

ably able to use both sulfide produced in the sediment

and/ormethane expelled in the fluids (Olu-LeRoy et al.,

in press). The chemosynthetic macro- and megafauna

are located at sites where fluids are expelled (Olu et al.,

1997; Sibuet and Olu, 1998), but there is currently no

statistical evidence for any direct relationship between

methane concentrations and living biomass. With the

J.P. Werne et al. / Chemical Geology 205 (2004) 367–390384

exception of the mytilid, such a relationship appears

indirect for Kazan mud volcano species where no

methylotrophic-like symbionts were observed. Within

Mediterranean mud volcano fields, it has been docu-

mented that concentrations of methane increase with

depth in the uppermost meter of the mud breccias. In

most cases, this appears to be associated with the depth

zone of sulfate reduction (de Lange and Brumsack,

1998) and results in the generation of inorganic carbon

and hydrogen sulfide which can be used as a substrate

by the sulfide oxidizing symbionts of the macrofauna.

6. Summary and biogeochemical synthesis

AOM is a significant biogeochemical process in

many cold-seep sediments, evident in everything from

pore water and sedimentary geochemical profiles to

authigenic carbonates to (chemosynthetic) macrofau-

nal abundance and diversity. In sediments from Kazan

mud volcano, measured and modeled pore water

profiles of the concentrations of sulfate, sulfide, dis-

solved inorganic carbon (DIC) and d13CDIC all indi-

cate that AOM is presently occurring in the sediments

of Kazan mud volcano (Fig. 4, Haese et al., 2003). It

is clear from these measured and modeled pore water

profiles that sulfate and methane are consumed in the

same depth interval, coincident with the production of

sulfide and 13C-depleted DIC. Indeed, d13CDIC values

of �35x(PDB) require the oxidation of methane to

CO2 (Whiticar and Faber, 1986; Alperin et al., 1988;

Whiticar, 1999). 13C depleted authigenic carbonates

and TOC are identified in the zone where AOM is

believed to be occurring based on pore water profiles

(Figs. 4–7). Further evidence of AOM is found in 13C

depleted biomarkers of methane-oxidizing archaea and

SRB (Table 2). Visual observation of microbial con-

sortium aggregates through FISH (Fig. 10) and che-

mosynthetic macrofauna such as bivalves and tube

worms (Fig. 11) confirm the existence of organisms

that are dependent on methane and the byproducts of

AOM (such as the sulfide utilized by bacterial sym-

bionts dwelling in the tissues of the macrofauna).

The porewater (sulfate, sulfide, DIC) and solid

phase (bulkOM, and selected biomarkers) geochemical

profiles suggest that this process is restricted to a

narrow depth interval, where methane is completely

oxidized. This hypothesis is supported by the lack of

MPN response for aerobic methane oxidizers in even

the most surficial sediments of Kazan. The lack of mi-

crobiological evidence for aerobicmethane oxidizers in

the sediments further suggests that there are no aerobic

organisms utilizing methane escaping the AOM zone

(Werne et al., 2002), despite the presence of aerobic

methane-oxidizer biomarkers (diploptene and di-

plopterol) in near surface sediments. An alternative

explanation is that these compounds are derived from

an early, rapid phase of aerobic methane oxidation that

took place immediately after mud breccia emplacement

rather than from presently active bacteria. This hypoth-

esis is more attractive given that microbiological data

indicates the absenceofmethane-oxidizingbacteria cur-

rently living in Kazan mud volcano surficial sediments.

It is also conceivable, however, that the biomarkers

attributed to aerobic methane oxidizing bacteria by

Werne et al. (2002) could be derived from other

bacteria in this environment. Indeed, these biomarkers

are fairly general bacterial biomarkers, and there is

strong evidence for the existence of colorless sulfur

bacteria, both in MPN counts (Table 4) as well as in

visual observations of white patches (which may have

been Beggiatoa-like white filamentous bacteria) made

by Nautile submersible dives (MEDINAUT/MEDI-

NETH Shipboard Scientific Parties, 2000). In addition,

other biomarkers identified in Kazan mud volcano

sediments and other cold seep environments, such as

bishomohopanol, have been attributed to sulfide-oxi-

dizing bacteria (this study; Pancost et al., 2002).

Under such conditions of complete (or near-com-

plete) methane oxidation, only small amounts of meth-

ane are released into the bottom waters. This release is

typically associated with major faults where highest

methane fluxes (due to advective flow) can be expec-

ted. This aspect of fluid fluxes and methane oxidation

processes explains why bottom-water methane concen-

trations are so variable over mud volcanoes in the

eastern Mediterranean and the highest methane con-

centrations in the deep Mediterranean are identified

above Amsterdam mud volcano (Charlou et al., 2003),

which is characterized by intense active tectonism. (Ac-

tive) faults also serve as preferred fluid pathways

(focused flow), which leads to a high spatial heteroge-

neity of solute fluxes, fluid flow, and microbial andma-

crobenthic abundances, as identified through visual

observations made during dives of the submersible

Nautile.

J.P. Werne et al. / Chemical Geology 205 (2004) 367–390 385

The production of authigenic carbonates as a by-

product of AOM represents one of the major terminal

sinks for methane-derived carbon in cold seep envi-

ronments. This process also keeps methane out of the

atmosphere thereby reducing the impact of methane

on global climate change. The high variability in the

carbon isotopic composition of these carbonates can

be explained by the fact that: (1) they form both as a

result of chemical supersaturation and microbial cal-

cification (Aloisi et al., 2000, 2002); and (2) slight

variations in the depth of precipitation of carbonates

within the sediments will substantially alter their

isotopic signature due to the steep gradients in d13Cof the available DIC (Aloisi et al., 2000, 2002). One

implication of these observations is that deriving rates

of carbonate formation from pore water gradients may

underestimate the true calcification rate due to the ad-

ditional contribution from microbes and the very steep

and likely variable gradients in the concentrations and

carbon isotope composition of DIC resulting from

AOM.

Series I diethers are higher in concentration and

more 13C depleted than series II diethers (ca. �97xcompared to �70x ; Table 1) in the authigenic

carbonate investigated here. This pattern differs from

that observed in carbonates from Amsterdam mud

volcano (also in the Anaximander Mountains area)

where the two series are approximately equivalent in

abundance, as well as to authigenic carbonates from

Napoli mud volcano (Olimpi area), where series I

diethers are lower in concentration than series II

diethers (Bouloubassi, unpublished results). Further-

more, in mud breccia sediments from Kazan mud

volcano, only series II diethers were identified. These

observations suggest that different SRB could be par-

ticipating in the microbial consortium with the archaeal

methane-oxidizers in different environments, possibly

as a result of the different salinities of the two environ-

ments. Furthermore, different SRB may be active

syntrophic partners with archaea in seep areas charac-

terized by carbonate precipitation compared to areas

not precipitating carbonate within the same mud vol-

cano environment.

Additional support for the hypothesis that very

different microbial consortia are carrying out AOM in

different environments is found in the differences

between GDGT compounds found on Kazan mud

volcano and those identified elsewhere. While the

isotopic composition of C40 isoprenoids released from

GDGTs of Kazan mud volcano are up to �22x,

significantly 13C-depleted C40 isoprenoids were iden-

tified in authigenic carbonate crusts from Olimpi mud

volcano (Bouloubassi et al., unpublished results), as

well as in seeps from Milano and Amsterdam mud

volcanoes, and crusts, bacterial mats, seeps, and brines

from Napoli mud volcano (Pancost et al., 2001a).

These contrasting results in different cold seep loca-

tions, even within eastern Mediterranean mud volca-

noes, strongly suggest that the specific microbial

communities vary depending on the geochemistry of

the environment, including factors such as methane

concentration and flux, and salinity.

The archaeal/SRB aggregates (as determined by the

Acridin Orange method) show the greatest abundance

in the deepest sediment layer analyzed (>22 cm). This

contrasts with the pore water geochemistry and solid

phase bulk geochemical data, which suggests present-

day maximum AOM rates in the region around 13–15

cm depth. The lipid profiles, however, display a max-

imum in AOM indicators (e.g., 13C depleted specific

biomarkers) at an intermediate sediment depth of 16–

18 cm. The discrepancies in the geochemical and

microbiological data suggest that the historical center

of AOM in this environment may have migrated

vertically in the sediments, likely as a result of varia-

tions in the rate of the methane flux. While such a hy-

pothesis is not unlikely, it should be noted that the

microbiological analyses were taken on a separate adja-

cent core, so slight variations in the depth of AOM are

expected due to the lateral spatial variability in all

aspects of a mud volcano, from fluid venting to surface

topography. Furthermore, it is generally acknowledged

that lipid profiles and bulk OM provide a more ‘‘time-

averaged’’ view of the sedimentary processes, while

pore water geochemistry is thought to give more of a

‘‘snapshot’’ view of present-day conditions. DNA has

been observed to degrade on time-scales from 10min to

100,000 years, depending on specific circumstances.

These differences in the interpretable time frame of the

different geochemical and microbiological indicators

can often make interpretation problematical.

Model calculations estimate a rate of AOM of f6

mol m�2 year�1. The advective flow velocity at Kazan

mud volcano was found to be 4 cm year�1. Compared

to other cold seep sites, the advective flow velocity and

the rate of AOM are rather moderate (Haese et al.,

J.P. Werne et al. / Chemical Geology 205 (2004) 367–390386

2003). Based on the modeled methane concentration

profile and the state of methane saturation with respect

to gas hydrate, hydrate formation is expected at a

sediment depth of 2 m and below at site MNLBC19.

Whereas gas hydrate stability is thermodynamically

favorable on Kazan mud volcano, the rate of hydrate

formation is rather slow (Haese et al., 2003). These

findings may best explain the rare occasions of gas

hydrate recoveries during two expeditions to Kazan

mud volcano.

Acknowledgements

We would like to thank the Captain and crew of the

R/V Nadir, the submersible Nautile, and the R/V

Professor Logachev for their tireless assistance during

sampling. We also thank M. Kienhuis, E. Hopmans,

pentamethylicosane (PMI)

dialkyl gly

OH

O

O

Archaeol: X = H, X’ = H

sn-3-hydroxyarchaeol: X = H, X’ = OH

O

O

X

OH

X’

Crocetane

SRB di

OY '

OY

OH

Se

Se

Both series,Y’ =

or

n = 14

O

HO

X O

OH

O

HO

X

Y O

O Y O

O O

OHor

Glycerol dialkyl glycerol tetraether

X and Y =

Methane oxidizing archaeal biomarkers

and E. Panoto for technical assistance, and D. Saint

Hilaire and B. Riviere for assistance with TEM sec-

tioning and inclusion. Financial support was provided

by the Dutch funding organization NWOALW

(MEDINAUT, MEDINETH, and MEDISED proj-

ects) and the French funding agencies IFREMER,

UPMC (Paris 6) and CNRS (UMR 7621). This

manuscript benefited from reviews by two anony-

mous reviewers. [LW]

Appendix A. Biomarker structures

List of abbreviations and acronyms

MEDINAUT Joint Dutch–French research expedi-

tion to MEDiterranean mud volcanoes using

the submersible NAUTile in 1998

MEDINETH Dutch follow-up expedition to MEDI-

cerol diethers

OH

OH

diplopterol

diploptene

β,β-bishomohopanol

HO tetrahymanol

ether 1

ries I, Y =

ries II, Y =

, 15, or 16

s (GDGTs)

hopanoids

Eukaryotic biomarker

J.P. Werne et al. / Chemical Geology 205 (2004) 367–390 387

NAUT, focused on higher resolution geo-

physical surveys and sediment sampling in

1999 (MEDiterranean NETHerlands)

SBP Sub-Bottom Profiler

DAPI 4V,6V-DiAmidino-2-PhenylIndole

hydrochloride

MPN Most Probable Numbers

PCR Polymerase Chain Reaction

HPLC High Performance Liquid Chromatography

t-RFLP terminal-Restriction Fragment Length

Polymorphism

ANAXIPROBE Two phase research program carried

out in 1995 and 1996 to investigate the

origin and evolution of the Anaximander

mountains

AOM Anaerobic Oxidation of Methane

DIC Dissolved Inorganic Carbon

FISH Fluorescence in situ Hybridization

d13Cx stable carbon isotope composition of sub-

stance x

(V)PDB (Vienna) Pee Dee Belemnite, international

standard against which carbon and oxygen

isotope ratios are calibrated

PMI Pentamethylicosane

SRB Sulfate-Reducing Bacteria

GDGT Glycerol Dialkyl Glycerol Tetraether

C40 Carbon chain with 40 carbons

TOC Total Organic Carbon

TEM Transmission Electron Microscopy

OM Organic Matter

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