cover lois maignien lr - ghent university · seas, fp7) programs, a phd scholarship from the agency...

FACULTEIT BIO-INGENIEURSWETENSCHAPPEN

2011Lo

ïs MA

IGN

IEN

ISBN 978-90-5989-464-8

MSc. Raquel Medeiros Vinci

Loïs MAIGNIEN

Microbial Ecology of Carbon and Sulphur Cycles

in Deep-sea Carbonate Mounds

and Mud Volcanoes

MICRO

BIAL ECO

LOG

Y OF CA

RBON

AN

D SU

LPHU

R CYCLESIN

DEEP-SEA

CARBO

NATE M

OU

ND

S AN

D M

UD

VOLCA

NO

ES

Marine sediments are the major reservoir of methane on earth, where it is

produced by organic matter mineralization and water-rock interactions.

Despite a gross production of ~400 Tg per year of this potent greenhouse

gas, sediments only contribute to a very minor part of the atmospheric

methane pool. Microorganisms mediating the Anaerobic Oxidation of

Methane (or AOM) coupled to sulphate reduction indeed retains over 80%

of this methane flux and are thus major players in sediment carbon and

sulphur cycles, and climate control.

During this doctoral research, I studied the links between the activity and

community structure of marine sediments microorganisms and their envi-

ronment in order to better understand the functioning of these microbial

ecosystems.

A JULIE, LOUISE, ET AVA.

UN CLIN D’OEIL DE DARWIN

Promotors Prof. Dr. Ir. Nico Boon Laboratory for Microbial Ecology and Technology (LabMET) Department of Biochemichal and Microbial Technology Faculty of Bioscience Enfineering Ghent University Prof. Emerit. Dr. Jean-Pierre Henriet Renard Center for Marine Geology (RCMG) Department of Geology andf Soil Sciences Faculty of Sciences Ghent University

Dean of the faculty

Prof. dr. ir. Guido Van Huylenbroeck

Rector of the University

Prof. Dr. Paul Van Cauwenberge

Loïs MAIGNIEN

MICROBIAL ECOLOGY OF CARBON

AND SULPHUR CYCLES IN DEEP-SEA

CARBONATE MOUNDS AND MUD

VOLCANOES

Thesis submitted in fulfilment of the requirements for the degree of Doctor (PhD) in

Applied Biological Sciences.

This work was funded by the European Commission EURODOM Training Through

Research (FP6) and HERMES (Hotspot Ecosystem Research along Margins of European

Seas, FP7) programs, a PhD scholarship from the Agency for Inovation by Science and

Technology (IWT, contract SB53575) and the European Science Fundation Eurocores

MicroSYSTEMS research and travel grants.

ISBN 978-90-5989-464-8

To cite this work: MAIGNIEN, L. (2011). Microbial ecology of carbon and sulphur cycles in

deep-sea carbonate mounds and mud volcanoes. PhD thesis. Ghent University, Belgium.

Examination Committee

Prof. dr. ir. Marc Van MEIRVENNE. Chairman Department of Soil Management

Faculty of Bioscience Engineering Ghent University

Prof. dr. ir. Pascal BOECKX. Secretary

Department of Applied Analytical and Physical Chemistry Faculty of Bioscience Engineering

Ghent University

Prof. dr. Ir. Colin JANSSEN Department of Applied Ecology and Environmental Biology

Faculty of Bioscience Engineering Ghent University

Prof. dr. Nadine LE BRIS

Benthic Ecogeochemistry Laboratory Banyuls Marine Station

University Pierre & Marie Curie Paris, France

Dr. Helge NIEMANN

Department of Environmental Sciences Institute for Environmental Geosciences

Basel University, Switzerland

Prof. dr. Ann VANREUSEL Laboratory of Marine Biology

Department of Biology Faculty of Sciences Ghent University

Acknowledgements

This PhD work could have never been completed without the help, the consideration

and the competence of the following persons. They have all been kind enough, at some point

or another, to dedicate a bit of their time, energy and brilliant minds to move this PhD work

forward. I would thus like to seize this oportunity for thanking them.

My first acknowledgements go to Prof. Nico Boon, promotor of this thesis: his

patience and the freedom he gave my to realize my work were extremely appreciated. My

warmest thanks to Prof. Jean-Pierre Henriet: he was of tremendous support by his faultless

trust and optmism, and provided an ocean of opportunities whithout which none of this work

would have been possible. I am gratefull to Prof. Verstraete for welcoming me in his Lab. I

would also like to thank Prof. John Parkes for accepting to share so much of his vast

knowledge with me, for inviting me in his lab and for dedicating a precious time to revise and

recommend my work. I learned a lot from Dr. Barry Cragg who not only shared his sea-going

skills of great marine microbiologist, but also his home, his office and his tastes for

exceptional food and beverage! Sailing with them has been a very fruitfull and enjoyable

experience. The contribution of Prof. Antje Boetius, who supported my initiatives and

welcomed me in her lab in several occasions, has been extremely valuable. Dr Helge

Niemann dedicated a considerable part of his time and experience to teach me how to work

with radiotracers, to help me prepare my cruises, and to revise my manuscripts. May he

receive my warmest thanks! I am grateful to Dr Katrin Knittel for teaching me fluorescence

microscopy and finding some time to thoroughly revise my work, Dr. Gunter Wegener for his

help in the radiotracer lab and his hospitality in Bremen, Gabi Schüßler for her patient

technical assistance and great humour, and more generally to the persons from the Max

Planck Insitute for Marine Microbiology in Bremen, with whom I had countless discussions,

from whom I learned so much and who were of considerable help with their competence and

their availability.

I want to thank all my colleagues during these years at LabMET! I had a great time in

Belgium, mostly thanks to you. There is a special place in LabMET called the Rotonde. Its

inhabitant, los rotonderos, are extremely friendly persons, and while writing these lines, I

have a special thought for each of them: Simon, Bart, Tom, Willem, Joeri, Haydée, Joachim,

and the formers inhabitant, Peter, Selin, Ilse, Lynn, Birgit, Siegfried, and many more…

“Louis, I think this is the beginning of a beautifull friendship” (Casablanca). Keep in

touch with the Yeti!

A sincere thank to the Marine Geologists at RCMG who tried hard to explain me the

most basic things about rocks and sound waves: David, Davy, Anneleen, Lies, Marc, Koen,

Jeroen, Peter, Hans and all past and present RCMG staff. If we finally managed to speak a

common comprehensible language, it’s mostly due to their patience.

The help of the crew and scientific party, as well as the dedication of the chief

scientists of several cruises in the Gulf of Cadiz made this work possible: the R/V Maria S.

Merian MSM 1/3 cruise led by O. Pfannkuche , the R/V James Cook JC10 cruise led by K.

Heeschen and P. Weaver, the R/V Marion Dufresnes MicroSYSTEM cruise led by D.

Blammard and D. Van Rooij. Many thanks to all of them.

Un grand Merci à Christine et Yanninck qui m’ont transmis le plaisir de naviguer …

sur cet ocean d’Idées. Merci a Noémie et Hortense pour toute leur gentillesse envers leur

grand frère. Un clin d’œil à Stella et Brita. Merci à Bernard, Jeanine et Gilberte pour leur

constante affection et bienveillance. Merci à Michèle, Jean-Pierre, Sebastien et Lena pour

leur aide et leur bonne humeur!

Un torrent d’amour a Louise et Ava, mes petits soleils (pour quand vous saurez lire)

Dr. Julie Reveillaud: un immense merci pour tout, tous les passés et tous les possibles

à venir! Derrière chaque femme d’exception, se cache un grand homme. J’espere rester a la

hauteur…

Reading this, one may think that this thesis is the result of a collective work.

Well, it truly is!

Loïs, September 2011.

“The value of having for a time rigorously

pursued a rigorous science does not rest especially in its

results: for in relation to the sea of worthy knowledge,

these will be but a negligible little drop. But it brings

forth an increase of energy, of deductive ability, of

persistence; one has learned to gain one's purpose

purposefully. To this extent, in respect to all one does

later, it is very valuable to have once been a scientific

man.”

F. NIETSZCHE

Human, all too human.

A book for free Spitits

Table of content

SUMMARY 1

CHAPTERI. GENERALINTRODUCTION 7

1. STRUCTUREANDPROPERTIESOFMETHANE 92. METHANERESERVOIRSANDFLUXES 112.1 MARINESEDIMENTSANDGLOBALMETHANEBUDGET 112.2 METHANESOURCESINMARINESEDIMENTS 113. METHANESINKSINMARINESEDIMENTS. 134. ANAEROBICOXIDATIONOFMETHANE:BIOGEOCHEMISTRYANDECOLOGY 174.1 STOICHIOMETRYANDENERGETICYIELD 174.2 GEOCHEMISTRY: 174.3 MICROORGANISMSMEDIATINGAOM 184.4 MECHANISMS. 184.5 METHANOGENICACTIVITYOFAOMCOMMUNITIES 194.6 AOMCOMMUNITYSTRUCTURE 204.7 MICROBIALECOLOGY 204.8 AOMANDAUTHIGENICCARBONATEPRECIPITATION. 214.9 OPENQUESTIONSANDCURRENTCHALLENGESINTHECOMPREHENSIONOFAOMMECHANISM,ECOLOGY

ANDMETHANEBUDGET:ASUMMARY. 215. TRACKINGTHEORIGINANDFATEOFMETHANEWITHCARBONSTABLEISOTOPES. 236. MUDVOLCANOES 256.1 SUBSEAFLOORORIGINANDRELATIONTOENVIRONMENTALSETTINGS 256.2 FLUIDCONDUIT 256.3 METHANEEMISSION 267. THEGULFOFCADIZ. 277.1 ORIGINANDGEOLOGYOFTHEGULFOFCADIZ. 277.2 HYDROLOGICREGIMEINTHEGULFOFCADIZ 297.3 ORIGINANDSEAFLOOREXPRESSIONOFHYDROCARBONMIGRATIONINTHEGULFOFCADIZ. 298. CARBONATEMOUNDS 318.1 ALIFESTRATEGYTHROUGHOUTEARTHHISTORY 318.2 MODERNCOLD‐WATERCORALCARBONATEMOUNDS 318.3 CARBONATEMOUNDSINTHEGULFOFCADIZ 349. GENERALGOALANDOUTLINEOFTHETHESIS: 36

CHAPTERII. ANAEROBIC OXIDATION OF METHANE IN A COLDWATER CORAL

CARBONATEMOUNDFROMTHEGULFOFCADIZ 39

1. INTRODUCTION 422. RESULTS 452.1 SITEDESCRIPTION 452.2 POREWATERGEOCHEMICALPROFILES. 462.3 ON‐MOUNDLOCATION:INVITROSRRATESANDCELLCOUNTS 483. DISCUSSION 493.1 ANAEROBICOXIDATIONOFMETHANEINALPHAMOUND. 493.2 ORIGINOFMETHANEANDDICBUDGETINTHEALPHAMOUND. 503.3 FLUXESANDRATES 513.4 COMPARISONWITHOTHERMOUNDSETTINGS 534. CONCLUSION 545. MATERIALANDMETHODS 555.1 SAMPLINGPROCEDURE 555.2 GASANALYSIS 565.3 CARBONISOTOPES 565.4 SULPHATEANDSULPHIDECONCENTRATION 575.5 FLUXCALCULATION 575.6 INVITROSRRATES 585.7 DIRECTCELLCOUNT 58

CHAPTERIII. HETEROGENEOUS METHANE GEOCHEMISTRY AND ANAEROBIC

OXIDATIONINTHECARBONATESEALEDDARWINMUDVOLCANO,GULFOFCADIZ. 63

1. INTRODUCTION 662. RESULTS 682.1 SAMPLINGSITE 682.2 INWESTERNRIMATTHESEEPSITE 692.3 INWESTERNRIM,2MAWAYOFTHESEEPSITE 762.4 SOUTHRIM 762.5 NORTHRIM 772.6 REFERENCESITE 783. DISCUSSION 803.1 AOMACTIVITYDISTRIBUTION 803.2 MICROBIALCOMMUNITYSTRUCTUREATTHESEEPSITE. 803.3 ACTIVITYCOUPLINGINDARWINMVSEDIMENTS. 81

3.4 PROPOSITIONOFADEVELOPMENTMODELFORTHEDARWINMV 824. CONCLUSION 84

CHAPTER IV. ANAEROBIC OXIDATION OF METHANE IN HYPERSALINE COLD

SEEP SEDIMENTS. 87

1. INTRODUCTION 902. RESULTS 922.1 GEOCHEMISTRY 922.2 MICROBIALACTIVITY. 962.3 MICROBIALCOMMUNITYSTRUCTURE 983. DISCUSSION 1033.1 AOMACTIVITYINHYPERSALINEANDNON‐HYPERSALINESEDIMENTS 1033.2 AOMCOMMUNITYSTRUCTUREINHYPERSALINESEDIMENTSOFMERCATORMV. 1043.3 REACTIONCOUPLINGBETWEENAOM,SRANDMG 1063.4 METHANOGENESISRATESANDZONATION. 1084. CONCLUSION 1105. ADDENDUMI:BATHYMETRY,SAMPLINGANDSEDIMENTDESCRIPTION 1125.1 SAMPLINGSITE,SEAFLOOROBSERVATIONSANDSEDIMENTOLOGY 1126. ADDENDUMII:ABUNDANCEANDCONSORTIASTRUCTUREINVOLVINGANME1INMETHANESEEPS

114

CHAPTERV. METHANEOXIDATIONATTHECARLOSRIBEIROMUDVOLCANO:EXSITU

MICROBIALACTIVITYVS.GEOCHEMICALMODELING. 121

1. INTRODUCTION 1242. DESCRIPTIONOFTHEMODELUSEDTOESTIMATEINSITUAOMRATES 1263. RESULTS 1283.1 SAMPLINGSITE 1283.2 GEOCHEMISTRY 1293.3 EXSITUMICROBIALACTIVITY 1303.4 ESTIMATIONOFINSITUAOMRATES 1333.5 AOMMICROBIALCOMMUNITYSTRUCTURE 1334. DISCUSSION 1384.1 MO,SRANDMGACTIVITYCOUPLINGINCRMVSEDIMENTS. 1384.2 ESTIMATEDINSITUMETHANECONSUMPTIONINCRMVSEDIMENTS. 1394.3 AOMCOMMUNITYSTRUCTUREATCRMV. 1405. CONCLUSION 142

CHAPTERVI. GENERALDISCUSSION 145

1. WHATISTHECONTRIBUTIONOFAOMTOCOLDWATERCARBONATEMOUNDSPROCESSES? 1462. COMPARISON OF THE MICROBIAL COMMUNITY DIVERSITY IN THE THREE MV AND POSSIBLE

ENVIRONMENTALCONTROLS. 1493. AREGOMARCICELLSNOVELAOMMEDIATINGARCHAEA? 1524. HOW TO BETTER EVALUATE METHANE PROFILE, FLUX AND TURNOVER FROM DEEPSEA MARINE

SEDIMENTS? 1545. SOLVINGABIOENERGETICGAP:OTHERMECHANISMSFORAOM? 1566. WHATCANTRIGGERTHEFORMATIONOFSHELLTYPEAGGREGATESOFANMEANDSRB? 1597. PERSPECTIVES 162

CHAPTERVII. EXPERIMENTALPROCEDURES 165

1. MICROBATHYMETRY. 1662. CORINGANDSAMPLING 1663. GEOCHEMISTRY 1674. EXSITURATEMEASUREMENTS. 1675. NUCLEICACIDEXTRACTIONANDAMPLIFICATION. 1696. CLONELIBRARIESCONSTRUCTION,SCREENINGANDPHYLOGENYINFERENCE. 1697. SEQUENCEACCESSIONNUMBERS. 1708. PHYLOGENETICTREERECONSTRUCTIONANDPHYLOTYPEINFERENCE. 1719. (CATALYZED REPORTERDEPOSITION)FLUORESCENCE IN SITUHYBRIDIZATION (FISHANDCARD

FISH)CELLSTAININGANDMICROSCOPICOBSERVATIONS. 171

REFERENCES 173

CURRICULUMVITAE 197

List of abreviations ANME Annaerobic Methanotroph

AOM(R) Anaerobic Oxidation of Methane (Rates) AVS Acid Volatile Sulphide BSR Bottom Simulating Reflectors CARD-FISH Catalyzed Reporter Deposition – Fluoresence In Situ Hybridization cm bsf centimeter below the seafloor Gt Giga Ton GoC Gulf of Cadiz GoM Gulf of Mexico IODP Integrated Ocean Drilling Program m bsf meter below the sea floor m bsl meter below sea level MG Methanogenesis

H-MG Hydrogenotrophic Methanogenesis Ac-MG Acetotrophic Methanogenesis Meth-MG Methyltrophic Methanogenesis

MV Mud Volcano CAMV Captain Arutyunov Mud Volcano CRMV Carlos Ribeiro Mud Volcano

HMMV Håkon Mosby Mud Volcano R/V Research Vessel ROV Remotely Operated Vehicle

SIMS Secondary Ion Mass Spectrometry

SMTZ Sulphate Methane Transition Zone SRB Sulphate reducing Bacteria

SR(R) Sulphate Reduction (Rates) TRS Total Reducible Sulphur

Cruises

MD04 R/V Marion Dufresne 2004 Gulf of Cadiz MSM1/3 R/V Maria S. Merian 2006 Gulf of Cadiz JC10 R/V James Cook 2007 Gulf of Cadiz MD08 R/V Marion Dufresne 2008 Gulf of Cadiz TTR R/V Prof. Logachev Training Through Research Cruises

1

SUMMARY

A large fraction of oceanic and continental organic matter is buried in marine sediments,

where it is degraded by the action of microorganisms or by heating along the geothermal

gradient. Methane is the major end product of this process, and ~107 Tg of this hydrocarbon

are stored in marine sediments, either in fossil reservoirs or encaged in ice-like solid gas

hydrates. Potentially, marine sediments thus constitute a vast source of this greenhouse gas,

which as a more than 20-fold higher radiative forcing effect than the one of CO2 on a 100 years

basis. However, methane is retained in the sediment during its migration toward the sediment

surface by the microbially mediated anaerobic oxidation of methane (AOM). This reaction uses

the seawater sulphate diffusing in the sediment to oxidise methane into carbonate before it

reaches the ocean water. Hence AOM is a key reaction for climate control as it considerably

mitigates methane content in the atmosphere. Marine sediments, despite being the main

methane reservoir on earth, only accounts for 2% of the global methane emissions, i.e. less than

termite gut, ruminant enteric fermentation or rice paddies emissions.

Further, in areas of intense upward flux, methane promotes the development of thriving

ecosystems on the seafloor that only rely on this chemical energy rather than on direct sunlight

and photosynthetic primary production. These ecosystems sustain a high diversity of micro-,

meio- and macrofauna that sharply contrasts with typical deep-sea environments. By

controlling the methane flux and releasing sulphide as a by-product of the associated sulphate

reduction, AOM is at the base of these ecosystems and exert a strong ecological control on the

rest of these chemosynthetic communities.

SUMMARY

2

Hence, microorganisms mediating AOM have a central place at the interface between

different biological, geological and geochemical processes, all influencing their activity and

distribution. The study of their ecology and biogeography is thus of importance to understand

global geochemical cycles and anticipate the effect of physical and chemical changes in the

oceans.

Surprisingly, despite the wide distribution of AOM and the complexity of these

processes, only three closely related Archaea have been identified as anaerobic methanotrophs

(ANME), acting in concert with a few groups of sulphate reducing bacteria (SRB). Despite the

seemingly simple biochemical reaction carried out by this microorganisms, and the

considerable scientific efforts dedicated to this process, the AOM biochemical mechanism

remains unknown and the microorganisms involved have resisted repeated isolation attempts

that would have allowed the elucidation of their functioning. In this context, the study of these

microorganisms in their natural environments remains a very efficient study approach.

The aim of the present work is thus to gain a better knowledge of the ecology, activity

and biogeochemical role of these microorganisms. In this work, an integrated microbial

ecosystem functioning approach was used to elucidate the different feedback mechanisms

between environmental parameters, microbial activity and community structure. Natural AOM

hot spot ecosystems on the seafloor such as mud volcanoes (MV) displaying a wide variety of

habitat characteristics were used as natural laboratories. This work has been undertaken during

three European scientific expeditions in the Gulf of Cadiz, between the southern Portuguese

and Spanish margins, and the western Moroccan margin. This zone of broad methane migration

toward the seafloor encompasses a wide diversity of geological, oceanographic and

geochemical conditions, setting up a large array of habitats suitable for comparative studies.

Marine carbonate mounds represent the geological fingerprint of a widespread strategy of

life through earth history. Modern carbonate mounds built by cold water coral represent

conspicuous examples of such strategy. In the first part of this work, we describe a new type of

cold-water coral carbonate mounds, where methane migration strongly overprints the

geochemistry and diagenetic processes of these sediments. AOM activity in the Alpha mound

has a significant impact on the sulphur cycle, on mound’s dissolved inorganic carbon (DIC)

budget and isotopic signature. Further, this reaction has the potential to modify the carbonate

chemistry and alter the coral preservation state in the subsurface. This study highlights the

SUMMARY

3

importance of diagenetic processes that followed the mound’s formation, but that were not

related to the biology of corals, the main mound builders.

The second part of this thesis shows that carbonate production during AOM can lead to

the formation of massive carbonate hardgrounds in the crater of the Darwin MV. In turn, the

self-sealing of these sediments by carbonate induced the relocation of the geofluid flux at the

rim of the carbonate crust. Methane was apparently channelled through discrete pathways, as a

very heterogeneous AOM activity distribution along the rim has been observed. As a result,

both zones of very high and no activity were found. The former exhibited the highest methane

turnover measured thus far in the Gulf of Cadiz. There, AOM was mediated by consortia of

ANME-2 and DSS sulphate reducers that were consistently forming shell type aggregates of

ANME cells surrounded by DSS cells. Based on our observations and on the current

knowledge on methane-derived carbonate, a development model for this original MV is

proposed

The third part of this thesis focuses on the AOM microbial ecology under hypersaline

conditions. Density driven faulting of the sediments by salt masses (i.e. salt diapirs) often

occurs and provides an important escape pathway for over-pressurised fluid. Very little is

known about the methane turnover in these conditions and AOM inhibition due to hypersalinity

can potentially release large amounts of methane in the ocean. This study of the hypersaline

Mercator MV shows that, despite the very low energetic yield of AOM, methane consumption

occurred at salinity up to halite saturation (5.8 M NaCl), i.e. about ten times seawater salinity.

Further, hypersaline conditions exerted a strong pressure toward ANME-1 cells, and no other

ANME cells where present. The comigration of evaporite-derived sulphate together with

methane was a distinct feature in this habitat and promoted AOM activity through extended

depth. This was contrasting with the standard salinity Captain Arutyunov MV where AOM

occurred at the discrete interface between methane migrating from below and sulphate

diffusing from seawater. Overall, these results are challenging our current view of hypersaline

conditions as a strong selection factor toward high-energy yield metabolisms.

In a companion paper, geochemical modelling permitted to constrain the methane flux at

different sites of the Carlos Ribeiro MV, in the deep part of the Gulf of Cadiz. AOM and SR

turnover rates were measured after recovery of the sediments at the same locations. These

yielded turnover estimates that where at least an order lower than the previously estimated

methane and sulphate fluxes. This underestimation of in situ activity of microorganisms may be

SUMMARY

4

attributed to the difference of methane solubility between surface and in situ hydrostatic

pressure. Rates calculated with realistic in situ methane concentrations were indeed similar to

the geochemical model outputs. In these sediments, AOM was mostly mediated by ANME-2

and -3 cells, but the dominance of cells from the GoM Arc I group in the AOM zone suggested

that these may also play a role in AOM.

This work shows that AOM mediating microorganisms can thrive in a wide range of

habitats where they efficiently retain methane carbon in the sediments. Despite the low energy

that can be conserved from AOM for maintenance metabolism and biomass production, ANME

cells have the ability to face highly energy-demanding conditions such as hypersalinity,

nitrogen fixation, formation of dense aggregates and a probable syntrophic metabolism with

SRB. Hence, these organisms are remarkably adapted to environments characterised by energy

stress. In low redox gradients that are close to the thermodynamic equilibrium, the presence of

available energy strongly depends on the relative concentration of substrates and products. The

ability to revert metabolic pathways according to environmental conditions, as proposed for

AOM, may thus constitute a distinct evolutionary feature of subsurface microorganisms.

7

CHAPTER I. General

Introduction

CHAPTER I

8

GENERAL INTRODUCTION

9

1. Structure and properties of methane

Methane is the simplest hydrocarbon molecule, composed of one carbon covalently

bound to four hydrogen atoms. Its molecular weight is of 16 g mol-1. The melting point of

methane is -183 ºC and its boiling point is at -164 ºC under standard conditions (273K, 1 bar).

The solubility of methane in seawater at 1 bar is only ~1.4 mM, and increases with pressure

and thus with water depth (Figure I-1). Methane solubility also strongly decreases with salinity

(Figure I-1)

Methane has the capacity to form gas hydrate, i.e. methane molecules encaged in a water

lattice (Figure I-2). Pressure/Temperature conditions of hydrate stability match an important

part of ocean subseafloor, and hydrates are thought to constitute an enormous reservoir of

organic carbon in marine sediments. The presence of hydrates in marine sediments can be

detected by seismic survey, as the interface between the lower hydrate layer and free gas in

sediment typically yield a bottom simulating reflectors or BSR in seismographs.

Methane is one of the most reduced organic carbon compounds, due to the four C-H

bonds with the C atom being in the –IV redox state, and the redox potential of the CO2 / CH4

couple being of E’0= -240 mV (Thauer et al., 1977). In addition, methane has a high energy of

Figure I-1 Maximum solubility of methane in seawater as a function of depth at temperatures (4 and 13˚C) relevant for the studies presented in this thesis (left), or as a function of salinity (right). Note the difference of scale between methane solubility at surface and at 350 m water depth (the latter is the depth of the hypersaline MV studied in Chapter IV). Data from thermodynamic models of (Duan and Mao, 2006)

CHAPTER I

10

activation (434 kJ mol-1

for the first C-H bound) and its oxidation thus requires the action of

oxygen radical chemistry (Strous and Jetten, 2004, and references therein) or specific enzyme

cofactors. Finally, methane is a potent greenhouse gas in the atmosphere and is over 20 times

more effective than CO2 in trapping radiative energy (IPCC, 2007) on a 100 year basis.


11

2. Methane reservoirs and fluxes

2.1 Marine sediments and global methane budget

Sediment methane and hydrates consitute the major methane pool on earth, with an

average estimated mass of between 500 and 10.000 Gt of methane carbon ( Figure I-3). Most

recent estimates indicate a distribution of ~3000 Gt as hydrates and ~2000 Gt as gas bubbles.

(Reeburgh, 2007, and references therein). In comparison, the methane hydrate carbon pool

present in permafrost soils or shallow sediments -the second major hydrate reservoir- is much

lower, with estimates of ~ 400 Gt (Macdonald, 1990b). Such reservoirs are not yet

commercially exploited but there is a strong ongoing research on the extraction methods that

could harvest methane from hydrates fields (Milkov and Sassen, 2002).

The greenhouse effect of methane, together with the hydrate reservoir sensitivity to ocean

temperature, are of concern for climate change studies as a small bottom water temperature

increase could cause the destabilisation of hydrates, liberate free methane that could potentially

further contribute to global warming. Hence, the release of a small fraction of the methane from

the hydrate pool could have a considerable effect on the atmospheric methane, which is only of

4 Gt C (Dlugokencky et al., 1998). Marine and permafrost are likely to respond differently to

global temperature increase: marine hydrates can be close to sediment surface and thus

immediately affected by slight temperature increase. In addition, Dickens (2001) estimated that

an increase of 5 ºC of ocean water would divide by a factor 2 the extent of the deep-sea hydrate

stability zone. Due to the depth of hydrate stability zones in permafrosts, these are less likely to

respond quickly to surface temperature changes, although permafrost thawing and warmer

water incursioncould accelerate the hydrate dissociation process. Hence, the understanding of

possible source and sinks of methane are of considerable importance to produce relevant

climate change scenarios

2.2 Methane sources in marine sediments

In marine sediments, methane originates from three different sources: abiotic, abiogenic

and biogenic. Abiotic methane is the result of seawater interaction with ultramafic rocks in

hydrothermal systems at mid oceanic ridges. There, olivine ((Fe,Mg)2SiO4) in recently erupted

CHAPTER I

12

rocks is transformed to serpentine (Mg3Si2O5(OH)4) and H2 is released during the Fe(II)

oxidation to Fe(III) (Charlou et al., 1998). Methane is apparently formed, from dissolved CO2

and H2, by the Fisher–Tropsch reaction at T>300 ºC and P>500 bar in the presence of metal

catalysts. Such mechanisms are thought to produce the methane found in hydrothermal fluid

such as in Lost City vent fields in the mid Atlantic ridge (Boetius, 2005).

Abiogenic methane is produced by the abiotic alteration of biologically produced organic

matter. In sediments, abiogenic methane mainly results from the heating of buried organic

matter along the geothermal gradient. This thermogenic methane is typically formed in

subductive convergent margins where thick sediment layers are progressively heated in contact

with the earth mantle.

Biogenic methane is exclusively produced by the archaeal group of methanogens. The

low potential of the CH4/CO2 redox couple indicates that the biological formation of methane

occur in reducing environment, and consistently, methanogens are generally strict anaerobes.

Methanogens can use H2 + CO2, acetate or methyl groups of compounds such as methylamines,

methylsulphides, and methanol as substrates. The biological formation of methane is thus the

terminal step in the degradation of organic matter after the cleavage of macromolecules to

monomers and the monomer fermentation resulting in CO2, H2, and volatile fatty acids. In

marine sediments, the depth distribution of terminal electron acceptors involved in organic

matter oxidation mirrors the decreasing energetic yield of the redox reactions involved. Hence,

most favourable acceptors such as O2, NO3-, Mn(IV), Fe(III) and SO4

2- are consumed in this

order in the upper sediment layers, and methanogenesis using the least favourable electron

acceptor CO2, is mostly restricted to the sulphate free environments (Figure I-4).


13

3. Methane sinks in marine sediments.

Unlike in the atmosphere where it is readily oxidised by hydroxyl radicals deriving from the

ozone photodissociation (Logan et al., 1981), biological processes are the main sinks of

methane in soil, sediments and oceans. In presence of oxygen, methane is oxidized by

methanotrophic bacteria possesing the methane monooxygenase. This enzyme involve oxygen

radical chemistry for the activation of methane and the cleavage of the first C-H bond. The

oxidation of methane with nitrite has been recently discovered and involves similar

mechanisms, since some bacteria are able to generate oxygen from nitrite disproportionation. In

anaerobic conditions, and in the absence of nitrite, the activation mechanism of methane is not

yet understood but probably involves metal catalytic properties of the Ni(I)-containing F430

cofactors present in methanogens and anaerobic methanotrophs (Kruger et al., 2003; Mayr et

al., 2008; Scheller et al., 2011)

Methane produced in the deep sediment strata migrates toward the surface and penetrates in the

gradient of electron acceptors that are diffusing downward from seawater (Figure I-4). The

anaerobic oxidation of methane (AOM) coupled to sulphate reduction (SR) is the major sink of

methane in marine sediments. This microbially mediated reaction consumes ca. 382 Tg of

methane per year, which correspond to ca. 80-90% of the global methane gross production

from this environment (Reeburgh, 2007, and references therein), thus reducing the net emission

by a factor 5 to 10.

Methane can also be oxidized aerobically in the sediments. However, the rapid consumption of

oxygen by heterotrophic microorganisms in the first centimetres of sediment restricts the niche

of aerobic methanotrophic bacteria to surface sediments and oxygenated water column.

Recently, two additional mechanisms have been proposed for the anaerobic oxidation of

methane. (Beal et al., 2009) have shown that Mn(IV) and Fe(III) could be used as terminal

electron acceptor. In addition, it has been demonstrated that nitrite can oxidize methane through

a novel oxygenic pathway (Raghoebarsing et al., 2006; Ettwig et al., 2008; Ettwig et al., 2009;

Ettwig et al., 2010). However, the significance of these pathways is not yet fully understood.

Concentration of nitrate, the precursor of nitrite, is very low in seawater (3 orders lower than

sulphate), and the presence of oxidized metal ions in anoxic sediments is possible (D'hondt et

al., 2004; Parkes et al., 2005) but has only been observed in specific geological settings so far.

Hence, due to the relatively small pool of oxidized N, Fe and Mn species, these pathways could

CHAPTER I

14

only play a minor role in the ocean methane budget.

These different mechanisms are apparently efficient methane sinks, as it is estimated that the

net contribution of ocean to the atmospheric methane only represents <2% () from the global

flux (Reeburgh, 2007).


15

Figure I-2 Left: molecular representation of methane hydrate, i.e. a methane molecule (black/green) encaged in a water lattice (red/white) (reproduced from the ecosystems course curriculum Texas A&M university). Right: The domain of hydrate stability is illustrated in function of temperature and water depth instead of pressure (reproduced from U.S. Nat. Energy Tech. Lab.). Here, a water depth of 1200 m is assumed, but theoretically methane hydrate can start to form below 300 m water depth.

Figure I-3: Summary of the global methane reservoirs, fluxes and turnover (adapted from Prof. Reeburgh personal page at the University of California, Irvine www.ess.uci.edu/~reeburgh/fig9.html and (Reeburgh, 2007). Despite that marine sediments are the principal methane reservoir, they only contribute to ~1% to the annual methane flux toward the atmosphere.

CHAPTER I

16

Figure I-4 In marine ssediments, the vertical stratification of redox reactions involved in organic matter oxidation follows the decreasing energy yield of these reactions. CO2 is the less favourable electron acceptor and is used below the sulphate reduction zone. (reproduced from Fenchel and Jørgensen, 1977)


17

4. Anaerobic oxidation of methane: biogeochemistry and ecology

4.1 Stoichiometry and energetic yield

The AOM reaction couples the reaction of methane oxidation to sulphate reduction

according to the following stoichiometry:

CH4 + SO42- ⇌ HS- + HCO3

- + H2O Equation (1)

The free energy that can be harvested by microorganisms from this redox reaction is

ΔG0’= -16.9 kJ mol-1 in standard conditions, but it is estimated that this yield can reach ~40 kJ

mol-1 in situ. In the particular case of syntrophy between a sulphate reducing Bacteria (SRB)

and an anaerobic methanotrophs (ANME) Archaea, the available energy has to be shared

between both partners. These values are very close to the minimum energy yield requirement to

ensure ATP production and maintenance metabolism (4 to 10 kJ mol-1) (Alperin and Hoehler,

2009). Such low-energy way of life has major implications for the ecology of AOM mediating

communities. These microorganisms have a very slow growth rate of 3 to 7 month (Girguis et

al., 2005; Nauhaus et al., 2005). This, together with the apparent syntrophic nature of AOM

hindered successful isolation attempts, making physiological studies difficult. Secondly, such a

low yield implies that the pathways involved can be extremely dependent on the substrate and

product in situ concentrations, i.e. the electron flow through dissimilatory energy conservation

pathway can be reverted in function of the available electron sources and sinks. (See

“methanogenic activity of AOM organisms section” below)

4.2 Geochemistry:

In most sedimentary settings, AOM mediating microorganisms are active in a narrow

depth interval, where both gradients of downward diffusing sulphate from seawater, and

upward migrating methane overlap. Hence, the presence of a sulphate to methane transition

zone (SMTZ) is typical for AOM activity. The depth and extend of this zone is primarily

governed by the flux of methane (Borowski, 1996). In diffusion-controlled environments, the

SMTZ can be located at hundreds of meter below the seafloor, and can spread over ten’s of

meters (see for instance Webster et al., 2009). In high fluid advection environments and high

methane flux, the gradients are very steep and the SMTZ is restricted to a few cm immediately

CHAPTER I

18

below the sediment surface (see for instance Niemann et al., 2006a; Niemann et al., 2006b).

Eventually, when advective fluid flux is too high to allow downward sulphate diffusion,

methane freely escapes the sediment and is partly oxidized by aerobic methanotrophs at the

surface (Niemann et al., 2006a).

4.3 Microorganisms mediating AOM

Three groups of Archaea have been shown to mediate the anaerobic oxidation of methane

(ANME-1, -2 and -3). These ANME cells all belong to the phylum Euryarchaeota and are

closely related to methanogens. Based on the observation of tight consortia of ANME and SRB

using in situ fluorescence labelling (FISH) (Boetius et al., 2000), SRB are thought to function

in syntrophy with ANME, by carrying the sulphate reduction half-reaction in AOM. SRB

associated with ANME cells are related to Desulfosracina Deltapropteobacteria (ANME-1 and

-2 Knittel et al., 2003; Knittel et al., 2005; Schreiber et al., 2010) or Desulfobulbus (ANME-3

Losekann et al., 2007). However, (Pernthaler et al., 2008) have shown that other bacterial

clades could be occasionally involved in consortia with ANME-2 cells.

4.4 Mechanisms.

The biochemical mechanisms involved in AOM is not yet elucidated, highlighting the

continuous need of environmental studies that can bring additional knowledge on the

functioning of these communities. Metagenomic studies have shown that ANME cells posses

all the genes (but mcrA) involved in the sequential reduction of CO2 to CH4 (Hallam et al.,

2004; Meyerdierks et al., 2010) and are thus thought to mediate AOM through reverse

methanogenesis (Hallam et al., 2004; Thauer, 2011). Besides, inhibition experiments have

demonstrated the obligate syntrophic nature of AOM with sulphate reduction, as none of the

two reactions proceeds further when the other one is inhibited (Alperin and Reeburgh, 1985;

Hoehler et al., 1994; Nauhaus et al., 2002). However, neither the metabolic link between AOM

and SR reactions, nor the physical link between ANME and SRB living in aggregates, has been

elucidated. Hypothetically, the diffusion of a chemical carrier would transfer electrons resulting

from methane oxidation in ANME cells to SRB cells where they would be involved in sulphate

reduction. In this hypothesis, which is similar to the well-known interspecies hydrogen transfer

(Stams and Plugge, 2009, and references therein), an increase of ANME-SRB cell distance has


19

a dramatic effect on the thermodynamic yield of the reaction. Ultimately the physical

separation of cells with distance in the µm order (depending on the in situ conditions, Sorensen

et al., 2001) would stop the AOM reaction. To date, the addition of a wide range of possible

electron carrier (Nauhaus et al., 2005) didn’t succeed in uncoupling AOM from SR. An

exception is the interesting proposition of (Moran et al., 2008) of methyl sulphide as a possible

intermediate. This scenario uses a reverse methyltrophic methanogenesis pathway (coupling

CO2 reduction and CH4 oxidation) to produce CH3-SH, the latter being used as electron donor

for SR. Accordingly, Moran et al. found that the addition of CH3-SH inhibited 68% of the

AOM activity. However, this result could apparently not be reproduced in other laboratories

(Knittel and Boetius, 2009). Hence, to date, and in spite of considerable efforts from many

different groups, the fundamental AOM mechanisms remain elusive.

4.5 Methanogenic activity of AOM communities

An increasing number of studies have reported a dual methanotrophic-methanogenic

activity in environmental samples (Orcutt et al., 2005) or in vitro (Treude et al., 2007; Orcutt et

al., 2008b; House et al., 2009). Using parallel 14C-CO2 and 14C-CH4 radiotracer experiments,

these authors found that methanogenesis (MG) was active in AOM zones with MG rates

corresponding to 5-20% of the AOM rates. This finding further support the hypothesis of a

reverse methanogenesis pathway involved in AOM. However, the role of this two-way reaction

is not known. House et al. (2009) found large heterogeneities of carbon isotopic values within

ANME-1 populations. The authors proposed that some cells could have a methanotrophic

metabolism (more negative ∂ 13C values, see section 5 below), whereas other could have a

methanogenic metabolism (less negative ∂ 13C values). Such interpretation is supported by

thermodynamic models (Alperin and Hoehler, 2009) predicting that the presence of

fermentation end products such as acetate or H2 could act as a thermodynamic switch between

AOM and MG: when fermentation processes release H2 and increase its concentration above a

certain threshold, then AOM becomes thermodynamically unfavourable whereas MG becomes

exergonic. Hence, if ANME cell can use the methanogenic pathway in both directions, AOM

should probably be very sensitive to H2 levels. Such energetic constrains have been invoked to

explain the lack of AOM activity in an H2-rich brine pool in the Gulf of Mexico (Joye et al.,

2009)

CHAPTER I

20

4.6 AOM community structure

Thermodynamic constraints on AOM predict that, assuming a chemical electron carrier

hypothesis, there is a direct link between AOM mediating community structure (ANME-SRB

distance) and their activity. ANME-2 cells are mostly found in tight association with SRB cells

in shell type (ANME-2b) or mixed-type (ANME-2a) consortia (Knittel and Boetius, 2009);

Figure I-5, and occasionally as monospecific clusters (Treude et al., 2005c). ANME-1 cells, on

the other hand, seem to be often found as single cells, chains of ANME-1 cells, monospecific

aggregates or in loose association with SRB cells (Figure I-5). It is thus unclear whether

ANME-1 cells require a tight association with SRB. Strikingly, only the mixed-type of ANME-

2a is a thermodynamically sound arrangement, maximizing exchange surface between ANME

and SRB (Alperin and Hoehler, 2009). Using microscale reaction / transport models, these

authors have shown that the formation of the conspicuous shell-type cluster has an energy cost

for both partners. Hence, the reason for the formation of such aggregates could be related to yet

unknown environmental parameters. The finding of ANME cells with a methanogenic activity

(previous section) has the potential to resolve the apparent ANME-SRB cell distance

conundrum, as isolated ANME cells or monospecific aggregates could have a methanogenic

activity, and would thus not need to be in the vicinity of electron sinks such as SRB.

4.7 Microbial ecology

In general, a given methane seep environment, or a depth horizon is dominated by a single type

of ANME cells, thus suggesting the existence of ANME ecotypes (Knittel and Boetius, 2009).

The environmental parameters controlling the AOM community structure are not always clear.

However, several patterns emerge from the numerous environmental studies of methane seeps.

(i) The structure of microbial community and the type of ANME involved seem to be governed

by methane flux. This was evident from studies in Hydrate Ridge for instance, where sediments

bearing high methane flux below Beggiatoa mats and lower flux below Calyptogena fields are

dominated by ANME-2a and ANME-2b respectively (Knittel et al., 2005). Similarly, the AOM

microbial community structure was dependant of the methane flux zonation at the Haakon

Mosby MV in the Barent Sea (de Beer et al., 2006; Niemann et al., 2006a). (ii) ANME depth

distribution (Knittel et al., 2005; Yanagawa et al., 2011) indicates a niche separation between

ANME-1 and ANME-2, as the former are consistently found in the deeper, methane-rich and

sulphate-poor or -depleted sediments, whereas ANME-2 are located at the transition zone. (iii)


21

Hypersaline environments seem to select for ANME-1 and exclude other ANME types (Lloyd

et al., 2006; Yakimov et al., 2007; Niederberger et al., 2010).

4.8 AOM and authigenic carbonate precipitation.

The precipitation of carbonate in methane seeps has been widely observed (Greinert et al.,

2002; Luff and Wallmann, 2003; Mazzini et al., 2004; Luff et al., 2005; Stadnitskaia et al.,

2008) and such carbonate constitute an indication for paleoseeps occurrences (Peckmann and

Thiel, 2004; Birgel and Peckmann, 2008). Striking examples of authigenic carbonate include

the vast chemoherms found off South Carolina on the Hydrate ridge or the massive carbonate

chimneys cropping out the Black Sea sediments (Michaelis et al., 2002). Such phenomenon is

thought to occur due to rapid alkalinity production at high AOM turnover (equation 1). In

addition, based on the analysis of alkalinity production and weak acids/bases exchanges,

(Soetaert et al., 2007) have shown that AOM reactions tend to force a pH evolution toward a

stable value around 7.9, which is above the critical pH for CaCO3 precipitation.

4.9 Open questions and current challenges in the comprehension of AOM mechanism,

ecology and methane budget: a summary.

Although research on methane turnover in marine sediment has occupied a very large part of

marine sciences during the last decade, the comprehension AOM at the ecological, biochemical

and geochemical level remains one of the largest challenges in (marine) microbiology.

• The elucidation of the metabolic pathway involved in the methane oxidation coupled to

sulphate reduction is probably the major question remaining unanswered to date. A growing

body of (meta-)genomic evidence suggest that AOM is mediated by the same enzymes

involved in methanogenesis, but acting in reverse (i.e. the reverse methanogenesis hypothesis).

However, 8 electrons are generated during AOM, and the transport mechanism as well as the

final destination (electron acceptor), are not yet clear. A chemical shuttle such as H2, acetate,

formate or methanol could not be identified. Moreover alternative electron acceptors other than

SO42- (NO2

-, FeIII and MnIV) have been recently discovered but their contribution to AOM

activity in sediments is not known.

• Another metabolic conundrum is the recent discovery of a dual AOM / methanogenic

activity of sediments microorganisms under methanotrophic conditions. Such activity could be

due to different cells having different activities, or a simple back reaction of the ANME

CHAPTER I

22

metabolic pathway involved in methanotrophy. Even more surprising was the conclusion of

microscale modeling studies suggesting that most Archaea at cold seep could in fact be normal

methanogens with few being methanotrophs. Methane production and consumption by methane

seep microorganisms will thus deserve more attention.

• The identification of several Archeal phylotypes that can apparently mediate AOM

raises the question of the existence of ANME ecotypes. The set of environmental conditions, or

niches, selecting for particular ecotypes are poorly constrained and it remains not clear why

certain group dominate particular AOM ecosystems. More generally, environmental factors

besides methane and sulphate concentrations, affecting AOM microbial communities

composition and activity are not well constrained.

• Conversely, the activity of AOM microorganisms can have an enormous impact on their

environments, mostly through the production of sulphide and solid carbonate. The exploration

of deep-sea methane seeps has only started to unveil the diversity and the extent of these

ecosystems, their structure and dynamics, but our current catalogue is probably far from

complete.

• Most of our knowledge on AOM ecophysiology derives from measures carried out at

the sea surface, on board of research vessels upon sediment recovery (ex situ) or in on-shore

laboratories (in vitro). However, very few data exist on the comparison between such data and

the in situ processes. In particular, the metabolic rate at which AOM occurs is proportinal to the

methane concentration in situ. Since such data is usually not directly measureable, a large

incertitude remains concerning the specific energy yield of the AOM reaction and the true

methane turnover in situ.

• Finally, if AOM microbial communities constitute an efficient benthic filter against

methane release in the water column, very little is known about their capacity to cope with

increasing methane flux in a context of ocean warming, acidification and methane hydrate

dissociation.


23

5. Tracking the origin and fate of methane with carbon stable isotopes.

The natural carbon pool contains both stable carbon isotopes 12C and 13C. Biological

assimilation or transformations during the carbon cycle tend to preferentially use the lighter 12C. Hence, organic matter derived from photosynthesis has a typical depletion in 13C (∂13C) of

about -20 ‰ compared to a reference value (0 ‰ is set according to the standard Vienna Pee

Dee Belemnite or VPDB value). Such value differs according to the carbon fixation pathways

used by plants and cyanobacteria: the more common C3 metabolism has an isotopic signature

comprised between -24 and -33‰ whereas the C4 metabolism has a signature between -16 and

-10‰. The crassulacean acid metabolism (CAM) used by plants in arid environments produces

a stable isope pool with values between -20 and -10‰. In turn, methane originating from

organic matter mineralization will further fractionate this pool differently according to the

mechanism of methane formation (Whiticar, 1999): biogenic methane will have a typical ∂13C

of between -50 and -100 ‰ VPDB, whereas methane thermogenesis has a lower fractionation

effect (-25 to -50 ‰). Hence, both the carbon assimilatory (biosynthesis of membrane lipids

and other macromolecules) and dissimilatory (mineralization, methanogenesis, AOM)

processes can be tracked based on ∂ 13C carbon values. In addition to geochemical evidences,

the presence of highly fractionated carbon in archaeal and bacterial membrane lipids provided

the first diagnostic of the microbial nature of AOM (Hinrichs et al., 1999) and is still a high

throughput method for AOM community analysis (Niemann and Elvert, 2008; Wegener et al.,

2008; Rossel et al., 2011). Stable isotope fractionation also potentially permits to discriminate

between methane derived inorganic carbon, dissolved (i.e. DIC) or as solid carbonates, which

have a lighter signature, and other DIC sources. In practice, a natural sample often contains a

DIC/carbonate pool of mixed origin of which the relative contribution can be determined if the

end members values are known.

CHAPTER I

24

Figure I-5 Epifluorescence micrographs of different ANME single cells and aggregates visualized by FISH or CARD- FISH. (a) Single ANME-1 cells living in a microbial mat from the Black Sea. (b) Mat-type consortia formed by ANME-1 (red) and DSS cells (green). (c–e) Mixed-type consortia of ANME-2a (red) and DSS (green) cells observed in different seep sediments. (f-g) Shell-type consortia of ANME-2c (red) and DSS (green) cells and (h) single ANME-2c cells observed in different seep sediments. (i) ANME-3/Desulfobulbus consortia. (picture and caption reproduced from (Knittel and Boetius, 2009)

Figure I-6 16S rRNA genes based phylogenetic tress showing the different deltaproteobacterial (left) and archaeal (right) partners known to be involved in AOM (shown in red). Phylotypes that are suspected to be involved in AOM are shown in blue. Several groups closely related to sulphate reducing bacteria (Seep SRB-1 to -4) are exclusively found in methane seeps, but only SeepSRB1-c (Schreiber et al., 2010) and a group related to Desulfobulbus (Losekann et al., 2007) have been shown to be involved in consortia with ANME cells. The trees where constructed with the maximum likelihood method (see Chapter VI: experimental procedures)


25

6. Mud Volcanoes

6.1 Subseafloor origin and relation to environmental settings

Mud volcanoes (MVs) are positive

relief formed by the extrusion of low-

density subsurface sediment on the

seafloor (Figure I-7). Most MVs are found

in compressional settings, such as

convergent margins (Kopf, 2002). The

pressure build up in the deep sedimentary

layers causes clay mineral dewatering and

tend to fluidize surrounding sediments,

allowing their extrusion through upper

layers. In additions, in convergent settings,

the progressive heating of the buried

sedimentary organic matter leads to the

formation of thermogenic gases and

further contribute to the pressure build-up

and to the fluidization and buoyancy of

the sediment masses through formation of

free gas inclusions. Hence, mud expelled

on the seafloor often contains high amounts of gas, mainly methane, in the form of solute, free

gas, or hydrates, and clasts that where mobilized during the ascent of the fluid through lithified

sediment horizons.

6.2 Fluid conduit

In general, upward fluid migration is allowed or facilitated by the presence of fault

systems (Kopf, 2002). These can either result from tectonic activity, or from diapiric motion of

sediment masses of lower density that are migrating upward and fracturing the overlaying

strata. Such low density masses includes evaporitic (gypsum, halite) or low grain density clay

Figure I-7 Section view of a mud volcanoes related to diapiric activity (left) or fault system (right) in relation to their deep source layer, and their conduit toward the seafloor. (Reproduced and adapted from (Kopf, 2002)

CHAPTER I

26

minerals (kaolinites, smectites, vermiculites) that have the potential to include high amount of

water.

6.3 Methane emission

Mud volcanoes are major escape conduits for deep methane reservoir. It has been

estimated that 30-70 Tg yr-1 of methane are transferred toward the MV surfaces (Kopf, 2002,

2005) However, the fate of this methane is not clear, as most of it can be oxidized by AOM,

and the fraction that escapes the sediment is probably oxidized aerobically in the water column.

In case of gas bubble formation however, considerable amount of methane can reach the

atmosphere (Solomon et al., 2009). Overall, in spite of numerous studies on the gross methane

emission, the contribution of deep-sea mud volcanoes to the atmospheric methane pool is not

yet fully constrained.


27

7. The Gulf of Cadiz.

7.1 Origin and geology of the Gulf of Cadiz.

The Gulf of Cadiz is located at the diffuse boundaries of oceanic and continental margins

on the one hand, and of the Iberian and African plate boundaries, in the prolongation of the E.-

W. Gloria fault, on the other hand. It is delimited by the South Portuguese and Iberian margins,

and the Moroccan margin in the North and East, by the Horseshoe abyssal plain and the

Gorringe Bank in the West, and by the Seine abyssal plain in the South (Figure I-8).

Since the inception of the Irish margin in the Triassic (~230 My), the Gulf of Cadiz

underwent many transformations due to interacting tectonic and depositional processes. The

margin evolved from a passive margin in the Mezozoic to an active margin in the Cenozoic

(Maldonado and Nelson, 1999, and references therein) due to the African and European plates

motion. Tectonic activity is at the origin of major features of the Gulf, such as the thick flysch

on the eastern part, developed during orogenesis of the Gibraltar Arc. Secondly, important

tectonically induced sediment displacement that occurred in the Tortonian led to the formation

of the large allochtonous unit of the Gulf of Cadiz or AUGC (Medialdea et al., 2004;

Medialdea et al., 2009) and constitutes the thickest unit of the -up to 14 km- sedimentary cover.

The AUGC, often referred as olistostrome and of which the origin is still debated (Gutscher et

al., 2002), is composed of huge volume of sediments from the Triassic to Neogene, including

mud and Triassic salt. Moreover, numerous faults and seabed morphologies resembling

acretionary wedges could also be related to compressive tectonic activities, including possible

currently active subduction (Gutscher et al., 2002). The region is undergoing current seismic

activity as witnessed by the >8.5 magnitude Lisbon earthquake in 1755, which together with

the resulting tsunami, almost razed the city.

CH

APTER

I

28

Figure I-8 Situation and geological map of the G

ulf of Cadiz (reproduced from

(M

edialdea et al., 2009)

37"

1::::> ()

10° w gow aow

Mud Volcano

Mud Volcano recently discovered in MVSEISOS Cruise I

rw

Saltdiapir

• •

6° W

Bathymetry (m)

Diapiric ridge

Strike slip fault

~ Thrust

~ AUGC fronts

Gravitational collapse

Fault


29

In term of deposition, seven different sedimentary regimes were identified, from Early

Mezozoic carbonate platform formation to contemporary human cultural regimes (<5000 y)

that influenced sediment deposition in major rivers basins located in the Gulf (Maldonado and

Nelson, 1999).

7.2 Hydrologic regime in the Gulf of Cadiz

The Gulf of Cadiz waters are dominated by two different water masses. The surficial

North-Atlantic waters generally flow eastward with a net inflow into the Mediterranean Sea

(Nelson et al., 1999), possibly as a compensation of a net loss of water by evaporation in the

later. This water mass, of lower temperature and salinity than the Mediterranean seawater has a

relatively lower density and is located in the first 300 m of the water column. Conversely, the

warmer (~13ºC) but more saline Mediterranean outflow waters (MOW) that are flowing

westward through the strait of Gibraltar have a higher density and occupy the zone below 300

m (Nelson et al., 1993). After the strait, the circulation of the MOW is strongly influenced by

the seabed topography that features numerous ridges and valleys, and by the continental slope

that induces a westward density driven current acceleration. Toward the deep abyssal plains in

the West, bottom water temperature decrease toward more typical deep-sea temperatures (~4

ºC).

7.3 Origin and seafloor expression of hydrocarbon migration in the Gulf of Cadiz.

The hydrocarbon source rocks in the Gulf of Cadiz are complex and not fully identified,

in part due to the composite sedimentary structure described above. A mixed thermogenic and

biogenic origin of gases in the migrating fluid, as indicated by the composite methane ∂ 13C

signature (~ -50‰ VPDB) and the relative content of higher hydrocarbons indicate that

multiple sources are involved (Mazurenko et al., 2003; Stadnitskaia et al., 2006; Hensen et al.,

2007) Hence, it is likely that methane and higher volatile hydrocarbons are produced by

thermal cracking of buried organic matter at great depth (between 5 and 14 km below the sea

floor or bsf), at temperature >150ºC, as well as by microbial methanogenesis (Stadnitskaia et

al., 2006; Hensen et al., 2007) possibly originating from Miocene black shales (Medialdea et

al., 2009). The upward migration of fluid and fluidized mud, with velocities up to cm yr-1

(Hensen et al., 2007) is provoked by a combination of compressive tectonic stress, presence of

CHAPTER I

30

deep-rooted faults deriving from tectonic or diapiric activity, and clay mineral dewatering

during the transformation of e.g. illite to smectite. During the ascending migration, fluid can

also be affected by secondary processes such as evaporite leaching leading to enrichment with

regard to Na+, Cl-, Ca2+, SO42-, and admixing with seawater in shallow subsurface (Hensen et

al., 2007).

As a result, numerous seafloor expressions of this fluid migration have been identified in

the Gulf of Cadiz. These have been classified in three main categories (Leon, 2006):

(i) Pockmarks: these circular negative reliefs on the seafloor that can reach up to 800 m

diameter and up to 18 m depth in the Gulf of Cadiz and are formed by the expulsion

of gas from surface sediments (Baraza and Ercilla, 1996; Somoza, 2003)

(ii) Over 50 mud volcanoes have been identified in the Gulf of Cadiz (Figure I-8). They are

characterized by the presence of reduced mud breccia containing gas, occasionally

in the form of hydrates, and hydrogen sulphide as a result of AOM. The

accumulation of erupted material form typical cone shaped positive relief on the

seafloor. These can reach 3500 m in diameter and reach 300 m height (Medialdea et

al., 2009). Seismic studies have shown that the formation of these mud volcanoes is

tightly linked to salt or marl diapiric activity underneath, and to compressive

tectonic-derived fault systems, both providing feeding conduits between the deep-

sited reservoirs and the seafloor (Medialdea et al., 2009).

(iii) Methane derived authigenic carbonate structures, in the form of slabs, crusts,

chimneys, or even massive carbonate mounds (Leon, 2006).


31

8. Carbonate mounds

8.1 A life strategy throughout earth history

The term carbonate mound broadly defines structures built by living organisms on the seafloor

by different processes, at different geological times, but that have in common to create massive

(m to tens of km wide) topographic height composed for a large part of carbonate. This brief

literature review is based on thorough review work of M. Hovland (Deep-Water Coral Reefs,

Unique Diversity Hot-Spots, 2008) and J.-P. Henriet and A. Foubert (Nature and Significance

of the Recent Carbonate Mound Record The Mound Challenger Code, 2009), and the reader is

redirected to this publications for “in-depth” review of the occurrence, distribution, geology

and biology of these structures. The formation of carbonate structures by living organisms, or

bioherms, was first recognised in Archaean rocks (~ -3.5 Gy), where microorganisms formed

carbonate stromatolites. Their number and diversity expended during the Proterozoic, and some

modern examples can still be observed today, e.g. in the Shark Bay (Australia). In these

structures, carbonate formation is related to the mineralisation of organic matter deriving from

cyanobacteria phototrophic activity (Taylor et al., 1993). With the onset of metazoan organisms

in the late Precambrian (~ -650 My), new types of carbonate mounds appeared, involving

sponges, corals or bryozoans. In these mounds, carbonate was derived from the accumulation

of skeletal carbonate, early diagenetic processes associated with biomass mineralization by

microorganisms, and photosynthesis. Hence, behind the relatively simple definition of

carbonate mounds provided a the beginning of this section, resides in fact a complex interplay

of biological activities, spanning from microorganisms photosynthesis and organic matter

mineralisation to ecosystem engineering metazoans, of oceanographic constraints such as

primary productivity, seawater level, temperature and mixing regimes, and of geological

properties of the underlying seabed. However, the unifying trait of these different mound forms

resides in the local accumulation of carbonate resulting from enhanced local biological activity.

8.2 Modern cold-water coral carbonate mounds

In contrary to their tropical counterparts, these corals are azooxanthellate, i.e. they do not

possess algal symbionts, but both reef types are comparable in term of productivity and

biodiversity (Roberts et al., 2006). The development of the cold-water coral reef like structures

CHAPTER I

32

along continental margins may lead to the formation of massive carbonate mounds. As for the

Devonian mounds shown in Figure I-9 and Figure I-10, these mounds seem to result from

various processes including coral growth controlled by hydraulic conditions, locally enhanced

organic matter supply, glacial interglacial alternations, and microbial biomineralisation. An

important step forward in understanding the distribution of these mounds along the continental

margins was the discovery by Dullo et al. (2008) that most thriving cold water corals were

lying within a seawater density envelope of sigma-theta (σθ) = 27.35 to 27.65 kg m-3. This

provided a strong support for a hydraulic forcing on the development of these mounds.

However, based on overlaps between mound distribution and possible subseafloor hydrocarbon

deposits and seepage routes, several authors also proposed an internal forcing hypothesis

connecting the two observations (Henriet et al., 1998; Naeth et al., 2005; Hovland, 2008).

Possible links between hydrocarbon seepage and mound distribution and persistence across

long geological times include methane and other hydrocarbons derived carbonate (See section

4.8), which could consolidate the mound sediment matrix, or provide a hard ground substratum

on the sea floor for the settlement of sessile cold-water corals. The recent drilling of the

challenger mound (Off Ireland) during the integrated ocean drilling program (IODP) leg 307

(Ferdelman et al., 2006) has shown that such mechanisms was unlikely, as a sulphate to

methane transition zone was only present below the mound’s base, and that on mound

carbonate isotopic composition did not support an hydrocarbon origin (Takashima et al., 2009).


33

Figure I-11 Major constituents of cold-water coral carbonate mounds. Left: Large Lophelia pertusa colony (Photo M. Roberts), and Madrepora oculata (Photo U.S. National Oceanographic and Atmospheric Administration / Coral Reef Information System).

Figure I-9 Beauchateau carbonate mound (A), and Fort Condé mud mound (B) in the south of Belgium near Couvin. These Devonian mounds were built by various processes involving sponges, corals, brachiopods, crinoids, iron oxidizing bacteria and probably organic matter mineralisation. Different geological sequences are apparently related to sea water level variations, and changes of bottom water conditions. Reproduced from (Boulvain, 2001).

Figure I-10 Kess-Kess Devonian mounds in the Moroccan anti Atlas. The reef or hydrothermal nature of the mounds is still debated (Cavalazzi et al., 2007). Picture from the Cocarde project. www.cocarde.eu.

CHAPTER I

34

8.3 Carbonate mounds in the Gulf of Cadiz

The El Arraiche mud volcano field is located in the shallow area (700-450 mbsf) of the

Moroccan margin. It features several active mud volcanoes such as Mercator MV, Al Idrissi

MV and Gemini MV, witnessing repeated event of fluid flow and mud extrusions over the last

2.4 Ma, as revealed by seismic data acquired during R/V Belgica and R/V Prof. Logachev TTR

12 cruises (Van Rensbergen, 2005). Mud volcanoes in the El Arraiche field are mainly

clustered along two ridges: Renard and Vernadsky ridges, rising about 300 m above the

seafloorand characterized by steep escarpments on their flanks (Figure I-12, Figure I-13).

These ridges were interpreted as related to fault systems, and thus constitute privileged pathway

for deep fluid up flow. In this context, the discovery of fossil cold-water coral carbonate

mounds on top of the Renard Ridge was of utmost interest, and offered a new opportunity to

address the question of an internal forcing of carbonate mounds. These carbonate mounds are

located close to the inferred hydrocarbon seeps resulting from over pressurized sediment zones

in contact with MV conduits (Figure I-14). In addition, patches of living or dead corals were

also reported near Captain Arutyunov MV (this thesis), Darwin MV (this thesis), Meknes MV

(Hovland, 2008), Rabat MV and Tasyo MV (Akhmanov, 2001), Pipoca MV and Almazan MV

(Somoza, 2003) and Al Idrissi MV (Henriet et al., 2009), suggesting that both cold water corals

carbonate mounds and fluid extrusion though the seafloor are often associated in this region.


35

(LdT: Lazarillo de Tormes MV; DQ: Don Quichote MV)

Figure I-13: Topography of the Pen Duick escarpment and the Renard Ridge. In the S.-W. of the Gemini MV, several carbonate mounds built by cold-water corals crop up from the escarpment (white dots) and apparently root on the base of the what has been interpreted as a fault bounded cliff (Foubert et al., 2008). Methane biogeochemistry was investigated in the Alpha mound (nbr. 292). Numbers refer to the cores recovered during the MSM1/3 cruise aboard the R/V Maria S. Merian (2006). Depth range: -300 mbsl (purple) –700 mbsl (yellow)

Figure I-14: Seismic profiles (data from T. van Weering, NIOOZ, The Nederlands) across the Pen Duick escarpment (PD) and the Gemini MV (GMV), indicating zones of overpressurized sediments (OP) in contact with MV conduits. Inferred seeps (IS) based on the seismic data apparently occur near carbonate mounds. Reproduced from (Hovland, 2008).

Figure I-12 The El Arraiche mud volcano field, located on the Moroccan Margin. The Pen Duick escarpment and the Renard ridge, where several cold-water coral carbonate mounds where discovered, are located within a zone of hydrocarbon seepage, as witnessed by numerous mud active mud volcanoes in the viscinity (reproduced from (Van Rensbergen, 2005)

CHAPTER I

36

9. General goal and outline of the thesis:

Microbial communities mediating AOM are key players in the control of greenhouse gas

emissions, benthic habitats ecology and ecosystem engineering. However, their study is

hampered by their slow growth, the difficulties associated with their isolation, the remoteness

of their deep-sea habitats, and the extreme conditions associated with these habitats. Hence, in

parallel to geochemical, metagenomic or in vitro approaches, integrated studies of natural

methane seep environments have proven to be a successful approach for the comprehension of

the ecology of AOM microbial communities.

This work aims at a better comprehension of the ecology of these communities by

studying AOM microbial ecosystem functioning in different natural environments, e.g.

understanding the feedbacks between environmental parameters, microbial community

structure and relevant microbial activities.

This approach is summarised in the opposite

scheme: with an extensive characterisation of the

different summits (environment, activity,

community) in a range of different deep sea

methane seeps habitats, we aim at understanding

the interactions between them (arrows).

Chapter II addresses the question of a possible internal forcing in the Alpha mound, a cold-

water coral carbonate mounds located in the Pen Duick Escarpment, in the Gulf of Cadiz. The

aim of this chapter is thus to evaluate the presence of methane migration through such

structure, and it’s possible impact on the carbonate budget, the DIC stable isotope signature,

and microbial abundance and activity.

Chapter III focuses on the recently discovered Darwin MV, which is covered for a large part

by a thick carbonate platform. We examine the impact of this unusual structure on the methane

flux and microbial activity distribution. In conclusion we provide a possible model that

integrate the results of this study and all previous observations of this MV. I show that the

concept of “the self-sealing nature of methane seeps” developed first by (Hovland, 2002) may


37

explain the current distribution of AOM microbial activity at this MV.

Chapter IV compares the microbial community structure and methane cycling in the

hypersaline sulphate rich sediments of Mercator MV with the standard salinity sediment of

Captain Arutyunov MV. Based on current knowledge on metabolism selection in hypersaline

environment, it was unlikely to find AOM activity at Mercator MV. I show however that AOM

and SR were active and in spite of an apparent inhibition, consumed a significant part of the

rising methane flux. The presence of sulphate from evaporite leaching stimulated AOM and

compensated the salinity inhibition. Further, the AOM community was remarkable in that

AOM was exclusively mediated by ANME-1 cells, which only proceeded in a methanotrophic

mode, and showed only distant interactions with SRB cells.

Chapter V focuses on the Carlos Ribeiro MV. Methane and sulphate fluxes estimated in a

companion paper by Vanneste et al. (in press) were compared to AOM and SR rates obtained

with radiotracer measurements. I show that reaction transport modelling exceeded microbial

activity by one order of magnitude. However, these conflicting data may result from an

underestimation of the in situ methane pool, as reassessment of the microbial activity based on

in situ methane concentration tend to converge toward modelled methane flux. In addition I

show that archaeal community in the crater centre was dominated by GoM Arc I cells, which

has been only found in mud volcanoes so far and thus constitute a candidate phylotype that

could be involved in AOM.

Chapter VI is a general discussion covering the recent studies on the Alpha mound and other

carbonate mounds in the vicinity in the perspective of the question raised in Chapter II. The

functioning of the different AOM ecosystems in the four MV studied in this thesis are also

reviewed, in term of microbial diversity, community structure and possible AOM mechanism.

Similar experimental procedures were used for the studies described in Chapter III to V. These

are extensively described in Chapter VII.

39

CHAPTER II. Anaerobic

Oxidation of Methane in a Cold-Water

Coral Carbonate Mound from the

Gulf of Cadiz

Redrafted after: L. Maignien, D. Depreiter, A. Foubert, J. Reveillaud, L. De Mol, P. Boeckx, D.

Blamart, J.-P. Henriet and N. Boon. Anaerobic oxidation of methane in a cold-water coral

carbonate mound from the Gulf of Cadiz. International Journal of Earth Sciences, In press

CHAPTER II

40

AOM IN THE CARBONATE MOUND ALPHA

41

ABSTRACT

The Gulf of Cadiz is an area of mud volcanism and gas venting through the seafloor. In

addition, several cold-water coral carbonate mounds have been discovered at the Pen Duick

escarpment amidst the El Arraiche mud volcano field on the Moroccan margin. One of these

mounds -named Alpha mound- has been studied to examine the impact of the presence of

methane on pore-water geochemistry, potential sulphate reduction (SR) rate and dissolved

inorganic carbon (DIC) budget of the mound in comparison with off-mound and off-

escarpment locations. Pore water profiles of sulphate, sulphide, methane and DIC from the on-

mound location showed the presence of a sulphate to methane transition zone at 350 cm below

the sea floor. This was well correlated with an increase in SR activity. 13C-depleted DIC at the

transition zone (-21.9 ‰ vs. Vienna Pee Dee Belemnite) indicated that microbial methane

oxidation significantly contribute to the DIC budget of the mound. The Alpha mound thus

represents a new carbonate mound type where the presence and anaerobic oxidation of

methane has an important imprint on both geochemistry and DIC isotopic signature and budget

of this carbonate mound.

CHAPTER II

42

1. Introduction

Cold-water corals are commonly found on the continental margins in a wide range of depth and

latitude (Roberts et al., 2006). Unlike their tropical counterparts, cold-water coral are

azooxanthellate and thus do not rely on the activity of a photosynthetic symbiont to develop,

but rather on local supply of organic matter. Under favorable environmental conditions (Dullo

et al., 2008), these organisms can form important carbonate structures or carbonate mounds

remaining in the geological records (De Mol, 2002; Kenyon, 2003; Wheeler et al., 2007; Frank

et al., 2009). They are mainly composed of the stony corals species Lophelia pertusa and

Madrepora oculata (De Mol, 2002), which constitute thriving habitats for numerous benthic

species through the development of reef-like frameworks (Costello, 2005). Cold-water

carbonate mounds have thus been recognized as hotspot ecosystems on the continental margins

(Weaver, 2004). Several mound provinces have been described on the southwest Irish margin

where they often host prosperous ecosystems with living corals on their surface (Porcupine

bank and Rockall through) (Henriet et al., 1998; Huvenne, 2002; Kenyon, 2003). In this area,

carbonate mounds are situated between 500 and 1000 meters water depth, rising above the

seafloor from a few meters to 250 meters and some of them are 2 kilometers in diameter. In the

Belgica mound province (Porcupine bank), the first scientific drilling of a carbonate mound

(Challenger mound) during the Integrated Ocean Drilling Program (IODP), leg 307 (Ferdelman

et al., 2006) showed that such mounds may constitute an important carbonate sink (Titschack et

al., 2009), and constitute a unique microbial habitat compared to other sub seafloor

environments (Webster et al., 2009). First results from this expedition indicated that the

geochemistry and diagenetic processes within the Challenger mound are not influenced by

methane and other hydrocarbons (Ferdelman et al., 2006).


43

The Gulf of Cadiz is an important zone of hydrocarbon-rich fluids migration. Tectonic

compression and Olistrostrome development led to the formation of numerous mud volcanoes,

diapiric cones and ridges, and pockmarks with several occurrences of gas hydrates (Kenyon,

2002 ; Pinheiro, 2003; Somoza, 2003; Van Rensbergen, 2003, 2005; Kenyon, 2006; Leon,

2006). Methane migration and its subsurface removal due to microbial anaerobic oxidation of

methane (Niemann et al., 2006b) led to the formation of authigenic carbonates as attested by

the presence of 13C depleted clasts, slabs or chimneys (Leon, 2006). Recently, several fossil

cold-water coral carbonate mounds -almost devoid of living corals- have been described in this

area suggesting that these margins once provided suitable conditions for the development of

cold-water coral carbonate mounds (Van Rensbergen, 2003, 2005; Foubert et al., 2008). The

discovery of such mounds on the top of the Pen Duick escarpment (Foubert et al., 2008) in the

Al Arraich mud volcano field is of particular interest to study mound formation processes and

sedimentary diagenesis in a context of hydrocarbon migration.

In marine sediments, microorganisms mediating Anaerobic Oxidation of Methane (AOM)

indeed consume methane at the expense of sulphate according to the equation (Hoehler et al.,

1994; Nauhaus et al., 2005):

CH4 + SO42- HCO3

- + HS- + H2O (equation 1)

When present, AOM has a strong impact on the overall geochemistry and diagenetic processes

in the sediment. In particular, the production of carbonate alkalinity can lead locally to

carbonate precipitation (Ritger et al., 1987; Luff and Wallmann, 2003; Luff et al., 2004;

Stadnitskaia et al., 2008)

In this study, we describe methane related geochemical processes in the Alpha mound, located

at the Pen Duick escarpment in the Gulf of Cadiz, as well as two reference locations off-mound

and off-escarpment. We show that unlike other carbonate mounds described so far, methane is

CHAPTER II

44

present in the sediments of the Alpha mound, and a front of AOM at 350 cmbsf has a profound

influence on both the geochemistry and the DIC budget of this mound.

Figure II-1 The Gulf of Cadiz (A) is a zone of mud volcanisms, spanning from Portuguese and Spanish margin in the north and Moroccan margin in the east, to Atlantic abyssal plains in the west. The zone of study (B, 5m contour line spacing) is located in the Al Arraich mud volcano field, on the Moroccan margin. Several cold-water coral carbonate mounds are located along the Pen Duick escarpment (delimited by dashed lines in B and C), a 4km long and 80 m high fault-bound cliff. The Alpha mound is a 30m high carbonate mound in the eastern part of the escarpment. Note the presence of Gemini and Lazarillo de Tormes mud volcanoes in the vicinity of the escarpment. C: maps of the escarpment showing that the cold-water carbonate mounds are rooting on the plateau in the N. E. side and on the foot of the escarpment in the S.W. side.


45

2. Results

2.1 Site description

The Pen Duick escarpment, a 4 km long fault-bounded cliff on the western part of the

El Arraiche mud volcano field (Moroccan margin, Figure II-1A and B), was mapped by

multibeam bathymetry during the CADIPOR Cruise 2002 on board of the R/V Belgica (Van

Rensbergen, 2005). The escarpment’s base is at about 700 mbsl (meters below the sea level),

and rises to a plateau at 520 mbsl. Fifteen carbonate mounds of height ranging from 20 to 60

meters crop out of the top of this cliff (Figure II-1C). The Alpha mound is located at the

southeastern end of the escarpment, near the Gemini mud volcano. It is 50 m high with a slope

inclination between 15° and 25°. The on-mound station was located near the mound summit,

and cold-water coral debris constituted a large part of the recovered sediments (Foubert 2008).

The off-mound station was located on the escarpment next to the base of the mound, and the

off-escarpment station, 2 km away from the escarpment (Figure II-1B).

Table II-1 Location and feature of the cores used in this study

CHAPTER II

46

2.2 Pore water geochemical profiles.

On-mound, sulphate concentration decreased with depth, forming a gradient from sea-

water concentration values (28 mM) at the sediment-water interface to complete depletion at

400 cm below the sea floor (cmbsf) (Figure II-2A). This concentration gradient corresponded

to a downward sulphate flux of 54.0 mmol m-2 yr-1 (Table II-2). Conversely, methane

concentration increased with depth, forming a gradient from complete depletion at 300 cmbsf

to 2.0 mM at the bottom of the core (580 cmbsf). This concentration gradient corresponded to

an estimated upward methane flux of 17.0 mmol m-2 yr-1. These two gradients defined a

sulphate to methane transition zone (SMTZ) between 300 and 400 cmbsf. Coinciding with this

SMTZ, a sulphide concentration peak was observed with maximum sulphide concentration of

3.6 mM at 350 cmbsf.

On-mound, Dissolved Inorganic Carbon (DIC) increased with depth from 1 mM at the

sediment surface up to 32.0 mM at the SMTZ whereas δ13C-DIC decreased with depth, with the

lowest value (–21.9 ‰ vs. Vienna Pee Dee Belemnite or VPDB) coinciding with the SMTZ.

Below, δ13C-DIC remained stable around -16‰.

In order to gain further knowledge on the origin of methane present on-mound, the

carbon stable isotopic signature of methane were measured at the bottom of the on-mound core:

methane 13C depletion was -51.1 ‰ at 580 cmbsf, -51.8 ‰ at 530 cmbsf and -52.2 ‰ at 500

cmbsf.


47

Figure II-2 On-mound (A) pore-water geochemical profiles of sulphate, methane, sulphide, DIC and δ13C-DIC as well as SR rates (SRr) and cell counts. Off-mound (B) and off-escarpment (C) pore-water sulphate, methane, sulphide profiles. : data from MD04 cores. : data from MSM1/3 core (Table II-1). The two datasets are similar and indicate that the cores used for geochemical measurement and activity test correspond to the same setting. Dashed lines indicating estimated gradients were fitted manually.

A SMTZ was also present at off-mound and off-escarpment locations (Figure II-2B

and C), but deeper in the sediment, in the 500-900 cmbsf , and 1000-1500 cmbsf respectively.

Sulphate concentration gradients corresponded to downward sulphate fluxes of 22.6 and 12.5

mmol m-2 yr-1 respectively (Table II-2). Methane was present in the deeper part of the core at

both locations, with maximum concentration of 0.9 mM and 0.6 mM respectively. At both

sites, sulphide concentration showed a maximum within the SMTZ.

CHAPTER II

48

2.3 On-mound location: in vitro SR rates and cell counts

The SR rate measurements revealed the presence of relatively high but scattered SR between

50 and 300 cmbsf. Relatively low or no SR activity was detected between 300 and 450 cmbsf

with a discrete peak (0.09 nmol/cm3/day) at 350 cmbsf. The latter coincided with the sulphide

increase at the SMTZ (Figure II-2). In the upper interval, depth integrated rates corresponded

to a sulphate conversion of 126.0 mmol m-2 yr-1, and 4.6 mmol m-2 yr-1 in the lower zone.

In order to assess the impact of the presence of an AOM activity zone on the microbial

community within the on-mound sediments, we performed cell counts by direct microscopic

observation of fluorescently stained cells (AODC). Direct cell counts showed that cell number

decreased between sediment surface and 50 cmbsf and then remained constant around 2x108

cells cm-3 of sediment, down to 350 cmbsf. Below, the cell number decreased to 6x107 cells cm-

3 at 420 cmsbf.

Table II-2 Estimated concentration gradients, diffusive fluxes and methane / sulphate flux ratio for the on-mound, off-mound and off-escarpment cores. Concentrations are given in mM in pore-water.


49

3. Discussion

3.1 Anaerobic oxidation of methane in Alpha mound.

Our results show that methane was present in shallow subsurface sediment of the Alpha

mound. On-mound, migrating methane was anaerobically consumed forming a SMTZ

associated with a peak of sulphide at the interface between 300 and 400 cmbsf. Such SMTZ

typically results from the microbialy mediated AOM reaction coupled to SR (Iversen, 1985;

Niewohner, 1998). The presence of a microbial community carrying AOM was supported by

the observation of a SR activity peak coinciding with the SMTZ. In addition, DIC was

produced at this depth and displayed a typical light 13C signature. Therefore, the presence of

methane and its consumption had a significant impact on both the geochemistry and microbial

activity of the Alpha mound sediment. Beyond the parameters analyzed in this study, AOM is

likely to alter other biogeochemical processes: previous report have indeed shown that i) AOM

derived sulphide can reduce Fe III and form stable complexes with Fe II, thus reducing the pool

of reactive iron available for diagenetic processes such as dissimilatory iron reduction

(Thamdrup et al., 1994; Jorgensen et al., 2004; Poulton et al., 2004; Lovley, 2006). ii/ AOM

derived DIC can precipitate and form authigenic carbonate, a well documented process at cold-

seep settings (Aloisi et al., 2002; Luff and Wallmann, 2003; Luff et al., 2004). However, less

is known about methane-derived carbonate in deeper SMTZ and lower methane flux. In such

situation, the effect of DIC production can be balanced by diffusion in sediment and

precipitation due to supersaturation is less likely to occur. Yet, authigenic carbonate (nodules)

in a geochemical environment comparable to Alpha mound has been described for instance in

the Gulf of Mexico (Ussler III, 2006; Chen et al., 2007) or as 13C depleted carbonate in the

sediments of the Chilean margin (Treude et al., 2005b). Therefore, carbonate formation due to

methane derived DIC is also probably occurring in Alpha mound.

CHAPTER II

50

3.2 Origin of methane and DIC budget in the Alpha mound.

Considering that thermogenic methane has a typical δ13C signature between -50 and -

20‰ (Schoell, 1980, 1988) and biogenic methane in marine sediment, a signature between -110

and -60‰ (Rice and Claypool, 1981; Schoell, 1983; Whiticar et al., 1986; Whiticar, 1999), we

can therefore take an average of -35‰ for the former and -85‰ for the latter. According to

these end-member values, on-mound methane with δ13C =-51.8‰ would thus originate for 66%

from a thermogenic source. These results are conform with previous studies on the origin of

methane in the Gulf of Cadiz methane seeps (Stadnitskaia et al., 2006), indicating a mixed but

dominantly deep thermogenic origin of migrating methane. Therefore, the main source of

methane responsible for the formation of a SMTZ and the AOM activity in the Alpha mound

does not derive from microbial methanogenesis in the mound itself, but rather from deeper

sources.

Further, the contribution of AOM to the total DIC budget in the on-mound core can be

estimated based on a simple two sources isotope-mixing model involving AOM derived DIC

and non-AOM derived DIC. The mixing of these two sources results in a bulk DIC with an

intermediate isotopic signature. In this model:

(Equation 2)

(Equation 3)

where FAOM and Fnon-AOM are the contribution of each source (in %) and δ13C-DICAOM and δ13C-

DICnon-AOM, the isotopic signature of each source. As shown in Figure II-2 , all methane was

consumed during AOM reaction, excluding fractionation effect between the source methane

and the resulting DIC. Hence, AOM derived DIC has a similar δ13C than its methane source,

e.g. δ13C-DICAOM= -51.7 ‰. Besides AOM, the main DIC sources derive from nanofossils

calcite oozes (coccolith and forams) and coral fragments (Foubert et al., 2008). The former

displays values between 0 and -2‰ as measured in two close locations (Foubert et al., 2008).


51

The later is mainly composed of aragonite and typically displays ∂ 13C values comprised

between 0 and -10‰ within single fragments (Blamart, 2005). Hence a range of 0 to -10‰ is a

safe approximation for non-AOM DIC isotopic signature in this carbonate mounds. Using these

values in the isotope-mixing model, AOM accounted for 28-42% of bulk DIC at the SMTZ,

and 14-30% below this depth. AOM thus significantly contributed to the DIC budget in the on-

mound core.

3.3 Fluxes and rates

On-mound, sulphate consumption was in good agreement with the sulphide production (54 and

47 mmol m-2 yr-1 respectively) based on diffusion model. Highest sulphide concentrations were

observed in the SMTZ and AOM was likely to provide most of sulphide observed in this

location. Based on the stoechiometry of the AOM reaction (equation 1) and on both methane

and sulphate flux estimation, it is also possible to determine the contribution of methane

oxidation to the overall SR in the on-mound location. Methane flux estimation should however

be interpreted with caution as degassing during recovery and sampling likely occurred and

because of a low-resolution sampling in the SMTZ. In these conditions, it was difficult to infer

both methane flux and migration regime and thus the actual AOM contribution to the overall

SR. Based on the data available, we estimated that 33% of the diffusing sulphate (e.g. 17.8

mmol m-2 yr-1) was consumed by the AOM reaction.

The peak of SR activity (4.6 mmol m-2 yr-1) at the SMTZ (-350 cmbsf) was interpreted as AOM

dependant SR, whereas the activity between 50 and 300 cmbsf was likely due to organoclastic

SR. The AOM dependant SR was certainly underestimated, as AOM strongly depends on

methane concentration (Nauhaus et al., 2005). During in vitro incubation, the methane

concentration corresponded to saturation at 1 bar and 4°C (~1.4 mM), whereas in situ this

concentration can potentially reach ~60 mM (Duan and Weare, 1992) This difference between

CHAPTER II

52

in situ and in vitro conditions can explain the discrepancies observed between AOM dependant

sulphate consumption based on potential rates measurements (4.6 mmol m-2 yr-1) and on

methane flux calculation (17.8 mmol m-2 yr-1).

The sulphate flux was higher on-mound than off-mound and off-escarpment (Figure II-2).

Downward sulphate fluxes are usually driven by both organoclastic and AOM dependant SR

when methane is present. Off-mound and off-escarpment, organic content due to sedimentary

processes were probably similar, given the proximity of these two stations. Hence, differences

between sulphate fluxes in these locations were mainly controlled by methane. Our results

suggest that the methane flux was thus higher on the escarpment than in the surrounding

sediments. Such enhanced methane migration could be related to the fault system underlying

the escarpment and/or to mud volcanic activity as mud breccia and clasts have been observed at

170 cmbsf in the flank of the mound (Core AT570G, Foubert et al., 2008). Sulphate profiles

presented in this study are comparable to other locations where a SMTZ due to the presence of

methane has been observed, such as in the Blake Ridge sediments (Borowski, 1996, 2000), in

the Kattegat and Skagerrak sediments (Iversen, 1985), in the upwelling of Namibia

(Niewohner, 1998), or on the Chilean margin (Treude et al., 2005b).

Parkes et al. (Parkes et al., 1994; Parkes et al., 2000) determined an average of the prokaryotic

cell number as a function of sediment depth based on their observations in various sedimentary

settings. Total cell counts in Alpha mound sediment remained close to this average and in all

cases, within the interval containing 95% of the counts by Parkes et al.. Moreover, cell counts

within the AOM zone did not significantly differ from counts above and below this zone.

Therefore, the presence of a microbial community mediating the AOM reaction did not

stimulate the overall cell number in the on-mound sediment as compared to sediment without

methane. These observations are coherent with several previous studies: cell counts did not

increase within a deep SMTZ (150-200 mbsf) in the Porcupine seabight (Webster et al., 2009),


53

nor did it in a shallower SMTZ’s (150-200 cmbsf) in the Black sea sediments (Knab et al.,

2009). These observations probably reflect the fact that AOM mediating microrganisms have

very low growth rate (Girguis et al., 2005; Nauhaus et al., 2007) and although they may

metabolize substrates at high rates, they do not induce an overall cell number increase in

sediment horizons where AOM occurs, at least in low methane flux environments.

3.4 Comparison with other mound settings

The recent drilling of the Challenger mound during the Integrated Ocean Drilling Program

(IODP) Expedition 307 on the S.W. Irish margin (Ferdelman et al., 2006; Webster et al., 2009)

constitutes a reference for comparison with the Gulf of Cadiz Alpha mound. Both mounds are

outcropping structures built by reef forming cold water corals and lie in similar depths on the

continental margin. Challenger mound is higher (130 m) and wider (1000 m at its base) than

the Alpha mound in this study (30 m and 200 m respectively). In both locations, sulphate

gradients from mound summit down to a SMTZ have been observed. In the Challenger mound

however, the transition zone is wider (50 mbsf) and much deeper (150-200 mbsf) than in Alpha

mound, and is located below the mound’s base. As a consequence, it has been suggested that

AOM did not participate in the mound formation processes, and organoclastic SR may drive

the carbonate diagenesis in the Challenger mound. In contrast, our results showed that AOM

driven SR is an important microbial process in the on-mound location. Hence, Alpha mound

represent a new mound type where reef-framework forming scleratinians and methane driven

DIC production -and probably carbonate precipitation- both contribute to the mound processes.

CHAPTER II

54

4. Conclusion

To our knowledge, we describe here the first occurrence of a cold-water

carbonate mound coininciding with current hydrocarbon migration. Methane migration and

anaerobic oxidation in this mound markedly shaped the geochemistry of the sediments and

participated to the DIC production in the on-mound location. Alpha mound therefore

constitutes an original mound setting where methane-related diagenesis participated to the

mound’s processes. Further studies on the Gulf of Cadiz carbonate mounds should provide new

insights on the initiation and development of cold-water coral carbonate mounds in a context of

hydrocarbon migration. In particular, the recovery of the entire mound sediment sequence and

the mound base would allow to study the time scale of both coral development an hydrocarbon

migration, and to examine if methane derived authigenic carbonate could facilitate the

settlement of these corals. Besides, to generalize the present results to the entire mound, other

on-mound coring is needed to assess the heterogeneity of such methane related processes

within the mound. These questions are part of the rational for the recently submitted Integrated

Ocean Drilling Program pre-proposal 673 for a scientific drilling of the Alpha mound:

“Atlantic Mound Drilling 2: Morocco Margin”.


55

5. Material and methods

5.1 Sampling procedure

For sulphate, methane and DIC measurements, three cores were taken in June 2004

during the oceanographic cruise PRIVILEGE by the R/V Marion Dufresne (Table II-1). On-

mound, a large square-section (25x25 cm) Kasten type core (CASQ core MD04-2804) was

recovered from the top of the Alpha mound. In addition, giant “calypso” piston cores were

deployed at two reference locations: on the escarpment (off-mound core MD04-2809) and at

the foot of the Pen Duick escarpment (off-escarpment core MD04-2806). The CASQ core was

sampled within 4 hours upon recovery at deck ambient temperature (15°C). Piston cores were

split in halves and sampled within 10 hours after recovery.

From each core, between 2 and 8 mL of pore-water were taken each 20 cm using Rhizon

membranes (standard pore size 0.1 µm) (Seeberg-Elverfeldt, 2005) plugged into glass vacuum

tubes (Eijkelkamp, Giesbeek, The Netherlands). Samples were then stored and transported at –

20°C.

In addition, gravity core MSM1/3-294 was taken at the same on-mound location during the

MSM1/3 cruise in may 2006, aboard the R/V Maria S. Merian. Upon recovery, the core was

cut in sections and stored in a cold room immediately. Pore-water was extracted by plugging

Rhizons membranes into predrilled sampling port in the plastic core liner. After pore-water

collections, the core sections where stored at 4°C in trilaminate PE/Al/PE bags under anaerobic

conditions for further in vitro studies in the home laboratory.

CHAPTER II

56

5.2 Gas analysis

For methane and light hydrocarbons measurements, a “headspace method” adapted to

Rhizon sampling was used: each pore-water tube was thawed and shaken to allow dissolved gas

to be transferred to the headspace. The tubes were weighed to determine the amount of pore-

water collected. After equilibration of the headspace to ambient pressure with Nitrogen, 1 mL

of headspace was injected in a gas chromatograph (Carlo Erba Instruments, Wigan, UK)

equipped with a Varian PoraplotQ (25m x 0.53mm) column and a flame ionization detector.

The column temperature was initially set at 30°C for 3 min. and was then increased to 90°C

with a 30°C/min. rate, and finally to 190 °C with a 10°C/min. Headspace concentrations were

determined using a standard mixture and pore-water gas concentrations were calculated

according to the amount of pore-water collected in each tube. For dissolved inorganic carbon

(DIC) measurements, 1mL of pore-water was acidified with 0.33 mL of phosphoric acid

(Borowski, 1996) in 5 mL gas-tight argon-flushed bottles. 1 mL of the headspace was injected

in a Shimazu gas chromatograph (GC-14B) equipped with a Hayesep D column (Alttech

Associates, Lokeren, Belgium) and a thermal conductivity detector.

5.3 Carbon Isotopes

Delta13C-CH4 was measured from pore-water sample headspace, and δ13C-CO2 from

DIC bottle headspace. One mL of gas at the appropriate dilution was analyzed, using gas

cryotraping and cryofocusing (and subsequent combustion to CO2 for CH4) on a trace gas

preparation unit (AHCA-TGII, PDZ-Europa, UK) coupled to an isotope ratio mass

spectrometer (IRMS) (20-20, Sercon, UK). The δ13C value of the laboratory reference was -

36.9 ‰ and was measured against reference CH4 (Air Liquide, Belgium) with a δ13C of -38.4


57

‰ determined gravimetrically. δ13C results are expressed in the Vienna Pee Dee Belemnite

(VPDB) scale.

5.4 Sulphate and sulphide concentration

Sulphate concentration in pore-water was measured by anion exchange chromatography

(Gieskes, 1991) with a IC 761 (Metrohm, Herisau, Switzerland). The sulphide concentration

was measured by the colorimetric method (: 0.5 mL of pore-water preserved in a zinc acetate

solution was mixed with 0.4 mL of Cline Reagent under anoxic conditions. After 20 minutes,

the optical density was measured at 670 nm (UVIKON930 Spectrophotometer).

5.5 Flux calculation

In order to estimate the flux of sulphate and sulphide in the three locations, the different

gradients were considered linear, corresponding to steady-state situations. In this case, the

estimated gradient obtained by linear regression of the curve is introduced in the Ficks’s first

law:

(1) With (equation 1)

Where J is the flux (mmol m-2 yr-1), Ds is the sediment diffusion coefficient (cm2 yr-1) corrected

according to porosity and sea bottom temperature (Boudreau, 1997), D0 is the molecular

diffusion coefficient, φ is the porosity of the sediments, and ΔC/Δx is the estimated gradient

(represented by dashed lines in pore-water profiles, Figure II-2). Porosity was determined

from the weight loss per volume of wet sediment after drying at 60°C for 48 hours. Porosity

values at the sulphate to methane transition zone were used for flux calculation.

CHAPTER II

58

5.6 In vitro SR rates

Sediment slurries (12 mL, final sediment dilution 1/6) were prepared with sediment

from core MSM1/3-294 from each 25 cm interval along the core, and each 10 cm within the

sulphate to methane transition zone (SMTZ) according to methods described by (Nauhaus et

al., 2002). Each slurry was incubated at 4°C in triplicate in presence of 5µl of 35S-SO42- (60

kBq) and dissolved methane as sole electron donor. After 118 days, sulphate turnover was

determined by the cold distillation method according to (Treude et al., 2003)and (Kallmeyer et

al., 2004). SR rates were calculated according to:

(equation 2)

where TRI35S is the activity (in Bq) of total reduced inorganic sulphur intermediate, 35SO42- the

activity of sulphate, [SO42-] the sulphate concentration and t the incubation time in days. Results

are given as potential rates per cm3 of undiluted sediment.

5.7 Direct Cell Count

Acridine Orange Direct Counts (AODC) on sediment from core MSM1/3-294 was

carried following the method described in (Fry, 1988): On board, 1 cm3 sediment plug was

taken with sterile 5 mL cut-off syringe and transferred in a sterile vial containing 4 mL of filter

sterilized (0.2 µm) 2% formaldehyde in artificial seawater. In the laboratory, 15 µl of this slurry

was filtered through a 0.2 µm black polycarbonate membrane (Millipore, Belgium) mounted on

a filtration column. The sediment was then stained on the membrane with 1 mL of a 5 mg.l-1

acridine orange solution (Sigma, Belgium) for 1 minute, and rinsed with 3 mL of sterile

artificial seawater. The membrane was dried and mounted on a microscope slide with a

minimum of paraffin oil. Slides were viewed under blue excitation light in an epifluorescence


59

microscope (Zeiss, Germany). A minimum of 100 cells was counted per membrane. At least

two membranes per sample were prepared and results are given as average of duplicate counts.

CHAPTER II

60

ACKNOWLEDGMENTS

This project was funded by the EURODOM research training network program from the

European Commission under the 5th framework program and by the European Science

Foundation “Microsystems” project. LM, DD, and LDM were recipient of a scholarship from

the Institute for the Promotion of Innovation through Science and Technology in Flanders

(IWT-Vlaanderen). The authors wish to thank the crew and shipboard scientific party of the

R/V Marion Dufresne led by Y. Balut (IPEV, Institut Paul Emile Victor) and of the R/V Maria

S. Merian led by O. Pfannkuche (IFM-Geomar, Kiel). We are grateful to A. Stadnitskaia for

reviwing this work and we thank L. Wehrmann, P. de Schryver, and W. de Muynck for their

help during manuscript preparation.


61

63

CHAPTER III. Heterogeneous

Methane Geochemistry and Anaerobic

Oxidation in the Carbonate-Sealed

Darwin Mud Volcano, Gulf of Cadiz.

To be submitted as: L. Maignien, R. J. Parkes, B. Cragg, H. Niemann, K. Knittel, S. Coulon, A.

Akhmetzhanov, P. Weaver, N. Boon. Anaerobic oxidation of methane and carbonate sealing of

the Darwin Mud Volcano (Gulf of Cadiz).

CHAPTER III

64

AOM ACTIVITY AT DARWIN MV

65

ABSTRACT

The Darwin mud volcano (MV) is a newly discovered cold seep in the Gulf of Cadiz (East

Atlantic margin) that displayed a conspicuous structure. The MV crater was covered by a thick

methane-derived carbonate platform of ca. 8000 m2, colonized by chemosynthetic bivalves. Such

structure prevented the methane-laden fluid to escape through the crater and induced seep

relocation at the rim of the carbonate platform. There, strong spatial variations in methane flux

and anaerobic oxidation (AOM) were observed, suggesting that relocation occurs through

preferential channeling pathways. In particular, we identified a microbial activity hot spot

bearing maximum AOM rate of 260 nmol cm-3 d-1 at 1.7 cm below the sediment surface. This

was twice as high than 2 m away, and three orders of magnitude higher than in other stations

around the central crater. There, the AOM microbial community was dominated by ANME-

2/DSS consortia that formed typical shell type aggregates, contrasting with other ANME cells

that were present as single cells. Our seafloor and biogeochemical observations are consistent

with the concept of cold seeps self-sealing by methane –derived carbonates, and a development

model for this MV is proposed in the context of this hypothesis.

CHAPTER III

66

1. Introduction

In marine cold seeps, methane migrating toward sediment surface is oxidized by anaerobic

oxidation of methane (AOM) coupled to sulphate reduction (SR):

CH4 + SO42- => HS- + HCO3

- + H2O equation 1

The formation of a sulphate to methane transition zone (SMTZ) resulting from the local

consumption of both substrates is a typical proxy for the presence and activity of AOM

mediating microorganisms. Moreover, the production of bicarbonate and alkalinity during the

AOM reaction supports carbonate precipitation (Berner, 1980):

Ca2+ + 2 HCO3- CaCO3(s) + CO2 + H2O equation 2

Authigenic methane-derived carbonate formation at cold seeps has been extensively

studied (Greinert et al., 2002; Luff and Wallmann, 2003; Mazzini et al., 2004; Luff et al., 2005;

Reitner et al., 2005; Stadnitskaia et al., 2008; Bahr et al., 2009): carbonate crusts are formed at

the AOM activity front, and grow downward due to the activity of microbial mats carrying AOM

on their lower surface. The accumulation of methane-laden fluid below the crust may lead to

horizontal fluid migration followed by seep relocation at the rim of the crust (Naudts et al.,

2010). Carbonate formations can also take the shape of slabs (Mazzini et al., 2004) or chimneys,

depending on the structure of the sediment layers or fluid channeling pathways (Mazzini et al.,

2008). In extended zones of high fluid flux, such mechanisms can lead to the formation of

massive carbonate chemoherms (Bohrmann et al., 1998; Greinert et al., 2001).

AOM is mediated by Archaea closely related to methanogens of the orders

Methanosarcinales (ANME-2 and -3) and Methanomicrobiales (ANME-1) often co-occuring in

consortia with sulphate reducing bacteria (SRB) of the genera Desulfosarcina/Desulfobulus

(Knittel and Boetius, 2009, and reference therein). These observations led to the hypothesis of a

syntrophic relationship between both cell types (Hoehler et al., 1994; Boetius et al., 2000).

In the Gulf of Cadiz, complex tectonic activity (Gutscher et al., 2002; Medialdea et al.,

2009), olistostrome development (Maldonado and Nelson, 1999; Medialdea et al., 2009), and salt

diapirism (Fernandez-Puga et al., 2007) are at the origin of hydrocarbon migration through the

seafloor, and of the formation of numerous seep related structures (Somoza, 2003) such as mud

volcanoes (MV) (Pinheiro, 2003; Van Rensbergen, 2005, Chapter IV and V; Niemann et al.,

2006b), and hydrocarbon-derived carbonate (Diaz-del-Rio et al., 2003; Leon, 2006; Magalhaes


67

and Pinheiro, 2009). Recent exploration (Kopf et al., 2003; Masson et al., 2006; Weaver and

Masson, 2007) shed light on the Darwin MV, a new seafloor structure at ~1100 m water depth

that differs from other Gulf of Cadiz MV studied to date in exhibiting a massive carbonate

platform in its center. Colonisation by chemosynthetic bivalves (Genio et al., 2008; Vanreusel et

al., 2009) suggests that fluid migration and sub seafloor microbial processes are still active.

The aim of this study is to assess the current geochemical and microbial activity in term of

methane turnover at this original MV, to relate these to the microbial community structure and to

the description of meiofaunal assemblages in the companion paper of (Pape et al., in press). We

propose a development model for this MV that accounts for the different observations of Darwin

MV and uses previous studies on carbonate development and fluid dynamics at cold seep.

CHAPTER III

68

2. Results

2.1 Sampling site

Darwin MV was located on the Moroccan Margin, at 1100 m water depth (Figure III-1A). This

structure was mapped by high-resolution bathymetry using the remotely operated vehicle (ROV)

ISIS during the JC10 cruise aboard the R/V James Cook in 2007. It was 55 m high, 900 m wide

at the base and had a distinct “crater” of about 100 m in diameter. Its structure was markedly

different from other MV’s described thus far in the Gulf of Cadiz in that a thick carbonate

platform covered the crater (Figure III-1B). Chemosynthetic bivalves (Bathymodiolus

mauritanicus, (Genio et al., 2008)) colonized the numerous fissures between slabs (Figure

III-1C) and boulders (Figure III-1D). In addition, white filamentous mats were occasionally

observed in the fissures and in the West Rim Site. Coring the MV centre was not possible due to

the presence of such hard ground, and only North, South and West Rim sites were sampled

Figure III-1B and Table III-1).

Figure III-1 Location, map and observation of the Darwin MV. (A.) The Darwin MV is located in the Gulf of Cadiz, on the Moroccan margin at 1100 m water depth. (B.). The positive relief structure of the Darwin MV shown in the high-resolution bathymetry map. The white circle broadly indicates the location of thick carbonate platform covering the crater center. (C.) Fissures between carbonate slabs are colonized by living chemosynthetic bivalves (in black) and empty shells (in white). (D.) Carbonate boulders in the crater center.


69

At this latter station, a small sediment area (100 cm2) was covered with white filamentous

mats, and coring by mean of a small push cores deployed with the ROV provoked a strong

release of gas bubbles from the sediment (video available online 1, 2). This site, called “Seep site”

in a companion paper studying the meiofaunal assemblage at Darwin MV (Pape et al., in press),

was surrounded by shell and Lophelia-like coral debris. A second push core was taken 2 m away

of the Seep site, as in the study of Pape et al. and a reference site located at 1 km was sampled

for comparison with non-seep sediments.

Table III-1 Coordinates and features of the cores used in this study, with the overview of the analysis performed on each core. Geochemistry (GC): porewater concentration of methane, hydrogen, sulphate, sulphide and acetate. AOM: anaerobic oxidation of methane. SR: sulphate reduction. Ac-MG: acetotrophic methanogenesis. Meth-MG: methylotrophic (methylamine) methanogenesis. H-MG: hydrogenotrophic methanogenesis. *: porewater geochemistry data reproduced from Pape et al. (in press).

2.2 In western Rim at the Seep site

At the BS site, AOM activity was present from sediment surface down to 16.5 cm below

the seafloor (cm bsf) (Figure III-2). Volumetric AOM rates (AOMR) were higher near the

surface (260 nmol cm-3 d-1 at 1.7 cm bsf) and broadly decreased with depth with scattered values.

SR activity followed a similar trend, with maximum SR rates (SRR) near the surface (197 nmol

cm-3 d-1 at 1.7 cm bsf). Below, SRR decreased with depth to a minimum rate of 8.2 nmol cm-3 d-1

at 18.5 cm bsf. Depth integrated rates ∑AOMR and ∑SRR were 4197 and 2477 mmol m-2 yr-1

respectively over the core length. At the same BS site, Pape et al. (in press) reported a steep

sulphate concentration gradient between the surface (29 mM) down to the 11 cm bsf (3 mM)

indicating a downward sulphate flux, and a methane gradient toward surface between 7 cm bsf (1

mM) and 1 cm bsf (0.1 mM) indicating an upward methane flux. Below, methane values were

more scattered, probably due to degassing during core recovery.

1 http://www.youtube.com/watch?v=K-U8sQ5nAxs&feature=channel 2 http://www.youtube.com/watch?v=ZCzZeCqeehQ

CHAPTER III

70

Figure III-2 Geochemistry and microbial activity at the Seep site. SR and AOM activity (grey area). Note the methane, sulphate and sulphide profiles are from a second push core located ~ 20 cm away from the core used for activity measurements; geochemical data are reproduced from Pape et al. (in press).

Bacterial and Archaeal clone libraries were constructed from sediment in the 1-2 cm bsf

interval, where AOMR and SRR were highest. In general, bacterial diversity was high, with

clones belonging to 9 different phyla, and with most OTU only containing one single

representative sequence. 15/67 sequences belonged to Deltaproteobacteria (Figure III-3), with

sequences falling in the Seep SRB1-a and -e groups (Schreiber et al., 2010), and in the short

chain hydrocarbon oxidizing (SCHO) SRB (Kniemeyer et al., 2007) closely related to

Desulfosarcina (DSS). In addition, 7/61 clones were closely related to aerobic methanotrophs


71

Figure III-3 Phylogenetic tree showing the affiliation of deltaproteobacterial sequences. The tree was based on a set of 1026 nearly full-length 16s rDNA sequences (>1400 bp) affiliated with Deltaproteobacteria and that had a high alignment score. The tree was reconstructed with the maximum likelihood method, in combination with filter excluding the most variable position among reported Deltaproteobacteria sequences. Calculations were performed on the PhyML server (Guindon et al. 2010) using the aLRT branch support calculation method.

CHAPTER III

72 010

( AF304195, Me1hy1obac1er lu1eus

AF304197, Methy1obaC1er mrulnus , AJ704656. marine sediment clone HMMV!IO(I~ J HMMV

~i... FJ813558.1, Gu~ of Codlz mud vol<onoes, Oorwin MV, clone GoC_Boc_36, 1 sequence(s)

AJ704654, manne seclomen1. clone HMMVI!eg- 1 Met 1 AB036711, Ba1hr::l't.::lus japonicus me1hanouophic a •llsymbion1

~ff11i4S:·~<i:~~ ~~~:a~~~Jo"~~~J>~~~ V, clone GoC_Boc:_89, 2 sequence(s)

AF304196, Me1hy1omonas me1hanica FN820313.1, Gul! ol Csdlz mud volconoes, Dorwin MV, clone GoC_Boc_90, 1 sequence(s) J HMMV

~~~~: 1~~~: ~~ ~~': J~::':;7;~n ~~~~~~~~b~B~~~~. 2 sequenee(s) MPH FJ712439. Kazan Mud Volcano. clono KZNMV-0-857

FJ813522.1, Gul! of Codlz mud volc:oonoes, Oorwin MV, clone GoC_Boc_82, 1 sequence(s) J HMMV

L- AB3~~~~~~:.~:n.:a:ari~~~~Beg-7 Met 11 '------- X872n. Leucothrix mucor r-C AY592885, Molano mud volcano, clone Milano-WF1 8-44 1 [ FJ813536.1, Gult of Cldlz mud voleanoes, Darwin MV, clone GoC_sae_ 153, 1 sequence(s)

AF420367, hydro1hermal sedimen1s, isola1e Cl 8038 FM213426. endosymbion1 o1 Pe1rasma sp.

AY542553. GuH ol Mexico seafloor sed men1. clone GoM GB425 688-15 FN820314.1 , Gult of Cadlz mud votcanoes, Darwin MV, clone GoC_Bac_93, 4 sequenee(s) FJ264668, me1hano soep sediment clone OrlgSod828

FJ813586.1, GuH of Cadlz mud voleanoes, Darwin MV, elone GoC_Bac_147, 1 HqUênee(s) FJ712589. Kazan Mud Volcano. clone KZNMV-25-856 FJ813578.1 , Gutf of Cadlz mud voanoes, Darwin MV, clone GoC_Bac_97, 1 sequence(s) AB476202. ventTal setae ol Shinkaia crosnieri, clone HATS 839 AB271124, Ollgobrachla mashikoo enclosymbion! F -

AF432145. Sed•monticola selenatireducens FJ813533.1, Gul! of Cedlz mud volcanoes, Darwin MV, clone GoC_Bac_126, 2 sequenee(s)

FJ81~~~5~u~ ~o".flz~':J":~i:.:'o':.~:~~n MV, clone GoC_Boc_162, 1 sequence(s) FJ712551 . Kazan Mud Volcano, clone KZNMV- 10- 890

00836238. Thiohalomonas nd.ralireducens AY225638. Mod-Adanloc Rodge hydroth&rmal $edomen1. clone AT -53-48

FJ813560.1, Gulf of Cediz mud volcenoes, Darwin MV, clone GoC_Bac_67, 1 sequence(s) EU373861, canyon and slopo sedimen1. clone HCM3MC80_5G_Fl EF605262, S1erOidobaC1er den~nficans

FJ712522. Kazan Mud Volcano. clone KZNMV-10-840 r--{ GU061310, Yellow Sea on1ertldal beach seawa1er a120 m dep1h. clone 20m- 65

-l_- FJ813568.1, Gulf of cadlt: mud volcanOês, Darwin MV, ctone GoC_Bac:_76, 1 uquenCé(s) U88044. Amancoccus tamworthenS&S

AJ430587, Caldi1hnx abyssi FJ813580.1 , Gul! ol Csdlz mud volcanoes, Darwin MV, clone GoC_Bac_159, 3 sequenct(s)

't_ _____ ~~~m: ~~~:,e,::,~;:;::;c'!""" FoLJveCon1rol_8_s

EU491556, sealloor lavas lrom 1h& Eas1 Paafoc Rose. clone EPR4059- B2- Bc54 FJ813529.1, Gulfot Cadlz mud volcanoes, Darwin MV, clone GoC_Bac_112, 1 sequence(s)

EU491432, seafloor lavas lrom Hawai'i Soulh Poin1 X4. clone POX4b3AOI

"t~?i~~.'i<a;:.:~r;:r;~=~ . clone KZNMV-0- 81 ê FN820300.1. Gul! of Cadlz mud volcanoes, Dorwin MV, clone GoC_Bac_35, 1 sequence(s) ABS30245. marine sediment. clone Tokyo. 16S 8ac.45

{ Fj~~!~~~~~1!::,~:~ Cr~.:.v:::x~«;,~·c~~~n~Yz c!~r;e 4~~cë~;c_155, 1 sequence(s)

EF06I9S2.~;:.~·=::;.'!:~~ë!~one Napoll-38-44; BC07- 3ll-44-

[{: FJ813585.1 , Gul! of Cadlz mud volcanoes, Darwin MV, clone GoC Boe 64, 1 sequence(s) 00070829. basah alass from Cobb Seamoun1 Juan do Fuca. clone Jdl'BGBac1 42 ,-1 FN820337.1 , ~uit of Cadlz mud volcanoes, Darwin MV, clone GoC_BaC::,164, 1 sequence(S)

-l FN820316.1. GulfoiCadizmudvolcanoes. CRMV. cloneGoC Bac 103 AY5923n. Ams1crdam mud volcano. clono Amslordam-28 -1 i ; 8c20-2B-17

FJ813575.1, Gu~ of Cadlz mud vol<anoes, Darwin MV, clone GoC Boc_163, 2 sequence(s) U385764. subseaflooo sedlmon1 of 1h& Sou1h Ch01a Sea. clone MD2896-:S170

00004678. marine hydroc:oorbon soep sediment clone CAMV3008934 AY592596. Napoli mud volcano, clone Napoli-1 8-41 ; 8C07 -1 8-41

AY029807. Lep101richóa goodfellowli FJ746265. ocean sedimenl. 5306 m waler doplh, during C<uise Knox02rr, clone SPGI2_273_283_8 19

FJ813587.1 , Gult of C.diz mud volcanoes, Darwin MV, clone GoC_Bac_S6, 1 sequence(s) G0356993. me1hane soepseclomon1. clone FeUveConboi_B_ IO

M FJ813S18.1, Gulfot Cadiz mud volcanoes, Darwin MV, clone GoC Bac SS, 1 sequence(s) EU385679. subseafloor seclomen1 of !he Sou1h Chona Sea. clone MD2896-S'58 FJ813535.1, Gu1f of Cadlz mud volcanoes, Darwin MV, clone GoC_Bac_139, 1 sequence(s)

FJ7~~:::.7.l!i~~.!t ~':,1:~~N~~:o-43: BC07-38-43 '-- AJ704718. manno seclomon!. clono HMMVP09-S4

'------ CPOO I472. Acidobac1arium capsula1um

~~~~~~K';:;~~v~~~~~ ~;:~,~~~~G- 11 FJ813590.1, Gu~ of Codlz mud volcanoes, Dirwin MV, clone GoC_Boc_&l , I sequence(s)

FJ264601, methane seep sediment, clone Mn3b-B2 FJ813532.1, Gult of Cadiz mud volcanoes, Darwin MV, clone GoC_Bac_125, 2 sequence(s) AY592082, Kazan mud vok:ano. clone Kazan- 1 B-05/BC 19-1 B-05

EU491064, sea11oor lavas trom 1ho Loo'hi Seamoun1 Seuth Roh X3, clone P7X3b4C02 FJ813583.1, Gulf of Cadlz: mud volcanoes, Darwin MV, cloneGoC_Bac_41, 1 sequence(s)

f ~Js;1S::1.~~~f, !~~~~ ~~d~~r::::.~.:rnt.~~:;;:~oC Bac 16, 1 sequence(s) EU385693, subsealloo< seclimen1 ot 1h& Sou1h Chona Sea. clone MD2896-818 FJ813531.1 , GuU of Cadlz mud volc:anoes, Darwin MV, clone GoC_BK_118, 1 sequence(s) FJ813516. 1, Gulf of Cadlz mud volcanoes, Darwin MV, clone GoC_Bac_73, 2 sequence(s) CU9221 56. mesophiliic: anacrobic digesrer whic:h lreats munic:ipaJ wastewarer sludoe. done

~J~~~~~~2~~~~~~~~on':,l~MJM~j~B42 FJ813524.1, Gul! of Csdfz mud votcanoes. Darwin MV, clone GoC_Boc:_86, 1 sequenct(s) AY592116, Kazan mudvolcano, clone Kezan- 18-39/BC19-18-39

~~m~:~~o.\.c~:~~~C:,:.f.:~.=· :,~.:;,~~~~,U';:,::r;'s~1~uence(s) 00451496. Fushan torest soil. Taiwan, clone FAC57

AB303221 , AcanthopleuribaC1eo pedis EU04U71 , marone seclimen1, Seu1h Slopo. Sou1h China Sea. clone MD2896-812 FJ813517.1, Gult of Ctdiz mud volcanoes, Dtrwin MV, clone GoC B.c_S, 1 sequence(s)

rl Ft~~~~~~~:~::~~=~.:O~:n.<i:ä~~~~&~~f~~~<;~ê_eac_160, 1 sequence(s) FJ712517. Kazan Mud Volcano. clone KZNMV-10-833

EU491299, sealloor lavas trom 1he 1.oo'hi Seamoun1 Piscos Peal< X2, clone P9X2b8811 FJ813584.1, Gulfot Cadlz. mud volcanoes, Darwin MV, clone GoC_Bac_58, 1 sequence(s) EF081294. Thermodesulfovibrlo hydrogenlp

EU491260, seafloor lavas from 1heloo'hi Seamoun1 Pisces Peal< X2. clone P9X2b2E08 FJ813592.1, Gutfot Cadlz mud volcanoes, Darwin MV, clone GoC_Bac_69, 1 sequence(s)

FJ205229. lnaC1ive hydrothermal flold secllmen1s. dop1h:2350m. clone A 12F X82558. Nrtrospira mosooviensis

X86776, Leplosp.rilum ferroo)Ctcfans AJ458462. Thermoleophilum al>um A8426211 . anaerobic benzene degrading ennchmenl culture. clone mbl- b29

FJ813527.1, Gulf of C1diz mud volcanoes, Darwin MV, clone GoC_Bac_101, 1 sequence(s) AY013608, sample laken lrom benea1h 1helancllill, clone 8VA77

)1.---- U65647. RubrobaCIO< radoo1olerans A8109437, Anaerol•nea thermohmosa

L FJ813515.1, Gul! ol Codlz mud volconoes, Oorwin MV, clone GoC_Bac_4, 1 sequence(s) FJ516926, upper seclomen1. clone TDNP_USbc97_217_1_91

FJ615412. anaerobic methane- oxichz.•ng membrane bioreactor. ctoneAOM- B-c4 FJ813520.1 . Gult of Cadiz mud volcanoes, Darwin MV, clone GoC_Bac_30, 1 sequence(s) AY592581 . Napoli mudvolcano. clone Napolo-18-26: 8C07-1B-26 FN820298.1, Gultot Cadlz mud volcanoes, Darwin MV, clone GoC_Bac 31, 1 sequence(a)

r AY592372, Amslerdam mud volcano, clone Amswdam-28-12; BC20-28-T2

Q EU488025, sibcic&aslic sedmonl •rom Thalassla sea grass bed, clone CK 1C3 47 J813570.1, Gul! of Codlz mud volcsnoes, Darwin MV, clone GoC_8aë 1o6, 1 sequence(s)

EU438091 , siliciclasbe sedment trom Thalassia sea grass bed, done CK_1 C4 _62 AB067647. Caldolinea aeropl1ila

M2tn4. Thermotoga marilima

~ CU

"t;) 0 c: 0

~ 0 E E CU \!)

"' ~ ~ E .9 <.o c: ~

Cl ~

J

co ~

~ 0


73

Figure III-4 Phylogentic tree showing the affiliation of bacterial sequences (except Deltaproteobacteria). The tree was obtained by inserting clone library bacterial sequences and their close relatives into the All-Species Living Tree LTP100 release (Yarza et al., 2008; 2010) using parsimony criteria and without allowing changes in the overall tree topology. A filter excluding the most variable position was used.

(Methylococacceae), and belonged to the HMMV Meth I, Meth II and MPH groups (Losekann et

al., 2007)(Figure III-4). Most known anaerobic methanotrophs (ANME) were represented in the

archaeal clone library (Figure III-5): ANME-2a (13/32) and -2c (2/36) (Boetius et al., 2000),

ANME-1b (6/36) (Hinrichs et al., 1999) and ANME-3 (1/36) (Niemann et al., 2006a).

Remaining Euryarchaea belonged to the GoM Arc 1 group (1/36), Marine Benthic Group E

(1/36), Deep Sea Euryarcheotic Group (DSEG, 1/36). Crenarchaeal sequences (4/36) were all

closely related to the Nitrosopumilus maritimus isolate (Konneke et al., 2005).

CHAPTER III

74

Figure III-5 Phylogenetic tree showing the affiliation of archaeal sequences. The tree was based on a set of 365 nearly full-length (>1400 bp) 16s rDNA genes with a high alignment score and was reconstructed using the maximum likelihood method, in combination with filter excluding the most variable position among reported archaeal sequences. Calculations were carried on the PhyML server (Guindon et al., 2009).

In the same sediment horizon, the presence and distribution of ANME cells and their

putative bacterial syntrophic partner were investigated by CARD-FISH labeling and microscopic

observations (Figure III-6). Most archaeal cells were involved in shell-type clusters, typically

forming the core of these aggregates (Figure III-6A). Using group-specific probes, cells forming

the inner core of these consortia were identified as ANME-2 cells (Figure III-6B and C), whereas


75

DSS cells were forming an outer layer (Figure III-6D). ANME-1 positive cells were present,

with a coccoid shape (Figure III-6E). ANME-3 cells were rarely observed and were found as

single or paired cells (Figure III-6F).

Figure III-6 Epifluorescence microscopy imaging of selected groups from the Seep site microbial community (1-2 cm bsf). DAPI stained cells appear in blue in each panel. Positive CARD-FISH cells (B. to F.) appear in green. (A.) FISH stained cells stained with the Cy3 monolabeled Arc-915 probe (red) specific for the domain Archaea. CARD-FISH stained cells with (B.) the Arc-915 probe, (C.) the EelMS-932 probe specific for ANME-2 cells, (D.) the DSS probe specific for Desulfosarcina/Desulfococcus including most Seep SRB cells, (E.) the ANME1-350 probe specific for ANME-1 cells, and (F.) the ANME3-1249 probe specific for ANME-3 cells. Scale bar: 5 µm.

CHAPTER III

76

2.3 In western rim, 2 m away of the Seep site

At short distance from the Seep site (2m), both AOM and SR activities were lower next to

the sediment surface and increased with depth, reaching maximum activity at 11.5 cm bsf Figure

III-7). At this depth, AOMR was of 88 nmol cm-3 d-1 and SRR of 148 nmol cm-3 d-1, which

translated into ∑AOMR of 1169 mmol m-2 yr-1 and ∑SRR of 1075 mmol m-2 yr-1. Pape et al. (in

press) reported sulphate, sulphide and methane profiles in an adjacent core. As for the BS site,

these are reproduced here as an indication of the probable geochemistry in these sediments.

Figure III-7 Geochemistry and microbial activity 2m away of the Seep site. SR and AOM activity (grey area). Note that methane, sulphate and sulphide profiles are from a second push core adjacent from the core used for activity measurements; the geochemical profiles are reproduced from Pape et al. (in press).

2.4 South Rim

At the Southern Rim Site (Figure III-8), sulphate and methane concentrations formed a

sulphate to methane transition zone (SMTZ) between 45 and 60 cm bsf: in this interval, sulphate

decreased from 24 mM to 0.25 mM and methane increased from 0.01 mM to 0.58 mM. Sulphide

concentration was maximum within the SMTZ (1.6 mM at 64 cm bsf) and steeply decreased

above and below. Peaks of both AOM and SR activity were present in the SMTZ with maximum

values of 2.4 and 0.7 nmol cm-3 d-1 respectively at 51 cm bsf. Within the SMTZ, ∑AOM and

∑SRR were of 17.3 and 44 mmol m-2 yr-1. Above the SMTZ (0-45 cm bsf), sulphate

concentration remained close to seawater values, between 24 and 28 mM, and SR was present

with scattered rates between 0 and 3.5 nmol cm-3 d-1. There, methane was below 0.01 mM and

AOMR were very low or zero. In this interval, ∑ SRR was of 109.8 mmol m-2 yr-1, which

translates into an overall ∑SRR of 153 mmol m-2 yr-1 in this site. Methanogenesis (MG) with


77

(H-MG) or acetate (Ac-MG) was absent in this site, in spite of the presence of these substrates:

an H2 concentration peak of 230 nM was present at 58 cm bsf, and acetate concentration was

between 22 and 51 µM throughout the core. Methylamine methanogenesis (Met-MG) was only

present above the SMTZ with maximum rates of 5.3 pmol cm-3 d-1.

Figure III-8 Geochemical and microbial activity profiles at the South Rim. AOM, SR and MG microbial activity rates (grey area) are plotted with their respective substrates (circles), i.e. sulphate, methane, H2, and acetate. DIC is expressed as dissolved CO2 (squares). Meth-MG rates were calculated based on an arbitrary methylamine concentration of 5µM, and should thus be considered as potential rates.

2.5 North Rim

At Northern Rim site (Figure III-9), no SMTZ was observed, as sulphate concentration

remained constant with depth, methane concentration was very low, between 0.5 and 2.5 µM,

and sulphide concentration was below detection limit at all depths. Acetate concentration linearly

increased with depth from 11.5 to 27.5 µM, at the exception of a peak of 68 µM at 37.5 cm bsf.

Significant methanogenesis was present at this site with H-MG rates of 6 pmol cm-3 d-1 at 7 cm

bsf and of 1 pmol cm-3 d-1, and Met-MG rates of 4.6 pmol cm-3 d-1 at 17.5 and 1.4 pmol cm-3 d-1

at 27.5 cm bsf, but no Ac-MG was observed.

CHAPTER III

78

Figure III-9 Geochemical and microbial activity profiles at the North Rim. Empty symbols: surface sediment recovered with a multicore. Plain symbol: data from the gravity core at the same site. Sulphate, methane, sulphide and DIC (expressed as dissolved CO2) concentration profiles. MG microbial activity rates (grey area) are plotted with their respective substrates (circles), i.e. H2, and acetate. Meth-MG rates were calculated based on an arbitrary methylamine concentration of 5µM, and should thus be considered as potential rates.

2.6 Reference Site

At the reference site, sulphate concentration decreased linearly with depth from 27 mM at

sediment surface down to 13,5 mM at 480 cm bsf. In this interval, SRR remained below 1 nmol

cm-3 d-1, and sulphide concentration was below detection limit. Methane concentration was very

low and decreased from deep sediments (0.97 µM at 480 cm bsf) to sediment surface (0.09 µM),

with AOMR values close to detection limits (max. rate of 0.04 nmol cm-3 d-1) as 14C-CH4

turnover was close to turnover blank values at this site. H2 and DIC were detected throughout the

core, with maximum concentration of 7280 nM at 169 cm bsf and 1.8 mM at 474 cm bsf

respectively. However, no H-MG activity was observed at all depths. Similarly, in spite of

acetate being present throughout the core (15-33 µM), a single peak of Ac-MG of 0.11 pmol cm-

3 d-1 was present at 30 cm bsf. No Met-MG activity was detected in this site.


79

Figure III-10 Geochemical and microbial activity profiles at the Reference site. Empty symbols: surface sediment recovered with a multicorer. Plain symbol: data from the gravity core at the same site. AOM, SR and MG microbial activity rates (grey area) are plotted with their respective substrates (circles), i.e. sulphate, methane H2, and acetate.

CHAPTER III

80

3. Discussion

3.1 AOM activity distribution

At the seep site, AOM and SR rates were the highest measured in the Gulf of Cadiz with

maximum volumetric rates one order higher than rates measured at the crater center of Capt.

Aryutinov MV ((Niemann et al., 2006b) and Chapter IV) or Carlos Ribeiro MV (Chapter V).

The relatively lower AOM and SR activity 2 m away from the seep site was consistent with the

observation of a strong horizontal geochemical gradient at this site (Pape et al., in press), as

methane and sulphide were absent 5 and 10 m away from the seep site. In addition, these results

support the conclusion of Pape et al. who proposed that a higher methane flux and AOM activity

could account for the differences of meiofauna composition and distribution between Seep site

and 2 m from Seep site. Moreover, AOM activity at Southern Rim site was three orders lower

than at the seep site and no indication of AOM activity was detected in the North rime site,

indicating that methane flux and its consumption in subsurface sediments strongly varied at both

the meter and decameters scales around the carbonate platform covering the MV center. These

observations are in sharp contrast with the typical MV structure of a focused fluid flow at the

MV crater center, with methane flux and AOM activity decreasing toward the crater rim. Such

structure is illustrated by the studies of the Carlos Ribeiro MV (Vanneste et al., 2011) and

Chapter V). The differences between sites that are equally distant from the MV center are

interpreted as a result of focused fluid flow through discrete channeling pathways towards the

rim of the MV.

3.2 Microbial community structure at the seep site.

In the high AOM activity sediment at the seep site, members of the ANME-2 phylotype

dominated the archaeal community, but most other known methanotrophs were present. In

addition, sequences belonging to the putative syntrophic SRB partner of ANME-2 cells (Seep

SRB1, Knittel et al., 2005; Schreiber et al., 2010) were also found. These results are consistent

with the ANME ecotype hypothesis of a single ANME phylotype dominating a given AOM

community (Knittel and Boetius, 2009). All the ANME-2 cells were involved in dense shell type

aggregates, whereas the more rare ANME-3 and ANME-1 positive cells were found as single

cells. The factors triggering the formation of dense consortia are unknown, but Alperin and

Hoehler (2009) predicted that the presence of organic matter fermentation end products such as

H2 and acetate could play an important role. Since elevated concentration of these end products


81

could make methanotrophic activity endorgonic, the presence of SRB cells that can efficiently

scavenge these products could promote the formation of such shell type aggregates. Although

these end products were not quantified at the seep site, the high microbial and meiofaunal

biomass are good proxies for the presence of organic matter and our observations are in line with

the Alperin and Hoehler hypothesis. The presence, in the same sediments, of aerobic

methanotrophs closely related to the ones found in the surface of Håkon Mosby Crater Center

(HMMV Met I, II and MPH groups) was remarkable, as these usually do not coexist with the

oxygen sensitive ANME cells (Boetius and Joye, 2009). Similarly, sequences closely related to

Nitrosopumilus maritimus, an Archaeon that uses oxygen for ammonium oxidation, were also

found. The presence of both cell types can be explained either by the reworking of surface

sediments during sampling, or by the presence of aerobic micro-niches in the upper 1.5 cm

sediments layer, possibly due to bioturbation by the dense meiofauna at the Seep site surface

sediment (Pape et al., in press).

3.3 Activity coupling in Darwin MV sediments.

The AOM reaction (equation 1) predicts that methane and sulphate turnover should follow

a 1:1 ratio in the AOM zones of cold seeps. In practice, SRR often exceeds AOMR due to the

presence of alternative electron donors for sulphate reduction, such as non-methane

hydrocarbons present in migrating fluid, or sedimentary organic matter (Bowles et al., 2011). At

Seep site and 2m from the Seep site, both AOM and SR activity profiles showed similar trends,

and 2m from the Seep site, the ∑SRR:∑AOM ratio was 1.08. These observations indicated a

strong coupling between both AOM and SR activities. However, at Seep site, AOM exceeded SR

with a ∑SRR:∑ AOMR ratio of 0.59, thus deviating from the 1:1 ratio predicted by AOM

stoichiometry. This could potentially be due to strong horizontal variations of the methane flux,

leading to different geochemistry and activities in sediments sampled for AOM and SR rates

measurements. At South Rim site, SR activity in the first 50 cmbsf was not coupled to AOM and

was interpreted as organotrophic sulphate reduction. Below, both AOM and SR peaks coincided

with a ∑SRR:∑AOMR ratio of 8.3 close the average ratio of 10.7 for methane seep site (Bowles

et al., 2011). This indicated that AOM only partly accounted for the overall SR activity and

downward sulphate flux at the South Rim site. The remaining sulphate flux is attributed to

organoclastic sulphate reduction, as shown by the presence of AOM-independent SR activity

above the AOM zone.

CHAPTER III

82

Besides, several in vitro studies have shown that AOM, in spite of a net methane

consumption, may also produce methane at a rate of ~10% of the AOM rate either in situ (Orcutt

et al., 2005) or in vitro (Treude et al., 2007; Orcutt et al., 2008b; House et al., 2009). The

absence of methane formation in the AOM zone of the South Rim site indicated that this AOM

side reaction does not necessarily occur. These results were similar to those obtained in other

Gulf of Cadiz MV such as Capt. Arutyunov MV, Mercator MV (Chapter IV) and Carlos Ribeiro

(Chapter V). This ability to both oxidize and produce methane does not seem to depend of

ANME types present, as these MV were dominated by different ANME cells (ANME-1 at

Mercator MV, ANME-2 at CAMV and ANME-2 and 3 at CRMV), and could thus be rather

triggered by environmental factors. At Darwin MV, methane production was rather interpreted

as conventional methanogenesis. Since methylamine is a non-competitive MG substrate

(Oremland and Polcin, 1982), the presence of Met-MG activity in the sulphate zone at South and

North Rim sites was coherent. However, H-Mg activity at the North Rim site, and Ac-MG at the

reference site were more unexpected as sulphate reducers usually outcompete methanogens for

these substrates (Kristjansson et al., 1982; Schonheit et al., 1982)

3.4 Proposition of a development model for the Darwin MV

Typical MVs, such as Carlos Ribeiro and Capt. Arutyunov MVs (next chapters), have a

rather predictable physical and biogeochemical structure: deep rooted-fluid migrating through a

central conduit formed a mud dome on the seafloor. Concentric rims formed by previous

eruptive events surround a “crater eye” indicating the most recent mud eruption. Both

geochemical and ex-situ rates measurement -(Vanneste et al., 2011), Chapter IV and V- have

shown that such structure was correlated with decreasing methane flux along a radial axis from

the crater eye towards the crater rim. The Darwin MV obviously deviated from this model and

the fluid dynamics in this MV was less predictable. Based on the current knowledge on methane-

derived carbonate formation at cold seep sites, we propose a development model for the Darwin

MV that can account for previous observations of this structure and for the methane flux and

microbial activity distribution described in this study. We posit that intense methane migration

first occurred in the crater center. Its anaerobic oxidation by AOM microorganisms led to the

formation of methane derived carbonate crust and boulders as observed on the MV center, which

then developed into a compact chemoherm, as for instance in the Hydrate Ridge (Greinert et al.,

2001). The methane origin of the carbonate matrix, demonstrated by its low ∂13 -C signature


83

(Nekhorosheva and Blinova, 2009), is supporting this interpretation. Intense AOM activity at the

base of the carbonate layer would lead to sulphide production and the development of a

chemotrophic fauna at the surface, such as the dense Bathymodiolus fields (Genio et al., 2008;

Rodrigues et al., 2010). As carbonate crust developed downward (Reitner et al., 2005; Mazzini et

al., 2008), the sulphate flux through the carbonate diminished, preventing further methane

oxidation at the base of the crust. The concurrent decrease of upward sulphide flux triggered the

decline of the Bathymodiolus population. Overpressure due to fluid accumulation below the

chemoherm, possibly at the origin of the observed fissure network, provoked the relocation of a

major part of the fluid flux toward the rim of the chemoherm through discrete channels. Such

fluid dynamics would follow the mechanism described by (Naudts et al., 2010) of cold seeps on

the New Zealand margin. In this study, the authors identified different stages of methane-derived

carbonate formation. The methane flux relocation was clearly visible in the form of gas buble

formation at the rim of the carbonate crusts exposed on the seafloor. Such mechanism could thus

explain the high and focused methane flux and the high AOM activity at the Seep site, the

relatively low AOM activity at the South site, and the apparent lack of significant methane flux

at the North rim site.

CHAPTER III

84

4. Conclusion

This study of the Darwin MV provides elements of the functioning of singular cold seep covered

by a thick carbonate layer and illustrates the “self-sealing” nature of some of these structures

(Hovland, 2002). This peculiar structure may explain the strong horizontal variations of methane

flux and AOM activity by focusing the migrating fluid flux to discrete hot spot at the rim of the

carbonate chemoherm. Such seep relocation may lead to very high microbial activity, as in the

Seep site and 2m away, with the highest AOM rates and methane turnover measured thus far in

the Gulf of Cadiz. The wealth of chemosynthetic fauna shells on the MV center, with only few

living specimen, has been interpreted as a decline of the fluid emission at this site (Vanreusel et

al., 2009). We argue that the global activity of this MV is difficult to estimate due to its structure,

and would require an intensive sampling effort around the rim in order to provide global estimate

of fluid emission and methane turnover.


85

87

CHAPTER IV. Anaerobic

oxidation of methane in hypersaline

cold seep sediments.

Submitted to Applied and Environmental Microbiology as L. Maignien, R. J. Parkes, B. Cragg,

H. Niemann, K. Knittel, S. Coulon, A. Akhmetzhanov, P. Weaver, N. Boon. Anaerobic oxidation

of methane in hypersaline sediments of Mercator Mud Volcano in the Gulf of Cadiz

CHAPTER IV

88

AOM IN AN HYPERSALINE ENVIRONMENT

89

ABSTRACT

Life in hypersaline environments could be limited by bioenergetic constraints. Microbial activity

at the thermodynamic edge, such as the anaerobic oxidation of methane (AOM) coupled to

sulphate reduction (SR) is thus unlikely to thrive in these environments. In this study, carbon and

sulphur cycling were investigated in the hypersaline cold seep sediments of Mercator mud

volcano (MV) and compared to the adjacent non-hypersaline Captain Arutyunov MV. AOM

activity - albeit partially inhibited- was still present at a salinity of 292 g L-1 (ca. 8 fold seawater

concentration) with rates of 2.3 nmol cm-3 d-1, and even in saturating conditions with rates of 0.5

nmol cm-3 d-1. Methane and evaporite-derived sulphate co-migrated in these sediments, which in

combination with the partial inhibition, resulted in an AOM activity that was spread over wide

depth intervals. Up to 79% of total cells in the AOM zone were identified as ANME-1 by

fluorescence in situ hybridisation (FISH). No other ANME type could be detected either by

FISH or in 16s rDNA gene libraries, indicating a possible salinity selection toward ANME-1.

The depth distribution of methanogenesis rates did not match AOM rates nor ANME-1 cells

counts, thus arguing for a strict methanotrophic role of these cells in Mercator MV sediments.

Most ANME-1 cells formed monospecific chains and were generally distant from sulphate

reducing bacteria or other cells. At all sites, AOM activity co-occurred with SR activity and

sometimes significantly exceeded it. Possible causes of these unexpected results are discussed.

This study demonstrates that in spite of a low energy yield, microorganisms carrying AOM can

thrive in salinity up to saturation.

CHAPTER IV

90

1. Introduction

Marine mud volcanoes (MV) are formed by the extrusion of fluidized sediments of deep

origin to the sea floor (Milkov, 2000; Dimitrov, 2002; Kopf, 2002; Niemann and Boetius, 2010).

Erupted material (mud breccia) often contains high amounts of methane, and occasionally higher

hydrocarbons (Kopf, 2002 and reference therein). The migration of hydrocarbons towards the

sediment surface typically fuels a great variety of microbes and symbiotic fauna that harvest

energy from these reduced molecules, directly or indirectly, using seawater electron acceptors

(e.g. O2, NO3, SO4). Hydrocarbon seepage therefore promotes the development of thriving

cold-seep ecosystems, which support high standing stocks of free living and symbiotic

chemosynthetic microbes. One of the most important processes at cold seeps is the anaerobic

oxidation of methane (AOM) coupled to sulphate reduction (SR) (Knittel et al., 2005), and

references therein:

CH4 + SO42- ⇌ HS- + HCO3

- + H2O ΔG0’=-16.9 kJ mol-1 equation 1

On a global scale, this reaction retains more than 80% of uprising CH4 in the sea floor

(Reeburgh, 2007), and references therein.

AOM is performed by three groups of Euryarchaea related to methanogens: ANME-1,

ANME-2, and ANME-3 (Hinrichs et al., 1999; Boetius et al., 2000; Orphan et al., 2001b, 2002b;

Knittel et al., 2005; Niemann et al., 2006a). These anaerobic methanotrophs often form consortia

with sulphate-reducing bacteria (SRB) of the genera Desulfosarcina / Desulfococcus (ANME-1

and -2) or Desulfobulbus (ANME-3) (Knittel et al., 2003; Losekann et al., 2007). Based on these

observations, a syntrophic relationship between these microorganisms has been postulated

(Boetius et al., 2000; Orphan et al., 2001a) with an up to now unidentified Interspecies Transfer

Agent (ITA) mediating electron transfer from methane oxidising ANME cells to their sulphate-

reducing partners. The typical geochemical signature of AOM activity in marine sediments is the

presence of a so-called sulphate-methane transition zone (SMTZ), i.e. opposed gradients of

downward diffusing sulphate and upward migrating methane (Reeburgh, 2007). Microorganisms

mediating AOM are usually present and active in a narrow depth interval at the interface

between these gradients (Knittel and Boetius, 2009). In addition to this sulphate dependent

mode, AOM may also be coupled to the reduction of oxidised metal species such as Fe(III) and

Mn(IV) (Beal et al., 2009) and N species (Raghoebarsing et al., 2006; Hu et al., 2009; Ettwig et


91

al., 2010). So far, evidence for an environmental significance of these pathways, particularly in

marine environments, is missing.

Based on an inventory of known metabolisms able to thrive in hypersaline environments,

it has been proposed that the upper limit of salt concentration at which energy conservation can

occur “primarily depends on bioenergetic constraints”, and secondly on the “mode of osmotic

adaptation used” (Oren, 1999; Oren, 2011). Balancing cell osmotic pressure by active ion

pumping or the production of compatible solutes is indeed energetically costly and apparently

excludes dissimilatory pathways characterized by low energy yields. From an energetic

standpoint, it is thus surprising that a few studies could detect AOM in hypersaline environments

(Oren, 2011). Yet, the influence of salinity on AOM activity and microbial community

composition is not well constrained. One study, for instance, found geochemical and molecular

evidence for AOM activity mediated by ANME-1/SRB in hypersaline sediment of the Gulf of

Mexico (Lloyd et al., 2006), while a recent study on a deep-sea brine pool and a MV, in contrast,

suggested that presence of hydrogen prevented AOM activity in spite of the presence of both

methane and sulphate (Joye et al., 2009). However, circumstantial evidence indicate a

dominance of ANME-1 over the other ANME groups in hypersaline settings, thus suggesting

that this clade might be more tolerant to high salinity (Daffonchio et al., 2006; Harrison et al.,

2009; Niederberger et al., 2010; La Cono et al., 2011). Nevertheless, significant ANME-1

populations are also found in non-hypersaline methane seeps, e.g. in the Eel River Basin, the

Black sea microbial mats, the Hydrate Ridge, or the Tommeliten seeps (Addendum II, p.114, and

(Orphan et al., 2002a; Knittel et al., 2005; Niemann et al., 2005; Treude et al., 2005c).

Recently, an hypersaline mud volcano (named Mercator MV after the Belgien

cartographer) was discovered by Van Rensbergen et al. (2005) in the Gulf of Cadiz, East Atlantic

Large amounts of methane were reported (Nuzzo et al., 2009; Scholz et al., 2009) in this

structure. This hence provides a very good opportunity to study the microbial ecology of

methane and sulphur cycling in an extreme hypersaline environment. Here, we document for the

first time the microbial activity and community structure in relation to geochemical parameters at

Mercator MV, with the aim to understand the functioning of AOM communities under extreme

hypersaline conditions. Our results are compared with those obtained from Captain Arutyunov

MV (CAMV), a non-hypersaline MV in the vicinity of the Mercator MV.

CHAPTER IV

92

2. Results

2.1 Geochemistry

Mercator MV was sampled in the crater centre as well as at three rim sites (Figure IV-1and

Figure IV-7p112). At Mercator MV crater centre site, gas was venting from the seafloor and

large halite (NaCl) and gypsum (CaSO4) crystals were recovered from the sediments between

150 and 200 cm below the sea floor (cmbsf). CAMV was sampled in the crater centre site. For

comparison with non MV sediments, a reference station was sampled 2.4 km N.-E. of Mercator

MV. Details of the high-resolution bathymetry method, sampling sites description and coring

devices are given in supplementary information.

Figure IV-1 Study location and sampling stations. (A) Gulf of Cadiz bathymetric map showing the location of the two mud volcanoes investigated in this study. (B) High-resolution bathymetric maps of the Mercator mud volcano, located in the shallow region of the Gulf of Cadiz. This MV was cored in the Crater Centre (JC10-09 and -19), Northern Rim (JC10-11), and Southern Rim (JC10-13 and -15). Depth range: -350 m (yellow), -500 m (blue).

The salinity in the sediments of Mercator MV and CAMV was aproximated by the chloride

concentration (Figure IV-2). At the Mercator MV crater centre (cores JC10-09 and -19), chloride

concentration at 10 cmbsf was already 3.5-fold (2011 mM) higher than seawater values and

further increased with depth along concave up gradients, up to halite saturation level (~5800

mM) at 220 cmbsf. At the rim stations (cores JC10-11, -13 and -15), chloride concentration

formed a quasi-linear gradient with depth from seawater concentration (~580 mM) at the surface

up to 3213 mM at 67.5 cmbsf. Both at the reference station (cores JC10-02 and -04) and CAMV

Northern Rim

Southern Rim

Crater Centre


93

crater Centre (core JC10-66), chloride concentration reflected seawater concentration at the

sediment surface and remained constant with depth.

Table IV-1 Sampling stations. Size, stations location and core description in the Mercator MV and the Captain Arutyunov MV (CAMV)

Three methane rich habitats with decreasing salt contents (chlorinity gradients) were

selected for further detailed studies on geochemistry and microbial activity: (i) hypersaline

sediments (core JC10-19) hereafter named Mercator MV Crater Centre, (ii) sediments with lower

but still elevated salinity (cores JC10-13 and JC10-15) hereafter named Mercator MV Rim 1 and

Rim 2 respectively, and (iii) sediments with regular seawater salinity (core JC10-66) hereafter

named CAMV Crater Centre.

Figure IV-2 Sediment salinity. Comparison of chloride concentration profiles used to estimate salinity in the sediments of Mercator MV and CAMV.

CHAPTER IV

94

Methane – At all three locations, an upward migration of methane in the sediment was

indicated by elevated methane concentrations in deeper section of the cores, generally decreasing

toward surface (Figure IV-3). These results are in agreement with previous publications on the

geochemistry of the chosen structures (Hansen et al., 1998; Van Rooij, 2005; Niemann et al.,

2006b; Nuzzo et al., 2009; Scholz et al., 2009). At Mercator MV Crater Centre, methane

concentration decreased from 3 mM at 75 cmbsf to 0.6 mM at the sediment surface. At Mercator

MV Rim 1, the methane decreased from 0.23 mM at 21 cmbsf to 5.10-3 mM at 11.5 cmbsf, and

similar trend was observed at Mercator MV Rim 2. At CAMV Crater Centre, methane

concentration steeply decreased from 1.2 mM at 26.5 cmbsf to near background concentrations

at 21.5 cmbsf, and methane was depleted in the upper 20 cm of the core.

Sulphate - Sulphate concentration was close to seawater values at the sediment surface (28-

30 mM) in all cores (Figure IV-3). At Mercator MV, sulphate first decreased with depth down to

13.2 mM at Crater Centre or to 24.4 mM at Rim 2 (at 162 cmbsf and 42 cmbsf respectively), but

then increased again with depth, reaching maximum values of 18.5 and 28.3 mM respectively.

At Mercator MV Rim 1, sulphate concentration remained constant down to 28 cmbsf. At CAMV

crater Centre, sulphate concentration steeply decreased and reached 2.8 mM at 22.5 cmbsf, and

was below detection limit below 55 cmbsf

Sulphide - At all Mercator MV sites, we could not detect any soluble sulphide (ΣH2S, HS-,

S2-) (data not shown), although the decreasing sulphate contents and sulphate reduction rates

(next section), indicated sulphide production. However, at least some of the sulphide could be

precipitated because acid volatile sulphide (AVS) content was elevated (0.5 mmol L-1 of

sediment) at Mercator MV Rim 2 (32 cmbsf, data not shown). In contrast, at CAMV Crater

Centre, we could detect substantial amounts of (dissolved) sulphide peaking with 3.2 mM at 22.5

cmbsf (Figure IV-3D), i.e. at the interface between the methane and sulphate gradients.

Acetate and hydrogen - Acetate was present in the pore water of all sites (Figure IV-3)

with decreasing concentrations from the deeper layers (between ~40 µM at Mercator Rim sites to

~90 µM at Mercator Crater Centre) toward the surface (5-20 µM). At CAMV Crater Centre, a

peak of acetate (107 µM) occurred at 40 cmbsf. Substantial amounts of dissolved dihydrogen,

mostly as discrete concentration peaks, could be detected in all four cores (Figure IV-3).

Maximum hydrogen concentration ranged from 0.7 nM at Mercator crater Rim 2 to 4.4 nM at

Mercator crater Centre.


95

Figure IV-3 Geochemistry and microbial activity at Mercator MV and CAMV. AOM, SR and MG activities (triangle) at each station plotted with respective substrate/product concentration profiles (methane,

E' ~ .c a .. 0

A Mercator WN erater center JC 10-19

-1

0 0

Sulphate (mM] 10 20 30

Methane ( mM) 2 3 4567

t-------t

_._ ANME·1 eens

H2 (nM]

5

=H-MG _._ H,

Acelate I1•MJ 0 20 40 60 60

l t t + , l + l

l • )

• =Ao·M ~ _._ Acel31e

10 0 2 4 6 8 10 0 20 40 60 00 u u ~ u u u u

100

SRR (nmol cm" d'') AOMR (nmol cm-> d'~ % total eelt number H·MG (pmol cm-> <f1) Ac-MG (pmol cm-> d'~

• • • • •

/. • • =Me<I>MG • o.o 0.5 1.0

Meth·MG (pmol cm-> d' 1)

B: Mercator WN erater rim 1 JC 10-13 Sulphate (mM) Methane (mM) H2 (nM] Acelate I1•MJ 10 20 30 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 20 40 60 60 100

::~~~~~-:--_____..~I-~s;::'"lÇ;~:::::-~ .. -"---T. ~~·

·30 ~ I?- I . ·40 t:+'t=O ~ .

2 4 6 8 10 SRR (nmol cm" d'')

= SRR _._ SUphale

2 • 6 8 10 AOMR (nmol cm-> <f~

= AOMR _._M _

20 40 60 00

% total eelt number

- • - ANME-1 cetls

10 20 30 40 50 0.0 0.5 1,0 1,5 2.0 2.5 0 2 4 6 8 10 H·MG (pmol cm" <f1

) Ac·MG (pmol cm-> d'~ Meth·MG (pmol cm-> d' 1)

= H-MG _._ H,

=Ao-MG _._ Acelate

=Meti>-MG

C: Mercator WNcra ter rim2 JC10-1 5

-10

-20

E' ~

·30

.c ·40 a .. ·50 0

·60

·70

Sutphate (mM] Methane (mM) H, (nM) Acelate h•MJ 10 20 30 o.o 0.2 0.4 0,6 0,8 1,0 1,2 1,4 0.0 0.2 0,4 0.6 0,8 20 40 60 60 100

2 4 6 8 10

SRR (nmol cm" d''l

~~~~~r-------------~ ~~~ ~-r-------------,

--·I

2 4 6 8 10 0 20 40 60 00

AOMR (nmol cm" d'l % total eelt number

•

• •

•

= Ac-MG _._ Acetate

c::::::J Meth-MG

10 20 30 40 50 0.0 0,5 1.0 1,5 2.0 2.5 0.00 O.Q1 0.02 0.03 0,04

H-MG (pmol cm" <f'l Ac-MG (pmol cm" d'l Meth-MG (pmol cm" d''l

D: CAMV erater center JC 10-66 Sulphide (mM] 1 2 3

Sulphate (mM) Methane (mM) 10 20 30 0.0 0.5 1,0 1.5 2.0 2.5

· 1

-150

-200

-250

·300 = SRR -350 _._ SUphale =AOMR

·400 ._----~-=su~~~-~------L---~===-L-----_J 5 10 15 20 25 0

SRR (nmol cm-> <fl 2 4 6 8 10

AOMR (nmol cm" d'1)

E' ~ .c a .. 0

H2 (nM] Acelate h• M) 20 40 60 60 100 0.0 0.2 0,4 0.6 0.8 1.0

o r·----~~~-.ot----~~--========~ ·10

· 20

·30

-60 I ·70

-~ ·1 I -150

-200

.40Q L-------~~--~---L----~~~--------------

o.o 0.2 0.4 0.6 0.8 0.00 0.05 0.10 0.150.0 0.5 1,0 1,5 2.0 2.5 H-MG (pmol cm-> d'l Ac-MG (pmol cm·' d'1) Meth-MG (pmol cm·' d'1)

CHAPTER IV

96

sulphate/sulphide, hydrogen, acetate). Methylamine methanogenesis rates are given based on a methylamine concentration of 5 µM in sediment pore water (see Material and Methods). Note the scale difference for methane at Mercator MV Crater Centre (A), and for SRR and methane at CAMV (D). Error bars correspond to ± one standard error of the mean (n=3) when replicates were taken.

2.2 Microbial activity.

(i) Anaerobic Oxidation of Methane

Significant rates of AOM (AOMR) could be detected in all habitats investigated in this

study (Figure IV-3). At Mercator MV Crater Centre (Figure IV-3A), AOM activity was present

throughout the entire core, with two zones of apparently higher activity: just below the surface,

with a maximum AOMR of 6 nmol cm-3 d-1 at 21 cmbsf, and at greater depth (170 cmbsf) with a

maximum AOMR of 2.3 nmol cm-3 d-1. Notably, both maxima coincided with a decrease in

methane concentrations (Figure IV-3). At Mercator MV Rim 1 and Rim 2 (Figure IV-3B and C),

AOM activity was present in near surface sediments with rates of 0.8 and 2 nmol cm-3 d-1

respectively, and decreased with depth to values close or below detection limit. At 25 cmbsf,

AOMR increased and two activity peaks of 3.0 and 3.6 nmol cm-3 d-1 were detected between 30

and 40 cmbsf at Rim 1, and a single peak of 8.5 nmol cm-3 d-1 at Rim 2. At the latter, AOM

activity extended down to the bottom of the core, with rates between 1 and 1.7 nmol cm-3 d-1. At

CAMV Crater Centre (Figure IV-3D), a distinct peak of AOM activity (11.4 nmol cm-3 d-1) was

found between 28 and 31 cmbsf, which vertically coincided with the sulphide peak and the depth

of the sulphate-methane transition. AOM was close to the detection limit above this active zone.

As already observed by (Niemann et al., 2006b), weak AOMR (0.5 to 1.3 nmol cm-3 d-1) were

detected below the SMTZ between 47 and 297 cmbsf.

(ii) Sulphate reduction.

At Mercator Crater Centre (Figure IV-3A), SRR showed three maxima: immediately below

the surface (SRR of 5.58 nmol cm-3 d-1at 5 cmbsf), at 56 cm (2.36 nmol cm-3 d-1), and at 220

cmbsf (7.47 nmol cm-3 d-1). At Mercator MV Rim 1 (Figure IV-3B), SR was scattered over

depth, but showed a general increase towards 30 cmbsf, broadly corresponding to depth of the

two AOM maxima. At Mercator MV Rim 2 (Figure IV-3C), SR was maximal just beneath the

sediment surface (9.6 nmol cm-3 d-1), and showed a second weaker maximum of 1.83 nmol cm-3

d-1 at the same depth (31 cmbsf) where also the AOM maximum was found. At CAMV Crater

Centre, in contrast to the rather weak coupling between AOM-SR found at Mercator MV, SR


97

peaked (24.13 nmol cm-3 d-1) just in the same sediment horizon (28-31 cmbsf) where also AOM

was maximal.

Depth integrated AOM rates (∑AOMR) and SR rates (∑SRR) were comprised between

147 and 898 mmol m-2 yr-1, and 326 and 1803 mmol m-2 yr-1 respectively (Table IV-2).

Remarkably, AOMR exceeded SRR in several sediments intervals. This difference was

significant (no overlap of 95% confidence intervals) at Mercator MV Rim 2 (37.5 cmbsf) and in

most sediment horizons between 7 and 29 cmbsf at Mercator MV Crater Centre. As a

consequence, ∑AOMR exceeded ∑SRR by a factor 2.8 at Rim 2 in the 27-77 cmbsf interval,

1.23 over the entire core (Table IV-2), and 2.2 at Crater Centre between 7 and 49 cmbsf.

Core Depth integrated rates

[mmol m-2 yr-1]

∑SRR ∑SRR ∑AOMR ∑AOMR(total) / ∑AOMR(total) /

Station JC10- (Total) (SMTZ) (Total) ∑SRR(SMTZ) ∑SRR(total)

Mercator

Crater

Centre 19 1803 n.a. 898 n.a. 0,50

MV Rim 1 13 326 n.a. 80 n.a. 0,25

Rim 2 15 458 n.a. 562 n.a. 1,23

CAMV

Crater

Centre 66 435 232 147 0,63 0,34

Table IV-2 Areal microbial activity rates. Areal rates ∑SRR and ∑AOMR were obtained by integrating volumetric anaerobic oxidation of methane rates (AOMR) and sulphate reduction rates (SRR) over the entire core (total) or the over the sulphate to methane transition zone (SMTZ). The AOM contribution to SR over the entire core (resp. over the SMTZ) is given by the ratios ∑AOMR(total)/ ∑SRR(total) (resp. ∑AOMR(total)/ ∑SRR(SMTZ). In the absence of an SMTZ in Mercator MV sediments, the latter ratio does not apply to these sites. n.d.: not determined. n.a.: not applicable.

(iii) Methanogenesis

With the exception of Mercator MV Crater Centre, methanogenesis (M) using different

substrates could be detected in all cores, albeit at very low rates and at discrete depth horizons.

No methanol methanogenesis was observed in any of the sites investigated. In principal, we

found a vertical succession of methylotrophic (Meth MG) with methylamine as a substrate in the

shallowest horizons, followed by acetotrophic (Ac) and then by hydrogenotrophic (H) MG. At

Mercator MV RIM 1 (Figure IV-3B), Meth-MG peaked with 8.1 pmol cm-3 d-1 at 3 cmbsf, Ac-

MG with 2.3 pmol cm-3 d-1 at 13 cmbsf and H-MG with 50.5 pmol cm-3 d-1 at 17 cmbsf. At RIM

2 (Figure IV-3C), a peak of Meth-MG was also present near sediment surface, but we could not

CHAPTER IV

98

detect H-MG or Ac-MG. Similarly, at CAMV Crater Centre, H-MG was absent, but we found

two peaks of Ac-MG at 55 and 300 cmbsf (~0.13 pmol cm-3 d-1) and a near surface peak in

Meth-MG (Figure IV-3D).

2.3 Microbial community structure

The structure of the microbial community involved in AOM in hypersaline sediments of

Mercator MV Rim 1 was investigated by constructing both archaeal (85 clones) and bacterial (75

clones) 16S rRNA gene clone libraries with DNA extracted in the 32-33 cmbsf interval where

AOM was maximum. Sequences belonging to the ANME-1b clade (Hinrichs et al., 1999) largely

dominated the archaeal library (n=69/85, Figure IV-4), followed by sequences belonging to the

GoM-Arc1 clade (n=14/85) (also called ANME-2d in Mills et al., 2003; Mills et al., 2004; Lloyd

et al., 2006). Most bacterial sequences were affiliated with sulphate-reducing bacteria (SRB)

belonging to the Deltaproteobacteria (n=50/75, Figure IV-5). Off these, sequences belonging to

the Seep-SRB1 cluster of putative syntrophic ANME partners these, sequences belonging to the

Seep-SRB1 cluster of putative syntrophic ANME partners (Knittel et al., 2003) constituted the

largest fraction (n=39/75) of the bacterial diversity, falling in the Seep-SRB1-c (n=30/75), Seep-

SRB1-d (n=7/75) and Seep-SRB1-a (n=2/75) subgroups (Schreiber et al., 2010).


99

Figure IV-4 Affiliation of Archaeal 16S rRNA sequences from Mercator MV Rim 1 (32-33 cmbsf). The phylogenetic tree was reconstructed based on a subset of 340 sequences using the maximum likelihood method (RaxML) filtering out positions according to base frequencies among Archaea (50% cutoff). Results from 1000 bootstrap analysis are indicated as percentage.

CHAPTER IV

100

Figure IV-5 Affiliation of Bacterial 16s rDNA sequences from Mercator MV Rim 1 (32-33 cmbsf). The phylogenetic tree was reconstructed based on a subset of 195 sequences using the maximum likelihood method (RaxML) filtering out positions according to base frequencies among Deltaproteobacteria (50% cutoff). Results from 1000 bootstrap analysis are indicated as percentage

~~~===============-- Gammaproteobactena (outgroup)

UXWu ---------=====---- DesuHovibrionales spp

~ AB2t 2873, Synlrophorhabdus aromatic:ivora, UASB granular sludge

28~

98

l~ llA<utfM>nrniAA "1'1'

_1 !l!lll(- G0246356, North Yellow Sea sediments, clone Cm1-19 _98Y" --- EU245341, hypersaline moeroblal mat. clOne MAT - CR- H6-C08 ~ FN820319, Gultol Cadiz Mud Volcanoes, MercatorMV, clone GoC_Bac_ 115, 5 sequence(s) -- FJ264598, methane seep sedtment, clOne Mn3b-B5

l~ Seep...SRB2

~ AY592401 , Amsterdam mud volcano, Eas1em Med•terranean, 1995m water de, clOne Amsterdam- 28-43; BC20- 2B .96Yo- FJ712616, Kazan Mud Volcano, Anaximander Mountains, East Medtterranean, clone KZNMV- 30-862

L 1~ FJ813561 , Gulf of Cadlz Mud Volcanoes, MercatorMV, clone GoC_Bac_70, 2 sequence(s) 1 AJ347756, Brine-seawater interface Shaban deep red sea. clone ST - 12K13

100o/~~ Seep...SRB4 36 Desultocapsa spp

~p:--;:::::-- Desullobulbus spp

sl~c;ï7" ANME3 aggregate group

0~ Seep-SRB3

64Y.tr FJ813554 , Gult of Cadiz Mud Volcanoes, MercatorMV, c lone GoC_Bnc_142, 1 sequence(s) too• AY542227, Gultol Mexico sealtoor sediments, clone GoM IDB- 15 100° AJ237601 , Desulfobacterium anilini

10 AM745210, marine sediments, clone GoM GC232 4463 Bac38

L FJ813591 , Gulf of Cadiz Mud Voiëanoes;-Mercä torMV, clone GoC_Bac_98, 1 sequence(s)

1 o• AF029048, benzene mineralizing consortium clone SB- 30 1 oo{Q AM682606, sediment trom oil polluted water trom petrochemical treatmen. clone 58 EDB 1

Sllo/q- FJ813555 , Gul! of Cadiz Mud Volcanoes, MereatorMV, clone GoC_Bac_144, 1 sequence(s) 2s;s- G0356990, methane seep sediment. clone Fe$04_8_7 _ 28"" FM179899, marine sediments, clone Gulllaks_b16

FM179888, marine sediments, clone TommOS 1274 3 Bac79 2~ , Y 17698, Desullococcus oleovorans Hxd3 - -

6~ ~ Desultatibacilum spp 9~ AY268891 , Desullofaba fastidiosa

AF099063, Desullofaba gelida 9 ~ AF32t820, Desullofaba hansenii

IOOY, 99'l![V Desulfobacterium spp

I 1

1 <m'; Olesullobacula spp

1 ~~_-=-- DesuiiO!ignum spp ~ -~~7, Desulfospira joergensenu

4 ~ DesuHobacler spp

ql~ DesuHonema spp

3 ~V':!DesuHoeoccus spp

9~~ AJ237603, Desulfosaroina cetonica eZ'l\ Y 17286, Desulfosarcina ovata

i M34407, Desulfosarcina variabilis

~0% EU542523, sediment and soit slurry, clOne Er-LLAYS-70

EU047530, benzene-degrading, sulfate-reducing enrichment culture, clone Bzn$082 Oo/'{_ FJ813556 , Gult of Cadlz Mud Volcanoes, MercatorMV, c lone GoC_Bac_146, 1 sequence(s)

4° FM242333, sediment, clone 35 T9d-oil 2Q'Yc~EU385937, subsealtoor sediment of the South China Sea, clone MD2902- B 149 } a• - G0356958, methane seep sediment, clone Fe_B_ I4 t (/)

• FN820296, Gult of Cadiz Mud Volcanoes, MercatorMV, clone GoC_Bac_19, 29 sequence(s) Cl> 10• EU385887, subseafloor sediment ol the Soulh China Sea, clone M02902- B69 Cl>

21'.~U385712, subsealtoor sediment of the South China Sea . clone MD2896- B101 "0 62• EU592467, hypersaline sediment, clone SSS3A en 55°/. AY771942, marine surface sediment, clone SL13 ;:o

--- FJ813564 , Gult of Cadiz Mud Volconoes, MercatorMV, clone GoC_Bac_81, 1 sequence(s) CD ~ EU245466, hypersaline microbial mat, clone MAT- CR- M1 - HIO ......

00109912, salt pond, nonlithilying microbial mat. clone E48B11cD '

56 00521790, Gult of Mexico sediments, clone SMII - GC205- Bac2d (/)

4~ A~J~~~: :;;;~~! =~~:~:s~~~~eH~~~~~~~12 en 8 !l~ EU04 7539, benzene-degrading, sultate-redÜcing enrichment cuhure, clone Bzn$327 ~ 4,..,_ AJ535249, marine sediment above hydra te tidge. clone Hyd89- 61 ......

4% AM745148, marine sediments, clone GoM161_BaC45 Ó. t l'l'q ~ Seep...SRBI- e

13,-?3'J'ç.r Seep...SRB 1- b

1 ~~'t:3 Seep...SRB1-f

54"• AJ535235, manne sediment above hydrate ridge, clone Hyd89-21 16'11( FN820317, Gullof Cadiz Mud Votcanoes, MercalorMV, clone GoC_Bac 105, 1 sequence(s) 8'1, FJ813550 , Gult of Cadiz Mud Volcanoes, MercatorMV, clone Goe_eac_79, 1 sequenee(s) ~~ FJ712482, Kazan Mud Volcano, Anaxomander Mountaons, East Medtumanean, clone KZNMV-5-870 4 7"!11 EU622295, Eel River Basin methane seep 520 m depth, clone BC B2 31 55~ 00004675, manne hydrocarbon seep sediment, clone CAMV3008922

AJ704697, marine sediment, clone HMMVBeg-45


101

The in-situ abundance and distribution of known archaeal anaerobic methanotrophs

(ANME-1, -2 and -3) was further investigated in the three Mercator MV cores applying CARD-

FISH. Confirming the results of the clone library, ANME-1 was the only group of known

methanotrophs detected at Mercator MV. Members of this clade were found at several depth

intervals. At Mercator MV Rim 1, ANME-1 cells were dominant at 29 cmbsf, accounting for

79% (±0.8 % standard error) of the DAPI stained (n=848) cells (Figure IV-3C). At the same

depth, 75% of the cells belonged to the domain Archaea, indicating that most of these were

ANME-1 cells (results not shown). Bacteria accounted for 21% of DAPI stained cells, of which

53% (e.g. 11% of the total cell number) belonged to the genus Desulfosarcina / Desulfococcus,

which includes the Seep-SRB cluster of putative ANME partners (results not shown). In general,

ANME-1 cells were rod-shaped, formed chains of 2 to 16 cells (Figure IV-6A and B) and were

not associated with other type of cells. Less than 10% of ANME-1 cells formed large clusters

(Figure IV-6C and D) comprising few non-ANME-1 coccoid cells. At Mercator MV Rim 2,

ANME-1 cells were only observed at 42.5 cmbsf and constituted 58% (±10%) of the DAPI

stained cells (n=180) (Figure IV-3C). There, cells were smaller (~0.7 µm), with coccoid or short

rod shapes and found as single cells or chains of 2 to 4 cells (Figure IV-6E and F). Very few

DAPI-stained cells and no ANME-1 cells were detected in the Mercator MV Crater Centre at the

investigated depths. In contrast, in the AOM zone (30 cmbsf) of the CAMV Crater Centre, the

presence of ANME-2 cells (previously found by Niemann et al., 2006b) was confirmed by FISH

assays. There, ANME-2 cells occurred as shell type aggregates, surrounded by other non-ANME

cells (Figure IV-6G and H).

CHAPTER IV

102

Figure IV-6 Direct microscopic observations of AOM communities of CARD-FISH and DAPI stained cells at Mercator MV. Monospecific chains of rod-shaped cells positive for the ANME-1 probe (A and B), or aggregates comprising ANME-1 positive cells (C and D) observed at Mercator MV crater rim 1 (32 cmbsf). E and F: small cocci or short rod-shaped ANME-1 positive cells observed at Mercator MV crater rim 2 (41 cmbsf). Overlay of DAPI stained (blue) and ANME-2/Cy3 (red) stained cells images (double stained cells appear in purple). White scale bars: 5 µm.


103

3. Discussion

3.1 AOM activity in hypersaline and non-hypersaline sediments

For a long time, hypersaline environments where considered as hostile or even

biogeochemical dead ends. Indeed, the immense ionic strength and osmotic pressure appear to

exclude most life on Earth, yet specialised microbes and a few eukaryotes have accomplished to

minimise the negative effects of hypersalinity, apparently allowing them to thrive in these

challenging environments (Boetius and Joye, 2009). Because of the many biochemical

constraints associated to life in hypersalinity, these environments are believed to select for

metabolisms associated with elevated energy yield (Oren, 2011). AOM is characterised by one of

the lowest known energy yields among microbial energy conserving reactions. In the particular

case of the putative syntrophic nature of AOM, the energy is shared, which further reduces the

energy yield for each syntrophic partner. Despite these constraints, previous studies could

already show that AOM can occur in hypersaline environments such as Mono- (Joye et al., 1999)

and Big Soda lake (Iversen et al., 1987) at salinity of 88-90 g L-1 (~1.6 M Cl-). In marine cold

seep sediments, Lloyd et al. (2006) provided geochemical and molecular evidence for AOM

activity at 146 g L-1 (~2.5 M Cl-). Our results significantly extend the salinity range for AOM,

showing that substantial rates (2.3 nmol cm-3 d-1 at Mercator MV Crater Centre) can be attained

under hypersaline conditions at 263 g L-1 (4.5 M Cl-), and even at halite saturating conditions

(340 g L-1 or 5.8 M Cl-) with rates of 0.5 nmol cm-3 d-. Hence, ANME-1 cells detected in

extreme hypersaline environments, such as the Bannock, l’Atalante, and Thetis deep sea brine

pools, with respective salinities of 148 g L-1 (Daffonchio et al., 2006), 230 g L-1 (Yakimov et al.,

2007) and 348 g L-1 (La Cono et al., 2011), could carry AOM. However, these results are in

contrast to the current concept that hypersaline conditions select for high-energy yield

metabolisms. It is not clear how microbial communities mediating AOM can implement energy

demanding osmoregulatory mechanisms (e.g. ion pumping, production of compatible solutes)

unless the low AOM energy yield is balanced by a very high methane turnover per cell.

In spite of high methane and sulphate concentrations at Mercator MV sites, AOMR was below

8.5 nmol cm3 d-1, i.e. up to 3 orders of magnitude lower than the maximum AOMR observed

elsewhere (Knittel and Boetius, 2009). Moreover, we found a rather wide vertical distribution of

AOM activity at all Mercator MV sites. In contrast, at CAMV, characterised by background

CHAPTER IV

104

salinity, AOM activity was confined to a discrete sediment horizon as already found previously

at this structure (Niemann et al., 2006b) and at other non-hypersaline seep systems (Knittel and

Boetius, 2009). This strongly suggests that AOM was inhibited at the level of cell activity or

population growth, probably as a result of the extreme hypersaline conditions at Mercator MV.

This interpretation is supported by the results of (Nauhaus et al., 2005), Figure III-2 showing that

AOM rates tend to decrease with increasing salt concentration above seawater salinity during in

vitro incubations with seep sediments.

Maximum AOMR at CAMV were higher compared to any of the Mercator MV stations, but

depth integrated areal rates (∑AOMR) were 6.1- and 3.8-fold lower than in Mercator MV Crater

Centre and Rim 2, respectively (Table IV-2). In contrast to regular ocean sediments, Mercator

MV hypersaline sediments were characterized by elevated sulphate levels due to the leaching of

sulphate-rich evaporite strata by the ascending fluid (Scholz et al., 2009). In this system, sulphate

was therefore not only transported from seawater downwards into the sediment, but also upward

together with other ions, hydrocarbons and fluids. Furthermore, the inhibition of AOM in these

hypersaline sediments prevented a rapid consumption of sulphate from both sources and upward

migrating methane, and therefore, the formation of a typical SMTZ. Instead, the hampering of

methane and sulphate turnover led to a very broad AOM zone where methane and sulphate

overlap and in which AOM-communities are spread-out vertically. Consequently, such vertical

extension of microbial activity resulted in higher turnover per unit area and thus compensated the

reduction of volumetric rates to some extend.

Our results provide new insights on the origin of the gas venting observed during video

surveys (Van Rooij, 2005). Shallow sub-seafloor internal seismic reflectors resembling bottom-

simulating reflectors (BSR) were identified in Mercator MV Crater Centre sediments, suggesting

the presence of gas hydrates near the sediment surface (Depreiter et al., 2005). Based on these

observations, it was proposed that such gas venting could be caused by methane hydrate

dissociation (Van Rooij, 2005). Our results show that the main trigger for gas venting was rather

the inhibition of the AOM microbial activity in this hypersaline environment.

3.2 AOM community structure in hypersaline sediments of Mercator MV.

The Mercator MV environment closely resembles the Gulf of Mexico gassy sediment

described by (Lloyd et al., 2006), in term of hypersalinity and by the fact that ANME-1 cells


105

dominated both environments. Therefore, extremely saline environments may well exert a

selective pressure toward ANME-1, as already proposed by (Lloyd et al., 2006) and (Yakimov et

al., 2007). This would also explain the findings of ANME-1 in brines in the eastern

Mediterranean (Daffonchio et al., 2006; Yakimov et al., 2007; La Cono et al., 2011). Since

ANME-1 cells have also been reported in the CAMV Crater Centre site (Niemann et al., 2006b)

and other non-hypersaline environments (see Addendum II of this chapter, p.114), a negative

selection towards other ANME groups thus appears to be the most probable ANME selection

mechanism in hypersaline cold seeps. Such selection pattern could be related to the

comparatively low effect of ionic strength on Archaea in general and ANME-1 in particular. The

permeability of archaeal membranes comprising isoprenoidal glycerol ethers is generally lower

than that of bacterial membranes comprising fatty acid glycerol esters (Valentine, 2007). Low

membrane permeability reduces the energy loss associated to random ion exchange between the

cyto- and ectoplasm. Specifically, ANME-1 comprises high contents of membrane spanning

lipids, so called glycerol dialcyl glycerol tetraethers - GDGTs (Niemann and Elvert, 2008).

Membranes composed of GDGTs are at the lower end of permeability when comparing typical

membrane lipids (Yamauchi et al., 1993; Valentine, 2007). This could also explain the

apparently negative selection of hypersalinity towards ANME-2 and -3 as these organisms

feature membranes composed of diethers characterised by higher membrane permeability. In

addition, genes coding for manosylglycerate and di-myo-inositol-phosphate synthesis pathways

were identified in the ANME-1 genome (Meyerdierks et al., 2010). These two compatible

solutes are widely employed by halophile microorganisms to increase their turgor pressure

(Roberts, 2004; da Costa and Empadinhas, 2008). These compounds may thus also confer

hypersalinity resistance to the ANME-1 cells.

In Mercator MV Rim 1 sediments, ANME-1 cells were mainly present as monospecific

chains, with no apparent contact with any other cell type. Such organization has already been

reported in most previous ANME-1 microscopic observations (excepted in the conspicuous

Black Sea mats and occasionally in Eel River basin sediments, see supplementary information).

This thus seems to be the dominant spatial organisation of ANME-1 cells, irrespective of the

hypersaline conditions found at Mercator MV. However, increasing ANME-to-SRB cell distance

has a detrimental effect on AOM energy yield (Sorensen et al., 2001; Alperin and Hoehler,

2009). In our microscopic observations, distances between ANME-1 cells and the closest non-

ANME-1 cell typically exceed 150 µm, the size of the observation grid. Such distance could be

an artefact from CARD-FISH sample preparation, but (Orphan et al., 2002b) found similar

CHAPTER IV

106

bacteria-free ANME-1 in the absence of disruptive treatments. Such existence of bacteria free

ANME-1 population is also supported by the observation of large patches of DSS free ANME-1

aggregates in subsurface microbial mats in the Black sea sediments (Treude et al., 2005c). In

addition, at 29 cmbsf in Mercator MV Rim 1 sediments, the microbial community was composed

of 79% of ANME-1 and 11% DSS cells, resulting in an ANME:SRB ratio of 7:1. Such ratio

strongly differs from the typical 1:3 ratio observed in shell type consortia (Orcutt and Meile,

2008a) and necessarily imposes distances between both partners. Consequently, our observations

suggest that monospecific chains of ANME-1 cells are able to carry out AOM even if their

distance to SRB cells exceeds the previously published critical distance (typically 5 µm under

the Hydrate Ridge conditions. Alperin and Hoehler, 2009). Hence, in this system, AOM could

involve an electron transport mechanism different that the commonly proposed AOM/SR

coupling involving a chemical ITA diffusion between both partners. Alternatively, ANME-1

cells could overcome this thermodynamic constraint by carrying out both parts of the redox

reaction, i.e. the oxidation of methane and the reduction of sulphate (Hansen et al., 1998; Orphan

et al., 2002b). However, (Meyerdierks et al., 2010) could not find known genes involved in

dissimilatory sulphate reduction in the ANME-1 genome (~80% coverage). Another possibility

explaining the apparent separation of both syntrophic partners could be a direct electron shuttling

between distant ANME and SRB cells through a conductive matrix. In mesocosms experiments

with marine surface sediments, diffusion-independent electron shuttling over 1.2 cm distance has

been shown (Nielsen et al., 2010). The expression of genes coding for membrane c-type

cytochromes by ANME-1b cells (Meyerdierks et al., 2010) would be in line with this hypothesis

of a direct electrons shuttling (Mehta et al., 2005). However, evidence for electroactivity of

ANME-1 or Seep-SRB cells are lacking.

3.3 Reaction coupling between AOM, SR and MG

At CAMV Crater Centre, we found that depth of SRR and AOMR maxima coincided and

areal SRR (∑SRR) exceeded areal AOMR (∑AOMR) within the SMTZ. Based on these areal

rates, 63% of the sulphate reduction in the AOM zone was due methane oxidation (Table IV-2).

This was thus compatible with a coupling between these reactions with an equimolar

consumption of both substrates (equation 1). Several factors can account for the remaining 37%

sulphate reduction activity in the AOM zone: organoclastic sulphate reduction through

mineralization of higher hydrocarbons co-migrating with methane (Niemann et al., 2006b;


107

Kniemeyer et al., 2007; Bowles et al., 2011), or sedimentary organic matter from microbial or

seep fauna necromass (Hilario and Cunha, 2008; Niemann et al., 2009; Sommer et al., 2009).

Relatively low concentrations of higher hydrocarbons were found at CAMV (Niemann et al.,

2006b). It is thus likely that the substantially higher ∑ SRR are fuelled by a combination of

AOM, hydrocarbons and necromass oxidation.

We also found the reverse situation where ∑AOMR consistently exceeded ∑ SRR over

large depth intervals at Mercator MV Crater Centre or Rim 2 and at CAMV Crater Centre

between 43 and 300 cmbsf. These remarkable results suggested the presence of alternative

electron acceptors, other than sulphate, in both MV sediments. Among potential electron

acceptors, Fe(III) and Mn(IV) or nitrate/nitrite were shown to be utilised in alternative modes of

AOM. Although speculative at present, it therefore seems possible that AOM at Mercator MV

might, partially, be independent from SR. Although the presence of oxidized Mn, Fe or N

species in subseafloor sediments and brines is possible (D'hondt et al., 2004; Parkes et al., 2005),

there is, to our knowledge, no such data available for Mercator MV that would substantiate this

aspect further. At CAMV, the intrusion of seawater within the ascending fluid (Hensen et al.,

2007) could be the source of such electron acceptors.

Interestingly, similar decoupling of AOM and SR were already observed in conditions of

very low or no sulphate, either in vitro (Hansen et al., 1998; Beal et al., 2011), or in

environmental samples, at the base of or below SMTZs (Hansen et al., 1998; Joye et al., 2004;

Niemann et al., 2006b; Parkes et al., 2007), which apparently correspond to the preferential

habitat for ANME-1 cells in non-hypersaline methane bearing marine sediments (Knittel et al.,

2005; Yanagawa et al., 2011). These observations suggest that ANME-1 dominated AOM

communities could potentially decouple AOM and SR, as observed in the present study.

Recent work (House et al., 2009) showing important isotopic carbon heterogeneities

among ANME-1 cells from Eel River Basin sediments, together with the presence of significant

methanogenesis rates, indicates that ANME-1 cells may switch from a methanotrophic to a

methanogenic metabolism. Similar conclusions arose from micrometer scale reaction/transport

modeling (Alperin and Hoehler, 2009) showing that the concentration of reduced fermentation

product -such as H2- could act as a thermodynamic switch between the two metabolisms. This is

in agreement with the increasing number of studies reporting a dual methanotrophic-

methanogenic activity in environmental samples (Orcutt et al., 2005) or in vitro (Treude et al.,

2007; Orcutt et al., 2008b). In contrast, since the vertical distribution of AOM and MG rates did

CHAPTER IV

108

not match in any of the sites investigated, it seems that ANME cells did not operated in such dual

mode within AOM zones of Mercator and CAMV, and have rather a strict methanotrophic

metabolism.

3.4 Methanogenesis rates and zonation.

MG rates observed in this study were thus attributed to “true” methanogenesis. These

were however too low and within too narrow sediment intervals to significantly contribute to the

methane pool at all investigated sites. In general, the presence of sulphate is thought to exclude

methanogenesis from marine sediments (Oremland and Taylor, 1978), as SRB generally

outcompete methanogens for the utilisation of H2 or acetate (Schonheit et al., 1982). In line with

this general rule was the presence of Ac-MG rates only below the SMTZ at CAMV Crater

Centre. However, departing from such zonation, peaks of Ac-MG and H-MG also occurred in

the sulphate zone in Mercator MV Rim 1 sediments. Similar observations were made in the

Napoli MV hypersaline sediments of the East Mediterranean sea (Lazar et al., 2011). It has been

shown that, in the presence of sulphate, methyltrophic methanogens can still metabolize methyl

moieties but mostly redirect them through the reverse methanogenesis oxidative pathway, with

CO2 as final product rather than CH4 (Finke et al., 2007). In this case, sulphate reduction act as a

sink for the electrons released during methyl oxidation via interspecies H2 transfer. Similar

process could occur with acetate as electron donor (Phelps et al., 1985; Achtnich et al., 1995)

and could explain the residual Ac-MG rate observed at Mercator MV Rim 1. This interpretation

of residual Ac-MG was supported by the very high peak of acetate oxidation to CO2 at 13 cmbsf

(7847 pmol cm-3 d-1, data not shown) compared to the CH4 formation (2.4 pmol cm-3 d-1) at the

same depth. The interpretation of the H-MG activity peak was more problematic, as SRB usually

maintain H2 levels that are inhibitory for methanogens (Kristjansson et al., 1982). However, it is

possible that energetic constraints imposed by hypersaline conditions prevented SRB to consume

hydrogen down to such low level, thus opening a narrow window for hydrogenotrophic

methanogenesis activity along salinity gradients. However, to our knowledge, H2 kinetics of

consumption by SR and MG in varying salinity that could support this interpretation is lacking.

The Met-MG activity in sulphate rich near-surface sediments at all sites excepted Mercator

MV Crater Centre was coherent with the fact that methylated amines are non-competitive

substrate that cannot be used by SRB (Oremland and Polcin, 1982). The lack of Met-MG activity

below was unlikely due to an increasing salt inhibition as methylotroph are among the most

halotolerant methanogens (McGenity, 2010). This rather suggests that the source of substrate for


109

Met-MG was near-surface diagenetic processes, such as for instance glycin and betain

degradation (Oren, 1990).

CHAPTER IV

110

4. Conclusion

In brine sediments of Mercator MV, the presence of both sulphate and methane in the

ascending fluid fuels substantial rates of AOM and SR. Unlike at regular ocean sediments, no

clear sulphate-methane transition zone is present, but AOM and SR are distributed over a wide

depth interval. However, most probably as a result of the hypersaline conditions, AOM is

partially inhibited, so that maximum volumetric rates are orders of magnitude lower in

comparison to other cold seeps. At Mercator MV, AOM is mediated by ANME-1, which mostly

occurs as monospecific cell-chains and constitutes up to 79% of the microbial community. This,

together with previous findings of ANME-1 in hypersaline settings, suggests that this ANME

cluster is well adapted to elevated salinity, despite the low energy conserved from the AOM

reaction. We also found that AOM and SR can be uncoupled with AOM significantly exceeding

SR over wide depth intervals. Hypothetically, this could be related to the utilisation of electron

acceptors other than sulphate for AOM. Finally, unlike in several other seep sites,

methanogenesis is apparently not coupled to AOM as such activity was not detected where AOM

was maximum.


111

ACKNOWLEDGMENTS

This work was supported by the “HERMES” project grant (Hot-Spot Ecosystems along

European Margins) under FP6 of the European Commission, the European Science Foundation

“MicroSystems” grant (FWO-506G.0656.05) and the Geconcerteerde Onderzoeksactie (GOA) of

Ghent University (BOF09/GOA/005). LM was recipient of a PhD grant from the “Institute for

the Promotion of Innovation by Science and Technology in Flanders” (IWT-Vlaanderen,

contract number SB-53575). We wish to acknowledge the crew and scientific party of the

MSM1/3 cruise led by O. Pfannkuche aboard the RV Maria S. Merian (2006), who largely

contributed to the preliminary results enabling this work. The authors would like to thank the

shipboard scientific party and crew of the Research Vessel “James Cook” (JC10 cruise) and the

ROV ISIS pilots/engineers. They contributed to this study with their valuable on-board

assistance and long seafloor video observations shifts. In particular, K. Heeschen and V.

Hühnerbach are thanked for cruise organisation and for on-board data handling respectively. We

thank W. Verstraete for helpful suggestions, and M. Mußmann for assistance with CARD-FISH

assays.

CHAPTER IV

112

5. ADDENDUM I: BATHYMETRY, SAMPLING AND SEDIMENT

DESCRIPTION

5.1 Sampling site, seafloor observations and sedimentology

Mercator MV was located in the shallow region of the Gulf of Cadiz, amidst the El Arraich

mud volcano province (Van Rensbergen, 2005) at 350 m water depth. CAMV was located

deeper (1300 m water depth), about 100 km N.-E. of Mercator MV (Figure IV-1A). In the Gulf

of Cadiz, thermogenic methane is formed at about 4000 m water depth and sediment fluidization

occurs due to clay mineral dewatering (transformation of smectite to illite) with typically low ion

contents of pore waters (Nuzzo et al., 2009; Scholz et al., 2009). However, the upward migrating

fluids at Mercator MV are enriched in sodium, chloride and sulphate, probably by mixing with a

subsurface evaporite strata. Evaporite leaching is much more pronounced in Mercator MV

compared to CAMV (Scholz et al., 2009). Mercator MV (Crater Centre and Rim sites) and

CAMV (Crater Center) as well as a reference site were sampled.

,

Figure IV-7 Gypsum (A) and halite (B) crystals recovered from a gravity core at Mercator crater centre station (JC10-19), between 200 and 240 cm below the sea floor (cmbsf). Scale bars approximately correspond to 5 cm.

At Mercator Crater Centre (core JC10-009 and -019), sediments recovered by gravity and

piston coring mainly consisted of mud breccia and were characterized by a very low porosity,

decreasing from 50% near the seafloor down to 15% at 32 cmbsf (JC10-009) or 115 cmbsf

(JC10-019). In addition, large crystals of Gypsum (Figure IV-7A) and Halite (Figure IV-7B),

identified based on their morphology, were recovered from the Mercator MV crater centre. At

Rim 1 (core JC10-013) and Rim 2 (core JC10-015) sites, sediments mainly consisted of an


113

alternation of compact and “foamy” mud breccia, with porosity comprised between 40% and

60%, overlain by a 10 cm layer of brown hemipelagic sediments. Observations carried out with

the ROV Isis at Mercator MV revealed an active methane seep in the centre of the crater: we

found gas venting from small holes (ca. 5 cm in diameter), confirming previous observations of

(Van Rooij, 2005). On the contrary, no gas seepage could be found at CAMV. The CAMV

Crater Centre core was recovered from the location previously sampled by (Niemann et al.,

2006b) (core GeoB 9041-12). Facies and fauna associated with these mud volcanoes are further

described in (Niemann et al., 2006b) and (Vanreusel et al., 2009).

CHAPTER IV

114

6. ADDENDUM II: Abundance and consortia structure involving ANME-1 in methane seeps

Location

cmbs

f

AN

ME

-1

(%)

DSS

(%)

AN

ME

-2

;AN

ME

-3

Cel

l Nbr

cm-3

x109

Structure of ANME-1 cells and ANME-

1/DSS associations

Salin

ity

‰

AO

MR

:SR

R (n

mol

cm-3

d-1

)

Reference

170 - - - - 292 2.3:1.9 Gulf of Cadiz

Mercator MV

Crater Centre 220 - - - - -

326 0.5:7.5

Mercator MV

Rim1 31 79 10 No -

Chains of 2 to 16 ANME-1 cells

<10% ANME-1 cells formed large

aggregates with non ANME-1 cells

104 3:5.2

Mercator MV

Rim2 32 59 - No -

Cocci or short rod pair of cells, positive

for ANME-1 probe 104 8.4:1.8

This study

0-2 5 11 No 20.45 2:1050

2-4 9 7 No 1.80 3:1050

East

Mediterranean sea

Napoli MV

10-12 22 0 No 1.99

116(4)

2:1000

0-2 >0 7 13;<1 3.52 -

2-4 >0 8 No 3.62 60:450

East

Mediterranean sea

North Alex MV

10-12 1 3 7;<1 2.70

Single ANME-1 cells or cell filaments, no

consortia

35(3)

30:250

(Omoregie et al.,

2009)


115

Lake Plußsee 8-15(1) 0.1-1 <1 / 0.1-

0.5; 0.005

Single cells or short chains of up to 5

cells, no consortia Freshwater (Eller et al., 2005)

3 1-15 Hydrate Ridge

Beggiatoa mat 9 9-29 Yes Yes

3 8 Calyptogena field

12 21 Yes Yes

-

Mostly as single cells without any directly

associated bacterial or archaeal partner.

Chains of 2 to 10 cells. Spherically

aggregated ANME-1 cells rarely detected.

33 (Knittel et al.,

2005)

Eel River basin 6-12

Yes

Dom-

nant

Yes Yes -

Single cell / filaments

Filaments near DSS aggregates

Interspersed ANME-1 filaments and DSS

33

(Orphan et al.,

2002b)

(Orphan et al.,

2004)

Eel River basin 2-15 Yes - Yes - ANME-1 rods and filaments 33 1-55: - (House et al.,

2009)

Haakon Mosby MV 9-10 <0.5 7 No Cell filaments 33 (Losekann et al.,

2007)

1 2.7 10 4.6 5.4 0.3:100 Gulf of Mexico

Brine Pool 9 45.9 4.6 2.4 6.8 31

1.2:700

1 2.5 18 0 3.7 40:10 Gulf of Mexico

Hydrate 7 19 25.9 3.0 2.2

Cell filaments

Dense monospecific ANME-1 aggregates 31

160:400

(Orcutt et al., 2005)

Black sea 250-

2000(1) 3 3 2 0.3 Single cells ~20 ~1.5:- (Durisch-Kaiser et

al., 2005)

CHAPTER IV

116

Water column

Lost Hammer

terrestrial Arctic

hypersaline seep

- 3.4 No No 0.00045 No DSS 240 - (Niederberger et

al., 2010)

Baltic sea

Eckernförde bay 0-40 low Yes Yes 0.14 Short filament of 4-6 rectangular cells SW 5-120: -

(Treude et al.,

2005a)

North sea

Tommeliten seep 170 13 - No 0.12

Single ANME-1 cells or short chains of

up to 3 cells, no consortia Seawater 2:2

(Niemann et al.,

2005)

Black sea

Carbonate

Chimney mat

0 - Yes Yes -

. Interspersed ANME-1 and DSS cells or

large aggregates

. Much lower DSS number compared to

ANME-1 and to ANME-2/DSS

aggregates

. Large area (up to 400 µm in diameter) of

DSS free ANME-1 aggregates.

Seawater -

(Michaelis et al.,

2002)

(Blumenberg et al.,

2004)

(Knittel et al.,

2005)

(Treude et al.,

2007)

Black sea

subsurface mat 11-13 40(2) 10 No - - ~20 1500:1800

(Treude et al.,

2005c)

Black sea

Sedimentary

Yes No ANME-1 in Large aggregates of short

chains ~20 -

(Reitner et al.,

2005)


117

carbonate crusts

mat

Long filaments surrounding dense DSS

cells containing Greigite and PHA

inclusions

Table IV-3 Studies using (CARD)-FISH to describe the abundance, distribution and community structures involving ANME-1.

(1): cm water depth. (2): evaluated by slot blot hybridization of rRNA. (3): data from (Feseker et al., 2010) (4): Chloride profile was not given in (Omoregie et al., 2009), and was thus estimated from the Cl- profile published in (Lazar et al., 2011).

CHAPTER IV

118

Publications on AOM communities involving ANME-1 cells, their quantification and

structure relative to DSS cells or other ANMEs are briefly reviewed in Table IV-3. In all

studies, ANME and DSS cells were quantified by (CARD-)FISH cell counts, excepted in

(Treude et al., 2005c) where dot slot was also used.

In these studies, 3 types of ANME-1 organisation were reported: (A) single cells or chains of

ANME-1 cells with no apparent association (i.e. contact or small distance) with other non-

ANME-1 cells, (B) large ANME-1 aggregates interspersed with fewer non-ANME-1 single

cells or aggregates (including DSS cells) and (C) large aggregates of interspersed ANME-1 and

DSS single cells. (A) was by far the dominant type and was observed in almost all methane

seep sites where ANME-1 were reported. It is possible however, that individual cells or

filaments are detached from large aggregates during FISH procedures. In (B) type of

aggregates, a syntrophic association between ANME-1 and DSS involving electron carrier

diffusion was not evident as ANME-1/DSS distances were often very large. (Treude et al.,

2007) reported for instance DSS free aggregates as large as 400µm in diameter. (C), which

would resemble the ANME-2/DSS type of association was only occasionally observed in Eel

River sediments and in Black sea chimneys mats. Hence, ANME-1 dominated communities,

tend to have higher ANME:DSS cells ratio, and higher ANME/DSS cell-cell distances.

Besides, these observations support two other characteristic patterns of ANME-1 distribution.

As already proposed by (Knittel et al., 2005), the proportion of ANME-1 cells, relatively to

ANME-2 cells, generally increases with depth and could possibly relate ANME-1 ecological

niche to low sulphate environments. Such pattern was observed in Napoli MV, Gulf of Mexico

brine pool and hydrate bearing sediments, Hydrate ridge Beggiatoa mat and Calyptogena

(Table 1) and was reported by recent studies using phospholipid (Rossel et al., 2011) and

molecular (Yanagawa et al., 2011) markers. Moreover, ANME-1 dominated the hypersaline

environments reviewed (Napoli MV, Lost Hammer terrestrial seep, Mercator MV) and other

ANME types were not present. A similar pattern was supported by (Lloyd et al., 2006) and

(Yakimov et al., 2007) using archaeal clone libraries. However, such pattern was also observed

in non hypersaline conditions, in a subsurface Black sea mat (Treude et al., 2005c)


119

121

CHAPTER V. Methane

oxidation at the Carlos Ribeiro Mud

Volcano: Ex-situ Microbial Activity vs.

Geochemical Modeling.

To be submitted as L. Maignien, R. J. Parkes, B. Cragg, H. Niemann, K. Knittel, S. Coulon, A.

Akhmetzhanov, P. Weaver, N. Boon. Methane oxidation at the Carlos Ribeiro Mud Volcano:

Ex-situ Microbial Activity vs. Geochemical Modeling.

CHAPTER V

122

METHANE TURNOVER AT CRMV

123

ABSTRACT

The determination of the methane flux and consumption rates by microorganisms is of

importance to constrain the methane budget and the carbon cycling in cold seep sediments. In

this study, the rates of methane oxidation (MO) and sulphate reduction (SR) measured in the

deep sea Carlos Ribeiro mud volcano (CRMV) sediments by ex situ radiotracers turnover were

one order lower than the modelled methane and sulphate flux resulting from reaction transport

geochemical models described in a companion paper (Vanneste et al., 2011). This apparent

inconsistency could be explained by estimating in situ microbial activity rates based on

modelled methane profiles. This suggested that the difference of methane solubility between in

situ vs. ex situ conditions can lead to a large underestimation of the in situ rates. A small

fraction of the methane flux was oxidised in the bioirigated shallow sediment subsurface,

probably by an aerobic process, but the largest part was consumed by anaerobic oxidation of

methane (AOM) coupled to sulphate reduction (SR), involving all known types of anaerobic

methanotrophs (ANME-1, -2 and -3). However, the AOM community in the MV crater centre

was different than other known seep sites as it was dominated by GoM-Arc1 Archaea and

butane/propane oxidising sulphate reducing Bacteria.

CHAPTER V

124

1. Introduction

Anaerobic Oxidation of Methane (AOM) coupled to sulphate reduction (SR) is the most

significant microbial process at methane cold seeps such as mud volcanoes (Knittel and

Boetius, 2009).

CH4 + SO42- ⇌ HS- + HCO3

- + H2O ΔG0’=-16.9 kJ mol-1 Equation 1

This reaction is performed by various Archaea closely related to methanogens forming

distinct phylogenetic groups: ANME-1, ANME-2, and ANME-3 (Hinrichs et al., 1999;

Boetius et al., 2000; Orphan et al., 2001a; Orphan et al., 2001b; Knittel et al., 2005; Lloyd et

al., 2006; Losekann et al., 2007). These anaerobic methanotrophs often form tight associations

with sulphate-reducing bacteria (SRB) of the genera Desulfosarcina / Desulfococcus (ANME-

1 and -2) or Desulfobulbus (ANME-3) (Knittel et al., 2003; Losekann et al., 2007), and a

syntrophic relationship between these microorganisms has been proposed (Boetius et al., 2000;

Orphan et al., 2001b). However, the presence of monospecific aggregates or single ANME

cells in active AOM zones (Losekann et al., 2007), CHAPTER III and IV, and the results from

reaction/transport models of ANME/SRB aggregates indicate that cell contact is not required

for AOM activity (Alperin and Hoehler, 2009). The typical geochemical signature of AOM

activity in sediments is the presence of a sulphate-methane transition zone (SMTZ), i.e.

opposed gradients of downward diffusing sulphate and upward migrating methane.

Microorganisms mediating AOM are usually present and active in a narrow depth interval at

the interface between these gradients.

Subsurface methane flux and turnover can be quantified by two complementary

approaches. On the one hand, reaction transport models (RTM) integrate sulphate diffusive

fluxes and fluid advection rates in order to constrain the methane concentration profile and

flux (Boudreau, 1997). On the other hand, the ex-situ approach, consisting in injecting

radiolabeled substrates in sediment samples upon core recovery (Iversen et al., 1987; Fossing

and Jørgensen, 1989), permits to measure methane and sulphate turnover and identify active

AOM sediments horizons. However, large discrepancies have been observed between AOM

turnovers estimated by these two approaches. This is illustrated by study of the Captain

Arutyunov MV (CAMV) in the Gulf of Cadiz: RTM resulted in a methane flux of 6268 mmol

m-2 yr-1 (Hensen et al., 2007; Vanneste et al., 2011), whereas ex-situ AOM and SR areal rates

were of 383 and 577 mmol m-2 yr-1 (Niemann et al., 2006b). We hypothesise that the


125

differences in methane solubility between in situ and ex situ conditions may explain these

discrepancies.

In this chapter, we describe the community structure involved in AOM at three different

sites of the CRMV and determined the major microbial activities in these sediments (AOM, SR

and Methanogenesis or MG). Further, we use realistic methane concentrations from a

geochemical model combined with the AOM rate dependency on substrate concentration (Jin

and Bethke, 2002, 2003, 2005; Dale et al., 2008; Knab et al., 2008) to estimate the in situ

methane turnover.

CHAPTER V

126

2. Description of the model used to estimate in situ AOM rates

The AOM rate (VAOM) dependency on substrate concentration (Jin and Bethke, 2002,

2003, 2005; Dale et al., 2008; Knab et al., 2008) can be expressed as a function of maximum

rate (Vmax):

Equation 2

According to this model, the maximum specific rate is modulated by a Michaelis–

Menten kinetic factor Fk,

Equation 3

and a thermodynamic factor FT accounting for activity inhibition when the reaction yield ΔG

approaches a threshold value ΔGBQ corresponding to the smallest amount of energy that must

be conserved for ATP formation (Jin and Bethke, 2005):

Equation 4

Based on Equations 2 to 4, in situ AOM rates (VAOM.IS) can thus be estimated from

measured ex situ rates (VAOM.ES) by:

Equation 5

Following the same assumptions than in (Dale et al., 2006; Knab et al., 2008), i.e

KCH4>>[CH4], Fk becomes:

. Equation 6

Replacing this expression of FK in Equation 5, and since VMAX, , and [SO42-]

do not vary between in situ and ex situ conditions, Equation 5 becomes:

Equation 7


127

ΔG is calculated according to:

Equation 8

where VAOM.ES is the ex situ AOM rate measured by radiotracer turnover, [CH4]IS is the in situ

methane concentration modelled in (Vanneste et al., 2011), [CH4]ES is the ex situ methane

concentration measured during the radiotracer turnover determination ((Treude et al., 2003).

For calculations, we used R=8.3144 J.mol-1.K-1 for the gas constant, T=277.1 K as the absolute

in situ temperature, χ=2 as a stoechiometric factor (Jin and Bethke, 2005; Knab et al., 2008),

ΔGBQ=10 KJ.mol-1 as the critical yield value for energy conservation (Jin and Bethke, 2005;

Knab et al., 2008). For ΔG calculation, we used the activity coefficient values provided in

(Alperin and Hoehler, 2009), i.e.

[HCO3-] and [HS-] values were taken from (Vanneste et al., 2011). At three locations

studied, our depth scale was corrected by fitting sulphate and chloride profile to the profiles

from (Vanneste et al., 2011). Note that unlike the Crater Eye and the Off-center sites, the Rim

site of our study had no counterpart in the study of Vanneste et al. However, their “Margin”

site presented similar sulphate and chloride profiles, and data from this site was used as a

comparison for the Rim site of our study.

Estimated in situ areal AOM rates were calculated by integrating (by the rectangle

method) the in situ volumetric rates resulting from this model within the boundaries of the

SMTZs.

CHAPTER V

128

3. Results

3.1 Sampling site

The CRMV is located on the south Portuguese margin, in the deep part of the Gulf of

Cadiz at water depth of 2200 m. The crater diameter was ca. 1500 m and mud accumulations

formed dome-like structure of ca. 80 m height. Successive mud eruptions formed concentric

rims that are visible on the high-resolution bathymetric map. The CRMV was sampled at the

Crater Eye and Off Centre sites that were also used for reaction transport modelling in

(Vanneste et al., 2011). In addition, we sampled a radial mudflow at the Crater Margin (Figure

V-1and Table V-1).

Figure V-1 The Carlos Ribeiro mud volcano (CRMV) was located in the deep basin of the Gulf of Cadiz (N.-E. Atlantic). The CRMV base was at 2300 m water depth. The crater was about 140 m high and 360 m wide. Circles indicate the location of the sampling sites. The mud volcano was sampled in the Crater Eye and Off Centre sites that were used for geochemical modelling in the study of (Vanneste et al., 2011). In addition, the Crater Rim site was also sampled. Circle colours correspond to sampling devices used at each site. (red: megacore, green: gravity core and blue: piston core). Depth range: - 2170 m (brown), -2200 m (green).


129

Table V-1 Core depth and position at the three CRMV sites of this study. Sediments were also taken 2.9 km S.-W. of CRMV for reference.

3.2 Geochemistry

Methane was present in the deeper sediment strata at the three locations investigated,

with maximum values around 2 mM (Figure V-2). Methane concentration decreased toward

sediment surface, indicating an upward methane flux. A large part of this methane flux was

consumed in the sediment subsurface, as shown by the steep concentration gradients between

26 and 6 cm below the sea floor (cmbsf) at Crater Eye, 44 and 14 cmbsf at Off Centre, and 41

and 21 cmbsf at Crater Margin sites. However, residual methane concentrations were still

present above these intervals (between 0.002 mM at Crater Rim and 0.022 mM at Crater Eye

station).

Sulphate concentration remained close to seawater values (~28 mM) along depth

intervals varying from 7 cmbsf (at Crater Eye and Rim sites) to 14.5 cmbsf (at Off Centre site)

(Figure V-2). Below, sulphate concentration steeply decreased down to values <1mM at depth

of 27, 32 and 42 cmbsf at Crater Eye, Off Centre and Rim sites respectively. Below these

depths, sulphate concentration gradually reached values below detection limits.

A peak of sulphide concentration was present at the interface of methane and sulphate

gradients, with maximum values of 0.3, 2.6 and 1.8 mM at Crater Eye, Off Centre and Rim

sites respectively.

In the three locations, opposed gradients of sulphate and methane as well as sulphide

concentration maxima defined so-called SMTZ. These SMTZ were located between 5 and 35

cmbsf at Crater Eye station, between 23 and 52 cmbsf at Off Centre station and between 11

and 50 cmbsf at Rim station.

Hydrogen gas was absent in all locations, excepted at the Crater Eye site were a discrete

peak (0.4 nM) was present at 56 cmbsf. The volatile fatty acid acetate was present in all

locations, with concentration generally decreasing from deeper strata toward sediment surface.

Maximum concentrations were of 107, 80 and 29 µM at Crater Eye, Off Centre and Crater

Rim respectively, and reached minima between 10 and 20µM near sediment surface.

CHAPTER V

130

3.3 Ex situ microbial activity

(i) SulphateReduction

Peaks of sulphate reduction activity (SR) were present in sediment subsurface within the

SMTZ at all locations (Figure V-2). Maximum SR rates (SRR) were of 23, 8.9 and 9.5 nmol

cm-3 d-1 at Crater Eye (11 cmbsf), Off Center (23 cmbsf) and Crater Rim (37 cmbsf) sites

respectively. At the later, SRR peaks of 5,4 and 7,8 nmol cm-3 d-1 were also present at 19 and

25 cmbsf. SRR were generally very low or null below the SMTZ and near sediment surface.

Areal SRR at Crater Eye, Off Centre and Crater Rim sites were of 584, 688 and 475 mmol m-2

yr-1 respectively when the entire core depth was considered for integration of the volumetric

rates (Table V-2).

(ii) MethaneOxidation

In general, three zones of methane oxidation (MO) could be distinguished. In the shallow

subsurface at Off Centre and Rim sites, MO rates were of 12,6 and 6,3 nmol cm-3 d-1 at 3 and 1

cmbsf respectively (Figure V-2). Below, MO rates decreased steeply down to values of 0,5

nmol cm-3 d-1 at 13 cmbsf and 0,1 nmol cm-3 d-1 at 11 bsf in the Off Centre and Rim stations

respectively. Such shallow subsurface activity was not present at Crater Eye station, possibly

due to loss of surface sediments during piston coring.

Within the SMTZ of the three locations, a peak of MO was present with maximum MO

rates of 11.2 nmol cm-3 d-1 in Crater Eye site at 13 cmbsf, 15.5 nmol cm-3 d-1 in Off Centre site

at 35.5 cmbsf and 5.1 nmol cm-3 d-1 in Crater Rim site at 19 cmbsf. In the Crater Eye site, the

depth of the MO maximum coincided with the depth of the SR peak. In the Off Centre site,

there was an apparent depth offset between MO and SR maxima. However, SR measurement

was lacking at the depth of the MO peak (35.5 cmbsf). In the SMTZ of the Rim site, the depth

of MO peak corresponded to the upper SR maximum (19 cmbsf).

Below the SMTZs, MO was present but at very low rates (around 1 nmol cm-3 d-1) at

Crater Eye and Off Centre sites. At Crater Rim sites, the bottom of the core corresponded to

the lower boundary of the SMTZ and MO data was not available below the SMTZ.

Areal MO rates were of 257, 787 and 206 mmol m-2 yr-1 over the entire core lengths and

of 208, 455 and 138 mmol m-2 yr-1 within the SMTZ at Crater Eye, Off Centre and Crater Rim

sites respectively (Table V-2).


131

Figure V-2 Microbial activity rates and geochemical profiles I. First column: Sulphate reduction rates plotted with sulphate and sulphide concentration profiles. Second column: methane oxidation rates plotted with methane concentration profile. Third column: estimated in situ methane oxidation rates based on ex-situ methane turnover and methane profile from reaction / transport models. Model results are presented in (Vanneste et al., 2011), and are reproduced here (methane and sulphate profiles). Fourth column: proportion of the three types of ANME cells observed at different sediment depth.

CHAPTER V

132

Figure V-3 Microbial activity rates and geochemical profiles II. Methanogenic substrate concentration (H2 and acetate) and hydrogenotrophic (H-MG), Acetotrophic (Ac-MG) and methylamine methyltrophic (Met-MG) methanogenesis rates.

(I) >w Q:; ~ (.)

> :2 a:: (.)

e ~ .c 15. "' 0

H2 (nM) 0,0 0,1 0,2 0,3

0 ~

-100

·200

·300

-400

Acelate (~ MJ 0,4 OC6 20 40 60 80 100 120 ~!

l ï

<( ·500

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~

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èri

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0

I == ~;MG = Mett>MG I -600 '-----'-----'·

o.o 0,5 1,0 1,.5 o.o 0.5 1.0 o.oo 0,05 0,10 0,15 0.20 0.25

H-MG (pmot cm-> d''l Ac-MG (pmot cm-> <f'l Meth-MG (pmot cm-> d''l

H2 (nM) Acelate I1•MJ 0,0 0,2 0,4 0,6 0,8 0 20 40 60 80 100 120 140 0 1~~~==~.~~~~~~~~

·20

·80

·100

-120 = H-MG ~H'

l = AG-MG l = Me<t>MG -Acetate .

· 140 L---------------~------J_ ________ _L ______________ __J

0 2 • 6 8 10 0 5 10 0.00 0.05 0.10 0.15 0.20

H-MG (pmot cm'3 d''l Ac·MG (pmot cm-> <f') Meth·MG (pmot cm" d''l

H2 (nM) Acelate (~MJ o.o 0.5 100 10 15 20 25 30 35

OI 1 · 10

-20

·30

-40

] ~H-MG = H·MG

~H' ~H2 =Me<t>MG

I 0,0 0,.5 ~.0 0,5 1,0 0,0 0,5 1,0

H-MG (pmot cm-> d'1) Ac-MG (pmot cm" <f1) Meth-MG (pmot cm-> d'1)


133

(iii) Methanogenesis

In general, methanogenesis (MG) rates were low and restricted to narrow depth intervals

(Figure V-3). Hydrogenotrophic methanogenesis (H-MG) and acetotrophic methanogenesis

(Ac-MG) were only present in the shallow subsurface sediments at Off Centre site (Figure

V-3), with a maximum H-MG rate of 7.8 pmol cm-3 d-1 at 1.5 cmbsf, and Ac-MG rate of 10.1

pmol cm-3 d-1 at 11 cmbsf. At Crater Eye site, a peak of methylamine methanogenesis (Meth-

MG) was observed in shallow subsurface sediments, with Meth-MG rate of 0.23 pmol cm-3 d-1

at 8.5 cmbsf. Below, Meth-MG was present at very low rates (<0.01 pmol cm-3 d-1). At the Off

Centre site, a Meth-MG peak of 0.18 pmol cm-3 d-1 was present at 25.5 cmbsf. No methanol

methanogenesis was observed in any of the sites investigated.

3.4 Estimation of in situ AOM rates

In situ MO rates within the SMTZ of each site were evaluated based on Equation 5.

Estimated rates presented a peak within the SMTZ of each of the three sites, with maximum

values of 157, 61 and 72 nmol cm-3 d-1 at Carter Eye, Off Centre and Crater Rim sites

respectively (Figure V-2). Below, significant rates (between 3 and 32 nmol cm-3 d-1) were

observed down to the lower boundary of the SMTZ at the three locations. Areal MO rates

based on these estimated in situ volumetric rates were of 2505 mmol m-2 yr-1 at Crater Eye site,

2235 mmol m-2 yr-1 at Off Centre site, and 2263 mmol m-2 yr-1 at Crater Rim site (Table V-2).

3.5 AOM microbial community structure

The structure of the microbial community involved in methane oxidation activity in the

SMTZ of the Crater Eye site was investigated by constructing both archaeal (65 clones) and

bacterial (71 clones) 16S rRNA gene libraries from the 15-17 cmbsf sediment interval.

Amplified Ribosomal DNA Restriction Analysis (ARDRA) resulted in 34 archaeal and 48

bacterial groups displaying different restriction patterns for which at least 2 representative

clones were sequenced. Among Archaea (Figure V-4), sequences belonging to the GoM Arc I

clade defined by by (Lloyd et al., 2006) constituted the largest group (n=27). In addition, most

known anaerobic methanotrophs groups were represented with sequences belonging to

ANME-1b (n=2), ANME-2a (n=7), ANME-2c (n=10) and ANME-3 (n=11). The remaining

sequences belonged to the class of Thermoplasmata (n=8). Most bacterial sequences were

affiliated with sulphate-reducing bacteria (SRB) belonging to the Deltaproteobacteria (n=39,

Figure V-5). Off these, sequences belonging to the short-chain hydrocarbon oxidiser group

CHAPTER V

134

(Butane12-GMe cells and Bus5 strain) defined by (Kniemeyer et al., 2007) constituted the

largest group (n=31). Sequences belonging to the Seep-SRB1 clade of putative SRB syntrophs

involved in AOM (Knittel et al., 2003; Schreiber et al., 2010) were also present in the library:

Seep-SRB1-b (n=1), Seep-SRB1-e (n=3) and Seep-SRB1-f (n=2)

Figure V-4 Affiliation of archaeal sequences from the Crater Eye AOM zone (15-17 cmbsf). The tree was reconstructed by the maximum likelihood method (RaxML), excluding most variable position from the alignment (base frequency below 50% among archaeal sequences).

FN820396.1, Gulf of Cadiz Mud Volcanoes, CRMV, clone GoC_Arc_69, 1 sequence(s) )> FN820363.1, Gulf of Cadiz Mud Volcanoes, CRMV, clone GoC_Arc_18, 7 sequence(s) z FN820433.1, Gulf of Cadiz Mud Volcanoes, CRMV, clone GoC_Arc_120, 2 sequence(s) I FN820378.1, Gulf of Cadiz Mud Volcanoes, CRMV, clone GoC_Arc_ 42, 1 sequence(s) ~ AY592032, Kazan mud volcano, Eastern Mediterranean, 1673m water , clone Kazan BC 19-3A-08 m AJ704631, marine sediment, clone HMMVBeg-36 w

CR937011, losmld library trom a sediment above gas hydrales at H, clone fos0625e3 AJ578119, marine sediments above gas hydrate, clone HydBeg92

AY592805, Milano mud volcano, Eastern Mediterranean, 1958m water, clone Milano-WF1A-09 AY592066, Kazan mud volcano, Eastern Mediterranean, 1673m water , clone Kazan BC19-3A-44 FN820426.1, Gulf of Cadiz Mud Volcanoes, CRMV, clone GoC_Arc_105, 1 sequence(s)

FJ555679, enrichment culture, submersed membrane bioreactor enrichment, clone AOM-Cione-E10 FN820392.1, Gulf of Cadiz Mud Volcanoes, CRMV, clone GoC_Arc_65, 6 sequence(s) AJ578097, marine sediments above gas hydrate, clone HydBeg46

8\NME-2b

AJ578092, marine sediments above gas hydrate, clone HydCal75 AY591995, Kazan mud volcano, Eastern Mediterranean, 1673m water, clone Kazan BC19-2A-17

FN820423.1, Gulf of Cadiz Mud Volcanoes, CRMV, clone GoC_Arc_103, 3 sequence(s) FN820383.1, Gulf of Cadiz Mud Volcanoes, CRMV, clone GoC_Arc_36, 1 sequence(s) FN820372.1, Gulf of Cadiz Mud Volcanoes, CRMV, clone GoC_Arc_33, 1 sequence(s) FN820374.1, Gulf of Cadiz Mud Volcanoes, CRMV, clone GoC_Arc_35, 5 sequence(s)

AB177276, PCR-derived sequence trom methane hydrate bearing subs, clone OOP1251 A20.3 00369741, anaerobic methane oxidation/denitrification reactor, clone 0-ARCH FJ982666, submarine permafrost, clone PASLSS0.5m_1 00301886, acidic peatland, clone CBd-465B FJ907179, archaeon enrichment culture, mixed inoculum bioreactor, clone LCB A 1 C7 AM851 080, alpine lake sediment, clone Cad24-73 -FN820380.1, Gulf of Cadiz Mud Volcanoes, CRMV, clone GoC_Arc_ 44, 27 sequence(s) FN820385, Gult of Cadiz Mud Volcanoes, MercatorMV, clone GoC_Arc_ 49, 14 sequence(s) AY592554, Napeli mud volcano, Eastern Mediterranean, 191 Om water, clone Napeli BC07-4A FJ649528, Amsterdam Mud Volcano saprepel sediment, East Mediterr, clone AMSMV-S1-A42 AM745181, marine sediments, clone GoM161 _Arch61

:11 Halobacterium

FN820360.1, Gulf of Cadiz Mud Volcanoes, CRMV, clone GoC_Arc_10, 2 sequence(s) FN820406, Gult of Cadiz Mud Volcanoes, MercatorMV, clone GoC_Arc_83, 68 sequence(s) G0356829, methane seep sediment, clone Fe_A_ 41 AJ578089, marine sediments above gas hydrate, clone HydCal61 AM745236, marine sediments, clone GoM GC232 4463 Arch60 FN428819, brackish-marine sediments, clone Aarflus Bay (Henry IV/04) clone Arch62

ANME-1a

FN820369.1, Gulf of Cadiz Mud Volcanoes, CRMV, clone GoC_Arc_30, 1 sequence(s) AF142982, clone BURTON20 A 00640164, marine sediment, clone SBAK-mid-55 G0356800, methane seep sediment, clone Fe_A_12

G0356808, methane seep sediment, clone Fe_A_20 FJ604791, cave water, clone MCAr230

FJ900695, oil field, clone WL-3 FN820417.1, Gulf of Cadiz Mud Volcanoes, CRMV, clone GoC_Arc_107, 4 sequence(s) FN820371.1, Gulf of Cadiz Mud Volcanoes, CRMV, clone GoC_Arc_32, 2 sequence(s) FJ712373, Kazan Mud Volcano, Anaximander Mountains, East Mediter, clone KZNMV-5-A24

FN820400.1, Gulf of Cadiz Mud Volcanoes, CRMV, clone GoC_Arc_73, 1 sequence(s) 00641824, Madovi Estuary sediment, clone MES-117

FN820390, Gult of Cadiz Mud Volcanoes, MercatorMV, clone GoC_Arc_61, 1 sequence(s) 00082935, deep sea hydrothermal vent environments, 13 degree Nor, clone metC10

Archaeoglobi_Archaeoglobales_Archaeoglobaceae

Methanococci_Methanococcales

Oeep Sea Euryarcheotic Group(OSEG)

Crenarchaeota

l )> z ~ m rZJ Ol

)> z ~ m rZJ 0

~ G) 0 ~ )>

~

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l ~ OJ G) 0

~ CD g. Ol ::J 0 en Ol

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-i :::r CD

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""0 öï en 3 Ol

5i


135

Figure V-5 Affiliation of deltaproteobacteria sequences from the Crater Eye AOM zone (15-17 cmbsf). The tree was reconstructed by the maximum likelihood method (RaxML), excluding most variable position from the alignment (base frequency below 50% among deltaproteobacteria sequences).

98°

AY696662, Escherichia albertii

Oesultovibrionales

Geothermobacter spp

AY177793 , Aniarctic sediment, clone LH2 14 FJ813557, Gulf of Cadiz mud volcanoes, CRMV, clone GoC_Bac_11, 1 sequence(s)

AY592722, Napoli mud volcano, Eastern Med., sediment layer 18-27 cm, clone BC07-3B-55 X79415, Oesulfuromusa succinoxidans

X77216, Pelobacter acidigallici AJ969448, chimney of Rainbow deep sea hydrothermal vent, clone PICO pp37 Rainbow 70

AY187303, Geopsychrobacter electrodiphil X85131, Syntrophus buswellii X85132, Syntrophus gentianae

EU542439, sediment and soil slurry , clone Er-MS-29 FJ813565, Gulf of Cadiz mud volcanoes, CRMV, clone GoC_Bac_87, 1 sequence(s)

00521796, Gulf of Mexico sediments, clone SMI1-GC205-Bac3j AB177162, methane hydrate bearing subseafloor Peru margin (OOP Leg 201 ), clone OOP1230B18.24

•;,.,.--.,....,..----..,=- Seep-SRB2

FN820344, Gulf of Cadiz mud volcanoes, CRMV, clone GoC_Bac_176, 2 sequence(s) FJ813594, Gulf of Cadiz mud volcanoes, CRMV, clone GoC_Bac_206, 1 sequence(s)

•;..------- AJ34 7756, Brine seawater interface, Shaban se a, clone ST -12K13 Y13672, Oesulfocapsa sultexigens

AF099062, Oesulfotalea psychrophila AJ237602, Oesulfobacterium catecholicum

L42613, Oesultorhopalus vacuolatus

Seep-SRB4

Oesulfotignum spp

AJ237607, Oesulfobacterium indolicum AJ237603, Oesultosarcina cetonica M34407, Oesulfosarcina variabilis G0356960, methane seep sediment, clone Fe_B_143 EF077225, Guaymas Basin, hydrothermal sediment, clone

EF077226, Gult of Mexico, gas seep sediment, enrichment on butane, clone Butane12-GMe FN820305, Gulf of Cadiz mud volcanoes, CRMV, clone GoC_ Bac_ 44, 1 sequence(s)


o FN820346, Gulf of Cadiz mud volcanoes, CRMV, clone GoC_Bac_177, 1 sequence(s) FJ813595, Gulf of Cadiz mud volcanoes, CRMV, clone GoC_Bac_178, 1 sequence(s)


00007534, marine hydracarbon seep sediment, clone Tommeliten_BAC57FL FJ813579, Gulf of Cadiz mud volcanoes, CRMV, clone GoC_Bac_165, 1 sequence(s)

]I a r:: '0

AB177195, methane hydrate bearing subseafloor sediment Peru margin (OOP Leg 201 ), clone OOP1230B3.29 G0356952, methane seep sediment, clone Fe_B_135

(/) ;o OJ

FJ712562, Kazan Mud Volcano, Anaximander Mountains, East Mediterranean Sea, clone KZNMV-25-B1 0 AJ535247, marine sediment above hydrate ridge, clone Hyd89-22 FN820349, Gult of Cadiz mud volcanoes, CRMV, clone GoC_Bac_172, 1 sequence(s)


FM179872, marine sediments, clone Tomm05 1274 3 Bac118 AJ535248, marine sediment above hydrate ridge-:- cloneHyd89-63

AF354163, clone 1427

Seep-SRB1-c

AY592383, Amsterdam mud volcano, Eastern Med., sediment layer 14-31 cm, clone BC20-2B-23 l>Al---- FJ813538, Gulf of Cadiz mud volcanoes, CRMV, clone GoC_Bac_9, 2 sequence(s)

AY592202, Kazan mud volcano, Eastern Med. , sediment layer 22-34 cm, clone BC19-3B-36 AY592189, Kazan mud volcano, East. , sediment layer 22-34 cm, clone BC19-3B-23

Seep-SRB1-a

...... & (/) ;o OJ ...... eb

]~ -

(/) CD CD '0

Cn ;o ~

tJ CD (/)

s_ i3' oOl ()

éii èJ () CD Ol CD

CHAPTER V

136

The presence of ANME-1, -2 and -3 cells in the CRMV sediments was confirmed by

direct observations of CARD-FISH labelled cells using group specific probes. At Crater Eye

site, ANME-2 and ANME-3 cells were present between 8.5 and 23.5 cmbsf (2,3 to 32,3 % of

the DAPI stained cells) and at 18.5 and 23.5 cmbsf (6.5 and 1.5% of the DAPI stained cells)

respectively (Figure V-2A). At Off Centre site, ANME-1 and ANME-2 cells were only present

at 40.5 cmbsf, coinciding with the MO peak (respectively 16 and 6.5% of DAPI stained cells.

(Figure V-2B). At Rim site (Figure V-2C), ANME-2 cells (1.4 to 37.5% of DAPI stained cells)

were found between 22.5 and 32.5 cmbsf, whereas ANME-3 cells were only observed at 22.5

and 29.5 cmbsf (5.9 and 14.5% of the DAPI stained cells respectively). ANME-2 and -3 cells

were both involved in very different structures: they occasionally formed dense shell-type

consortia surrounded by non-ANME cells (Figure III-6A and B), but were mostly present as

large size (Figure III-6C and D) or small size (Figure III-6E and F) loose associations with

non-ANME cells, or as isolated single cells (Figure III-6G and H). In addition, ANME-2 were

also found associated as dense cell aggregates (Figure III-6I), as paired cells (Figure III-6J), or

as dense monospecific aggregates (Figure III-6K and L)

Table V-2 Reaction rates and geochemical fluxes. (a): modeled flux calculated by reaction / transport models. Data from (Vanneste et al., 2011). (b): Methane and sulphate turnover based on ex situ methane and sulphate rates at 1 bar and after recalculation using in situ methane concentrations. ∑SRR and ∑AOMR correspond to depth integrated volumetric rates of SR (integrated over the entire core) and AOM (integrated over the SMTZ)


137

Figure V-6 Microscopic observations of fluorescently labeled ANME cells. Overlay of CARD-FISH and DAPI stained cells (green) and DAPI only stained cells (blue). (A) ANME-2 cells in Off Center at 40.5 cmbsf; (B) ANME-3 cells Crater Rim, 28 cmbsf; (C) ANME-2 cells in Off Centre at 40.5 cmbsf; (D) ANME-3 cells in Crater Rim at 33 cmbsf; (E) ANME-2 cells in Crater Rim at 13.5 cmbsf; (F) ANME-3 cells in Crater Eye at 18.5 cmbsf; (G) in Crater Rim at 28 cmbsf; (H) ANME-3 cells in Crater Eye at 13.5 cmbsf. ANME-2 cells in Crater Rim at 33 cmbsf (I and J), and in Off Centre at 40.5 cmbsf (K and L).

CHAPTER V

138

4. Discussion

4.1 MO, SR and MG activity coupling in CRMV sediments.

Ex situ MO rates indicated that methane was mostly consumed in the SMTZ of the three

sites at CRMV. In addition MO and SR activity peaks coincided, thus showing that methane

oxidation coupled to sulphate reduction (i.e. AOM) was the main sink for methane in the three

zones of the MV. At Crater Eye and Margin sites, methane oxidation accounted for 29-36% of

the sulphate reduction (Table 2). However, these ratios between methane and sulphate turnover

rates may be overestimated, as ex situ rates were measured at atmospheric pressure (see next

section). The higher ratio (66%) in the Off Centre site probably resulted from a sampling

artefact: SR rate was not measured at 35.5 cmbsf where MO rates were highest. This likely led

to an underestimation of the areal ex situ SRR. As discussed elsewhere (Bowles et al., 2011),

non-AOM SR at cold seeps sites has been commonly observed and may exceeds methane

turnover, on average, by a factor 10. Such activity could result from organic matter

mineralization or oxidation of hydrocarbons containing 2 or more carbons (C2+) that are co-

migrating with methane. The latter was supported by the presence of C2+ hydrocarbons in

CRMV sediments (Vanneste et al., 2011) and by the presence of butane / propane oxidizing

sulphate-reducing bacteria in the SMTZ of the Crater Eye site (see below).

Besides AOM, low but significant MO rates were present in the shallow subsurface (0-

14 cmbsf) at Off Centre and Crater Margin sites, with no corresponding SR peaks. These rates

mostly resulted from high methane turnover, since methane concentration was very low. The

presence of a methane tail between the SMTZ and the surface was probably due to a

thermodynamic inhibition of AOM at low methane concentration (Dale et al., 2008). In these

zones, oxygen rather than sulphate was possibly the terminal electron acceptor for methane

oxidation. Such situation of deep (10 cm) penetration of oxygen in the sediment has already

been described at the CAMV (Sommer et al., 2009) where chemosyntetic tubeworms irrigate

shallow subsurface sediments with oxic seawater. Using 13C-methane profiles, Sommer et al.

(2009) have shown that such bioirrigation was responsible for aerobic methane oxidation in the

shallow subsurface. Large populations of chemosynthetic tubeworms have been observed in

the CRMV sediments (Hilario and Cunha, 2008; Vanreusel et al., 2009). Moreover, the

constant sulphate concentration in the 0-10 cmbsf interval, above the sharp gradients of the

SMTZ, indicated that bioirrigation occurred in the CRMV sediments at these two CRMV sites.


139

Weak MO rates (0-3 nmol cm-3 d-1) were present in the deep part of the cores, whereas

sulphate was entirely depleted. Similar rates were also observed at CAMV Crater Centre site

(Niemann et al. 2006a; Chapter IV). This activity cannot be attributed to trace methane

oxidation during methanogenesis, as described in (Zehnder and Brock, 1979), as MG rates

were below detection limit at the corresponding depths. Possible pathways for such MO

activity thus remain elusive.

Similarly to Mercator MV and CAMV in the Gulf of Cadiz (Chapter III and IV), MG

rates were very low or null in the sediment of CRMV. Several studies have shown that seep

sediments also produced methane during AOM, at rates of up to 10% of the AOM rates

(Treude et al., 2007; Orcutt et al., 2008b). The lack of significant MG rates in the AOM zones

of CRMV indicated that such coupling is absent in CRMV sediments and thus does not always

occur.

4.2 Estimated in situ methane consumption in CRMV sediments.

We used modelled in situ methane profiles and measured ex situ methane turnover in

order to provide an estimation of the in situ AOM rates. With this method, the estimated in situ

areal AOM rates were one order higher than the measured ex-situ areal AOM rates, but were in

the same order than the downward sulphate flux estimated by RTM (Table V-2; (Vanneste et

al., 2011)). Besides AOM, heterotrophic sulphate reduction is the other probable sulphate-

consuming pathway in these sediments. However, several observations suggested that this

pathway was a very minor sulphate sink in CRMV sediments: the low organic content of the

Gulf of Cadiz mud volcanoes sediments (Stadnitskaia et al., 2006), the quasi absence in

CRMV sediments of hydrogen that typically results from organic matter fermentation, the low

SR rates outside AOM zones, and the peak of sulphide coinciding with AOM activity peaks.

Therefore, the major part of the sulphate flux should be attributed to AOM dependant sulphate

reduction. However, the ex-situ areal AOM rates at the three CRMV stations were one order

lower than the RTM estimated sulphate fluxes, suggesting that these rates were most probably

underestimating the in situ rates. On the other hand, estimated in situ areal AOM rates were on

the same order than the sulphate flux estimated by (Stadnitskaia et al., 2006) As a

consequences, AOM-dependant sulphate reduction rates were also probably underestimated by

ex situ measurements and our method would thus predict a ~10 fold increase of estimated in

situ areal SRR. Whether this method provided accurate estimates of the true in situ AOM rates

is still debatable. The method used to estimate in situ turnover rely on modeled methane

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profiles that can vary according to several partially constrained parameters, such as boundary

conditions, rate constant or microbial activities distribution. Better geochemical profiles will

probably result from the development of in situ methods, e.g. submersible mass spectrometer

devices (Wankel et al., 2010)

Most of the differences between ex situ and estimated in situ rates were due to the kinetic

inhibition of the lower methane concentration at ambient pressure. The thermodynamic

correction factor (FkIS/FkES) indeed only contributed for 0.6 to 7.4% of the estimated in situ

turnover at CRMV Off Canter and Crater Eye respectively. This was in agreement with the

observations from (Dale et al., 2008; Knab et al., 2008; Wankel et al., 2010) that a kinetic

inhibition modulated the AOM rates amplitude, whereas a thermodynamic inhibition rather

controlled the microbial activity depth distribution in Skagerrak sediments (Norwegian trench).

4.3 AOM community structure at CRMV.

In the three locations investigated, ANME cells represented a large part of the microbial

community, with ANME-2 cells dominating in term of both cell number and depth

distribution. In addition, the ANME distribution correlated well with the AOM activity peaks.

Sequences belonging to the Seep-SRB1 group of putative syntrophic SRB (Knittel et al., 2003;

Schreiber et al., 2010) were also detected in the AOM zone of the Crater Eye. These

observations are thus in agreement with previous studies of cold seep ecosystems showing that

AOM is mediated by ANME cells (Knittel and Boetius, 2009). ANME-2 and -3 cells were

mostly found as single cells, monospecific ANME aggregates or mixed-type consortia, and

very few shell-type consortia were present in the three locations. Based on bioenergetics

considerations, (Alperin and Hoehler, 2009) proposed that high hydrogen concentration

resulting from fermentative processes in organic sediments might promote the formation of

shell-type consortia, with ANME cells having a H-MG activity rather than AOM. Following

this hypothesis, the generally low organic content of the Gulf of Cadiz MV sediments (0.2-0.5

wt%) (Stadnitskaia et al., 2006), may thus explain this low abundance of shell-type consortia.

Sequences belonging to the GoM Arc I Archaea clade belonging to the methanogen

branch of Archaea formed the most abundant archaeal group in the AOM zone of CRMV

Crater Eye. However, a methanogenic role, at least using the conventional substrates tested in

this study, can be excluded since MG rates were very low or zero at this site. GoM Arc I

sequences distribution showed a strong bias toward AOM zones off mud volcanoes from the

Gulf of Cadiz (Chapter IV), East Mediterranean Sea (Heijs et al., 2005; Omoregie et al., 2008;


141

Pachiadaki et al., 2010) and Gulf of Mexico (Lloyd et al., 2006). Further, the GoM Arc I clade

is a sister group of the AOM Associated Archaea (AAA) clade (Fig. 6; (Knittel and Boetius,

2009). A methanotrophic role of AAA has been recently suggested by Schubert et al. (2011),

describing large aggregates of 13C-depleted AAA cells in the putative AOM zone of an alpine

lake. These observations, together with our finding of GoM Arc I sequences in the AOM zone

of CRMV, thus provide some motives to further explore the potential of GoM Arc I cells as

possible methane oxidizers in cold seeps environments.

A large part of the bacterial sequences (31/71) found in the AOM zone of the CRMV

Crater Eye site formed a monophyletic group that included the Butane12-GMe cells and the

Bus5 strain: two butane and propane oxidising sulphate reducers (Kniemeyer et al., 2007). To

our knowledge, environmental sequences belonging to this group have not yet been reported

(in the Silva 104 database). Our results thus provide additional evidence that AOM zones of

methane cold seeps are suitable environments for the Butane12-GMe cells. Their presence in

the Crater Eye sediments was probably due to the co-migration of butane and propane together

with methane, as shown by the measurements of C2+ hydrocarbons concentration at Crater

Eye and Off Centre sites (Vanneste et al., 2011).

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5. Conclusion

The sediments of CRMV displayed some typical features of cold seep sites, such as shallow

SMTZ with AOM coupled to SR consuming the major part of the ascending methane.

However part of the methane flux was apparently oxidised aerobically in the bioirigated

shallow subsurface zone of the sediment. Microbial communities involved in AOM included

all known ANME types and most of the putative SRB syntrophic partners, but the dominance

of GoM-ArcI Archaea and of butane / propane oxidising SRB at the Crater Eye site were

remarkable attributes compared to other AOM communities described thus far. In these

sediments, ANME-2 and -3 cells were mostly found as loosely organized consortia or single

cells, rather than typical shell-type clusters. This could be due the low content of organic

matter and fermentation by-products. Finally, the combination of realistic methane profiles

from geochemical models with ex situ microbial activity turnover, provided AOM rates

compatible with the estimated sulphate and methane fluxes at the CRMV. The systematic use

of such approach should thus help to better constrain the methane flux and turnover in deep-

sea sediments.


143

145

CHAPTER VI. General

Discussion

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146

Recent development of Gulf of Cadiz carbonate mound studies based on the results in

Chapter II contributed to a more complete picture of the mound processes in this location.

Besides, the comprehensive description of methane related microbial processes in Chapters III

to V provides the basis for a comparative microbial ecology overview in these cold seeps

environments. These aspects will be further developed in this section and evaluated along the

larger ecological concept of life in low-energy environments.

1. What is the contribution of AOM to cold-water carbonate mounds

processes?

In the Chapter II of this thesis, it has been demonstrated that the geochemistry and the

microbial activity in the Alpha carbonate mound is strongly influenced by the presence of a

methane source in the underlying sediments. These results raised new questions on the role of

AOM in carbonate mound environments. What is the influence of AOM on the sediment

lithology in a carbonate mound? How does it change the stable isotope record and influence

carbonate precipitation/dissolution reactions? Does it influence other microbial processes and

diagenetic processes? Are methane and AOM common features of all carbonate mounds on the

Pen Duick escarpment? To address these questions, a new multidisciplinary research cruise was

organized on board of the R/V Marion Dufresne in July 2008 (MD 169 MICROSYSTEMS

cruise). In addition to Alpha Mound, two other mounds were sampled along the escarpment and

were named Beta and Gamma mounds (number 252 and 254 in the map Figure I-13). A brief

review of these new results (Larmagnat and Neuweiler, 2011; Templer et al., 2011; Van Rooij

et al., 2011; Wehrmann et al., 2011) and their significance is discussed in this section.

i) Alpha mound was sampled in its summit, with an ~70 m offset comparing to the core

used in chapter II. Unexpectedly, the sediment geochemistry of the two cores was very

different: the sulphate profile gradient was smaller and did not reach complete depletion in the

new core. Hence, assuming that the sulphate profile is controlled by methane and AOM, this

observation implies that methane distribution is very heterogeneous in the Alpha mound and

suggest the existence of preferred channelling pathways rather than uniform upward diffusion.

ii) Methane and AOM rates where detected (AOMR of <1 nmol cm-3 d-1) in the Beta

mound but not in the Gamma mound, showing that methane flux is not a common feature of all

these mounds. This observation questions the links between methane and carbonate mound

development in this region. If cold-water coral would have benefited from the development of

GENERAL DISCUSSION

147

methane derived carbonate on the sea-floor, then all mounds should witness of at least

paleoseepage proxies. However, more compelling evidence for a genetic link between mounds

and methane should be sought in the deeper strata underlying these structures. Hence the

scientific drilling proposal currently in review at the Integrated Ocean Drilling Program (IODP

673, ‘‘Atlantic Mound Drilling 2: Morocco Margin’’) for a scientific drilling of the Alpha

mound will should document this aspect further.

iii) Pen Duick carbonate mounds have been found to be extremely rich with regard to

oxidised iron. In Gamma mound with no AOM, organic matter mineralisation is thus probbly

coupled to dissimilatory iron reduction by heterotrophic iron reducing bacteria (Lovley, 1991)

In Alpha and Beta mound however, where AOM produced sulphide, iron precipitates as iron

sulphide, and mineralization mostly occurred through the sulphate reduction pathway.

iv) In term of subseafloor microbial metabolism, AOM appears to be the most active

compared to other processes, as ATP measurements showed significant values only in the

AOM zones. This further confirms that the in vitro sulphate reduction rates observed in Chapter

II, that were lower in the AOM zone than in the organoclastic sulphate reduction zone, are

probably largely underestimated due to the lower methane solubility in vitro. As expected,

microbial community structure profiles evaluated by fingerprinting techniques show significant

shifts in the AOM zones toward the presence of AOM mediating microorganisms.

v) These studies provided a compelling explanation for the apparent coral dissolution in

methane bearing carbonate mounds. Such observations were counterintuitive at first sigh since

AOM tend to induce carbonate precipitation. However AOM derived sulphide reoxidation, a

process that lowers the pH, has been shown to induce carbonate dissolution. Hence, periods of

sediment oxidation by seawater following periods of methane flux decline may provoke such

cold water coral fossils dissolution in methane bearing sediments.

Based the Chapter II and these new results, the Alpha and Beta mounds were clearly

identified as new cold water coral carbonate mound types distinct from the numerous mounds

found along the European margins, in term of biogeochemistry and diagenetic processes.

Hence, these new mound types can serve as comparison for the comprehension of the processes

involved in the numerous paleomound systems that have thrived during most of the earth

history. In particular, this shows that the inorganic carbon pool isotopic signature is not

necessarly coupled to the trophic regime of the main mound builders. The low ∂ 13C values

observed in alpha mound could have indeed suggested that corals had relied on methane or

other hydrocarbon as a food source. Rather, our results are highlighting the importance of post-

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formation microbial diagenetic processes such as AOM in the geochemistry of the mound

sediments.

GENERAL DISCUSSION

149

2. Comparison of the microbial community diversity in the three MV and

possible environmental controls.

The factors influencing the microbial diversity in natural ecosystems is a central question

in current microbial ecology, and the principles underlying patterns of biodiversity of Bacteria

and Archaea are only starting to be unveiled (Horner-Devine et al., 2004; Martiny et al., 2006;

Prosser et al., 2007; Ramette and Tiedje, 2007; Fierer, 2008; Green et al., 2008). Moreover, the

significance of functional redundancies within microbial communities is still poorly

understood: if indeed several phylotypes are able to carry out the same reaction -as for ANME-

1 -2 and -3 cells- then what factor determines the presence and dominance of each of these

phylotypes? Here, we compare both archaeal and bacterial diversity (Shannon Index), observed

richness and estimated richness (Chao1 index) in all mud volcanoes investigated in Chapter III,

IV and V. The three diversity parameters were clearly highest at Darwin MV West Rim site

than in the other MV’s (Figure VI-1). Compared to other sites, the Darwin West Rim habitat

was more complex or heterogeneous, as shown by the presence of both aerobic methanotrophs

bacteria and ammonium oxidizing Archaea, along with oxygen sensitive SRB and AOM

microorganisms within the same sediment sample (1 cm depth interval), thus indicating a wide

diversity of ecological niches at this site. This habitat also sustains a higher energy flux /

productivity, as shown by the higher AOM activity at this site and the presence of gas at

saturation concentration in near surface sediment indicating a high methane flux. Conversely,

both archaeal and bacterial observed diversity (Sobs, Figure VI-1) and estimated richness

(Chao I) were lowest at Mercator Rim site, where ANME-1b strongly dominated. At this site,

habitat conditions exerted a strong selective pressure due the extreme hypersaline conditions.

Therefore, our results are in agreement with the propositions that habitat heterogeneity and

higher productivity can promote higher bacterial and archaeal diversity, whereas diversity are

lower in more extreme and challenging habitats (Horner-Devine et al., 2004; Fierer, 2008).

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Figure VI-1 At top: representation at each location of the bacterial and archaeal diversity at the relevant phylogenetic level. At bottom: Non parametric richness estimator (Chao1; (Chao, 1984), observed richness (Sobs) and Shannon diversity index H calculated with the program EstimateS (Colwell and Coddington, 1994). H is given as OTU equivalent e.g. exp(H), as recommended by (Jost, 2006)

Assessing the microbial diversity with clone libraries is a powerful tool when studying

low diversity environments such as the Mercator MV. Most OTU where represented by a large

number of sequences and estimated richness was close to observed richness. These were

indications of good coverage of both archaeal and bacterial diversity. However, in

heterogeneous environments bearing large number of different microbial cells, good diversity

coverage necessitates larger sequencing efforts that may become costly and time consuming.

Hence if this approach remains valid to determine the major taxa present, high throughput

sequencing techniques should be used when the goal is to compare diversity patterns based on

statistical clustering analysis between high diversity habitats. In the last phase of this PhD

work, multiplex pyrosequencing of 16S phylotags was used for the microbial community

fingerprinting of all AOM zones investigated in this thesis. Based on this large number of

sequences (~250 000), each representing a single cell of the community, it is likely that this

approach will yield a better picture of each community and some yet undetected patterns in

AOM community structure.

GENERAL DISCUSSION

151

At the phylotype level, our results at Mercator MV, together with previous studies of

hypersaline methane seeps mentioned in Chapter IV, indicate that hypersalinity has a selective

effect toward ANME-1 cells.. The fact that only ANME-1 cells were found in clone libraries

and microscopic observations (Chapter IV) also indicated that hypersalinity excluded other

ANME phylotypes.

In this work, the presence of other selective factors was less clear. For instance, methane

flux toward the sea floor has often been invoked as a determinant parameter in selecting

different AOM microbial communities and ANME cell types (Knittel et al., 2005; Niemann et

al., 2006a). In this line, (Niemann et al., 2006a) demonstrated that the amplitude of methane

flux clearly caused an ecological zonation at Hakon Mosby Mud Volcano (HMMV) in the

Barent Sea, with different ANME’s and aerobic methanotrophs mediating methane oxidation.

In our study, such influence could not be clearly observed as ANME-2 cells constituted a large

part of the microbial communities in both high flux (Darwin MV) and lower flux (CRMV). In

addition, Chapter V and (Vanneste et al., 2011) have shown that methane flux was decreasing

from the crater centre toward the rim of the mud volcanoes. However, no clear ANME

distribution pattern could be observed within such natural gradient (Chapter V). Hence, it is

possible that methane flux do exert a role in shaping cold seep microbial communities, but

probably at larger flux amplitude than the one observed in the investigated mud volcanoes.

Finally, the ability of AOM communities to form massive carbonate structure on the

seafloor has an indirect influence of the community structure. As shown by the study of Darwin

MV (Chapter III), the formation of such carbonate hard grounds induces not only methane flux

relocation at the rim of these hard grounds, but also the focusing of this flux. Hence, this

process promotes the formation of very active and very diverse microbial communities, but in

discrete hotspots.

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3. Are Gom-Arc I cells novel AOM mediating Archaea?

At CRMV, the archaeal community involved in AOM was dominated by sequences of

the GoM-Arc1 phylotype. Members of this group were also abundant in Darwin MV west Rim.

Such dominance of GoM-Arc1 has no analogues among reported AOM communities. In this

section we discuss the possibility for these cells to form a novel ANME type.

The closest relative of the GoM Arc I group is the AOM associated Archaea group, or

AAA (Knittel and Boetius, 2009) (Figure I-6). Members of AAA were initially found together

with NC10 bacteria in an enrichment mediating the limnic anaerobic oxidation of methane

coupled to denitrification N-AOM (Raghoebarsing et al., 2006). Despite the report (Ettwig et

al., 2008; Ettwig et al., 2010) of NC10 bacteria mediating N-AOM alone, a methanotrophic

role of AAA is supported by the reports of (Hu et al., 2009) showing the enrichment of AAA

cells in a reactor performing N-AOM at 35°C, and of (Schubert et al., 2011) describing large

aggregates of 13C-depleted AAA cells in the putative AOM zone of an alpine lake. Hence,

despite the fact that GoM Arc I are not monophyletic with other ANME groups, their strong

affiliation with AAA argues for a methanotrophic role of GoM Arc I.

Secondly, the GoM Arc I group falls within the large branch of methanogens. This

specialized branch of Euryarchaeaota is characterized by the presence in its members of the

unique methanogenesis pathway as the sole energy conservation metabolism. The only

exception is the presence of anaerobic methanotrophs (ANME), which also possess such

pathway (Hallam et al., 2004; Meyerdierks et al., 2010), but apparently use it in the reverse

order (reverse methanogenesis hypothesis). Hence, It is very likely that GoM Arc I are either

methanotrophs or methanogen. However, in CRMV sediments and Mercator MV sediments

where GoM arc I constituted a large part of the microbial community, methanogenesis activity

based on a wide range of substrate was absent, but AOM activity was high. Hence these

observations also strongly argue for a methanotrophic role of GoM Arc I cells.

Thirdly, environmental molecular surveys indicate that GoM Arc I distribution show a

strong bias toward AOM zones off mud volcanoes from the Gulf of Cadiz (Chapter III, IV and

V), East Mediterranean sea (Heijs et al., 2005; Heijs et al., 2007; Omoregie et al., 2009;

Pachiadaki et al., 2010) and Gulf of Mexico (Lloyd et al., 2006, Mills et al., 2003, 2004). This

pattern of distribution thus also argues for a participation in AOM.

The formation of typical consortia of GoM Arc I with DSS cells would have provided

further evidence for their role in AOM. In spite of repeated attempts to observe GoM Arc I

GENERAL DISCUSSION

153

cells by fluorescence microscopy using group specific probes, these cells could not be detected.

In parallel however, a clear fluorescence signal of recombinant E. coli expressing the GoM Arc

I 16s rDNA gene was observed, demonstrating that the probe hybridisation was not affected by

steric hindrance or 16s rRNA secondary structure. Since ANME cells resisted all cultivation

attempts thus far, the demonstration of methane assimilation based on 13C stable isotope

profiles of the ANME cell content by FISH and NanoSIMS (secondary ion mass pectrometry)

is currently the golden standard to identify a methanotrophic metabolism (Orphan et al., 2002b;

Treude et al., 2007). Hence, FISH labelling of GoM Arc I cells clearly deserves further efforts,

as their successful identification by fluorescence microscopy would open the way for

NanoSIMS experiments and the elucidation of the metabolism of this conspicuous archaeal

group.

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4. How to better evaluate methane profile, flux and turnover from deep-

sea marine sediments?

The Chapters I to V of the thesis clearly illustrated the difficulty to quantify deep-sea

benthic processes when gas solutes are involved. The very large difference of methane

solubility between in situ and ex situ conditions indeed hampers the reconstruction of methane

concentration profile and the evaluation of methane flux and turnover rates. Two methods are

currently in use to estimate these parameters in sediment: reaction-transport models and

microbial activity measurement using labelled substrates. Each method bears some

uncertainties. On the one hand, the former do not involved true microbial activity but only infer

possible activity based on solute profiles. The limit of such method can be well illustrated by

the situation at Mercator MV (Chapter IV): in the quasi-absence of sulphate gradient and of

sulphide due to its precipitation as AVS, a reaction transport modelling approach would have

concluded that AOM was completely inhibited hypersaline conditions. On the contrary, our

results have demonstrated that AOM was active with significant areal rates. The major

drawback of the ex situ method, on the other hand, is the very low methane solubility at

ambient pressure compared to the deep sea (see Figure I-1), which may lead to a large

underestimation of the in situ methane pool size. Since AOM rates are calculated by

multiplying the measured turnover by the size this methane pool, ex situ rates underestimate in

situ rates when the in situ methane pool exceeds the maximum methane solubility at ambient

pressure. In the Chapter V, we have proposed a recalculation method for AOM rates measured

ex situ that permitted to reduce the gap between rates with the two methods.

Recent development in deep-sea instrumentation should permit gaining precision in these

estimates. Firstly, using a submersible in situ membrane inlet mass spectrometer, (Wankel et

al., 2010) could accurately measure methane in a deep-sea brine pool from the Gulf of Mexico.

Similar to our conclusions in Chapter V, they found that AOM rates corrected for in situ

methane concentration were 35-40 fold higher than ex situ rates. Hence, if coupled to an in situ

sediment pore water extraction system, such apparatus has the potential to provide more

accurate methane profiles. Secondly, (Parkes et al., 2009) described a set of tools for deep-sea

sediments sampling, recovery and in vitro incubation without depressurisation during the entire

process. With some modifications, such tools could be used for the injection of radiolabeled

GENERAL DISCUSSION

155

substrates in sediment maintained at in situ conditions of pressure and temperature as well as

for the recovery of the in situ methane pool.

The determination of accurate activities is important not only for the estimation of

methane turnover and emission from methane seep sites, but is also determinant for the

understanding of this metabolism that has a critical energy yield under methane concentration

at ambient pressure. The determination of AOM and SR cell specific rates is thus a key

parameter to assess how much energy is truly gained by these microorganisms.

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5. Solving a bioenergetic gap: other mechanisms for AOM?

AOM coupled to SR has a weak energy yield under standard conditions (-16.9 kJ mol-1),

and a slightly higher yields under in situ conditions (up to ~40 kJ mol-1). Such yields are higher

than the minimum energy quantum that can support the formation of ATP, and has thus the

potential to support at least cellular maintenance metabolism (3-10 kJ mol-1) (Hoehler et al.,

1994; Alperin and Hoehler, 2009). However, in the case of AOM, and assuming a syntrophic

mechanism between ANME and SRB, the conserved energy has to be shared by both partners.

Moreover, the dominant hypothesis for AOM functioning invokes the electron transfer via a

chemical shuttle such as H2, acetate, formate, or methylsulphide (Nauhaus et al., 2002;

Nauhaus et al., 2005; Moran et al., 2008). Hence, increasing distance between ANME and SRB

further decreases the overall energy yield of the AOM reaction ((Figure VI-2). In other words,

AOM reaction stops if SRB do not maintain the level of the reduced shuttle produced by

ANME at a very low level. This constrain is common with known cases of interspecies electron

transfer (Stams and Plugge, 2009): for instance, butyrate can be fermented in acetate + H2 by a

bacteria only if H2 is maintained very low (1 Pa) by the activity of a methanogen: 4H2 + CO2

=> CH4 + 2H2O within tight aggregates of both syntrophs.

(Figure VI-2 Free energy yield of methane oxidation (MO, plain line) and sulphate reduction (SR, dashed line) as a function of H2 as a possible interspecieas transfer agent. Assuming a minimum free energy yield of -10 KJ mol-1 for cell maintenance, this garph defines a hydrogen concentration range (∆ITA) for which the coupling of MO to SR is energetically favorable (reproduced from (Alperin and Hoehler, 2009). Since hydrogen concentration gradient between the hydrogen producer and the hydrogen scavenger is directly dependant on the cell-cell distance, AOM energetic yield is strongly constrained by such distance. (Alperin and Hoehler, 2009) calculated that, in the favorable conditions of the Hydrate Ridge methane seep sediments, an increase of 5 µm between an ANME and a SRB cell correspond to a free energy loss 30%.

GENERAL DISCUSSION

157

Without formally disproving it, previous observations and results from this thesis do not

argue for interspecies hydrogen transfer in AOM. Firstly, the morphology of ANME and SRB

do not conform to such model. In shell type of aggregates (Figure I-5), the ANME-SRB

exchange surface is minimal and (Alperin and Hoehler, 2009) calculated that such aggregates

has a high energy cost for both partner. Secondly, most ANME-1 cells (Chapter IV and

addenda of this chapter), and many ANME-2 and -3 cells found at CRMV (Chapter V) have

been observed to live without direct bacterial partner. Thirdly, none of the electron shuttle that

has been tested can uncouple AOM and SR, nor enrich for a single partner.

We thus propose that, with such a lack of coherence between these observations and the

proposed mechanism, one should continue to explore alternative mechanisms for AOM. For

instance, a single cell could mediate both reactions. None of the known sulphate reduction

genes has been found in metagenomic studies of ANME cells (Hallam et al., 2004;

Meyerdierks et al., 2010), but it is possible that such genes are parts of the genomes that were

not sequenced, or that sulphate could be reduced by a novel pathway. Alternatively, there are

growing evidences that microbial cells can exchange electrons directly through conductive

appendages (nanowires) (Reguera et al., 2006; Reguera, 2011), or through membrane bound

cytochomes (Mehta et al., 2005; Reguera, 2011; Summers et al., 2011). In mesocosms

experiments with marine surface sediments, diffusion-independent electron shuttling over 1.2

cm distance has been shown (Nielsen et al., 2010). The expression of genes coding for

membrane c-type cytochromes by ANME-1b cells (Meyerdierks et al., 2010) would be in line

with this hypothesis of a direct electrons shuttling, i.e. electrical current between different

microbial cells (Mehta et al., 2005). The nature of such conductive matrix is not clear, but the

high amounts of AVS at Mercator mud volcano (Chapter IV) indicated the presence of the

conductive iron sulphide. However, evidence for electroactivity of ANME-1 or Seep-SRB cells

are lacking. To address this question, we built a microbial fuel cell able to host AOM

communities from the investigated mud volcanoes. In absence of sulphate or other electron

acceptors besides the anode, the anodic potential of the fuel cell remained very low around -200

mV during three months. Considering that the electrical circuit was closed with a 10 kΩ

resistance and that methane was the sole electron donor, these preliminary results indicated that

methane was probably oxidised and electrons directly flowing through the fuel cell circuit.

Hence, this line of research should certainly be continued.

Finally, different ANME phylotypes may widely differ in term of phylogenetic affiliation

(Figure I-6), morphology (Chapter II, IV and V), genomics (Hallam et al., 2004; Meyerdierks

et al., 2010), ecological niches (see discussion of Chapter IV), and capacity to form organized

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aggregates with SRB (Chapter III, IV and V). There is thus a good possibility that different

ANME groups could find different strategies to carry out the AOM reaction. This was the

rational of the study of Nauhaus and colleagues (Nauhaus et al., 2005) studying the impact of

different environmental parameters on ANME-1 or ANME-2 dominated communities.

However, the Black Sea ANME-1 community of this study also bears a large amount of

ANME-2 cells, which may have interfered with the results of this comparative study. In

Chapter IV, we have found that hypersalinity is probably a very good selective parameter for

ANME-1 cells. We thus argue that salt concentration should be used to cultivate and maintain

stable ANME-1 communities in vitro, and perform physiological analysis to elucidate the

specific metabolism of this group.

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159

6. What can trigger the formation of shell type aggregates of ANME and

SRB?

Since its discovery (Boetius et al., 2000), conspicuous aggregates of Archaea and

Bacteria have raised large interests and fuelled numerous studies (Knittel and Boetius, 2009).

The reason is that such high level of organisation has to be strongly supported by an ecological

or thermodynamic determinism, making these aggregates an interesting model for microbial

ecology. Initially, the formation of such aggregates has been attributed to the syntrophic nature

of AOM consortia between a methanogen functioning in reverse and a sulphate reducer.

However, microscale reaction transport models (Alperin and Hoehler, 2009) have demonstrated

that not only is contact between partners unnecessary for AOM functioning, but also that shell

type organisation is detrimental to the overall AOM energy yield in a syntrophic perspective.

This finding thus calls for alternative models.

Alperin and Hoehler (2009) have proposed that organic matter fermentation could be

responsible for the formation of these aggregates. H2 in sediment impedes AOM functioning, as

suggested by in situ studies (Joye et al., 2009). Hence, by consuming fermentation-derived

hydrogen in the outer layer, sulphate reducers would maintain low H2 levels within the

aggregates and allow AOM. Further, this model implies that most ANME cells would function

as hydrogenotrophic methanogens, and only a minor part of the inner core would mediate

AOM, relegating this reaction to a side reaction of organic matter mineralization. In this view,

the high yield of organic matter mineralisation would fuel the low-yield AOM reaction.

Alternatively, based on a comparison of AOM communities in different environments,

and previous work on ANAMMOX (anaerobic ammonium oxidation), one could speculate that

oxygen could be considered as a selection factor for AOM microorganisms. Under controlled

aerobic conditions, OLAND (Oxygen-Limited Autotrophic Nitrification/Denitrification)

community is organized in clusters where layers of ammonium- and nitrite- oxidising bacteria,

both consuming oxygen, surrounds a core of oxygen-sensitive ANAMMOX cells (Vlaeminck

et al., 2010) Figure VI-3. In this case, the shielding of ANAMMOX cells from oxygen has

been invoked to explain such organisation. This hypothesis also considers that SRB are acting

as a shield for the ANME cells of the inner core, as suggested by the aggregates geometry. By

producing large amounts of sulphide, which spontaneously reacts with oxygen, SRB would

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prevent any oxygen incursion inside the aggregates and protect the very oxygen sensitive

ANME cells.

We argue that similar processes could lead to the formation of highly organized

ANME2/SRB clusters in near surface, bioturbated gassy sediments. The consistent position of

SRB cells outside of the shell-type clusters, whereas ANME-2 cells are forming the cluster core

(Chapter III) would be in agreement with such protective mechanisms. At CRMV, where the

AOM zones are deeper and well below the possible oxygen incursion zone by bioturbation or

bioirrigation, shell type consortia were only rarely observed (Chapter V).

Moreover, (Dekas et al., 2009) have shown that ANME-2 cells are capable of nitrogen

fixation. This highly oxygen-sensitive enzymatic pathway is only performed in the very inner

core of the shell type aggregates (Figure VI-4). These observations are thus in line with the

oxygen shielding hypothesis.

GENERAL DISCUSSION

161

Figure VI-3 Microbial consortia performing the oxygen limited autotrophic denitrification (OLAND) provide a good model for the study of microbial aggregate formation. Such consortia are formed when oxygen is present. Oxygen is consumed by ammonium (green) and nitrite (blue) oxidizing bacteria. Such geometry prevents oxygen to penetrate in the inner core of the aggregate where oxygen-sensitive cells perform the anoxic ammonium oxidation (ANAMMOX). Reproduced from (Vlaeminck et al., 2010)

Figure VI-4 Patterns of nitrogen fixation in shell type AOM consortia involving ANME-2 cells (red) and DSS cells (green). Nitrogen fixation revealed by NanoSIMS and fluorescence microscopy occurs mostly in the inner core of the aggregates and is mediated by ANME-2 cells. Reproduced from (Dekas et al., 2009)

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162

7. Perspectives

Despite low AOM energy yield, ANME cells live in extremely hypersaline, or in negative

temperature habitats. They are able to fix nitrogen for biomass synthesis, one of the most

energy-demanding metabolisms. They are also able to invest energy in the formation of highly

organized consortia. Hence, all of the currently available data fits with the concept developed

by (Valentine, 2007) of adaptation of Archaea to energy stress (illustrated in Figure VI-5). In

this review of archaeal energy conservation metabolisms, cellular biochemistry and

environmental distribution highlights that Archaea are well adapted to energy stress. In the

particular case of AOM, energy stress is intrinsic of the reaction yield itself and of its

syntrophic functioning. Such considerations constitute an incentive to reconsider our views of

microbial ecology in low-energy environments. This domain of research has indeed built upon

observations of the surface world where energy is abundant due to the presence of

photosyntesis: this reaction maintains a very large redox gradient by reducing carbon to organic

molecules and oxidizing water into oxygen. This considerable amount of energy dictates the

ecology of surface microorganisms, with generally fast metabolism turnover and relatively

short generation times. In this “hyperenergetic” environment, energy-conserving reactions are

mostly irreversible because of the very high energetic steps involved. In a “hypoenergetic”

environment such as the deep subsurface biosphere, microorganisms have to conserve energy

from electrons flowing along very small redox gradients. Maintenance energy should be kept

minimal, with generation times orders higher than those in surface life. Biological energy

quantum, the minimum energy that can be conserved to sustain life, has to be lower. Yet, deep

subsurface constitute the major reservoir of microrganisms on earth, estimated to amounts up to

two third of the earth prokaryotic biomass (Whitman et al., 1998). ANME cells have been

found in 111 Myrs old sediments at 1600 mbsf (Roussel et al., 2008). Hence, in conditions

close to thermodynamic equilibrium, the capacity to revert metabolic pathways to harvest

energy from small environmental changes is probably a distinct feature of the deep biosphere

microorganisms.

GENERAL DISCUSSION

163

Figure VI-5 Temperature and pH requirements for growth distinguish thermophilic bacteria and archaea. 72 archaeal species that represent 32 genera are included (pink dots), as are 107bacterial species that represent 61 genera (blue dots). Methanogenic archaea are excluded. Only the type strain for each species is shown, and an average temperature is given for species that have a range for the optimal growth temperature. The overlaid blue ‘zone’ comprises environmental conditions to which bacteria are best adapted, the overlaid pink zone comprises conditions to which archaea are best adapted, and the grey zone represents those conditions to which both archaeal and bacterial species are well adapted. Data were compiled from Bergey’s Manual of Systematic Bacteriology and from the primary literature. Reproduced from (Valentine, 2007)

Figure VI-6 Schematic view of the latent redox mechanisms that might impact methane biogeochemistry and microbial activity in subsurface sediments. At top: radiolysis of water can potentially produce oxidants for methane. Note that a balanced material cycle is possible, with the potential to regenerate all starting material, but also note that the relative proportions of chemicals such as methane and acetate (Ac) could vary. At bottom, a potential scheme by which halogenated organic matter might be liberated from sediment and draw reducing equivalents away from methanogenesis. Reproduced from (Valentine, 2011)

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Radiolysis of water as source of hydroxyl radicals, or halorespiration using halogenated

compounds released during refractory organic matter slow degradation reviewed in (Valentine,

2011); Figure VI-6), have the potential to sustain such metabolisms. There is no doubt that new

ecological principles, novel metabolic pathways and energy conserving reactions, as intriguing

as in AOM microorganisms, will be revealed by studying this environment.

165

CHAPTER VII. Experimental

Procedures

CHAPTER VII

166

Since the same approach has been employed to study the four different mud volcanoes,

the Material and Methods of Chapter III to V display large overlaps. Hence, the methods that

were common to these chapters are reviewed in this single separated section.

Bathymetric data and sediment samples were acquired during the JC-10 cruise aboard the RV

James Cook (2007) using the Remote Operated Vehicle (ROV) ISIS, sediment surface corer

(mega corer), as well as gravity and piston corer.

1. Microbathymetry.

The microbathymetry data processing utilized the following field data produced by the

ROV ISIS: the sonar head data, ultra-short baseline acoustic positioning, Doppler navigation

data and altitude data from the vehicle’s sensors. The processing was done with the Caraibes

package (V 3.3) developed by the IFREMER and custom-tailored for the ROV ISIS

specifications. The processing workflow included, integration of ISIS sonar head data with

attitude sensors data and conversion into the CARAIBES native format, calculation of the true

water depth using vehicles altitude and pressure sensors data, cleaning of the bathymetry data

(removal of extreme values), filtering and smoothing of navigation data, merging of sonar head

and navigation data and gridding and output of XYZ data. The resulted grids were then

imported into Gilden Software’s Surfer (TM) and ESRI’s ArcGIS (TM) for further processing,

mapping and visualization. Surfer was used for corrections of obvious depth and lateral misfits

of adjacent swaths, and merging of the individual swaths into a complete coverage. The

coverage was then imported into ArcGIS and used for the production of maps, data

visualization and sampling plans.

2. Coring and Sampling

Sediments were recovered by gravity-, piston-, or mega corer coring (GC, PC and MC,

respectively). Subsequently to recovery, GCs and PCs were cut into 1 m sections and stored at

4°C until further processing (less than10 h after recovery). MCs as well as GC and PC core

sections were sub-sampled by extruding sediments from the core using a plunger.

EXPERIMENTAL PROCEDURE

167

3. Geochemistry

Pore water was extracted from 25 ml sediment using a pore water press (Reeburgh, 1967)

equipped with 0,45 µm pore size nitrocellulose filters. Porewater was then filtered through 0.1

µm pore size filters prior to sample storage. For sulphide measurement, 1 ml of pore water was

preserved in 500 µL zinc acetate solution (10% w/v). Acid volatile sulphide (AVS) was

extracted from sediment sealed in a glass jar by adding 6N HCl under continuous N2 flow

collected in a 7 mL Zn acetate trap, using the experimental setup described in (Kallmeyer et al.,

2004). Concentration of both dissolved and AV sulphide was measured calorimetrically using

the methylene blue method (Cline, 1969). Sulphate, chloride and acetate were measured by ion

chromatography as described in (Parkes et al., 2007).

Methane dissolved in sediments/porewater was analysed according to (Parkes et al.,

2007). In short, 2 cm3 of sediment (sampled with a 5 ml cut-off syringe) were transferred into a

glass vial containing 6 mL NaOH (2,5%, w/v), which was then immediately sealed with a butyl

rubber septum and stored upside down. Methane collected in the headspace was analysed by

gas chromatography and flame ionisation detection (Perkin Elmer/Arnel Natural Gas

Analyser).

4. Ex situ rate measurements.

Radiolabeled tracer turnover during short-term sediments incubations is a very efficient

method to determine how much substrate is metabolized per unit of time. The turnover is then

given by the radioactivity of the product divided by the initial radioactivity contained in the

substrate, assuming that radioactivity of the sediment prior to the experiment is null or

neglectible compared to the one introduced by the radiolabeled tracer. Alternatively, substrate

turnover is sometimes determined by stable isotope labelled substrate, thus avoiding

inconvenience and safety measures associated with the use of radioactive products. Such

approach is often used to follow substarte turnover in long term sediment incubation in the lab

(see for instance (Beal et al., 2009). In these homogenized and slurried sediment incubations,

the substrate turnover is determined by the evolution of the stable isotope profile with time.

However, in the case of short-term incubations with undisturbed sediments, of which the aim is

to study the microbial metabolism as close as possible to in situ conditions, such approach

would introduce incertainities linked with the stable isotopic profile of inorganic carbon and

sulphur pools already present in sediments prior to incubation with labelled substrates.

Radiolabeled substrates have thus been widely used for the determination of AOM SR and MG

CHAPTER VII

168

turnover rates in marine sediment in ex situ (on board, immediately after sediment recovery)

conditions.

For AOM and SR rates measurements, core-sections were sub-sampled with mini cores

(polycarbonate tube, diameter 2,5 cm, length 20 cm) with silicon-sealed side injection ports

spaced every 2 cm, or with glass barrels (diameter 1 cm, length 6 cm) with a syringe plunger at

one end, and a butyl rubber stopper at the other end as injection port (for details see (Treude et

al., 2005b). Within 2 hours after sampling, methane radiotracer (25 µl of saturated aqueous 14CH4 solution, 5 KBq) or sulphate radiotracer (5 µl of aqueous 35SO4

2- solution, 60 KBq) was

injected through the injection ports, and samples were incubated for 24 hours in the dark at in

situ temperature (e.g. 13° for Mercator MV stations and 4°C for CAMV, T was measured

during ROV dives). After incubation, AOM and SR reactions were stopped by transferring the

samples into glass flasks containing 25 ml NaOH (2.5% w/v), or 50 mL plastic tubes

containing 20 mL zinc acetate solution (20% w/v) respectively. Methane and sulphate turnover

were determined as described elsewhere (Jørgensen, 1978; Treude et al., 2003; Kallmeyer et

al., 2004). Briefly, the methane pool present in each incubation ([CH4]) was determined by gas

chromatograpy using a thermal conductivity detector. The radioactive methane pool 14CH4 in

each sample was then determined by stripping the incubation headspace out with natural air

flow, oxidising it through 850 ºC in line furnace, and traping the resulting carbon dioxide in

phenylethylamine. The radioactive inorganic carbon resulting from microbial methane

oxidation was then extracted from the samples by acidification with 6N HCl and trapping the

evolving 14CO2 gas in a phenylethylamine trap. For sulphate reduction turnover measurement,

the total reduced inorganic sulphur (TRIS) fraction in sediment incubation was reduced to

sulphide by addition of Cr(II). The TRIS fraction was extracted by addition of 6N HCl under a

flow of N2, and collected in a Zn-Acetate trap. The radioactivity in the different fractions was

measured by adding scintillation cocktail (UltimaGold, PerkinElmer). AOM and SR rates were

determined as a product of the turnover and methane or sulphate concentration respectively:

€

AOM =14CO2

14CO2+14CH4

×CH4[ ]t

SRR =TRI35S

TRI35S+35SO42− ×

SO42−[ ]t

where t is inbation time. For AOM rates, methane concentration was corrected for

maximum solubility at ambient pressure and in situ salinity (Duan and Mao, 2006).

Rates of methanogenesis were measured as described in (Parkes et al., 2007). Briefly, 14C labeled carbonate (10 KBq/µl), acetate (6 KBq/µl), methanol (2.05 KBq/µl), or


169

methylamine (7.25 KBq/µl) were injected into either mini cores (diameter 1.5 cm, length 10

cm) through silicone-filled side injection holes (4µl substrate per 1 cm depth interval), or 10 ml

cut off syringes closed with rubber stoppers (7.5 µl substrate per syringe), and incubated at in

situ temperature for 7 to 21 hours in nitrogen flushed gas tight bags. Microbial reactions were

then stopped by transferring incubations into glass jars containing 7 mL of 1M NaOH. In the

laboratory, 14C methane was determined as for AOM. Substrate turnover was determined

according to the radioactivity of the trapped CO2 and the amount of added label in the product

pool. Rates were calculated as for AOM and SR rates by multiplying the radiolabeled substrate

turnover per time unit by the substrate concentration (see above). For methylamine

methanogenesis, rates were obtained by multiplying the substrate turnover value by an arbitrary

methylamine concentration of 5µM (as methylamine concentration in pore water was

consistently below detection limit). Me-MG rates should thus be considered as maximum

potential rates rather than actual ex situ rates.

5. Nucleic acid extraction and amplification.

Environmental DNA was extracted from five replicates, each of 0.3 g sediment, using the

PowerSoil DNA extraction Kit (MoBio Labconsult, Brussels, Belgium) according to

manufacturer recommendations. 16S rRNA genes were amplified using Arch20f/Uni1392r

primer pair for Archaea (Kane et al., 1993; Massana et al., 1997) and GM3f/GM4r primer pair

for Bacteria (Muyzer et al., 1995). PCR conditions were as in (Losekann et al., 2007). PCR

were carried out in triplicate, amplicons were pooled and purified (PCR purification kit,

Qiagen).

6. Clone libraries construction, screening and phylogeny inference.

Purified PCR products were ligated in the PCR-2.1 vector and cloned into Top10

chemocompetent cells using TOPO-TA cloning kit (Invitrogen, Merelbeke, Belgium). The size

of the insert was verified with a colony PCR on overnight grown positive transformants using

M13 vector specific primer pair (Invitrogen). M13 PCR amplicons were used for amplified

ribosomal DNA restriction analysis (ARDRA) of the libraries with AluI and RsaI (Fermentas,

St. Leon-Rot., Germany). Digested DNA was run on a 3% agarose gel and images were

analyzed with the Bionumerics software (Bionumerics, Kortrijk, Belgium). Clones were

clustered according to restriction pattern using the Jaccard similarity index, and when a cluster

contained multiple clones, at least 2 representative clones were sequenced. Bidirectional

CHAPTER VII

170

sequencing was carried on ABI-3730xl sequencer (Applied Biosystem, Lennik, Belgium) at the

Genetic Service Unit of the Ghent University Hospital using M13 vector primers.

7. Sequence accession numbers.

Sequences from this study are published in GenBank under the following accession numbers:

Darwin MV: FN820339, FN820337, FN820336, FN820323, FN820318, FN820314,

FN820313, FN820300, FN820298, FN820295, FJ813592, FJ813590, FJ813587, FJ813586,

FJ813585, FJ813584, FJ813583, FJ813582, FJ813581, FJ813580, FJ813578, FJ813577,




FJ813522, FJ813520, FJ813519, FJ813518, FJ813517, FJ813516, FJ813515 for bacterial 16s

rDNA genes and FN820437, FN820436, FN820431, FN820428, FN820427, FN820425,

FN820424, FN820422, FN820415, FN820414, FN820413, FN820412, FN820373, FN820366,

FN820362, FN820356.

Mercator MV: FJ813536, FJ813545-FJ813556, FJ813561-FJ813564, FJ813569, FJ813573,

FJ813576, FJ813591, FN820294, FN820296, FN820299, FN820302, FN820303, FN820307,

FN820308, FN820310, FN820311, FN820312, FN820317, FN820319, FN820320-FN820327,

FN820331-FN820335, FN820338, FN820355, FN820359, FN820367, FN820370, FN820372,


FN820390, FN820402-FN820411, FJ813544.

Carlos Ribeiro MV: FN820354, FN820353, FN820352, FN820351, FN820350, FN820349,



FN820311, FN820310, FN820306, FN820305, FN820304, FN820301, FN820297, FJ813597,



FJ813539, FJ813538, FN820433, FN820426, FN820423, FN820421, FN820420, FN820419,




FN820364, FN820363, FN820361, FN820360, FN820357.


171

8. Phylogenetic tree reconstruction and phylotype inference.

All sequences were aligned with the online SINA webaligner of SILVA (Pruesse et al.,

2007); http://www.arb-silva de), and imported together with closely related sequences in the

ARB SILVA database release 102. Sequences were further clustered by operational taxonomic

unit (OTU, 98% similarity cutoff) with the MOTHUR software (Schloss et al., 2009).

Phylogenetic trees were reconstructed with the ARB software package (Ludwig et al., 2004)

using the maximum likelihood method RaxML implemented in ARB (Chapter IV) or the

PhyML server (http://www.atgc-montpellier.fr/phyml/) (Guindon et al., 2009) in combination

with filters removing the most variable position of the alignments (maximum frequency filter

implemented in the ARB software package)filtering out most variable positions of the

alignment (50% base frequency filter).

9. (Catalyzed reporter deposition)-fluorescence in situ hybridization

(FISH and CARD-FISH) cell staining and microscopic observations.

Upon core retrieval, 2 cm3 of sediment were fixed in filter-sterilized (0.2 µm)

formaldehyde in seawater solution (3% v/v final concentration) for 4 hours at 4°C. FISH and

CARD-FISH procedures were carried out according to (Pernthaler et al., 2001; Pernthaler et al.,

2002). For CARD-FISH, the following modifications were applied: for permeabilisation of

rigid archaeal cell walls, 15 µg/mL proteinase K was used (3 min. at room temperature); for

bacterial cell wall permeabilisation, 10 mg/mL lysozyme (15 min. at 37 °C) was used.

Horseradish-peroxidase- (CARD-FISH) or Cy3-labeled (FISH) probes targeting the following

groups were used: probes Eub338 I-III (Daims et al., 1999) specific for most Bacteria (35%

formamide in hybridisation buffer or FA), probe Arch915 (Stahl and Amann, 1991 )specific for

most Archaea (35% FA), probe ANME1-350 (Boetius et al., 2000) specific for ANME-1 (40%

FA), probe EelMS-932 (Boetius et al., 2000) specific for ANME-2 (40% FA), probe ANME-3-

1249 with unlabeled helper oligonucleotides ANME3-1243h and 1249h (Losekann et al., 2007)

specific for ANME-3 (40% FA) and probe DSS658 (Manz et al., 1998) specific for the

Desulfosarcina/Desulfococcus branch of Deltaproteobacteria (60% FA), including Seep-SRB

subgroups. Cells were counterstained with 1 µg/mL 4',6-diamidino-2-phenylindole (DAPI) for

10 min. Hybridized cells were examined under an epifluorescence microscope (Carl Zeiss

Axioplan, Jena, Germany). For each sample and probe, 50 independent microscopic fields of

view were counted, corresponding to approximately 400-800 DAPI-stained cells.

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172

173

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Personalia

Loïs Maignien Vrijheidslaan 21, 9000 Ghent, Belgium [email protected] [email protected] Nationality: French. Gender: male Date of Birth: 06/27/1978. 2 children

Education & Positions

2005-today: PhD student in Applied Biological Sciences. Lab. of microbial ecology and technology, Ghent University, Belgium. 2003-2004: Research assistant in Lab. of Fungal Genomics. Wageningen University and Research Center, The Netherlands. 2002-2003: Master of Science in cellular and molecular biotechnology. Wageningen University and research center, The Netherlands. 1999-2003: Master of Science in Bioprocess engineering. Institute for engineering sciences of Montpellier (ISIM), France. 1997-1999: General biology diploma (DEUG), biochemistry and genetics. Paris faculty of science (Paris VI). 1996-1997: Preparation to Bioengineer school entrance exams. Lycée Lakanal, Sceaux, France 1996: A level in sciences, specialization biology.

Training & experiences

- STAMPS: Strategies and Approaches for the Analysis of Microbial Population Stucture MBL, Woods Hole, USA. Analysing microbial community structure with next generation DNA sequencing methods. August 2011, 10 days,. - Advanced Bioinformatics course, Ghent University:

Building of a BioPerl/MySQL based script to handle high throughput (454) 16s phylotags datasets, access it via a web interface (apache/php), and analyze it with the “R”/Vegan packages. 15 days, Feb 2010.

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- ARB software and molecular phylogeny course, Max Planck Institute – Bremen, Germany Bioinformatics tools for molecular phylogeny and probe design. Aug. 2009, 1 week.

-Microbial activity measurement using radiotracers Max Planck Institute – Bremen. Guest scientist. Aug 2008, 1 month.

- Microbial ecology and ecosystem functioning workshop, Lunz, Austria. European Science Foundation exploratory workshop on the developments in the field of theoretical microbial ecology and ecosystem functioning. Sept. 2007.

- Microbial Diversity Course, Marine Biology Laboratory, Woods Hole, MA, USA. The aim of this 6 weeks course was to study microbial processes in their environmental context, including a very broad range of microbial way of life. Emphasis was put on cultivation and in vitro technique, as well as molecular methods and bioinformatics tools. (06/2005 to 08/2005)

- Lab of Microbial ecology and technology, Gent University, Belgium. (PhD position) Microbial ecology of carbonate mounds, cold seeps and mud volcanoes (gulf of Cadiz): linking environmental parameters with microbial activity and community structure. Keywords: Ecosystem functioning, Anaerobic oxidation of methane, radiotracer, (CARD-) FISH, DGGE, archaeal and bacterial 16s clone libraries, phylogenetic tree reconstruction, 16s phylotags pyrosequencing. (Current project). Participation to other projects in marine geochemistry and microbial ecology (GI tract, aquaculture, Microbial Fuel Cells, high pressure reactors).

- Lab of fungal genomics, Wageningen University, The Netherlands. (predoc. Position) Fundamental studies on the A. niger secretion pathway using transcriptome analysis with microarrays and protein fluorescence tagging approaches. (02/2004 to 12/2004) Keywords: secretion pathway / microarray / ER protein / FRET.

- Lab of molecular immunology and biotoxins, CICESE, Ensenada. Mexico. (Msc. thesis) Creation and selection of shark NAR antibodies and human scFv antibody variable domain against a bee toxin using phage display technique. (01/2002 to 01/2003). Keywords: Antibody Library / Recombinant protein expression / Library panning / Novel Antigen Receptor. Report available

Language

- French: mother tongue - English: read, spoken and written (TOEFL result: 560pts) - Spanish: read, spoken and written - German and Dutch: good notions

Scientific expeditions

- R/V Marion Dufresne, 10 days, July 2008, Gulf of Cadiz. Coord. of Microbiology team - R/V Darwin and ROV Isis: 20 days, May 2007, Gulf of Cadiz. Microbiology team - R/V Maria S. Merian: 50 days, May 2006, Gulf of Cadiz. Microbiology team

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IT knowledge

- Molecular ecology and bioinformatics: proficiency in ARB, unifrac, xploreseq, EstimateS, EbioX, ImageJ, Pintail, Mothur, Qiime… Good notions of Unix, Perl, and MySQL scripting and applications. - Geo Information Systems: ArcGIS, GrassGIS.

Publications

L. Maignien, R. J. Parkes, B. Cragg, H. Niemann, K. Knittel, S. Coulon, A. Akhmetzhanov, P. Weaver, N. Boon. Heterogeneous methane geochemistry and anaerobic oxidation activity at Darwin Mud Volcano (Gulf of Cadiz). In prep. L. Maignien, R. J. Parkes, B. Cragg, H. Niemann, K. Knittel, S. Coulon, A. Akhmetzhanov, P. Weaver, N. Boon. Comparison between rates of anaerobic methane oxidation and geochemical modeling at Carlos Ribeiro Mud Volcano (Gulf of Cadiz). Submitted. L. Maignien, R. J. Parkes, B. Cragg, H. Niemann, K. Knittel, S. Coulon, A. Akhmetzhanov, P. Weaver, N. Boon. Anaerobic oxidation of methane in hypersaline sediments. In prep Marzorati, M., Maignien, L., Verhelst, A., Luta, G., Sinnott, R., Boon, N., Van de Wiele, T., and Possemiers, S. Use of the barcoded pyrosequencing in an ecological survey of a simulator of the human gastrointestinal tract to follow up a prebiotic treatment. Submitted Y. Zhang, L. Maignien, N. Stadnitskaia, P. Boeckx, N. Boon. Stratification in Ginsburg Mud Volcano determines microbial community structure and activity in response to methane and sulfate supply. Submitted Y. Zhang, L. Maignien, X. Zhao, F. Wang, N. Boon. Enrichment of a microbial community performing anaerobic oxidation of methane in a continuous high-pressure bioreactor. BMC Microbiology, 11:137 (2011) L. M. Wehrmann , S. P. Templer, Stefano M. Bernasconi, B. Brunner, L. Maignien,T. G. Ferdelman. 2009. The imprint of methane seepage on the geochemical record and early diagenetic processes in cold-water coral mounds on Pen Duick Escarpment, Gulf of Cadiz. Marine Geology. In press. D. Van Rooij, D. Blamart, L. De Mol, F. Mienis, H. Pirlet, L.M. Wehrmann, R. Barbieri, L. Maignien, S.P. Templer, H. de Haas, D. Hebbeln, N. Frank, S. Larmagnat, A. Stadnitskaia, N. Stivaletta, T. van Weering, Y. Zhang, N. Hamoumi, V. Cnudde, P. Duyck, et al. Cold-water coral mounds on the Pen Duick Escarpment, Gulf of Cadiz: The MiCROSYSTEMS project approach. Marine Geology. In press. L. Maignien, D. Depreiter, A. Foubert, J. Reveillaud, L. De Mol, P. Boeckx, D. Blamart, J.-P. Henriet and N. Boon. Anaerobic oxidation of methane in a cold-water coral carbonate mound from the Gulf of Cadiz. International Journal of Earth Sciences, In press. S. E. Vlaeminck, A. G. Hay, L. Maignien and Willy Verstraete. In quest of the nitrogen oxidizing prokaryotes of the early Earth. Environmental Microbiology 13, 285-296 (2011)

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Y. Liu, P. De Schryver, B. Van Delsen, L. Maignien, N. Boon, P. Sorgeloos, W. Verstraete, P. Bossier, T. Defoirdt. PHB-degrading bacteria isolated from the gastrointestinal tract of aquatic animals as protective actors against luminescent vibriosis. FEMS Microbial Ecology, 74, 196-201. (2010) Vanreusel, A., Andersen, A. C., Boetius, A., Connelly, D., Cunha, M. R., Decker, C., Hilario, A., Kormas, K. A., Maignien, L., Olu, K., Pachiadaki, M., Ritt, B., Rodrigues, C., Sarrazin, J., Tyler, P., Van Gaever, S., and Vanneste, H.,. Biodiversity of Cold Seep Ecosystems Along the European Margins. Oceanography 22, 110-127. (2009). C. Boeckaert, D. P. Morgavi, J.-P. Jouany, L. Maignien, N. Boon and V. Fievez. Role of the protozoan Isotricha prostoma, liquid-, and solid-associated bacteria in rumen biohydrogenation of linoleic acid/ Animal Science 3, 961-971. (2009). Foubert, A., Depreiter, D., Beck, T., Maignien, L., Pannemans, B., Frank, N., Blamart, D., and Henriet, J. P. Carbonate mounds in a mud volcano province off north-west Morocco: Key to processes and controls. Marine Geology 248, 74-96. (2008). Boeckaert, C., Vlaeminck, B., Fievez, V., Maignien, L., Dijkstra, J., and Boon, N., Accumulation of trans C-18:1 Fatty Acids in the Rumen after Dietary Algal Supplementation Is Associated with Changes in the Butyrivibrio Community. Applied and Environmental Microbiology 74, 6923-6930. (2008). Rabaey, K., K. Van de Sompel, L. Maignien, N. Boon, P. Aelterman, P. Clauwaert, L. De Schamphelaire, H. T. Pham, J. Vermeulen, M. Verhaege, P. Lens and W. Verstraete. "Microbial fuel cells for sulfide removal." Environmental Science & Technology 40(17): 5218-5224. (2006).

Conference Abstracts of Oral Presentations Maignien, L; Boon, N; RV James Cook JC10 Shipboard Scientific Party. Environmental constrains on microbial methane oxidation activity and community structure in Gulf of Cadiz mud volcanoes. 19th Annual VM Goldschmidt Conference, JUN 21, 2009 Davos SWITZERLAND. GEOCHIMICA ET COSMOCHIMICA ACTA Vol. 73 Is. 13 P. A819-A819 L. Maignien, J.R. Parkes, B. Cragg, N. Boon, and the RV James Cook JC10 shipboard scientific party. Environmental constrains on anaerobic methane oxidation and microbial community structure in marine sediments: the case of Cadiz mud Volcanoes. 9th International Conference on Gas in Marine Sediments”, September 09, Bremen, Germany. L. Maignien, A. Foubert , D. Depreiter, N. Boon, D. Blamart , W. Verstreaete, J.-P. Henriet. Biogeochemical evidence for anoxic oxidation of methane occurrences in the juvenile carbonate mounds from the gulf of Cadiz. Geophysical Research Abstracts, Vol. 8, 06630, 2006

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Other interests and activities.

- Alto Saxophone (“Jour de Fête” Big Band, Brussels). - Sailing Race on 470 and cruiser/racer (Tour de France Voile, Spi Dauphine, World Student Championship, etc…). - Diving. (PADI Open Water Diver)

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cover lois maignien lr - ghent university · seas, fp7) programs, a phd scholarship from the agency...

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