Microbial communities performing

anaerobic oxidation of methane:

diversity of lipid signatures and habitats

Dissertation

zur Erlangung des Doktorgrades

der Naturwissenschaften

- Dr. rer. Nat. -

Am Fachbereich Geowissenschaften

der Universität Bremen

vorgelegt von

Pamela E. Rossel Cartes

Bremen

Februar 2009

1. Gutachter: Prof. Dr. Kai-Uwe Hinrichs, University of Bremen, Germany

2. Gutachter: Prof. Dr. Antje Boetius, Max Planck Institute for Marine Microbiology,

Bremen, Germany

No viniste de lejos, ni siquiera has llegado. Estabas desde siempre, como un lenguaje

escrito en el fondo de mí…

Para Xavi con mucho amor

TABLE OF CONTENTS

Abstract Thesis abstract……………………………………………………..I

Kurzfassung……………………………………………………...III

Acknowledgements………………………………………………………………………V

List of Figures……………………………………………………….............................VII

List of Tables………………………………………………………................................IX

List of Abbreviations………………………………………………………....................X

Chapter I: Introduction…………………………………………………….................1

General introduction………………………………………………………2

I.1. Properties and importance of methane………………………………..2

I.2. Production and consumption of methane……………………………..4

I.3. Microbial communities performing AOM…………………..............11

I.4. Distribution/Habitats of AOM communities………………………...13

I.5. Lipid signatures of communities performing AOM…………………18

I.6. Intact polar membrane lipids (IPLs)…..……………………………..21

I.7. Methods……………………………………………………………...28

I.8. Hypothesis and objectives…………………………………………...29

I.9. Contribution to publications…………………………………………30

I.10. References………………………………………………………….33

Chapter II: Intact polar lipids of anaerobic methanotrophic archaea and……………45

associated bacteria

II.1. Printed manuscript…………………………………………………..46

II.2. Supplementary online material……………………………………...61

Chapter III: Factors controlling the distribution of anaerobic………………………...63

methanotrophic communities in marine environments:

evidence from intact polar membrane lipids

III.1. Manuscript…………………………………………………………64

III.2. Supplementary material………..…………………...…………….106

Chapter IV: Experimental approach to evaluate stability and reactivity…………….111

of intact polar membrane lipids of archaea and bacteria in

marine sediments

Chapter V: Diversity of intact polar membrane lipids in marine…………………...125

seep environments

Chapter VI: Concluding remarks and perspectives………………………………….149

VI.1. Conclusions……………………………………………………….150

VI.2. Future perspectives……………………………………………….155

VI.3. Presentations and other activities…………………………………159

Thesis abstract ________________________________________________________________________

I

THESIS ABSTRACT

The main aim of this thesis was to study different microbial communities

involved in the process of anaerobic oxidation of methane (AOM) using lipid analysis.

During this work a variety of globally distributed methane-bearing systems characterized

by different environmental factors and anaerobic methanotrophic consortia were analyzed

for intact polar lipid (IPL) and apolar lipid composition. Moreover, an experiment was

designed in order to evaluate the stability of archaeal and bacterial IPLs in marine

sediments.

The three phylogenetically distinct clusters of Euryarchaeota called ANME-1, -2

and -3, which have been observed in association with sulfate-reducing bacteria of the

Desulfosarcina/Desulfococcus group (‘‘ANME-1/DSS and -2/DSS aggregates”) or

Desulfobulbus spp (‘‘ANME-3/DBB aggregates”) could be clearly distinguished by IPL

composition but not by apolar lipids. ANME-1/DSS was characterized by

glyceroldialkylglyceroltetraethers (GDGTs) with glycosidic, phospho, as well as mixed

of both , whereas diagnostic IPLs of ANME-2/DSS were archaeols with both glycosidic

and phospho headgroups. Distinctly, ANME-3/DBB contained neither glycosidic-

archaeols nor GDGT-based IPLs, but the phospho-archaeol composition was very similar

to ANME-2/DSS. The main and distinguishing feature of ANME-3/DBB was the high

contribution of the bacterial IPLs phosphatidyl-(N)-methylethanolamine (PME) and

phosphatidyl-(N,N)-dimethylethanolamine (PDME). Other bacterial IPLs that were

mainly found in ANME-2/DSS-dominated carbonate mats were IPLs with non-phospho

headgroups such as ornithine lipids, surfactins and betaine lipids, the latter with odd fatty

acid chains. In contrast, IPLs with phospho headgroups were generally more abundant in

sediment environments. The high contribution of glycosidic archaeal IPLs and the

presence of bacterial IPLs with non-phospho headgroups in carbonate mats can be

explained by adsorption of phosphate onto calcium carbonate.

In addition to the general differences in IPL composition of each of three AOM-

community types, the IPL distribution was also associated with several environmental

factors, allowing the characterization of their different habitats. ANME-1/DSS dominates

Thesis abstract ________________________________________________________________________

II

habitats with high temperature and low oxygen content in bottom waters. For ANME-

2/DSS systems, it was possible to differentiate between carbonate reef habitats and

sediment settings, with the former characterized by low temperature, high oxygen content

in bottom waters and high methane and sulfate concentrations, whereas the latter was

associated with higher sulfate reduction rates. ANME-3/DBB presented similar

environmental characteristics to ANME-2/DSS.

Furthermore, degradation of archaeal and bacterial IPLs was evaluated in marine

sediments, showing a loss of 80% for the archaeal and ~50% for the bacterial IPL at 5°C

after 465 days of incubation under sterile conditions. However, in non-sterile conditions

at 5°C, an increase in concentration of both IPLs at the end of the experiment was

observed. Therefore, biotic degradation of IPLs could not be proved because the pools of

produced and degraded IPLs in the non-sterile conditions were indistinguishable.

The results obtained during this thesis support the distinction of microbial

communities performing AOM based on IPL diversity and address the role of

environmental factors in the distribution of three major AOM-community types. This

work contributes substantially to the understanding of the distribution of AOM systems

on a global scale.

Kurzfassung ________________________________________________________________________

III

KURZFASSUNG

Der Schwerpunkt dieser Doktorarbeit liegt auf der Untersuchung von

unterschiedlichen Mikrobengemeinschaften, die an der anaeroben Oxidation von Methan

(AOM) beteiligt sind mit Hilfe von Lipidanalysen. Die Zusammensetzung von apolaren

und intakten polaren Lipiden (IPLs) wurde an einer breitgefächerten Auswahl von

methangeladenen Systemen analysiert, die durch verschiedene Umweltfaktoren und

anaerobische methanotrophische Konsortien charakterisiert sind. Außerdem wurde ein

Experiment konzipiert, um die Stabilität von bakteriellen und von Archaeen stammenden

IPLs in marinen Sedimenten zu untersuchen.

Die drei phylogenetisch unterschiedlichen Cluster von Euryarchaeen namens

ANME-1, -2 und -3, die oft zusammen mit sulfatreduzierenden Bakterien der Gruppe

Desulfosarcina/Desulfococcus (‘‘ANME-1/DSS und -2/DSS Aggregate”) oder

Desulfobulbus spp (‘‘ANME-3/DBB Aggregate”) beobachtet worden sind, konnten

eindeutig anhand der Zusammensetzung ihrer IPLs unterschieden werden, aber nicht

durch ihre apolaren Lipide. Charakteristisch für ANME-1/DSS sind

Glyceroldialkylglyceroltetraether (GDGT) mit sowohl glykosidischen, phospho und

gemischten Kopfgruppen, wohingegen diagnostische IPLs für ANME-2/DSS Archaeole

mit sowohl glycosidischen als auch phospho Kopfgruppen waren. Im Gegensatz dazu

zeigten ANME-3/DBB weder glykosidische Archaeole noch GDGT-basierte IPLs, aber

dafür eine zu ANME-2/DSS sehr ähnliche Zusammensetzung der Phosphoarchaeole. Der

größte Unterschied von ANME-3/DBB waren die bakteriellen IPLs phosphatidyl-(N)-

methylethanolamine (PME) und phosphatidyl-(N,N)-dimethylethanolamine (PDME).

Andere bakterielle IPLs, die hauptsächlich in ANME-2/DSS dominierten Karbonatmatten

gefunden wurden waren IPLs ohne phosphatbasierende Kopfgruppe wie Ornithinlipide,

Surfactin und Betainlipide, letztere mit ungeraden Fettsäureketten. Im Gegensatz dazu

hatten Lipide mit phosphatbasierenden Kopfgruppen einen höheren Anteil in

sedimentären Umgebungen. Der hohe Anteil von glykosidischen Archaeenlipiden und

bakteriellen IPLs ohne phosphatbasierende Kopfgruppen in Karbonatmatten kann durch

die Adsorption von Phosphat an Kalziumcarbonat erklärt werden.

Kurzfassung ________________________________________________________________________

IV

Zusätzlich zu den allgemeinen Unterschieden der IPL Zusammensetzung der drei

AOM-Gemeinschaften, war die Verteilung der IPLs auch mit verschiedenen

Umweltfaktoren verknüpft, was die Charakterisierung deren unterschiedlichen

Lebensräume ermöglicht. ANME-1/DSS dominiert Umgebungen mit hoher Temperatur

und niedrigem Sauerstoffgehalt im Bodenwasser. Für ANME-2/DSS Systeme war es

möglich zwischen Karbonatriffen und Sedimenten zu unterscheiden, wobei Erstere durch

niedrige Temperaturen, hohen Sauerstoffgehalt im Bodenwasser und hohe Methan- und

Sulfatkonzentrationen charakterisiert sind, während Letztere mit hohen

Sulfatreduktionraten verbunden waren. ANME-3/DBB zeigte ähnliche

Umweltcharakteristika wie ANME-2/DSS.

Zusätzlich wurde die Degradation von bakteriellen und von Archaeen

stammenden IPLs in marinen Sedimenten untersucht. Nach Inkubation für 465 Tage

unter sterilen Bedingungen bei 5°C wurde ein Abbau von 80% des Archaeen- und ~50%

des Bakterienlipids beobachtet. Unter nicht sterilen Bedingungen bei 5°C hingegen

wurde ein Anstieg der Konzentration von beiden IPLs am Ende des Experiments

festgestellt. Deshalb konnte der biologische Abbau von IPLs nicht belegt werden, da die

Pools von produzierten und abgebauten IPLs unter nicht-sterilen Bedingungen

ununterscheidbar waren.

Die Ergebnisse dieser Doktorarbeit zeigen, dass es möglich ist die verschiedenen

Mikrobengemeinschaften die an AOM beteiligt sind anhand ihrer IPL Zusammensetzung

zu unterscheiden und deuten auf die Rolle von Umweltfaktoren bei der Verteilung der

drei Typen von AOM Gemeinschaften hin. Diese Studie trägt wesentlich zum

Verständnis der Verteilung von AOM Systemen im globalen Maßstab bei.

Acknowledgements ________________________________________________________________________

V

ACKNOWLEDGEMENTS

I started my scientific career as a marine biologist, followed by a master in

oceanography, period during which I acquired the first knowledge about organic

geochemistry. This small background was widely extended during the realization of my

PhD under the supervision of Prof. Kai-Uwe Hinrichs, who gave me the opportunity to

join his working group. Thanks Kai for providing me support and inspiration during these

over three and half years. I would also like to thank the co-supervision of Marcus Elvert,

who contributed to my knowledge in GC and GC-MS and for the interesting and helpful

discussions. I am also grateful to Julius Lipp and Helen Fredricks for guiding my first

steps with HPLC-MS and in the analysis of IPLs. I would also like to thank the thesis

committee members for their review of my dissertation.

Additionally, I would like to thank all the colleges from the MPI in Bremen

involved in the MUMM project especially Antje Boetius, Tina Treude, Katrin Knittel,

Julia Arnds, Helge Niemann, Gunter Wegener, Janine Felden and Thomas Holler, for

supplying samples and for the useful discussions. I am also indebted to Julia Arnds,

Katrin Knittel, Antje Boetius and Alban Ramette for contributing in great part to the

work included in this thesis. Moreover, I would like to thank my friend Beth! Orcutt for

providing me samples from the Gulf of Mexico, together with some unpublished data

from this setting. Thanks also to Helge Niemann, Tina Treude and Janine Felden for

providing me some unpublished data. Thanks also to Florence Schubotz who helped me

with her expertise in bacterial IPLs and also for sharing unpublished data from the Black

Sea.

Thanks to Birgit Schmincke for being always so helpful with the administrative

paper work.

A special thank to all my colleges and friends from the Organic Geochemistry and

Geobiology groups in Bremen for providing a nice and pleasant working atmosphere.

Thanks for the interesting collaboration work with our lab guests John Pohlman and

Maria Pachiadaki. Thanks to Marcus and Xavi for technical support in the lab. I would

like to thanks also my friends Marcos Yoshinaga, Julius Lipp and Julio Sepulveda for

reading and reviewing part of my work.

Acknowledgements ________________________________________________________________________

VI

Thanks to Julio to be my brother all these years, to share so many histories and

experiences that I will never forget (gracias peladito espero que nuestros caminos se

junten nuevamente). Thanks also to Annette and Amaya; you have been my family in

Bremen, thanks for always being there in the good and bad moments, I will miss all of

you very much.

Thanks to my German teacher and good friend Ursula, who made me enjoy so

much the two hours of German lessons every Friday. I am very glad that I decided to stay

in Bremen, so I will be able to continue with that.

Thanks to my family in Barcelona, Montserrat, Julià and Jordi, for receiving me

as my own family, for taking care of me and giving me support during this PhD.

Thanks to my friends from South America, which despite the distance are always

so close to me: Lilian Nuñez, Andrea Elgueta, Jaime Letelier, Klaudia Hernandez,

Pamela Vaccari, Carlos Tapia and Marcelo Ayala. Thanks to my friends in Bremen for

giving me many great moments and to make me feel at home: Claudia & Sven, Petra,

Luisa, Elvan & Jerome, Cécile & Rick, Flo & Julius, Mathias & Susanne, Barbara &

Marius, Xavier & Gulnaz, Catalina, Ilham and Jeroen. To my former advisors and

friends Silvio Pantoja and Carina Lange, thanks for being always there.

A word of thanks to my family in Chile, Margarita, Gabriel, Soledad, Camila,

Aylin and Gabriel son, thanks for believe in me and give me your support during these

years. Especially to you mother for being a great friend and inspiring woman so strong

and perseverant, despite all the things you have being through, without you I wouldn’t be

this person.

Finalmente a Xavi, gracias por quererme tanto y por ser tan paciente en especial

este ultimo año. Gracias por tu compañía y atenciones. Por tu risa, tus miradas y caricias.

Espero seguir siendo tu compañera de viaje siempre en el polvo del tiempo. Este trabajo

te lo dedico a ti.

List of Figures ________________________________________________________________________

VII

LIST OF FIGURES

Figure I.1. Three-dimensional structure of the methane molecule………………..2

Figure I.2. Gas hydrate stability zone in the marine environment...………………3

Figure I.3. Model of methane hydrate structure…...……...………………………3

Figure I.4. Methane, temperature and past climate changes…...……….………....4

Figure I.5. Sources of atmospheric methane…………………………….……......5

Figure I.6. Classification of natural methane sources……………...………….......6

Figure I.7. Redox sequence in marine sediments………….……………………...7

Figure I.8. Phylogeny of archaea……………………………….………………....8

Figures I.9. Enzymatic pathway of CO2 reduction……………….………...............9

Figure I.10. Production and consumption of methane in marine sediments...........10

Figure I.11. Phylogenetic tree of Euryarchaeota including

anaerobic methanotrophic archaea (ANME)…………………...……12

Figure I.12. Methane-dependent sulfate reduction in ANME-1 and

ANME-2 in response to temperature variability..................................13

Figure I.13. Community distribution in relation to fluid flow……….….………...14

Figure I.14. Global distribution of ANMEs based on phylogenetic data..………..15

Figure I.15. Apolar lipids derived from ANME-1 and ANME-2............................20

Figure I.16. Phospholipid membrane bilayer.………….…………....……………22

Figure I.17. General features of archaeal and bacterial membranes…………........23

Figure I.18. HPLC-MS chromatogram from an IPL mixture………...…………...25

Figure I.19. Diversity of IPLs..……………………..……...……………………...26

Figure I.20. Characteristic mass spectra of PE in positive and

negative ion modes…………………………………..………………27

Figure II.1. Composite mass chromatograms of samples dominated by

different ANME communities…………………………………….…51

Figure II.2. Distribution of IPLs in AOM communities………………………….54

Figure II.3. Structure of IPLs…………………………..…………………………61

Figure III.1. Grouping of samples according to the dominance of

GDGT- and AR-based IPLs………………………………………….78

List of Figures ________________________________________________________________________

VIII

Figure III.2. Principal Component Analysis showing the distribution

of IPLs among the analyzed samples………………...………………81

Figure III.3. Redundancy Analysis in function of environmental data……………89

Figure III.4. Location of the samples included in the global survey……………..106

Figure III.5. Principal Component Analysis showing the distribution

of bacterial IPLs………………………………...…..………………107

Figure III.6. Principal Component Analysis showing the distribution

of apolar lipids among the samples…………………………………108

Figure IV.1. Experimental design of the degradation study……………………...115

Figure IV.2. Degradation of archaeal and bacterial IPLs at 5°C and 40°C

in sterile sediments………….…………………………….………...117

Figure IV.3. Degradation of archaeal and bacterial IPLs at 5°C and

40°C in active sediments………………………………….………...119

Figure IV.24 Variability of GDGT cores in sediments incubated at 5°C

in active sediments …………..………………………..….………...120

Figure V.1. MS2 positive ion spectra of glycosidic archaeols...………………...130

Figure V.2. MS2 positive ion spectra of glycosidic GDGTs.....………………...132

Figure V.3. MS2 positive ion spectra of phospholipid archaeols…………....….134

Figure V.4. MS2 positive ion spectra of phospholipid GDGTs…...…………….135

Figure V.5. MS2 positive ion spectra of the phospholipids PE

and its methyl derivates...…………………………………………..136

Figure V.6. MS2 positive ion spectra of ornithine lipids………………….....….137

Figure V.7. MS2 positive ion spectra of betaine lipids……………………....….138

Figure V.8. MS2 positive ion spectra of surfactins…...……………………...….139

Figure V.9. MS2 positive ion spectra of unknown IPLS a and b…….……...….141

List of Tables ________________________________________________________________________

IX

LIST OF TABLES

Table I.1. General guidelines to distinguish phospholipids…………………….27

Table II.1. Overview of analyzed samples and IPLs…………………………….50

Table III.1. Overview of analyzed samples, with sample location

and AOM-phylotypes……………………………………………..….68

Table III.2. Environmental data selected for redundancy analysis……………….72

Table III.3. Lipid code and source assignment of detected IPLs…………………75

Table III.4. Relative abundance of IPLs in percentage……………….………....109

Table III.5. Concentration of apolar lipids……………………….……………...110

Table IV.1. Frequency of analysis in experiments performed to test IPLs

stability…………….……………………………………………......116 Table V.1. IPL diversity in seep environments………………………….……...142

List of Abbreviations ________________________________________________________________________

X

LIST OF ABBREVIATIONS

16S Rrna Small ribosomal ribonucleic acid unit with a sedimentary unit of 16

ANME Anaerobic methanotrophic archaea

AOM Anaerobic oxidation of methane

APCI Atmospheric pressure chemical ionization

APT Phosphoaminopentatetrol

AR Archaeol

AS Arabian Sea

Beg Beggiatoa

BL Betaine lipids

BS Black Sea

Calyp Calyptogena

CARD-FISH Catalyzed reporter deposition fluorescent in situ hybridization

CH4 Methane concentration

Da Dalton

DAG Diacylglycerol

DAGEs sn-1,2-di-O-alkyl glycerol ethers

DCM Dichloromethane

DEG Dietherglycerol

DNA Desoxyribonucleic acid

EMS Eastern Mediterranean Sea

ER Eel River Basin

ESI Electrospray ionization

FA Fatty acid

FAME Fatty acid methyl esters

FISH Fluorescent in situ hybridization

GB Guaymas Basin

GC-MS Gas chromatography-mass spectrometry

GDGT Glyceroldialkylglyceroltetraether

GF Gullfaks oil field

List of Abbreviations ________________________________________________________________________

XI

Gly Glycosyl

GOM Gulf of Mexico

HMMV Håkon Mosby Mud Volcano

HPLC-MS High performance liquid chromatography mass spectrometry

HR Hydrate Ridge

IPL Intact polar membrane lipid

m/z mass to charge ratio

MAGE sn-1, mono-O-alkyl glycerol ether

MAPT Phosphomethylaminopentatrol

MAR Macrocyclic archaeol

MeOH Methanol

MS1 Primary order mass spectrometry stage

MS2 Secondary order daughter ion mass spectra

MSn Higher order daughter ion mass spectra

MUMM Methane in the Geo/Bio-System-turnover, metabolism and microbes

O2 Oxygen concentration in bottom waters

OH-AR Hydroxyarchaeol

OL Ornithine lipids

OM Organic matter

PAF Platelet activation factor (1-O-hexadecyl-2-acetoyl-sn-glycero-3-

-phosphatidylcholine)

PC Phosphatidylcholine

PCA Principal component analysis

PDME Phosphatidyl-(N,N)-dimethylethanolamine

PE Phosphatidylethanolamine

PG Phosphatidylglycerol

PI Phosphatidylinositol

PME Phosphatidyl-(N)-methylethanolamine

PMI 2,6,15,19-pentamethylicosane

PS Phosphatidylserine

RDA Redundancy analysis

List of Abbreviations ________________________________________________________________________

XII

rDNA Ribosomal ribonucleic acid

SMTZ Sulfate methane transition zone

SO42- Sulfate concentration

SOB Sulfide oxidizing bacteria

SR Sulfate reduction

SRB Sulfate reducing bacteria

SRR Sulfate reduction rate

Thio Thioploca

TLE Total lipid extract

TOC Total organic carbon

TOF-SIMS Time of flight mass spectrometry

VFA Volatile fatty acids

Chapter I ________________________________________________________________________

1

CHAPTER I

Introduction

Chapter I ________________________________________________________________________

2

GENERAL INTRODUCTION

The first chapter provides an overview about the significance of methane in the

global carbon cycle and a description of different processes during methane production

and consumption. Furthermore, this section will give an introduction to the role of the

oceans and the microorganism inhabiting marine sediments in the global methane budget.

A dominant part is dedicated to the identification of diverse microbial communities

involved in the anaerobic oxidation of methane (AOM) from widely distributed

hydrocarbon rich sediments. Finally, the last part of this section includes the main

objectives of this work.

I.1. Properties and importance of methane

Fig I.1. Three-dimensional tetrahedron of the methane molecule.

Methane is the simplest organic

molecule and the most reduced form of

carbon. Methane represents the main

component of natural gas, although this

can occur with other hydrocarbons such

as ethane, propane and butane. Methane

has a molecular weight of 16.04 and

consists of a central carbon atom

covalently bonded to four hydrogen

atoms (tetrahedron, Fig. I.1).

Methane solubility in water is rather low (~2,5 mM at 0°C and 1 atm of pressure)

and it is negatively affected by temperature (Duan et al., 1992) and salinity (Yamamoto et

al., 1976). Contrary to salinity and temperature, pressure has a positive effect on methane

solubility according to Henry’s law. However, in the marine environment, the

combination of low temperature and high pressure conditions enables the mixture of

Chapter I ________________________________________________________________________

3

methane and water molecules resulting in hydrate formation (Fig. I.2), which is a

crystalline, ice-like structure known as methane clathrate (Fig. I.3). Three different

methane clathrate structures have been described (I, II and H) and among these, structure

I is based on pure methane, while the other ones also include ethane, propane or butane

(Buffett, 2000). The stability of methane hydrates is also affected by the inclusion of

various ions and additional gases such as hydrogen sulfide or carbon dioxide (Fig. I.2).

Fig. I.2. Gas hydrate stability zone in the marine environment in relation to pressure and temperature (after Kvenvolden, 1998).

Fig. I.3. Model of methane hydrate structure I. Gas and water molecules are displayed in green and blue, respectively (Rehder and, Suess, 2004).

Methane is an important greenhouse gas due to its ability to absorb and re-emit

radiation, trapping the heat 25 times more efficiently than carbon dioxide (Lelieveld et

al., 1998). Thus, several studies focused on the relation between methane inventory, i.e.

fluctuations in atmospheric methane concentration, and temperature during glacial-

interglacial cycles (Petit et al 1999, Wuebbles and Hayhoe 2002, Kasting, 2004). These

studies provided strong evidence for the positive correlation of the greenhouse gas

content in the atmosphere (CO2 and CH4) and the temperature record of Antarctica during

the past four glacial-interglacial cycles (Fig. I.4).

Chapter I ________________________________________________________________________

4

Fig. I.4. Variations of methane, CO2 and temperature recorded in the Vostok ice core (Petit, 1999).

Past global warming events have been related to an increase in the emissions of

methane gas to the atmosphere. Among the responsible sources for these releases,

methane hydrate dissociation has been discussed. Dickens (2004) suggests that the

depleted �13C values from several sediment cores from north and central Atlantic Ocean

during the warming period of the initial Eocene maximum (IETM), at about 55 million

years ago, can be explained by a methane release from gas hydrate source. Similarly,

Kennett et al. (2002), based on the light �13C values of benthic and planktonic

foraminifera recorded in a core from the Santa Barbara basin, proposed that the end of the

last glacial maximum was caused by a big methane release due to a destabilization of gas

hydrates, idea which is know as the clathrate gun hypothesis.

I.2. Production and consumption of methane According to the Intergovernmental Panel on Climate Change (IPCC), methane

concentration in the atmosphere has increased by ~150% since pre-industrial times

(IPCC, 2001). Several sources have been identified which contribute to the release of

methane to the atmosphere (Fig. I.5, Reeburgh, 2007). Among these, human-related

sources such as rice cultivation contribute with 20%, production of coal with 7%, and

Chapter I ________________________________________________________________________

5

ruminant animals with 16%. Additionally, incomplete combustion of organic matter and

degradation of organic carbon in landfills contribute with 11% and 8%, respectively.

Fig. I.5. Sources of atmospheric methane in Tg (1012g) and relative contribution presented in percentages (in parentheses) of the total (Reeburgh, 2007).

Natural sources of methane

include wetlands, termites, oceanic and

geological sources. Wetlands contribute

with 23%, while termites contribute only

with 4% (based on cellulose utilization

by methanogens living in their guts).

Ocean and freshwater contributes with

2%, while geological sources, like

hydrates and gas production (including

seeps) contribute with 1% and 8% to the

atmosphere methane budget,

respectively. However, the real

contribution of hydrates is still not very

well constrained.

Several of the identified sources of methane release are not affected by microbial

consumption such as animal production, biomass burning, coal production and venting or

methane flaring. Contrary to these sources, the oceans play an effective role in

controlling methane emissions to the atmosphere with only 2% of contribution in the

methane global budget, although they cover 70% of the Earth surface (Reeburgh, 2007).

The use of stable isotopes to distinguish natural methane sources is a very

common approach. The isotopic value of methane in nature can be affected by the

contribution of the different isotopomers (12C, 13C and 1H, 2H). During the utilization of

carbon by living organisms a discrimination against the heavier isotope (13C) results in

products enriched in 12C (lower or more negative �13C value, Eq. 1). However, different

metabolic pathways can discriminate differently against 13C. The �13C value is expressed

as per mil (‰) deviation from VPDB (Vienna Pee Dee Belemnite standard) according to

equation 1.

Chapter I ________________________________________________________________________

6

� �� �

31213

121313 101

Standard/Sample /

��

���

�

CCCCC� Eq. 1

Fig. I.6. Bernard-diagram used for the classification of natural methane sources (Whiticar, 1999).

Sources of methane can be

classified as thermogenic or

biogenic/bacterial (Fig. I.6, Whiticar,

1999 and references therein).

Thermogenic methane is formed during

thermocatalytic degradation of kerogen

at temperatures above ~120°C (Tissot

and Welte, 1984) and it is generally

more enriched in 13C (�13C > -50‰) than

the methane from biogenic sources (�13C

< -50‰; Whiticar, 1999).

Methane derived from bacterial sources is restricted to lower temperatures (< 60°C,

Ziebis and Haese, 2005) and shows carbon isotopic compositions which are dependent on

the environment (freshwater and marine or saline sediments). Bacterial methane from

marine environments is generally more depleted in 13C compared to freshwater

ecosystems, resulting from the dominance of CO2-reduction as opposed to acetoclastic

methanogenesis. Furthermore, the relation between �13C values and the occurrence of

longer chain hydrocarbons relative to methane expressed by the ratio C1/(C2+C3) also

provides information about the methane source, with values of less than 50 and more than

100 for thermogenic and microbial origin, respectively (Whiticar, 1999).

During the microbial degradation of organic matter in sediments, macromolecular

organic compounds are broken down into smaller molecules in a sequence of redox

reactions (Fig. I.7, Jørgensen, 2001). This redox sequence ends with the generation of

methane by methanogenic archaea, which either use carbon dioxide or other low

molecular weight compounds (formate, acetate, methanol and methylated amines) as

substrates under anaerobic conditions. Among the metabolic pathways used to produce

methane (Eq. 2a-e), the production of methane by CO2 reduction (Eq. 2a) and acetoclastic

metanogenesis (Eq. 2d) are the most important.

Chapter I ________________________________________________________________________

7

Fig. I.7. Redox sequence during the degradation of organic matter in marine sediments (Jørgensen, 2001).

Methanogenic reactions:

CO2 reduction:

OHCHHCO 2422 24 � , �G0= -135.6 Eq.2a

Methanol reduction:

OHCHHOHCH 2423 � , �G0= -112.5 Eq.2b

Disproportionation of formate:

OHCOCHHHCOO 224 2344 � , �G0= -130.1 Eq.2c

Acetoclastic methanogenesis:

243 COCHHCOOCH � , �G0= -31.0 Eq.2d

Disproportionation of methylamines: � 424233 4324 NHCOCHOHNHCH , �G0= -75.0 Eq.2e

Methanogens are strictly anaerobic microorganisms, due to instability of the

hydrogenase enzyme complex F420 in the presence of oxygen, nitrate and nitrite

(Schönheit et al., 1981). This coenzyme works as electron donor during the reduction of

different one-carbon intermediates involved in CO2 and methanol reduction (Hedderich

Chapter I ________________________________________________________________________

8

and Whitman, 2006). Methanogens are represented by five orders of the Euryarchaeota:

Methanobacteriales, Methanococcales, Methanomicrobiales, Methanosarcinales and

Methanopyrus (Fig. I.8). Among these groups, different metabolic pathways have been

described. The utilization of CO2, formate or methanol (Methanobacteriacea), CO2 or

formate (Methanococcacea), CO2, formate or alcohols (Methanomicrobiacea), as

substrate has been observed (Blotevogel and Fisher, 1985; Jones et al., 1987; Hedderich

and Whitman., 2006). Additionally, Methanosarcinales can also disproportionate

methanol, use acetate, methylamines and other methylated compounds to produce

methane (Eq.2b, d and e) (Ferguson and Mah, 1983; Jones et al., 1987; Hedderich and

Whitman., 2006).

Fig. I.8. Phylogeny of archaea. Euryarchaeotal methanogens are displayed in red (Kasting, 2004).

Chapter I ________________________________________________________________________

9

Fig. I.9. Enzymatic pathway of CO2 reduction (Hedderich and Whitman, 2006). Abbreviations: MFR, methanofuran; H4MPT, tetrahydromethanopterin, S-CoM, coenzyme M and B, CoM-S-S-CoB; reduced coenzyme F420H2

During methanogenic

reactions a complex series of

enzymes are involved (e.g., CO2

reduction, Fig. I.9). However,

besides the different carbon sources

used during methanogenesis, all

methanogens share the same final

step in which the methyl-coenzyme

M reductase (mcr) catalyzes the

reaction between the methyl-

coenzyme M and the coenzyme B

promoting the reduction of the

methyl group into methane.

Methane oxidation in the troposphere and stratosphere is caused by the production

of hydroxyl radicals during UV degradation of ozone (Lelieveld et al., 1998). In the

biosphere, methane consumption is microbially-mediated under both aerobic and

anaerobic conditions (Eq. 3a and b), thus reducing the escape of methane to the

atmosphere.

10

2224 842,22 �� � molkJGOHCOOCH Eq. 3a

1023

244 25, �� � molkJGOHHSHCOSOCH Eq. 3b

Aerobic methanotrophy is performed by bacteria utilizing the methane

monooxygenase enzyme. Aerobic methanotrophs are members of the �, � and �

subdivision of the Proteobacteria (Hanson and Hanson, 1996). These bacteria are

ubiquitously occurring in soils, sediments, water and also as endosymbionts of mussels.

Based on different metabolic pathways used during the oxidation of methane and

assimilation of formaldehyde, aerobic methanotrophs are classified as type I, II or X

(Hanson and Hanson, 1996). Type I methanotrophs use the ribulose monophosphate

(RuMP) pathway, whereas type II methanotrophs use the serine pathway. Methanotrophs

Chapter I ________________________________________________________________________

10

of the type X can use both pathways. The utilization of other carbon sources besides

methane, such as chlorinated hydrocarbons, has also been observed in methanotrophs.

The utilization of chlorinated hydrocarbons by this group of bacteria makes these

microbes commercially interesting (e.g., Hanson and Hanson, 1996).

The recognition of anaerobic oxidation of methane (AOM) was reported for the

first time in the mid 70’s in anoxic marine sediments (Martens and Berner, 1974; Barnes

and Goldberg, 1976; Reeburgh, 1976). For a long time, oxidation of methane was

assumed to take place only under oxic conditions. However, due to the rapid utilization of

oxygen during the organic matter degradation, aerobic oxidation of methane is very

limited in marine sediments.

The diffusion of methane from deep sediments and its disappearance before

reaching the oxygen layer pointed to the utilization of methane in the presence of another

electron acceptor.

Fig. I.10. Scheme showing production and consumption of methane in marine sediments (figure obtained from ifm-geomar.de after Whiticar, 1999 and DeLong, 2000).

Barnes and Goldberg

(1976) proposed sulfate as most

possible electron acceptor in this

process due to the simultaneous

consumption of both methane and

sulfate in the sulfate methane

transition zone (SMTZ) of marine

sediments (Fig. I. 10). The

utilization of sulfate as electron

acceptor during AOM was later

confirmed by the detection of

radioactively labeled products (i.e.,

sulfide and CO2) formed during

turnover of artificially labeled

substrates (i.e., 14CH4 and 35SO42-)

in sediments from the SMTZ (Devol, 1983; Iversen and Jørgensen, 1985).

The process of AOM, contrary to aerobic methanotrophy, results in increased

alkalinity (Eq. 3b, Barnes and Goldberg 1976), which favors the precipitation of

Chapter I ________________________________________________________________________

11

carbonate. The precipitates formed during AOM are mainly aragonites and Mg-rich

calcites, which can vary in shape and size ranging from small crystals (Aloisi et al., 2000)

to carbonate chimneys (Michaelis et al., 2002) and are preserved in time back to the

Carboniferous (~300 My; Birgel et al., 2008).

OHHSCaCOCaSOCH 2322

44 � Eq. 4

After the first reports of AOM three decades ago, subsequent investigations have

provided detailed evidence of Archaea and Bacteria involved in AOM. Based on field

and laboratory studies, Hoehler et al. (1994) proposed for the first time the presence of a

consortium of methanogenic archaea and sulfate reducing bacteria (SRB) in sediments of

Cape Lookout Bight, North Carolina. These authors suggested that AOM is

thermodynamically favorable at hydrogen concentrations below 0.3 nM. Because the

energy yield produced during AOM is approximately half of the energy necessary to

produce an ATP molecule (Eq. 3b), the growth rates of methanotrophic communities in

natural environments has been of controversial debate. However, the discovery of large

amounts of AOM biomass from different methane-rich environments has provided

indisputable evidence for the feasibility of this process (Boetius et al., 2000; Michaelis et

al., 2002).

I.3. Microbial communities performing AOM During the last ten years subsequent studies have reported different microbial

groups responsible for AOM in marine sediments. Because ANaerobic MEthanotrophs

(ANME) have not been successfully isolated so far, information has been dominantly

obtained from cultivation-independent techniques. Among these, the analysis of 16S

rRNA and lipid biomarkers have been mostly applied, providing evidence for the

occurrence of three main clusters in the Euryarchaeota named ANME-1, ANME-2 and

ANME-3 (Fig. I.11). These cluster were found in close association with two dominant

groups of SRB (SEEP-SRB1 and 4) involved in AOM (Hinrichs et al., 1999; Boetius et

Chapter I ________________________________________________________________________

12

al., 2000; Orphan et al., 2001 and 2002; Knittel et al, 2005; Niemann et al., 2006;

Lösekann et al., 2007).

Fig. I.11. Phylogenetic tree of Euryarchaeota, including some methanogens and the groups involved in AOM (Boetius et al., 2000; Knittel et al., 2005; Lösekann et al., 2007; MUMM project).

ANME-1, which is distantly related to Methanosarcinales and

Methanomicrobiales, occurs in association with SRB of the Desulfosarcina-

Desulfococcus (DSS) group from the �-proteobacteria (Michaelis et al., 2002; Knittel et

al., 2005), as monospecific aggregates or as single cells (Orphan et al., 2001; Knittel et

al., 2005). Both ANME-2 and ANME-3 belong to the order Methanosarcinales. ANME-

2 has been observed in physical association with DSS (Boetius et al., 2000; Knittel et al.,

2005), while ANME-3 has been found in syntrophic partnership with Desulfobulbus sp.

(DBB) (Niemann et al., 2006; Lösekann et al., 2007).

Physiological characteristics of AOM communities are based on a few in vitro

studies (Nauhaus et al., 2002 and 2005) and mesocosm experiments (Guirguis et al., 2003

and 2005). Based on in vitro experiments Nauhaus et al. (2005) reported that changes in

sulfate concentration, pH and salinity seem not to influence AOM activity, contrary to

temperature. They concluded that ANME-2 is better adapted to cold temperatures than

ANME-1, which shows highest methane-dependent sulfate reduction rates between 16°C

Chapter I ________________________________________________________________________

13

and 24°C (Fig. I.12). Furthermore, higher activity of ANME-2 community was observed

at pH values of 7.4, whereas the pH optimum of ANME-1 showed a wide range between

6.8 and 8.1 (Nauhaus et al., 2005).

Fig. I.12. Methane-dependent sulfate reduction rates in ANME-1 and ANME-2 in response to temperature variability (Nauhaus et al., 2005).

Mesocosm studies performed by Guirguis and collaborators (2005) evaluated the

effect of fluid flow during growth of AOM consortia in sediments from seep and non-

seep areas. They specifically observed that at higher fluid flows, AOM communities were

stimulated by the advective methane, which induced higher growth rates of ANME-1

compared to ANME-2.

I.4. Distribution/Habitats of AOM communities AOM can take place in a wide variety of environments in which methane and

sulfate co-occur. Originally, AOM was studied in diffusive systems where low AOM and

SR rates in the order of a few nmol cm-3 d-1 had been observed (Martens and Berner,

1977; Iversen and Blackburn, 1981; Iversen and Jørgensen, 1985; Hoehler et al., 1994).

In these systems, the low rate of methane-rich fluids homogenously transported to the

surface (Ziebis and Haese, 2005) enables AOM-communities to oxidize the methane

almost completely (Iversen and Blackburn, 1981; Iversen and Jørgensen, 1985).

Contrary, seeps or vents are controlled by advective fluid flow leading to much higher

AOM and SR rates of the order of a few μmol cm-3 d-1 (Treude et al., 2003; Boetius and

Chapter I ________________________________________________________________________

14

Suess, 2004). AOM and SR rates are usually coupled in a 1:1 ratio (Hinrichs and Boetius,

2002; Nauhaus et al., 2002 and 2005). However, due to the fact that SR can as well be

fueled by other carbon substrates, a decoupling of both processes has been observed in

places where seepage of oil and higher hydrocarbon gases, such as ethane and propane,

are detected (e.g., Gulf of Mexico, Joye et al., 2004).

Methane-rich fluids in advective systems are transported along permeable

pathways (faults, cracks, scarps) induced by pressure gradients (Ziebis and Haese, 2005),

which result in varying fluid flow regimes. This affects the small scale heterogeneity of

seep communities which are dependent on hydrogen sulfide produced during AOM (Fig.

I.13).

Fig. I.13. Community distribution in relation to fluid flow in sediments from Hydrate Ridge (Sahling et al., 2002; Torres et al., 2002).

The input of methane, together with the sulfide rich fluids advected as a result of

AOM, is the basis for the abundant communities of organism living in seeps such as

sulfide oxidizing microbial communities and diverse benthic macrofauna with

methanotrophic symbionts (Sahling et al., 2002; Levin, 2005).

Cumulative molecular data provide evidence of a global distribution of AOM

communities (Fig. I.14). The occurrence of different AOM communities is observed in a

wide range of natural habitats, which are dominated by one of the consortia described

above. Hot spots of AOM communities are cold seep environments from globally-

Chapter I ________________________________________________________________________

15

distributed habitats including anoxic water bodies, mud volcanoes and oil fields, all of

which are often found in conjunction with methane gas hydrates. Moreover, AOM has

been observed at hydrothermal vent systems. A description of these environments is

provided below.

Fig. I.14. Global distribution of AOM communities based on fluorescence in situ hybridization (FISH) microscopy obtained during the projects MUMM I and II.

Cold seeps. Cold seeps are habitats where seepage of gases and methane-rich

fluids are transported by advective forces without a considerable increase in temperature.

In contrast to hydrothermal vents, the fluid rates and temperatures at hydrocarbon seeps

are dependent on the accumulation and burial of organic matter (Campbell, 2006). Since

the first report of cold seeps 20 years ago (Paull et al., 1984), several new cold seeps have

been found in passive (e.g., Suess et al., 1985, 1998; Yun et al., 1999) and active

continental margins (e.g., Paull et al., 1995). In this environment, the supply of methane

enables growth of diverse microbial communities such as methanotrophic archaea and

SRB.

Hydrothermal vents. Hydrothermal vents are observed at mid-ocean ridges,

where abiotic methane is produced by serpentinization of iron and manganese minerals

during the contact of basaltic material with sea water (Eq. 5a and b, Reeburgh et al.,

Black Sea

Haakon Mosby Mud Volcano

Eel River Basin

Hydrate Ridge

Wadden Sea

Eckernförder Bight

Congo Basin

Gulf of Mexico

Guaymas Basin

Chapter I ________________________________________________________________________

16

2007). Once the sulfide- and sulfate-rich vent fluids get in contact with the cold seawater

the precipitation of minerals produce the characteristic black smokers observed in

hydrothermal systems (Haymon, 1983). Characteristic features of hydrothermal vent

fluids are high temperatures (Lutz et al., 1994) and typically acidic pH values, although

higher pH values have also been reported (pH >10, von Damm et al., 1985). Due to the

presence of chemical and thermal energy produced in hydrothermal systems, this habitat

is a major focus of interest because it represents an analog for the origin of life.

� �� � � �� �)(magnetite e)(serpentin (olivine) HOFeOHOSiMg30H7SiOFeMg6 2434523245.05.1 � Eq. 5a

O2H4 2422 HCHCO � Eq.5b

Hydrothermal vent fluids sustain diverse communities including tube worms,

shrimps, clams and chemosynthetic microorganisms (Levin et al., 2005). Moreover,

AOM has also been reported in the Guaymas Basin hydrothermal field where ANME-1

and ANME-2 communities occur (Teske et al., 2002).

Anoxic water bodies. The largest anoxic marine basin is the Black Sea

(Reeburgh et al., 1991). Concentration of methane in the anoxic water column are in the

micromolar range (Reeburgh et al., 1991), which seems to facilitate the build-up of

chimney-like structures that harbors carbonate-rich microbial mats of AOM communities

(Michaelis et al., 2002; Treude et al., 2005). Both, lipid biomarkers strongly depleted in 13C and FISH data confirm the presence of ANME-1/DSS and AMME-2/DSS utilizing

methane as a carbon source (Michaelis et al., 2002; Blumenberg et al., 2004). Besides

these structures, the occurrence of pockmarks, mud volcanoes and gassy sediments is also

observed in the Black sea. Similarly, the occurrence of AOM in sediments and water

column of Cariaco Basin has been documented (Reeburgh, 1976; Ward et al., 1987),

although no evidence of chimney-like structures has been provided.

Mud volcanoes. Mud volcanoes are another important habitat, with high, but

episodic gas escape (Reeburgh et al., 2007). Most mud volcanoes are found as submarine

structures close to subduction zones and orogenic belts, in which high sedimentation rates

and the formation of hydrocarbons and fluids occur (Dimitrov et al., 2002; Milkov et al.,

Chapter I ________________________________________________________________________

17

2003). Methane release from these structures is estimated in the order of 13 Tg and 15 Tg

during inactive and eruptive periods, respectively (Milkov et al., 2003). At distinct mud

volcanoes, such as the Haakon Mosby Mud Volcano (HMMV), up to 40% of the released

methane is oxidized by aerobic and anaerobic methonotrophs (Niemann et al., 2006).

Distinctive from other seep environments is the dominance of ANME-3/DBB

communities at HMMV (Lösekann et al., 2007). A relative higher abundance of ANME-

3, although accompanied by other ANME groups, has been also reported at the mud

volcano from the Nile deep sea fan at the eastern Mediterranean Sea (Omoregie et al.,

2008).

Oil fields. Shallow and deep oil fields have been observed at Gullfaks and in the

Gulf of Mexico, respectively. Gullfaks is a big Norwegian oil and gas field located in the

northern North Sea at 140 m water depth (Hovland, 2007). This area is covered by sand,

which was deposited during the last glacial maximum (Hovland and Judd, 1988).

Microbial mats of sulfide oxidizing bacteria provide evidence of the occurrence of AOM

just a few centimeters below the seafloor, in which ANME-2a and -2c dominated

communities inhabit (Wegener et al., 2008). The northern Gulf of Mexico is a

hydrocarbon gas reservoir positioned over salt deposits of Jurassic age (Roberts et al.,

1999). The tectonic characteristics of this location produce conduits that allow the

transport of gas through seeps, brine pools and mud volcanoes, as well as the formation

of methane hydrates (Sassen et al., 1994). Large amounts of sulfide oxidizing bacteria,

inhabiting surface of sediments, together with a high abundance of ANME-1/DSS have

been observed at Gulf of Mexico seeps (Orcutt et al., 2005).

Gas hydrate environments. The occurrence of methane hydrates in cold seeps is

very well documented from several locations such as the Gulf of Mexico (Sassen et al.,

1994), the Eel River Basin (Kvenvolden and Field, 1981) and the Cascadia continental

margin (Suess et al., 1999). Among these locations, one of the most studied is Hydrate

Ridge, a geological feature discovered at the Cascadia Margin in the mid ‘80s (Suess et

al., 1985). Hydrate Ridge is characterized by high fluid flow and shallow deposits of gas

hydrates (Suess et al., 1999; Torres et al., 2002). In this habitat, the consortium of

ANMEs and SRB responsible of AOM was visually observed for the first time (Boetius

et al., 2000) in agreement with previous findings of huge amounts of AOM-derived

Chapter I ________________________________________________________________________

18

carbonate structures (Ritger t al., 1987) and 13C-depleted lipid biomarkers (Elvert et al.,

1999).

Besides the fact that AOM communities are widely distributed in various habitats

in which methane and sulfate co-occur, the dominance of single communities has been

reported. For example, ANME-1/DSS seems to dominate in subsurface sediments

(Knittel et al., 2005) and microbial mat structures (Michaelis et al., 2002), ANME-2/DSS

occurs in surface sediments related to methane hydrates (Knittel et al., 2005), and

ANME-3/DBB in mud volcanoes (Niemann et al., 2006, Lösekann et al., 2007). This

indicates that the selection of the respective groups depends on a yet unknown

environmental conditions found at the sites.

I.5. Lipid signatures of communities performing AOM The first description of a biomarker related to AOM came from the irregular tail-

to-tail isoprenoid crocetane (2,6,11,15-tetramethylhexadecane), which was observed in

the SMTZ of sediments in the Kattegat (Bian, 1994; Bian et al., 2001). Moreover,

crocetane was reported from recent and ancient cold seep environments associated with

marine gas hydrates (Elvert et al., 1999) and limestone formation (Peckmann et al., 1999;

Thiel et al., 1999), respectively. In all of these studies, crocetane was suggested to be a

biomarker of anaerobic methanotrophic archaea due to its structural characteristic and

strong depletion in 13C relative to the assimilated methane. Together with the occurrence

of crocetane in AOM environments, subsequent studies have provided a series of other

biomarkers characterized by very low �13C values as a consequence of methane

utilization. The first unambiguous evidence of archaea mediating AOM was the presence

of archaeol and sn-2-hydroxyarchaeol with �13C values < -100‰, which were found in

concert with ANME-1 sequences in methane rich sediments from the Eel River Basin

(Hinrichs et al., 1999). In a following study, Hinrichs et al. (2000) provided evidence for

not only archaeol and sn-2-hydroxyarchaeol as indicators of ANMEs but also bacterial-

derived fatty acids as well as straight-chain monoalkyl and dialkyl glycerol ethers

(MAGEs and DAGEs, respectively), which were less depleted in 13C compared to the

archaeal lipids. The presence of these non-isoprenoidal lipid biomarkers was attributed to

Chapter I ________________________________________________________________________

19

the SRB partners associated with the ANMEs (Hinrichs et al., 2000). The occurrence of

these and other biomarkers in various cold seep systems, including methane-hydrate

environments (Elvert et al., 1999, 2003 and 2005; Boetius et al., 2000), hydrothermal

vents (Teske et al., 2002), mud volcanoes (Pancost et al., 2000 and 2001; Niemann et al.,

2006), carbonate reefs (Thiel et al., 2001; Michaelis et al., 2002; Blumenberg et al., 2004)

and oil fields (Wegener et al., 2008), support the extensive distribution of these

communities performing AOM.

Several diagnostic biomarkers have been related to the dominance of the different

AOM communities in the marine environment. ANME-1 microbial mats from the Black

Sea were characterized by a high abundance of GDGT-derived biphytanes and higher

amounts of archaeol as opposed to hydroxyarchaeol (Fig. I.15A). In contrast, ANME-2

dominated mats were found to contain crocetane and crocetenes, and a higher abundance

of hydroxyarchaeol relative to archaeol (Fig. I.15B). Similar conclusions were drawn by

Elvert et al. (2005) who reported the diversity of biomarkers occurring in sediments from

Hydrate Ridge off the coast of Oregon. Biomarker patterns observed were specifically

related to different fluid flow regimes causing the development of distinct seep provinces,

namely Beggiatoa mats, Calyptogena fields and Acharax fields (Fig. I.13). Besides

archaeal biomarkers, high amounts of DSS-specific fatty acids (i.e., C16:1�5c and

cyC17:0�5,6) were detected at the Beggiatoa site (Fig. I.15C), where also high numbers of

ANME-2a/DSS aggregates were observed, whereas ANME-1 in deeper horizons of the

Calyptogena site showed higher contents of the fatty acid ai-C15:0 (Fig. I.15D). Generally,

sediments from the Calyptogena site were dominated by ANME-2c and characterized by

the additional occurrence of GDGTs containing 1 and 2 cyclopentyl rings, which have

been frequently detected in AOM environments (e.g., Pancost et al., 2001; Wakeham et

al., 2003). Carbon isotopic values of the biomarkers from ANME-2 were usually 20‰

more negative than the ones from ANME-1 dominated sediment horizons (Elvert et al.,

2005). This carbon isotopic difference between the two communities was previously

indicated in other studies (Hinrichs et al., 2000; Orphan et al., 2001; Blumenberg et al.,

2004).

Chapter I ________________________________________________________________________

20

Fig. I.15. Characteristic apolar lipids derived from ANME-1 and ANME-2 dominated chimney-like structures in the Black Sea (A and B, Blumenberg et al., 2004) and sediments underneath a Beggiatoa mat from Hydrate Ridge (C and D, Elvert et al., 2005).

The differentiation of ANME-3 from ANME-1 and -2 is less obvious and was

characterized by the sole presence of highly unsaturated 2,6,10,15,19-

pentamethylicosanes (PMI:4 and PMI:5) together with archaeol and hydroxyarchaeol, but

the absence of both crocetane and GDGTs (Niemann et al., 2006). The bacterial partner

of the Desulfobulbus group, however, was indicated by the high abundance of the

specific fatty acid C17:1�6c.

Chapter I ________________________________________________________________________

21

In summary, the occurrence of strongly 13C-depleted archaeal biomarkers in

AOM studies is accompanied by the presence of slightly 13C-enriched bacterial lipid

biomarkers. Among these bacterial lipids, the occurrence of complex fatty acids with 14-

18 carbon atoms, with and without double bonds, methyl-branches and cyclopropyl

isomers has been observed (Hinrichs et al., 2000; Elvert et al., 2003 and 2005). Also the

presence of MAGEs and DAGEs with similar patterns to the ones detected in the fatty

acids has been reported (Hinrichs et al., 2000; Elvert et al., 2005). However, all of these

previous biomarker studies targeted GC-amenable lipids, which are assumed to represent

only a minor fraction in living cells and may have only been found as a relict of deceased

microbial communities. To reduce the obstacles associated with apolar lipids, we

therefore targeted intact polar lipids (IPLs) which are the building blocks of the

cyctoplasmic membrane of all living cells and which can be directly related to

microbiological investigations using FISH or other techniques.

I.6. Intact polar membrane lipids (IPLs) The cytoplasmic cell membrane acts as a semi-permeable barrier and protects the

cell from the external environment. The membrane is composed of proteins and a lipid

bilayer (Fig. I.16).

Proteins can play different roles in the cell membrane such as recognizing

substrates, performing enzymatic activity and transporting substances (nutrients, ions and

waste) between the cytoplasm and the exterior of the cell (Madigan et al., 2003). On the

other hand, lipids are indispensable for the membrane structure due to their chemical

properties (hydrophobicity and hydrophilicity), which directly involve these molecules in

membrane permeability (Madigan et al., 2003). Because the cell membrane regulates the

transport between the exterior and interior of the cell, it is also important in the

conservation of cell energy (Madigan et al., 2003).

According with the fluid mosaic model, the cell membrane is composed of a

double layer or bilayer of lipids. The bilayer formed by phospholipids contains a fatty

acid tail (hydrophobic side) and a phosphate group in the polar part of the molecule

(hydrophilic side). The hydrophobic side is oriented inwards, while the hydrophilic side

Chapter I ________________________________________________________________________

22

or head group is facing outwards (i.e. the aqueous cytosol of the cell or the environment)

(Fig. I.16).

Fig. I.16. The phospholipid membrane bilayer (Tortora et al., 2004).

Lipids in the cell membrane of prokaryotes are represented by phospholipids,

glycolipids and sometimes hopanoids (e.g., in methanotrophic bacteria, Madigan et al.,

2003). In total, they represent up to 6% of the cell dry weight (Langworthy et al., 1983).

Membrane lipids are good candidates to distinguish Bacteria and Archaea. Bacteria

generally contain a phospholipid bilayer composed of fatty acids linked to a glycerol

backbone via ester bonds in sn-1 and sn-2 position (ester-bond acyl chains, Fig. I.17). In

sulfate reducers, these fatty acids may include methyl branching, double bonds and

cyclopropyl isomers (Taylor and Parkes, 1983; Dowling et al., 1986). Archaeal

membranes can occur both as a bilayer or monolayer (Fig. I.17). The bilayer of archaeal

cells contains isoprenoidal chains linked to the glycerol backbone in sn-2 and sn-3

position via an ether bond (i. e., isoprenoidal alkyl chains) and is generally formed by two

C20 hydrocarbon chains (phytanyl ethers) (Langworthy and Pond, 1986). Archaeal

monolayer membranes are composed of glycerol tetraethers, in which two glycerol

molecules are linked via two C40 hydrocarbon chains (biphytanyl ethers) (Langworthy

Chapter I ________________________________________________________________________

23

and Pond, 1986). Generally, ether bonds from archaeal membranes are more resistant to

higher temperature, pressure and pH (De Rosa et al., 1989) than the ester bonds present in

bacteria.

Fig. I.17. General features of archaeal and bacterial lipid membranes (Valentine, 2007).

Because the cell membrane is affected by external conditions such as temperature,

pH, pressure or salinity, several adaptations in prokaryotic cell membranes are related to

cell evolution, physiology, biogeochemistry and ecology (Langworthy, 1982). Among

these adaptations, changes in fatty acid compositions have been observed depending of

the habitat temperatures. In contrast to shorter saturated and unsaturated fatty acids in

psychrophilic bacteria, evidence of longer and saturated fatty acids, predominantly iso-

branched, is found in thermophilic bacteria (Langworthy, 1982). Additionally, the effects

of pH and temperature in a thermoacidophile were evaluated (De Rosa et al., 1974). At

lower pH and increasing temperature, the proportion of iso- and anteiso-fatty acids

Chapter I ________________________________________________________________________

24

increases, whereas at higher pH and increasing temperature cyclohexyl fatty acids

increase (De Rosa et al., 1974). Furthermore, the effect of temperature on polar head

group compositions of a thermophilic organism (i.e., Bacillus caldotenax) has been

investigated by Hasegawa et al. (1980). These authors reported a decrease in the amount

of PE (from 57% to 37%) and increase of PG (from 27% to 46%) in the total

phospholipid content induced by a temperature decrease from 65°C to 45°C.

Modifications observed in the hydrocarbon chains of archaeal-based tetraether

lipids include the increase in membrane stability at higher growth temperatures by the

formation of cyclopentane rings (Langworthy and Pond, 1986).

All the modifications in the membrane described above intent to protect the cell

from the environment. In general, archaeal membranes are less permeable, thus they may

be better adapted to hostile environments than bacterial ones (Valentine, 2007). Due to

this characteristic of Archaea, these microorganisms were assumed to live in extreme

environments in which low pH and high temperatures occur (Rothschild and Mancinelli,

2001). However, cumulative evidence shows that Archaea are not only prevalent in the

deep biosphere (Biddle et al., 2006; Lipp et al., 2008), hydrothermal vents (Teske et al.,

2002; Reysenbach et al., 2000; Schouten et al., 2003) and cold seeps (Boetius et al.,

2000; Knittel et al., 2005), but are also widely distributed in ocean waters (Karner et al.,

2001; DeLong, 2003).

The investigation on the diversity of intact polar membrane lipids (IPLs) from

both Bacteria and Archaea was extended by the utilization of high-performance liquid

chromatography mass spectrometry (HPLC-MS). Contrary to the other techniques (e.g.,

gas chromatography), the advantage of HPLC-MS is the possibility to study the intact

membrane lipid molecules instead of core or side chain products. During the analysis, the

chromatographic separation of IPLs is based on their polarity, which is mainly related to

the molecule’s head groups (Fig. I. 18).

Chapter I ________________________________________________________________________

25

Fig. I.18. HPLC-MS chromatogram (A) and density map (B) of an IPL mixture of commercially available standards mixed with an extract of microbial mat from the Black Sea. IPLs elution depends on their polarity, with less polar compound eluting at early retention times. Density map is a representation of the IPL peaks in relation to the retention time and the mass to charge ratio (range scanned from 500 to 2000 m/z). In it, the intensity of the black lines is correlated to the concentration of the IPL in the sample mixture. Bacterial-derived IPLs (PE, PG and PDME) in the density map are displayed in series due to the presence of different fatty acid chain lengths. Abbreviations of IPLs according to Fig. I.19

Diversity of polar head groups in IPLs has been described from cultures and

environmental samples based on HPLC-ESI-MS (Fig. I.19A), providing taxonomic

information that allows the distinction of different microorganisms (e.g., De Rosa et al.,

1986; Koga et al., 1998; Sturt et al., 2004; Koga and Morii, 2005; Van Mooy et al., 2006;

Koga and Nakano, 2008). HPLC-ESI-MS is equipped with an electrospray ionization

source (ESI) that produces a soft ionization of the analytes, which is particularly

appropriate for polar molecules like IPLs. Using this technique, the diversity of IPLs

characteristic of archaea from marine systems has been reported, including archaeol- and

GDGT-based IPLs with glycosidic head groups (Fig. I.19B, Sturt et al., 2004; Biddle et

al., 2006; Lipp et al., 2008). Furthermore, a variety of phospholipids from Bacteria has

been documented, including ether and ester phospholipids (Fig. I.19C) with diverse types

of head groups (Rütters et al., 2002; Sturt et al., 2004; Van Mooy et al., 2006).

Chapter I ________________________________________________________________________

26

OOPOH

O OO

OOPOH

O OO

OOPOH

O OO

R'

R''

R''

R''O

R'O

R'O

Diacylglycerophospholipid DAG

Acyl/ether glycerophospholipid AEG

Dietherglycerophospholipid DEG

OO

O

X=H, Diglycosyl archaeolX=OH, Diglycosyl hydroxyarchaeol

X

O

OO O

OHO

Diglycosyl glyceroldialkylglyceroltetraether GDGT with 0 cyclopentyl rings

OHOOH O

POH

O

OHONH2

O

Phosphatidylserine PS

Phosphatidylglycerol PG

OPOH

O

OOHHO

HOHO OH

Phosphatidylethanolamine PE

OPOH

OON

Phosphatidylcholine PC Phosphatidylinositol PI

OPO

O

OH2N

OPOH

O

ON

OPOH

O

ONH

OPOH

O

Phosphatidyl-(N)-methylethanolamine PME

Phosphatidyl-(N,N)-dimethylethanolamine PDME

O

HO OHHOO

OHOHO

HO OH

O

HO OHHOO

OHOHO

HO OH

OHO

HO OH

O OHO

HOHO

HO OH

OO O

OHO

Diglycosyl glyceroldialkylnonitoltetraether GDNT with 0 cyclopentyl rings

HOOPOH

O

Phosphatiddic acid PA

OOPOH

OHO

OH

NH2OH

Phosphoaminopentatetrol APT

OOPOH

OH2N

O

OHHOHO O

OPOH

OH

Glyco-phosphoethanolamine GPE

HOO

OHHOHO O O

O

O

O

n=1 Monogalactosyldiacylglycerol MGDG n=2 Digalactosyldiacylglycerol DGDG

n

HO3SO

OHHOHO O O

O

O

O

Sulfoquinovosyldiacylglycerol SQDG

A C

B

Fig. I.19. Diversity of IPL-head groups present in Bacteria and Archaea (A), glycolipids commonly observed in Archaea (B), and ester and ether linkages observed in phospholipids (C).

Structural information of IPLs can be obtained by ion-trap mass spectrometry (IT-

MS) configured to trap ions of interest which are later fragmented producing daughter ion

mass spectra (MSn). Identification of IPLs is based on fragmentation patterns obtained

from MSn experiments in positive and negative modes, and by comparison with

previously reported mass spectral data (Table I.1) (Sturt et al., 2004) and molecular

structures (Koga and Nakano, 2008 and references therein). Most of the structural

characteristics of IPLs can be obtained in MS2 (Fig. I.20). However, additional

information is obtained by analyzing the sample under positive and negative ionization

modes. IPLs positively ionized frequently loose the head groups providing information of

the lipid class (Fig. I.20A), whereas IPLs negatively ionized loose the fatty acid chain

located in the sn-2 position (Fig. I.20B). Structural information of diverse IPLs from

Archaea and Bacteria observed in this study are provided in the Chapter V of this work.

Chapter I ________________________________________________________________________

27

Positive ion mode [M +H]+ Negative ion mode [M -H]- Headgroup AEG, DAG DEG AEG, DAG DEG

PE 141 Da loss (phosphoethanolamine)

43 Da loss (ethanolamine) 43 Da loss

(ethanolamine)

APT 231 Da loss (phospho-APT) 133 Da loss (APT)

AEG-P; loss of sn-2 fatty

acid 133 Da loss (APT)

PG 189 Da loss

(phosphoglycerol + NH4

+ adduct) 75 Da loss (glycerol)

DAG-P; loss of head

group+ sn-2 fatty acid

75 Da loss (glycerol)

PI 162 Da loss hexose Major ion m/z 241 (phosphoglycosyl –

H2O)

PS 185 Da loss (phosphoserine) 87 Da loss (serine) 87 Da loss (serine)

PC All give a major ion m/z 184 (phosphocholine) All show 60 Da loss (CH3+ HCOO- adduct)

Table I.1. Characteristic headgroup losses of common phospholipids under HPLC-ESI-MS conditions in positive and negative ion modes (Sturt et al., 2004).

Fig. I.20. Mass spectra of phosphatylethanolamine (PE) diacylglycerol (DAG). Difference of mass between the positive (A) and negative ion mode (B) are explained by the addition and lost of one proton in the molecule, respectively. MS2 data in positive ion mode indicate the lost of 141 Da (PE) from the glycerol and fatty acid core with C31:2 (sum of both fatty acids). Negative ion mode indicates the lost of C15:2 from sn-2 position of the glycerol first (lyso fragment 434 Da) and the presence of the fatty acid C16:0 in the sn-1 position of the glycerol (fragment 255 Da).

Chapter I ________________________________________________________________________

28

I.7. Methods Most samples analyzed in this study were freeze-dried and extracted according to

a modified Bligh and Dyer protocol (Sturt et al., 2004) by microwave-assisted extraction

system (MARS-X, CEM, USA) for 15 min at a temperature of 70°C, while a few others

were extracted by ultrasonication. A mixture of standards covering different lipid classes

was added to the samples. The standards included cholestane (hydrocarbons), behenic

acid methyl ester (ketones), C-19 alcohol (alcohols) and C19-fatty acid (fatty acids) for

GC-amenable lipids, and C16-PAF for IPL analysis. The solvent mixture used during the

extractions was methanol:dichloromethane:buffer in a proportion of 2:1:0.8. The volume

of the solvent mixture used was 40 mL per every 10 g of dry sediment and 1 g of dry mat.

The first two extraction steps were performed with phosphate buffer, whereas the last two

were performed with trichloroacetic acid buffer (TCA). After collection of all

supernatants, the organic phase was separated from the aqueous one by multiple additions

of dichloromethane (DCM) and milli-Q water. This liquid-liquid extraction was

performed by using the same amount of water and DCM than the total solvent mixture

added during the extractions, starting with DCM (3 times) and then with water (3 times).

The organic phase or total lipid extract (TLE) was evaporated to dryness under a stream

of nitrogen and re-dissolved in a mixture of DCM:methanol (1:1), which was finally

injected into the HPLC-ESI-MS.

Due to the nature of the sample (e.g., oily etc.), additional clean-up steps were

performed on Eel river Basin, Guaymas Basin and two sediment samples from Gulf of

Mexico. Here, separation of the TLE into apolar, glyco- and phospholipids was carried

out on activated silica column (2 g of silica for 50-200 mg of extract) by elution with 20

mL of DCM, 40 mL of acetone and 40 mL of methanol, respectively. Acetone and

methanol eluted fractions were combined and evaporated under a nitrogen stream and re-

dissolved in DCM:methanol (1:1) prior to analysis. This procedure allows the detection

of IPLs previously not observed in the TLE probably due to matrix problems and ion

suppression. It is well documented that ESI signal can be affected by the sample matrix,

which, if contain endogenous material (in this case hydrocarbons), could interfere in the

ionization of the analytes of interest (Mallet et al., 2004). This problem can be solved to

some degree by additional clean-up steps (Mallet et al., 2004).

Chapter I ________________________________________________________________________

29

Parallel analyses of apolar lipid biomarkers were performed in order to compare

both intact (IPLs) and non-intact lipids (GC-amenable lipids). For the analysis of apolar

lipids, a fraction of the TLE was added to a Pasteur pipette with glass wool and separated

into maltene and asphaltene fraction, eluting the first of them with 2.5 mL hexane and the

second with 4 mL of DCM. The maltene fraction was further separated into four fractions

of increasing polarity on Supelco LC-NH2 glass cartridges (500 mg sorbent) using 4 mL

of hexane (hydrocarbons), 6 mL hexane/DCM (3:1; ketones/esters), 7 mL DCM/acetone

(9:1; alcohols) and 8 mL of 2% formic acid in DCM (free fatty acids). Each fraction was

evaporated to dryness under a stream of nitrogen and re-dissolved in hexane prior to

analysis. Previously alcohols were derivatized into trimethylsilylesters (TMS-derivatives)

by addition of N,O-bis(trimethylsilyl) fluoracetamide (BSTFA) and pyridine. Similarly,

fatty acids were transformed to methylesters (FAME) before analysis, using 20% Boron

trifluoride (BF3) in methanol. Both reactions were performed at 70°C for 1h. All

fractions were analyzed via gas chromatography-mass spectrometry (GC-MS) and GC-

flame ionization detection (GC-FID). Identification of GC-amenable lipids was based on

the comparison of retention times, mass spectra of commercial standards and from

literature.

I.8. Hypothesis and objectives The aim of this PhD work is the elucidation of the microbial community

structures in different marine methane-rich environments based on the diversity of lipid

signatures. This work is part of the MUMM II (Methane in the Geo/bio-System-

Turnover, Metabolism and Microbes) project, a multidisciplinary BMBF project which

started in a first phase already in January 2001.

AOM is, based on current knowledge, associated with the presence of three

phylogenetic clusters of methanotrophic archaea (ANME) and two groups of SRB (DSS

and DBB) in various marine environments (gas hydrate, mud volcanoes, hydrothermal

sediments and coastal subsurface environments). Different biogeographical patterns of

these clusters are probably related to varying environmental conditions found in a wide

range of settings (e.g., Arabian Sea, Black Sea, Eastern Mediterranean Sea, Eel River

Chapter I ________________________________________________________________________

30

Basin, Guaymas Basin, Gulf of Mexico, Gullfaks oil field, Häkon Mosby Mud Vulcano

and Hydrate Ridge). In order to evaluate the global distribution of these AOM

communities, this study reviews the diversity of lipids, known so far from the analysis of

characteristic apolar lipids, and extends the knowledge to intact polar membrane lipids

that provide valuable information of both Archaea and Bacteria. Additionally, the

combination of lipid biomarkers, available microbiological data, together with the

environmental characterization of each setting should improve our understanding of the

distribution of AOM communities and the factors controlling them.

Specifically, the present study addresses the following questions regarding AOM:

� What is the diversity of IPLs present in AOM environments?

� Is it possible, using IPL diversity to distinguish between ANME-1/-2/-3,

their SRB partners?

� Is it possible to assign the dominant ANME group in a sample without

molecular information?

� Is the IPL composition of an AOM community inhabiting carbonate

chimneys the same as the one found in the corresponding AOM

community in sediments?

� Is it possible to identify the most important environmental variables that

define the ecological niches of AOM communities?

� Do classical apolar lipid biomarkers provide the same information as

IPLs?

� What is the relation between IPLs and apolar lipids?

I.9. Contribution to publications

This thesis includes the complete version of two manuscripts. Chapter II is a

published manuscript and Chapter III is a manuscript version close to submission.

Chapter IV is a draft of a degradation experiment and Chapter V is a draft in which a

deeper insight into the diversity of intact polar membrane lipids observed during this

study is provided.

Chapter I ________________________________________________________________________

31

CHAPTER II - full manuscript Intact polar lipids of anaerobic methanotrophic archaea and associated bacteria

Pamela E. Rossel, Julius S. Lipp, Helen F. Fredricks, Julia Arnds, Antje Boetius, Marcus

Elvert, Kai-Uwe Hinrichs

Pamela E. Rossel extracted membrane lipids from three samples and identified

diverse archaeal and bacterial lipids with the support of Helen F. Fredricks and Julius S.

Lipp. Helen F. Fredricks extract membrane lipids from Hydrate Ridge sediments. Julia

Arnds and Antje Boetius provided phylogenetic data of the two microbial mats from the

Black Sea. Pamela Rossel, Marcus Elvert and Kai-Uwe Hinrichs wrote the paper jointly

with editorial input from all co-authors.

Published in Organic Geochemistry vol. 39, page 992-999,

doi:10.1016/j.orggeoche.2008.02.021.

CHAPTER III - full manuscript Factors controlling the distribution of anaerobic methanotrophic communities in

marine environments: evidence from intact polar membrane lipids

Pamela E. Rossel, Marcus Elvert, Alban Ramette, Antje Boetius and Kai-Uwe Hinrichs

Pamela E. Rossel extracted sediment and microbial mat samples, identified

diverse archaeal and bacterial polar and apolar lipids and compiled diverse environmental

data from literature to characterize the environments analyzed. Alban Ramette provided

expertise in multivariate analyses. Antje Boetius provided phylogenetic data and supplied

several samples analyzed in this study. Pamela Rossel, Marcus Elvert and Kai-Uwe

Hinrichs wrote the paper jointly with editorial input from all co-authors.

The manuscript is prepared for submission.

Chapter I ________________________________________________________________________

32

CHAPTER IV - draft Experimental approach to evaluate stability and reactivity of intact polar

membrane lipids of archaea and bacteria in marine sediments

Pamela E. Rossel, Julius S. Lipp, Verena Heuer and Kai-Uwe Hinrichs

Pamela E. Rossel prepared the experiment, extracted sediment samples and

quantified both archaeal and bacterial membrane lipids and

glyceroldialkylglyceroltetraether cores over the time of the experiment. Verena Heuer

performed acetate analysis. Julius S. Lipp gave support in the lab and with the membrane

lipids quantification. Pamela Rossel and all co-authors participated in the experimental

design. Unfortunately, due to several uncertainties in the results of this work, a new

experiment is indispensable, in which several problems related to the actual experimental

design should be overcome. Therefore this draft is just a guideline for further

experiments.

CHAPTER V - draft Diversity of intact polar membrane lipids in marine seep environments

Pamela E. Rossel, Marcus Elvert and Kai-Uwe Hinrichs

Pamela E. Rossel extracted sediment and microbial samples, identified diverse

archaeal and bacterial polar lipids and provided the molecular structures identified in seep

environments based on the mass spectral interpretation. All co-authors provided expertise

on lipid identification. Pamela Rossel wrote the paper jointly with editorial input from all

co-authors.

Chapter I ________________________________________________________________________

33

I.10. References Aloisi, G., Pierre, C., Rouchy, M. J., Foucher, J. P., Woodside, J., MEDINAUT scientific

party., 2000. Methane-related authigenic carbonates of eastern Mediterranean Sea

mud volcanoes and their possible relation to gas hydrate destabilization. Earth and

Planetary Science Letters 184, 321-338.

Barnes, R., Goldberg, E., 1976. Methane production and consumption in anoxic marine

sediments. Geology 4, 297-300.

Bian, L., Hinrichs, K. -U., Xie, T., Brassell, S. S., Iversen, N., Fossing, H., Jørgensen, B.

B., Hayes, J. M., 2001. Algal and archaeal polyisoprenoids in a recent marine

sediment: molecular isotopic evidence for anaerobic oxidation of methane.

Geochemistry Geophysics Geosystems 2, 2000GC000112.

Bian, L., 1994. Isotopic biogeochemistry of individual compounds in a modern coastal

marine sediment (kattegat, Denmark and Sweden). MSc thesis, Indiana University.

Biddle, J. F., Lipp, J. S., Lever, M. A., Lloyd, K. G., Sörensen, K. B., Anderson, R.,

Fredricks, H. F., Elvert, M., Kelly, T. J., Schrag, D. P., Sogin, M. L., Brenchley, J.

E., Teske, A. House, C. H., Hinrichs, K. -U., 2006. Heterotrophic archaea dominate

sedimentary subsurface ecosystems off Peru. Proceedings of the National Academy

of Science U.S.A. 103, 3846-3851.

Birgel, D., Himmler, T., Freiwald, A., Peckmann, J., 2008. Anew constrain on the

antiquity of anaerobic oxidation of methane: Late Pennsylvanian seep limestones

from southern Namibia. Geology 36, 543-546.

Blotevogel, K. -H., Fisher, U., 1985. Isolation and characterization of a new thermophilic

and autotrophic methane producing bacterium: Methanobacterium

thermoaggregans spec.nov. Archives of Microbiology 142, 218-222.

Blumenberg, M., Seifert, R., Reitner, J., Pape, T., Michaelis, W., 2004. Membrane lipid

patterns typify distinct anaerobic methanotrophic consortia. Proceedings of the

National Academy of Science U.S.A. 101, 11111-11116.

Boetius, A., Suess, E., 2004. Hydrate Ridge: a natural laboratory for the study of

microbial life fueled by methane from near-surface gas hydrates. Chemical Geology

205: 291-310.

Chapter I ________________________________________________________________________

34

Boetius, A., Ravenschlag, K., Schubert, C. J., Rickert, D., Widdel, F., Gieseke, A.,

Amann, R., Jørgensen, B. B., Witte, U., Pfannkuche, O., 2000. A marine microbial

consortium apparently mediating anaerobic oxidation of methane. Nature 407, 623-

626.

Buffett, B. A., 2000. Clathrate Hydrates. Annual Reviews of Earth and Planetary Science.

28, 477-507.

Campbell, K. A., 2006. Hydrocarbon seep and hydrothermal vent paleoenvironments and

paleontology: past developments and future research directions. Palaeogeography,

Palaeoclimatology, Palaeoecology 232, 362-407.

De Rosa, M., Gambacorta, A., Bu’Lock, J. D., 1974. Effects of pH and temperature on

the fatty acid composition of Bacillus acidocaldarius. Journal Bacteriology 117,

212-214.

De Rosa, M., Gambacorta, A., Gliozzi, A., 1986. Structure, biosynthesis, and

physicochemical Properties of Archaeabacterial Lipids. Microbiological Reviews

50, 70-80.

De Rosa, M., Gambacorta, A., Huber, R., Lanzotti, V., Nicolaus, B., Stetter, K. O.,

Trincone, A., 1989. Lipid structures in Thermophaga maritima. In: Microbiology of

Extreme Environments and its Potential for Biotechnology (eds. da Costa, M. S.,

Duarte, J. C., Williams, R. A. D.), 167-173, Elsevier Applied Science, London and

New York.

DeLong, E., 2003. Oceans of Archaea. American Society for Microbiology News 69, 503-

511.

Devol, A. H., 1983. Methane oxidation rates in the anaerobic sediments of Saanich Inlet.

Limnology and Oceanography 28, 738-742.

Dickens, G., 2004. Hydrocarbon-driven warming. Nature 429, 513-515.

Dimitrov, L. I., 2002. Mud volcanoes - the most important pathway for degassing deeply

buried sediments. Earth-Science Reviews 59, 49-76.

Dowling, N. J. E., Widdel, F., White, D. C., 1986. Phospholipid ester-linked fatty acid

biomarkers of acetate-oxidizing sulphate-reducers and other sulphide-forming

bacteria. Journal of General Microbiology 129, 3303-3309.

Chapter I ________________________________________________________________________

35

Duan, Z., Möller, N., Greenberg, J., Weare, J. H., 1992. The prediction of methane

solubility in natural waters to high ionic strengths from 0° to 250°C and from 0 to

1600 bar. Geochimica et Cosmochimica Acta 56, 1451-1460.

Elvert, M., Boetius, A., Knittel, K., Jørgensen, B. B., 2003. Characterization of specific

membrane fatty acids as chemotaxonomic markers for sulfate-reducing bacteria

involved in anaerobic oxidation of methane. Geomicrobiology Journal 20, 403-419.

Elvert, M., Hopmans, E. C., Treude, T., Boetius, A., Suess E., 2005. Spatial variations of

methanotrophic consortia at cold methane seeps: implications from a high-

resolution molecular and isotopic approach. Geobiology 3, 195–209.

Elvert, M., Suess E., Whiticar, M. J., 1999. Anaerobic methane oxidation associated with

marine gas hydrates: superlight C-isotopes from saturated and unsaturated C20 and

C25 irregular isoprenoids. Naturwissenschaften 86, 295–300.

Ferguson, T. J., Mah, R. A., 1983. Effect of H2-CO2 on Methanogenesis from acetate or

Methanol in Methanosarcina spp. Applied and Environmental Microbiology 46,

348-355.

Guirguis, P. R., Cozen, A. E., DeLong, E. F., 2005. Growth and population dynamics of

anaerobic methane oxidizing archaea and sulfate.reducing bacteria in a continuous-

flow bioreactor. Applied and environmental microbiology 71, 3725-3733.

Guirguis, P. R., Orphan, V. J., Hallam, S. J., DeLong, E. F., 2003. Growth and methane

oxidation o anaerobic methanotrophic archaea in a continuous-flow bioreactor.

Applied and environmental microbiology 69, 5472-5482.

Hanson, R. S., Hanson, T. E., 1996. Methanotrophic bacteria. Microbiological Reviews

60, 439-471.

Harvey, R. H., Fallon, R. D., Patton, J. S., 1986. The effect of organic matter and oxygen

on the degradation of bacterial membrane lipids in marine sediments. Geochimica

et Cosmochimica Acta 50, 795-804.

Hasegawa, Y., Kawada, N., Nosoh, Y., 1980. Change in Chemicals composition of

membrane of Bacilus caldotenax after shifting the growth temperature. Archives of

Microbiology 126, 103-108.

Haymon R. M., 1983. Growth history of hydrothermal black smoker chimneys. Nature

301, 695-698.

Chapter I ________________________________________________________________________

36

Hedderich, R., Whitman, W. B., 2006. Physiology and biochemistry of the methane-

producing archaea. Prokaryotes 2, 1050-1079.

Hinrichs, K. -U, Summons, R. E, Orphan, V., Sylva, S. P., Hayes, J. M., 2000. Molecular

and isotopic analyses of anaerobic methane-oxidizing communities in marine

sediments. Organic Geochemistry 31,1685-1701.

Hinrichs, K. -U., Boetius, A., 2002. The anaerobic oxidation of methane: New insights in

microbial ecology and biogeochemistry. In: Ocean Margin Systems (eds. Wefer G.,

Billett D., Hebbeln D., Jørgensen B. B., Schlueter M., van Weering T. C. E.), 457-

477. Springer-Verlag.

Hinrichs, K. -U., Hayes, J. S., Sylva, S. P., Brewer, P. G., DeLong, E. F., 1999. Methane-

consuming archaebacteria in marine sediments. Nature 398, 802-805.

Hoehler, T. M., Alperin, M. J., Albert, D. B., Martens, C. S., 1994. Field and laboratory

studies of methane oxidation in an anoxic marine sediment - evidence for a

methanogen-sulfate reducer consortium. Global Biogeochemical Cycles 8, 451-463.

Hovland, H., Judd, A. G., 1988. Seabed pockmarks and seepages: impact in geology,

biology and marine environment. Graham and Trotman, London, 1-293.

Hovland. M., 2007. Discovery of prolific natural methane at Gullfaks, northern North

Sea. Geo-Marine Letters 27, 197-201.

Iversen, N., Blackburn, H. T., 1981. Seasonal rates of methane oxidation in anaerobic

marine sediments. Applied and Environmental Microbiology 41, 1295-1300.

Iversen, N., Jørgensen, B. B., 1985. Anaerobic methane oxidation rates at the sulfate-

methane transition in marine sediments from Kattegat and Skagerrak (Denmark).

Limnology and Oceanography 30, 944-955.

Jones, W. J., Nagle Jr, D. P., Whitman, W. B., 1987. Methanogens and the diversity of

archaebacteria. Microbial Reviews 51, 135-177.

Jørgensen, B., 2001. Bacteria and marine biogeochemistry. In: Marine Geochemistry

(eds. Schulz H. D., Zabel M.), 169-206, Springer Verlag.

Joye, S. B., Orcutt, B. N., Boetius, A., Montoya, J. P., Schulz, H., Erickson, M. J., Lugo,

S. K., 2004. The anaerobic oxidation of methane and sulfate reduction in sediments

from at Gulf of Mexico cold seeps. Chemical Geology 205, 219-238.

Chapter I ________________________________________________________________________

37

Karner. M. B., DeLong, E. F., Karl, D. M., 2001. Archaeal dominance in the mesopelagic

zone of the Pacific Ocean. Nature 409, 507-510.

Kasting, J. K., 2004. When methane made climate. In: Scientific American, 78-85.

Kennett, J. P., Cannariato, K. G., Hendy, I. L., Behl, R. J., 2002. Methane Hydrates in

Quaternary Climate Change: The Clathrate Gun Hypothesis. American Geophysical

Union, Washington, USA.

Knittel, K., Lösekann, T., Boetius, A., Kort, R., Amann, R., 2005. Diversity and

Distribution of Methanotrophic Archaea at Cold Seeps. Applied and Environmental

Microbiology 71, 467-479.

Koga, Y., Morii, H., 2005. Recent Advances in Structural Research on Ether Lipids from

Archaea Including Comparative and Physiological Aspects. Bioscience

Biotechnology Biochemistry 69, 2019-2034.

Koga, Y., Morii, H., Akagawa-Matsushita, M., Ohga, M., 1998. Correlation of polar lipid

composition with 16S rRNA phylogeny in methanogens. Further analysis of lipid

component parts. Bioscience Biotechnology Biochemistry 62, 230-236.

Koga, Y., Nakano, M., 2008. A dendrogram of archaea based on lipid component parts

composition and its relationship to rRNA phylogeny. Systematic and Applied

Microbiology 31, 169-182.

Kvenvolden, K. A., 1998. A primer on the geological occurrence of gas hydrate. In: Gas

hydrates: Relevance to World Margin Stability and Climate Change 137 (eds.

Henriet, J. P., Mienert, J.), 9-30. Especial Publications Geological society, London.

Kvenvolden, K. A., Field, M. E., 1981. Thermogenic hydrocarbons in unconsolidated

sediment of Eel River, offshore northern California. American Association of

Petroleum Geologist Bulletin 65, 1642-1646.

Langworthy, T. A., 1982. Lipids of bacteria in extreme environments. In: Current Topics

in Membranes and Transport, Membrane lipids of prokaryotes 17 (eds. Razin, S.,

Rottem, S.), 45-77. Academic press.

Langworthy, T. A., Holzer, G., Zeikus, J., Tornabene, T. G., 1983. Iso- and anteiso-

branched glycerol diethers of the thermophilic anaerobe Thermodesulfotobacterium

commune. Systematic and Applied Microbiology 4, 1-17.

Chapter I ________________________________________________________________________

38

Langworthy, T. A., Pond, J. L., 1986. Archaeal ether lipids and chemotaxonomy.

Systematic and Applied Microbiology 7, 253-257.

Lelieveld, J., Crutzen, P. J., Dentener, F. J., 1998. Changing concentration, lifetime and

climate forcing of atmospheric methane. Tellus 50B, 128-150.

Levin, L., 2005. Ecology of cold seep sediments: interactions of fauna with fluid flow,

chemistry and microbes. In: Oceanography and Marine Biology: An annual Review

43 (eds. Gibson, R. N., Atkinson, R. J. A., Gordon, J. D. M), 1-46. Taylor and

Francis.

Lipp, J. S., Morono, Y, Inagaki, F., Hinrichs, K. -U., 2008. Significant contribution of

Archaea to the extant biomass in marine subsurface sediments. Nature 454, 991-

994.

Lösekann, T., Knittel, K., Nadalig, T., Fuchs, B., Niemann, H., Boetius, A., Amann, R.,

2007. Diversity and abundance of aerobic and anaerobic methane oxidizers at the

Haakon Mosby Mud Volcano, Barents Sea. Applied and Environmental

Microbiology 73, 3348–3362.

Lutz, R. A., Kennis, M. J., Pooley, A. S., Fritz, L. W., 1994. Calcium carbonate

dissolution rates in Hydrothermal vent fields of the Guaymas Basin. Journal of

Marine Research 52, 969-982.

Madigan, M. T., Martinko, J. M., Parker, J., 2003. Brock - Biology of Microorganisms.

10th Edition, Pearson education Inc. Upper Saddle River, New Jersey.

Mallet, C. R., Lu, Z., Mazzeo, J. R., 2004. A study of ion suppression effects in

electrospray ionization from mobile phase additives and solid phase extracts. Rapid

Communications in Mass Spectrometry 18, 49-58.

Martens, C. S., Berner, R. A., 1974. Methane production in the interstitial waters of

sulfate-depleted marine sediments. Science 185, 1167-1169.

Martens, C. S., Berner, R. A., 1977. Interstitial water chemistry of Long Island Sound

sediments, 1: Dissolved gases. Limnology and Oceanography 22, 10-25.

Michaelis, W., Seifert, R., Nauhaus, K., Treude, T., Thiel, V., Blumenberg, M., Knittel,

K., Gieseke A., Peterknecht F., Pape T., Boetius A., Amann R., Jørgensen B. B.,

Widdel, F., Peckmann, J., Pimenkov, N., Gulin, M. B., 2002. Microbial reefs in the

Black Sea fueled by anaerobic methane oxidation. Science 297, 1013-1015.

Chapter I ________________________________________________________________________

39

Milkov, A. V., Sassen, R., Apanasovich, T. V., Dadashev, F. G., 2003. Global gas flux

from mud volcanoes: A significant source of fossil methane in the atmosphere and

the ocean. Geophysical Research Letters 30, doi:10.1029/2002GL016358.

Nauhaus, K., Boetius, A., Krüger, M., Widdel, F., 2002. In vitro demonstration of

anaerobic oxidation of methane coupled to sulfate reduction from a marine gas

hydrate area. Environmental Microbiology 4, 296-305.

Nauhaus, K., Treude, T., Boetius, A., Krüger, M., 2005. Environmental regulation of the

anaerobic oxidation of methane a comparison of ANME-1 and ANME-II

communities. Environmental Microbiology 7, 98-106.

Niemann, H., Lösekann T., de Beer, D., Elvert, M., Nadalig, T., Knittel, K., Amann, R.,

Sauter, E., Schlüter, M., Klages, M., Foucher, J. -P., Boetius, A., 2006. Novel

microbial communities of the Haakon Mosby mud volcano and their role as a

methane sink. Nature 443, 854-858.

Omoregie, E. O., Mastalerz, V., de Lange, G., Straub, K. L., Kappler, A., Røy, H.,

Stadniskaia, A., Faucher, J. -P., Boetius, A., 2008. Biogeochemistry and community

composition of iron-and sulfur-precipitating microbial mats at the Chefren Mud

Volcano (Nile deep sea fan, Eastern Mediterranean). Applied and Environmental

Microbiology 74, 3198-3215.

Orcutt, B. N., Boetius, A., Elvert, M., Samarkin, V. A., Joye, S. B., 2005. Molecular

biogeochemistry of sulfate reduction, methanogenesis and the anaerobic oxidation

of methane at the Gulf of Mexico cold seeps. Geochimica et Cosmochimica Acta

69, 4267-4281.

Orphan, V. J., House, C. H., Hinrichs, K. -U., McKeegan, K. D., DeLong, E. F., 2001.

Methane-consuming archaea revealed by directly coupled isotopic and phylogenetic

analysis. Science 293, 484-487.

Orphan, V. J., House, C. H., Hinrichs, K. -U., McKeegan, K. D., DeLong, E. F., 2002.

Multiple archaeal groups mediate methane oxidation in anoxic cold seep sediments.

Proceedings of the National Academy of Science U.S.A. 99, 7663-7668.

Pancost, R. D, Sinninghe Damsté, J. S, Lint, S. D., van der Maarel MJEC, Gottschal J. C.,

Shipboard Scientific Party., 2000. Biomarker evidence for widespread anaerobic

Chapter I ________________________________________________________________________

40

methane oxidation in Mediterranean sediments by a consortium of methanogenic

archaea and bacteria. Applied Environmental Microbiology 66, 1126-1132.

Pancost, R. D., Bouloubassi, I., Aloisi, G., Sinninghe Damste, J. S., Party M. S. S., 2001.

Three series of non-isoprenoidal dialkyl glycerol diethers in cold-seep carbonate

crusts. Organic Geochemistry 32, 695-707.

Paull, C. K., Hecker, B., Commeau, R., Freman-Lynde, R. P., Neumann, C., Corso, W.

P., Golubic, S., Hook, J. E., Sikes, E., Curray, J., 1984. Biological communities at

the Florida escarpment resemble hydrothermal vent taxa. Science 206, 965-967.

Paull, C. K., Ussler III, W., Browski, W. S., Spiess, F. N., 1995. Methane- Rich plumes

on the Carolina continental rise: associations with gas hydrates. Geology 23, 89-92.

Peckmann, J., Thiel, V., Michaelis, W., Clari, P., Gaillard, C., Martire, L., Reitner, J.,

1999. Cold seep deposits of Beauvoisin (Oxfordian; southeastern France) and

Marmorito (Miocene; northern Italy): microbially induced authigenic carbonates.

International Journal of Earth Sciences 88, 60-75.

Petit, J. R., Jousel, J., Raynaud, D., Barkov, N. I., Barnola, M. -J., Basile, I., Bender, M.,

Chappellaz, J., Davis, M., Delaygue, G., Delmotte, M., Kotlyakov, V. M., Legrand,

M., Lipenkov, V. Y., Lorius, C., Pépin, L., Ritz, C., Saltzman, E., Stievenard, M.,

1999. Climate and atmospheric history of the the past 420,000 years from the

Vostok ice core, Antarctica. Nature 399, 429-436.

Reeburgh, W. S., 1976. Methane consumption in Cariaco trench waters and sediments.

Earth Planetary Science Letters 28, 337-341.

Reeburgh, W. S., 2007. Oceanic methane biogeochemistry. Chemistry Reviews 107, 486-

513.

Reeburgh, W. S., Ward B. B., Whalen S. C., Sandbeck K. A., Kilpatrick K. A., Kerkhof

L. J., 1991. Black Sea methane geochemistry. Deep-Sea Research 38 (suppl. 2),

S1189-S1210.

Rehder, G., Suess, E., 2004. Marine gas hydrates. IFM-geomar report 2002-2004, 35-38.

Reysenbach, A. -L., Longnecker, K., Kirshtein, J., 2000. Novel bacterial and archaeal

lineages from an in situ growth chamber deployed at a Mid-Atlantic Ridge

hydrothermal vent. Applied and Environmental Microbiology 66, 3798-3806.

Chapter I ________________________________________________________________________

41

Ritger, S., Carson, B., Suess, E., 1987. Methane-derived authigenic carbonates formed by

subduction-induced pore-water expulsion along Oregon/Washington margin.

Geological Society of America Bulletin 98, 147-156.

Roberts, H. H., McBride, R. A., Coleman, J., 1999. Outer shelf and slope geology of the

Gulf of Mexico: an overview In: The Gulf of Mexico Large Marine Ecosystems:

Assesment Sustainability, and Management (eds. Kumpf, H., Steidinger, K.,

Sherman, K.), 93-112. Black Science Ltd.

Rothschild, L. J., Mancinelli, R. L., 2001. Life in extreme environments. Nature 409,

1092-1101.

Rütters, H., Sass, H., Cypionka, H., Rullkötter, J., 2002. Phospholipid analysis as a tool

to study complex microbial communities in marine sediments. Journal of

Microbiological Methods 48, 149-160.

Sahling, H., Rickert, D., Lee, R. W., Linke, P., Suess, E., 2002. Macrofaunal community

structure and sulfide flux at gas hydrate deposits from the Cascadia convergent

margin, NE Pacific. Marine Ecological Progress Series 231, 121-138.

Sassen, R., MacDonald, I. R., Requejo, A. G., Guinasso, N. L., Kennicutt, M. C., Sweet,

S. T., Brooks, J. M., 1994. Organic geochemistry of sediments from chemosythetic

communities, Gulf of Mexico slope. Geo-Marine Letters 14, 110-119.

Schönheit, P., Keweloh, H., Thauer, R. K., 1981. Factor F420 degradation in

Methanobacterium thermoautotrophicum during exposure to oxygen. Federation of

European Microbiological Societies Microbiology Letters 12, 347-349.

Schouten, S., Wakeham, S. G., Hopmans, E. C., Sinninghe Damsté J. S., 2003.

Biogeochemical evidence that thermophilic archaea mediate the anaerobic

oxidation of methane. Applied and Environmental Microbiology 69, 1680-1686.

Sturt, H. F., Summons, R. E., Smith, K. J., Elvert, M., Hinrichs, K. -U., 2004. Intact polar

membrane lipids in prokaryotes and sediments deciphered by high-performance

liquid chromatography/electrospray ionization multistage mass spectrometry-new

biomarkers for biogeochemistry and microbial ecology. Rapid Communications in

Mass Spectrometry 18, 617-628.

Chapter I ________________________________________________________________________

42

Suess, E., Bohrmann, G., Huene, R., Linke, P., Wallmann, K., Lammers, S., Sahling, H.,

1998. Fluid venting in the eastern Aleutian subduction zone. Journal of

Geophysical research 103, 2597-2614.

Suess, E., Carson B., Ritger S. D., Moore, J. C., Jones, M. L., Kulm, L. D., Cochrane, G.

R., 1985. Biological communities at vent sites along the subduction zone off

Oregon. Biological Society of Washington Bulletin 6, 475-484.

Suess, E., Torres, M. E., Bohrmann, G., Collier, R. W., Greinert, J., Linke, P., Rehder,

G., Trehu, A., Wallmann, K., Winckler, G., Zuleger, E., 1999. Gas hydrate

destabilization: enhanced dewatering, benthic material turnover and large methane

plumes at the Cascadia convergent margin. Earth Planet Science Letters 170:1-15.

Taylor, J., Parkes, R. J., 1983. The cellular fatty acids of the sulphate-reducing bacteria,

Desulfobacter sp., Desulfobulbus sp. and Desulfovibrio desulfuricans. Journal of

General Microbiology 129, 3303-3309.

Teske, A., Hinrichs, K. -U., Edgecomb, V., de Vera Gomez, A., Kysela, D., Sylva, S. P.,

Sogin, M. L., Jannasch, H. W., 2002. Microbial diversity of hydrothermal

sediments in the Guaymas Basin: Evidence for anaerobic methanotrophic

communities. Applied and Environmental Microbiology 68, 1994-2007.

Thiel, V., Peckmann, J., Richnow, H. H., Luth, U., Reitner, J., Michaelis, W., 2001.

Molecular signals for anaerobic methane oxidation in Black Sea seep carbonates

and a microbial mat. Marine Chemistry 73,97-112.

Thiel, V., Peckmann, J., Seifert, R., Wehrung, P., Reitner, J., Michaelis, W., 1999. Highly

isotopically depleted isoprenoids: Molecular markers for ancient methane venting.

Geochemica et Cosmochemica Acta 63, 3959-3966.

Tissot, B. P., Welte, D. H., 1984. Petroleum formation and occurrence. pp. 527, Springer-

Verlag, Heidelberg, 1984.

Torres, M., McManus, J., Hammond, D. E., de Angelis, M. A., Heeschen, K., Colbert, S.

L., Tyron, M. D., Brown, K. M., Suess, E., 2002. Fluid and chemical fluxes in and

out of sediments hosting methane hydrate deposits on Hydrate Ridge, OR, I:

hydrological provinces. Earth and Planetary Science Letters 201, 525-540.

Tortora, G. J., Funke, B. R., Case, C. L., 2004. Microbiology: An introduction. 8th

Edition, Pearson education Inc. Benjamin/Cumminmgs, San Francisco, USA.

Chapter I ________________________________________________________________________

43

Treude, T., Boetius, A., Knittel, K., Wallmann, K., Jørgensen B. B., 2003. Anaerobic

oxidation of methane above gas hydrates at Hydrate Ridge, NE Pacific Ocean.

Marine Ecology Progress Series 264: 1-14.

Treude, T., Knittel, K., Blumenberg, M., Seifert, R., Boetius, A., 2005. Subsurface

microbial methanotrophic mats in the Black Sea. Applied and environmental

microbiology 71, 6375-6378.

Valentine, D. L., 2007. Adaptations to energy stress dictate the ecology and evolution of

the Archaea. Nature Reviews Microbiology 4, 316-323.

Van Mooy, B. A. S., Rocap, G., Fredricks, H. F., Evans, C. T., Devol, A. H., 2006.

Sulfolipids dramatically decrease phosphorus demand by picocyanobacteria in

oligotrophic marine environments. Proceedings of the National Academy of Science

U.S.A. 103, 8607-8612.

Von Damm, K. L., 1990. Seafloor hydrothermal activity: black smoker chemistry and

chimneys. Annual Reviews Earth Planetary Science 49, 2197-2220.

Von Damm, K. L., Edmond, J. M., Grant, B., Measures, C. I., 1985. Chemistry of

submarine hydrothermal solutions at 21°N, East Pacific Rise. Geochimica et

Cosmochimica Acta 49, 2197-2220.

Wakeham, S. G., Lewis, C. M., Hopmans, E. C., Schouten, S., Sinninghe Damsté J. S.,

2003. Archaea mediate anaerobic oxidation of methane in deep euxinic waters of

the Black Sea. Geochemica et Cosmochimica Acta 67, 1359-1374.

Ward, B. B., Kilpatrick, K.A., Novelli, P. C., Scranton, M. I., 1987. Methane oxidation

and methane fluxes in the ocean surface layer and deep anoxic waters. Nature 327,

226-229.

Wegener, G., Shovitri, M., Knittel, K., Niemann, H., Hovland, M., Boetius, A., 2008.

Biogeochemical processes and microbial diversity of the Gullfaks and Tommeliten

methane seeps (Northern North Sea). Biogeosciences 5, 1127-1144.

White, D. C., Davis, W. M., Nickels, J. S., Kind, J. D., Bobbie, R. J., 1979. Oecologica

40, 51-62.

Whiticar, M. J., 1999. Carbon and hydrogen isotope systematics of bacterial formation

and oxidation of methane. Chemical Geology 161, 291-314.

Chapter I ________________________________________________________________________

44

Wuebbles, D. J., Hayhoe, K., 2002. Atmospheric methane and global change. Earth

Science Reviews 57, 177-20.

Yamamoto, S., Alcauskas, J. B., Crozier, T. E., 1976. Solubility of methane in distilled

water and seawater. Journal of Chemical Engineering Data 21, 78-81.

Yun, J. W., Orange, D. L., Field, M. E., 1999. Subsurface gas offshore of Northern

Califorornia and its link to submarine geomorphology. Marine Geology 154, 357-

368.

Ziebis, W., Haese, R. R., 2005. Interactions between fluid flow, geochemistry, and

biogeochemical processes at methane seeps. In: Macro- and Microorganisms in

Marine Sediments (eds. Kristensen E., Kostka J., Haese R. R.), 267-298, AGU

Coastal and Estuarine Studies.

Chapter II ________________________________________________________________________

45

CHAPTER II

Intact polar lipids of anaerobic methanotrophic archaea

and associated bacteria

Pamela E. Rossela, Julius S. Lippa, Helen F. Fredricksb, Julia Arndsc, Antje Boetiusc,

Marcus Elverta, Kai-Uwe Hinrichsa

Published in Organic Geochemistry.

vol. 39, page 992-999, doi:10.1016/j.orggeoche.2008.02.021

aOrganic Geochemistry Group, Department of Geosciences, University of Bremen, 28334 Bremen,

Germany bWoods Hole Oceanographic Institution, Department of Marine Chemistry and Geochemistry, Woods Hole,

MA 02543, USA cMax-Planck-Institute for Marine Microbiology, 28359 Bremen, Germany

Chapter II ________________________________________________________________________

46

II.1. PRINTED MANUSCRIPT

ABSTRACT

Previous biomarker studies of microbes involved in anaerobic oxidation of

methane (AOM) have targeted non-polar lipids. We have extended the biomarker

approach to include intact polar lipids (IPLs) and show here that the major community

types involved in AOM at marine methane seeps can be clearly distinguished by these

compounds. The lipid profile of methanotrophic communities with dominant ANME-1

archaea mainly comprises diglycosidic GDGT derivatives. IPL distributions of microbial

communities dominated by ANME-2 or ANME-3 are consistent with their phylogenetic

affiliation with the euryarchaeal order Methanosarcinales, i.e., the lipids are dominated by

phosphate-based polar derivatives of archaeol and hydroxyarchaeol. IPLs of associated

bacteria strongly differed among the three community types analyzed here; these

differences testify to the diversity of bacteria in AOM environments. Generally, the

bacterial members of methanotrophic communities are dominated by

phosphatidylethanolamine and phosphatidyl-(N,N)-dimethylethanolamine species; polar

dialkylglycerolethers are dominant in the ANME-1 community while in ANME-2 and

ANME-3 communities mixed acyl/ether glycerol derivatives are most abundant. The

relative concentration of bacterial lipids associated with ANME-1 dominated

communities appears significantly lower than in ANME-2 and ANME-3 dominated

communities. Our results demonstrate that IPL analysis provides valuable molecular

fingerprints of biomass composition in natural microbial communities and enables

taxonomic differentiation at the rank of families to orders.

Abbreviations: ANME, anaerobic methanotrophic archaea; AR, archaeol; AOM, anaerobic oxidation of

methane; CARD–FISH, catalyzed reporter deposition–fluorescence in situ hybridization; GDGT,

glyceroldialkylglyceroltetraether; IPL, intact polar lipid; SRB, sulfate-reducing bacteria; OH-AR,

hydroxyarchaeol; PC, phosphatidylcholine; PDME, phosphatidyl-(N,N)-dimethylethanolamine; PE,

phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PME, phosphatidyl-(N)-

methylethanolamine; PS, phosphatidylserine; 2-Gly, diglycosyl; 2OH-AR, dihydroxyarchaeol.

Chapter II ________________________________________________________________________

47

INTRODUCTION

Anaerobic oxidation of methane (AOM) in the marine environment is mediated

by three phylogenetically distinct clusters of Euryarchaeota called ANME-1, -2 or -3 (cf.

Hinrichs et al., 1999; Boetius et al., 2000; Hinrichs and Boetius, 2002; Niemann et al.,

2006) that form consortia with sulfate-reducing bacteria (SRB) (Boetius et al., 2000;

Orphan et al., 2001a, 2002; Lösekann et al., 2007). ANME-2 are phylogenetically

affiliated with the order Methanosarcinales and are typically observed in physical

association with SRB of the Desulfosarcina/Desulfococcus group (Boetius et al., 2000;

Orphan et al., 2001b, ‘‘ANME-2/DSS aggregates”). ANME-3 are closely related to the

genera Methanococcoides and Methanolobus and have been found in association with

SRB related to Desulfobulbus spp. (Lösekann et al., 2007, ‘‘ANME-3/DBB aggregates”).

ANME-1 are not directly affiliated with any of the major orders of methanogens

(Hinrichs et al., 1999; Orphan et al., 2001b; Knittel et al., 2005). These archaea have been

observed in physical association with SRB of the Desulfosarcina/Desulfococcus group in

microbial mats (Michaelis et al., 2002) but also frequently as monospecific aggregates or

as single cells without a clear bacterial partner (Orphan et al., 2002).

Previous biomarker studies of AOM communities have focused on non-polar

lipids, such as hydrocarbons of archaeal origin, bacterial fatty acids, and archaeal and

bacterial glycerol-based ether lipids (e.g., Elvert et al., 1999, 2005; Hinrichs et al., 1999,

2000; Pancost et al., 2000; Blumenberg et al., 2004; Niemann et al., 2006). However, an

interpretation of the lipid profiles with regard to the distribution and composition of

active methanotrophic communities is limited by their relatively low taxonomic

specificity and the likelihood of incorporating signals from the past. The latter point is

particularly crucial due to the temporally highly dynamic physical–chemical conditions

encountered in many of the intensely studied AOM environments. By contrast, intact

polar lipids (IPLs) offer a more detailed view of microbial communities due to their

higher taxonomic specificity and property to select for live biomass (Rütters et al., 2002;

Sturt et al., 2004; Biddle et al., 2006).

Here we report the composition of IPLs in environmental samples dominated by

either one of the three major ANME groups and associated bacteria. We show that IPL

Chapter II ________________________________________________________________________

48

profiles can serve as valuable community fingerprints and relative indicators of biomass

of ANME archaea and associated bacteria in natural systems.

MATERIAL AND METHODS

IPL analysis

Samples from four different seep environments were analyzed, each dominated by

one distinct ANME group (Table II.1 and Fig. II.1): two microbial mats from the

northwestern Black Sea, one sediment sample from Hydrate Ridge and one sediment

sample from Häkon Mosby Mud Volcano. Both surface sediment samples from Hydrate

Ridge and Häkon Mosby Mud Volcano were covered by Beggiatoa mats.

IPL analysis was performed with a HPLC–ESI–MSn system using protocols

described previously by Sturt et al. (2004) and Biddle et al. (2006). Total lipid extracts

from microbial mats from the Black Sea and the sediment from Häkon Mosby Mud

Volcano were obtained with an automated microwave-assisted extraction system

(MARS-X, CEM, USA) at a temperature of 70°C, while the sediment from Hydrate

Ridge was extracted via ultrasonication. The latter sample was analyzed after

chromatographic separation as glyco- and phospholipids fraction (Sturt et al., 2004),

while the former samples were analyzed as total lipid extracts. Structural assignments

were based on mass spectral interpretation (cf. Sturt et al., 2004) and by comparison with

IPL inventories of cultured archaea and bacteria (e.g., Koga et al., 1998; Koga and Morii,

2005; Hinrichs et al., unpublished data). Chain length assignment, degree of unsaturation,

and determination of ether and ester bond linkages of bacterial IPLs were based on

molecular masses and fragments according to Sturt et al. (2004). Due to the limited

availability of commercial standards, we did not use response factors for IPL

quantification. Based on calibration curves we observed response factors for various

commercially available IPLs that can differ up to a factor of three. Thus, reported relative

distributions are semi-quantitative. Only compounds with a signal-to-noise ratio higher

than 6 were reported. The least concentrated reported compounds amounted to 0.12%,

0.06%, 5.0% and 5.2% of the total quantified IPLs in samples from Black Sea (two

samples), Hydrate Ridge and Häkon Mosby Mud Volcano, respectively. Composite

Chapter II ________________________________________________________________________

49

chromatograms of extracted quasi-molecular ions of individual IPLs were obtained from

the full scan (m/z 500–2000) from each sample (Fig. 1). Structural details of hexoses

linked via glycosidic bonds to archaeal ether lipids are not resolved; hence hexoses are

designated as glycolipids.

Catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH)

analyses

Microbial mats were fixed as described previously (Treude et al., 2007) and

homogenized. In situ hybridizations with horseradish peroxidase (HRP)-conjugated

probes followed by tyramide signal amplification were carried out as described by

Pernthaler et al. (2002) with slight modifications: endogenous peroxidases were inhibited

with methanol (30 min) and rigid archaeal cell walls were permeabilized with proteinase

K(15 μg ml-1, for 2 min at room temperature). Total cell counts were determined by 4’,6’-

diamidino-2-phenylindole (DAPI)-staining.

Hybridized and DAPI-stained samples were examined with an epifluorescence

microscope (Axiophot II microscope; Carl Zeiss, Jena, Germany). For each probe and

sample 700 DAPI-stained cells in 70 independent microscopic fields were counted. Probe

sequences and formamide concentrations required for specific hybridization were:

ARCH915 (35% formamide), EUB338 I–III (35% formamide), ANME-1–350 and

EelMS932 (40 and 60% formamide) (Amann et al., 1990; Daims et al., 1999; Boetius et

al., 2000).

Cha

pter

II

____

____

____

____

____

____

____

____

____

____

____

____

____

____

____

____

____

____

____

____

____

____

____

____

____

____

____

50

Tab

le II

.1:

Ove

rvie

w o

f AO

M sa

mpl

es a

naly

zed,

rela

tive

dist

ribut

ion

of b

oth

arch

aeal

and

bac

teria

l cel

ls a

nd IP

Ls, a

nd c

ore

lipid

dis

tribu

tion

of d

igly

cosy

l-G

DG

Ts.

Loca

tion

Sam

ple

Archaea

vs.

Bacteria

(%

of t

otal

cel

ls) a

AN

ME-

1/-2

/-3

(% o

f tot

al

arch

aeal

cel

ls)

Sour

ce

Arc

haea

l gly

co-

and

phos

phol

ipid

s

(% o

f tot

al a

rcha

eal I

PLs)

Bac

teria

l ph

osph

olip

ids

(% o

f to

tal b

acte

rial I

PLs)

b

Cor

e lip

id ri

ng

dist

ribut

ion

of

2-G

ly-G

DG

Ts

Nor

thw

este

rn

Bla

ck S

ea,

Dni

epr a

rea

Mat

795

, pin

k

(189

m w

ater

de

pth)

33

/16

100/

0/0

this

stu

dy

2-G

ly-G

DG

T (>

99),

2-G

ly-A

R (<

1)

DE

G-P

E C

30:0

(31)

, D

AG

/AE

G-P

E C

33:2

(19)

, D

EG

-PE

C31

:1 (1

8),

DA

G/A

EG

-PE

C35

:2 (1

1),

DA

G/A

EG

-PE

C32

:2 (1

1),

othe

r PE

s (1

0)

3>2>

>1>>

0>5

Nor

thw

este

rn

Bla

ck S

ea,

Dni

epr a

rea

Mat

822

, ree

f to

p (1

90 m

w

ater

dep

th)

31/4

3 35

/65/

0 th

is s

tudy

PG

-AR

(35)

, 2-G

ly-G

DG

T (3

3), t

enta

tive

phos

pho-

AR

(1

9), 2

-Gly

-AR

(5),

PS

-OH

-A

R (2

), 2-

Gly

-OH

-AR

(1),

PE

-OH

-AR

(1)

DA

G/A

EG

-PE

C31

:2 (3

2),

DE

G-P

E C

32:1

(11)

, D

AG

/AE

G-P

E C

31:1

(10)

, D

AG

/AE

G-P

E C

32:2

(9),

DE

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2)

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0 c

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rate

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men

t (S

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31/6

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0 K

nitte

l et

al. (

2005

)

PG

-OH

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(23)

, PE

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R (1

5), P

I-OH

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(13)

, P

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(12)

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(44)

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007)

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(53)

, PS

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sum

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ecte

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D N

o G

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Ts w

ith 0

and

5 ri

ngs d

etec

ted.

Chapter II ________________________________________________________________________

51

RESULTS AND DISCUSSION

Fig. II.1. Composite mass chromatograms of molecular ions of IPLs in samples dominated by ANME-1 (A), a mixed ANME-1/ANME-2 community (B), both from microbial mats collected in the Black Sea (BS), and sediments dominated by ANME-2 (C) and ANME-3 (D) from Hydrate Ridge (HR) and Ha�kon Mosby Mud Volcano (HMMV), respectively. Extracted m/z of quasi-molecular ions for archaeal IPLs are 1632-1645, 994, 807, 956, 823, 792, 820, 836, 852, 911 for the identified 2-Gly-GDGT, 2-Gly-AR, PG-AR, tentative P-AR, PGOH-AR, PE-OH-AR, PS-AR, PS-OH-AR, PS-2OH-AR, PI-OH-AR, respectively. The major bacterial IPLs are represented by the following quasi-molecular ions: m/z 674 and 688 for DAG/AEG-PE, 662 and 660 for DEG-PE, 764 and 736 for DAG/AEG-PG, 760 for DAG/AEG PC, 716 for DAG/AEG-PDME and 702 for PME.

IPLs of ANME-1

In the microbial mat from the trunk of a microbial reef in the Black Sea, all

archaeal cells were affiliated with ANME-1 (Table II.1). In this sample, diglycosyl

glyceroldialkylglyceroltetraethers (2-Gly-GDGTs) were the most abundant IPLs (Fig.

II.1A). Only small amounts of 2-Gly-archaeol (2-Gly-AR) were detected (Fig. II.2A).

The main GDGT core lipids in 2-Gly-GDGT were the di- and tri-cyclopentyl derivatives

Chapter II ________________________________________________________________________

52

(Table II.1). No polar derivative of hydroxyarchaeol (OH-AR) was detected. In terms of

the IPL composition, ANME-1 are distinct from other methanogens (e.g., Koga and

Morii, 2005), i.e., all major families of methanogens produce significant amounts of AR

and multiple types of phosphate-based IPLs. In fact, ANME-1 is most similar to members

of the hyperthermophilic Archaeoglobales that largely produce 2-Gly-GDGT, combined

with lower amounts of both 1-Gly-GDGT and small quantities of Gly-AR (Hinrichs et

al., unpublished data). Our results are consistent with evidence provided by Thiel et al.

(2007) who applied molecular imaging techniques based on ToF-SIMS to mat sections

obtained from the same reef system and dominated by cells of the ANME-1 morphology;

these sections consisted mainly of free GDGTs and 2-Gly-GDGTs.

The absence or extremely low relative abundance of OH-AR in ANME-1 archaea

was not apparent in earlier studies that focused on its non-polar derivatives (Hinrichs et

al., 1999; Blumenberg et al., 2004). However, in the ANME-1 dominated mat, the

concentration of non-polar OH-AR of 15 μg/g mat was very low compared to the mat

dominated by ANME-2 (436 μg/g mat). Low ratios of OH-AR/AR have been used as

indicator signatures of active ANME-1 (Blumenberg et al., 2004; Niemann and Elvert, in

press), but probably have to be interpreted with caution. We suggest that in ANME-1-

dominated environments lacking polar OH-AR, non-polar OH-AR is a relict from the

past, when environmental conditions selected for ANME-2.

IPLs of ANME-2

The mat from the top part of a reef structures in the Black Sea was characterized

by a mixture of ANME-1 and ANME-2 (Table II.1, 35% and 65% of total archaeal cells,

respectively), while the surface sediment sample from Hydrate Ridge was dominated by

ANME-2 (Table II.1, 97% of total archaeal cells, Knittel et al., 2005). Archaeal IPLs of

ANME-2 were largely based on AR and OH-AR with either glycosidic or phosphate-

based headgroups. Specifically, these included 2-Gly-AR, 2-Gly-OH-AR,

phosphatidylglycerol- (PG-) OH-AR, PG-AR, phosphatidylinositol- (PI-) AR, PI-OH-

AR, phosphatidylserine- (PS-) AR, PS-OH-AR, PS-2-OH-AR and a tentatively identified

AR with a phosphate-based headgroup of unknown structure (Fig. II.1B and C). This

phospho-AR, present in both samples containing ANME-2, but not in the ANME-1 and

Chapter II ________________________________________________________________________

53

ANME-3 dominated communities, was tentatively assigned based on information

obtained in negative ionization mode, which yielded an intense fragment of 433.5 Da

(interpreted as dehydrated lyso fragment with one phytanyl chain and without head

group). The unknown compound is formed by two masses 956.0 and 939.3 that probably

correspond to the ammonium adduct and the protonated lipid, respectively. None of the

corresponding ions yielded intense, interpretable fragments during MS2 experiments in

positive ionization mode. 2-Gly-GDGT was also detected in the two ANME-2 dominated

communities from Black Sea and Hydrate Ridge; relative concentrations of this

compound are consistent with the relative amounts of ANME-1 cells in these samples

(Table II.1 and Fig. II.1).

IPLs of ANME-3

The sediments from Häkon Mosby Mud Volcano were dominated by ANME-3

(Table II.1, 99% total archaeal cells, Lösekann et al., 2007). The sample contained the

most diverse distribution of archaeal and bacterial IPLs (Fig. II.1D). The main archaeal

IPLs were various phospholipids of AR and OH-AR, similar to those observed in the

ANME-2 system of Hydrate Ridge. In contrast to the ANME-1 and ANME-2 dominated

communities, neither GDGT-based IPLs nor glycosidic archaeol derivatives were

present. Likewise, the tentatively identified phospho-AR from the ANME-2 community

(Fig. II.1B and C) was not detected.

Bacterial IPLs

Compositional differences of bacterial IPLs reflect differences in the phylogenetic

affiliation of the bacterial members of AOM communities such as SRB (Fig. II.1A–D).

Bacterial IPLs vary in both structural diversity and relative abundance; ANME-3 and

ANME-2 dominated samples displayed both the highest abundance and highest diversity

of bacterial IPLs (Table II.1 and Fig. II.2). The Black Sea ANME-1 system was

dominated by phosphatidylethanolamine (PE) derivatives of dietherglycerol (DEG) lipid

types (Table II.1), while the ANME-2 systems contained mainly PE of mixed acyl/ether

glycerol (AEG) lipids or diacyl glycerol (DAG) lipids, although the corresponding DEG

types were also present. The high relative amounts of DAG/AEG lipids in combination

Chapter II ________________________________________________________________________

54

with PE and PG is consistent with the IPL composition of Desulfosarcina variabilis

(Rütters et al., 2001), a close relative of the sulfate reducers in ANME-2 communities,

although the chain length distribution and degree of unsaturation differ (Table II.1; cf.

Rütters et al., 2001; Sturt et al., 2004). In ANME-2 dominated communities, we also

observed phosphatidylcholine (PC) and PG (the latter observed in the Hydrate Ridge

sample only). With respect to the bacterial IPLs, the ANME-3 community is

distinguished from the ANME-2 community by a higher abundance of phosphatidyl-(N)-

methylethanolamine (PME) and phosphatidyl-(N,N)-dimethylethanolamine (PDME).

Fig. II.2. Compositional variation of IPL groups in the four AOM communities. (A) Distribution of archaeal IPLs (Gly-GDGT, Gly-AR and Gly-OH-AR, P-AR and P-OH-AR [P = phospho]), (B) relative amounts of archaeal and bacterial IPLs.

In all samples, PE was a major bacterial IPL and contributes between ~1 and 15%

to total IPLs in Black Sea mat samples, and between 15% and 40% in the sediments from

Häkon Mosby Mud Volcano and Hydrate Ridge, respectively (cf. Sturt et al., 2004, for

detailed discussion of bacterial IPLs in Hydrate Ridge sample). The total number of

carbon atoms in glycerol-bound acyl and/or alkyl moieties further distinguished the three

ANME community types. In the ANME-1 dominated sample, the dominant bacterial IPL

Chapter II ________________________________________________________________________

55

was a C30:0 DEGPE; in samples from Hydrate Ridge and Häkon Mosby Mud Volcano,

C32:2 DAG/AEG-PE and C32:2 DAG/AEG-PDME, respectively, were more abundant

(Table II.1).

Lipid taxonomy of uncultured AOM archaea

The presence of AR and OH-AR based core lipids in ANME-2 and ANME-3

archaea is consistent with their affiliation with the methanogenic orders Methanococcales

and Methanosarcinales (cf. Kates, 1997). When considering the presence and/or absence

of polar headgroups in ANME-2 and ANME-3 communities, the taxonomic relationship

is narrowed down to the Methanosarcinales: PI and PG derivatives are abundant in the

Methanosarcinales and but are absent in the Methanococcales (Koga and Morii, 2005).

Notably, no clear chemotaxonomic relationship exists between the phylogenetically

distinctive ANME-1 and any of the cultured methanogens (cf. Koga et al., 1998; Koga

and Morii, 2005). Closest relatives of ANME-1 in terms of IPL composition are the

Archaeoglobales (Hinrichs et al., unpublished data). Environmental IPL fingerprints that

resemble those from ANME-1 are those related to uncultured marine sedimentary

archaea (Biddle et al., 2006).

Archaeal vs. bacterial biomass

Relative amounts of archaeal vs. bacterial IPLs strongly varied in the samples

dominated by a single ANME type (percentages of archaeal IPLs are ~99%, 85%, 31%

and 52% for samples from the Black Sea, Hydrate Ridge, and Häkon Mosby Mud

Volcano, respectively; Fig. II.2B). These pronounced differences probably reflect

similarly large differences in archaeal vs. bacterial biomass among active AOM

community members, which in turn probably relate to ecophysiological characteristics of

the three types of AOM communities sampled here. Notably, the IPLs partly provided a

different picture of the relative abundance of archaeal vs. bacterial biomass than cell

counts by CARD–FISH (Table II.1). For example, for the Black Sea ANME-1 sample,

IPL analysis suggests a lower bacterial contribution to the microbial community than

CARD–FISH, while in the Häkon Mosby Mud Volcano ANME-3 sample, CARD–FISH

detected more archaea than IPL analysis (Fig. II.2B and Table II.1). Possible causes

Chapter II ________________________________________________________________________

56

include varying cellular IPL contents, e.g., due to differences in cell size and

morphology, and/or differences in physiological status of the bulk community that in turn

may affect both cellular IPL abundance and the ability to bind to CARD–FISH probes.

CONCLUSIONS

This study provides an unprecedented view of the lipid diversity of the three

globally relevant anaerobic methanotrophic communities, that is, communities dominated

by ANME-1, ANME-2, ANME-3 populations, and associated bacteria. The diversity and

relative amounts of both archaeal and bacterial IPLs differ remarkably between the three

community types. While lipid analysis is unable to capture the entire microbial diversity,

our results demonstrate that quantitative differences in microbial community structure

can be effectively resolved. Specifically, IPL analysis enables the differentiation of the

major players in natural microbial communities at the rank of taxonomic orders or higher.

ACKNOWLEDGMENTS

We thank the crew and shipboard scientist of R/V SONNE SO 148-1, R/V

L’Atalante, and R/V Poseidon for support during sample collection. Tina Treude is

gratefully acknowledged for providing microbial mat samples from the Black Sea, Katrin

Knittel for helping with analysis of FISH data, and Philippe Schaeffer and Richard

Pancost for their constructive reviews. This study was part of the program MUMM II

(grant 03G0608C), funded by the Bundesministerium für Bildung und Forschung

(BMBF, Germany) and the Deutsche Forschungsgemeinschaft (DFG, Germany). Further

support was provided from the Center of Marine Environmental Sciences (MARUM) at

the University of Bremen funded by the DFG. This is publication GEOTECH-316 of the

R&D program GEOTECHNOLOGIEN and MARUM-publication 0573.

Chapter II ________________________________________________________________________

57

REFERENCES

Amann, R.I., Binder, B.J., Olson, R.J., Chisholm, S.W., Devereux, R., Stahl, D.A., 1990.

Combination of 16S rRNA targeted oligonucleotide probes with flow cytometry

for analyzing mixed microbial populations. Applied and Environmental

Microbiology 56, 1919–1925.

Biddle, J.F., Lipp, J.S., Lever, M.A., Lloyd, K.G., Sörensen, K.B., Anderson, R.,

Fredricks, H.F., Elvert, M., Kelly, T.J., Schrag, D.P., Sogin, M.L., Brenchley,

J.E., Teske, A., House, C.H., Hinrichs, K.-U., 2006. Heterotrophic archaea

dominate sedimentary subsurface ecosystems off Peru. Proceedings of the

National Academy of Sciences of the USA 103, 3846–3851.

Blumenberg, M., Seifert, R., Reitner, J., Pape, T., Michaelis, W., 2004. Membrane lipid

patterns typify distinct anaerobic methanotrophic consortia. Proceedings of the

National Academy of Sciences of the USA 101, 11111–11116.

Boetius, A., Ravenschlag, K., Schubert, C.J., Rickert, D., Widdel, F., Gieseke, A.,

Amann, R., Jørgensen, B.B., Witte, U., Pfannkuche, K., 2000. A marine microbial

consortium apparently mediating anaerobic oxidation of methane. Nature 407,

623–626.

Daims, H., Bruhl, A., Amann, R., Schleifer, K.H., Wagner, M., 1999. The domain-

specific probe EUB338 is insufficient for the detection of all Bacteria:

development and evaluation of a more comprehensive probe set. Systematic and

Applied Microbiology 22, 434–444.

Elvert, M., Suess, E., Whiticar, M.J., 1999. Anaerobic methane oxidation associated with

marine gas hydrates: superlight Cisotopes from saturated and unsaturated C20 and

C25 irregular isoprenoids. Naturwissenschaften 86, 295–300.

Elvert, M., Hopmans, E.C., Treude, T., Boetius, A., Suess, E., 2005. Spatial variations of

methanotrophic consortia at cold methane seeps: implications from a high-

resolution molecular and isotopic approach. Geobiology 3, 195–209.

Hinrichs, K.-U., Boetius, A., 2002. The anaerobic oxidation of methane: new insights in

microbial ecology and biogeochemistry. In: Ocean Margin Systems (eds. Wefer,

Chapter II ________________________________________________________________________

58

G., Billett, D., Hebbeln, D., Jørgensen, B.B., Schlüter, M., van Weering, T.C.E.,

57–477, Springer-Verlag

Hinrichs, K.-U., Hayes, J.M., Sylva, S.P., Brewer, P.G., Delong, E.F., 1999. Methane-

consuming archaebacteria in marine sediments. Nature 398, 802–805.

Hinrichs, K.-U., Summons, R.E., Orphan, V., Sylva, S.P., Hayes, J.M., 2000. Molecular

and isotopic analysis of anaerobic methane-oxidizing communities in marine

sediments. Organic Geochemistry 31, 1685–1701.

Kates, M., 1997. Diether and tetraether phospholipids and glycolipids as molecular

markers for archaeabacteria (archaea). In: Molecular Markers in Environmental

Geochemistry (ed. Eganhause, R.P.), 35–48, Oxford University Press.

Knittel, K., Lösekann, T., Boetius, A., Kort, R., Amann, R., 2005. Diversity and

distribution of methanotrophic archaea at cold seeps. Applied and Environmental

Microbiology 71, 467–479.

Koga, Y., Morii, H., 2005. Recent advances in structural research on ether lipids from

archaea including comparative and physiological aspects. Bioscience

Biotechnology and Biochemistry 69, 2019–2034.

Koga, Y., Morii, H., Akagawa-Matsushita, M., Ohga, I., 1998. Correlation of polar lipid

composition with 16S rRNA phylogeny in methanogens. Further analysis of lipid

component parts. Bioscience Biotechnology and Biochemistry 62, 230–236.

Lösekann, T., Knittel, K., Nadalig, T., Fuchs, B., Niemann, H., Boetius, A., Amann, R.,

2007. Diversity and abundance of aerobic and anaerobic methane oxidizers at the

Haakon Mosby Mud Volcano, Barents Sea. Applied and Environmental

Microbiology 73, 3348–3362.

Michaelis, W., Seifert, R., Nauhaus, K., Treude, T., Thiel, V., Blumenberg, M., Knittel,

K., Gieseke, A., Peterknecht, K., Pape, T., Boetius, A., Amann, R., Jorgensen,

B.B., Widdel, F., Peckmann, J.R., Pimenov, N.V., Gulin, M.B., 2002. Microbial

reefs in the Black Sea fueled by anaerobic oxidation of methane. Science 297,

1013–1015.

Niemann, H., Elvert, M., 2008. Diagnostic lipid biomarker and stable carbon isotope

signatures of microbial communities mediating the anaerobic oxidation of

methane with sulphate. Organic Geochemistry 39, 1668-1677.

Chapter II ________________________________________________________________________

59

Niemann, H., Lösekann, T., Beer, D., Elvert, M., Nadalig, T., Knittel, K., Amann, R.,

Sauter, E.J., Schlüter, M., Klages, M., Foucher, J.P., Boetius, A., 2006. Novel

microbial communities of the Häkon Mosby mud volcano and their role as a

methane sink. Nature 443, 854–858.

Orphan, V.J., Hinrichs, K.U., Ussler, W., Paull, C.K., Taylor, L.T., Sylva, S.P., Hayes,

J.M., Delong, E.F., 2001a. Comparative analysis of methane-oxidizing archaea

and sulfatereducing bacteria in anoxic marine sediments. Applied and

Environmental Microbiology 67, 1922–1934.

Orphan, V.J., House, C.H., Hinrichs, K.-U., McKeegan, K.D., Delong, E.F., 2001b.

Methane-consuming archaea revealed by directly coupled isotopic and

phylogenetic analysis. Science 293, 484–487.

Orphan, V., House, C.H., Hinrichs, K.-U., McKeegan, K.D., Delong, E.F., 2002.

Multiple archaeal groups mediate methane oxidation in anoxic cold seep

sediments. Proceedings of the National Academy of Sciences of the USA 99,

7663–7668.

Pancost, R.D., Sinninghe Damsté, J.S., de Lint, S., van der Maarel, M.J.E.C., Gottschal,

J.C.and the Medinaut Shipboard Scientific Party, 2000. Biomarker evidence for

widespread anaerobic methane oxidation on Mediterranean sediments by a

consortium of methanogenic archaea and bacteria. Applied and Environmental

Microbiology 66, 1126–1132.

Pernthaler, A., Pernthaler, J., Amann, R., 2002. Fluorescence in situ hybridization and

catalyzed reporter deposition for the identification of marine bacteria. Applied and

Environmental Microbiology 68, 3094–3101.

Rütters, H., Sass, H., Cypionka, H., Rullkötter, J., 2001. Monoalkylether phospholipids in

the sulfate-reducing bacteria Desulfosarcina variabilis and Desulforhabdus

amnigenus. Archives of Microbiology 176, 435–442.

Rütters, H., Sass, H., Cypionka, H., Rullkötter, J., 2002. Phospholipid analysis as a tool

to study complex microbial communities in marine sediments. Journal of

Microbiological Methods 48, 149–160.

Sturt, H.F., Summons, R.E., Smith, K., Elvert, M., Hinrichs, K-U., 2004. Intact polar

membrane lipids in prokaryotes and sediments deciphered by high-performance

Chapter II ________________________________________________________________________

60

liquid chromatography/electrospray ionization multistage mass spectrometry –

new biomarkers for biogeochemistry and microbial ecology. Rapid

Communications in Mass Spectrometry 18, 617–628.

Thiel, V., Heim, C., Arp, G., Hahmann, U., Sjövall, P., Lausmaa, J., 2007. Biomarkers at

the microscopic range: ToF-SIMS molecular imaging of Archaea-derived lipids in

a microbial mat. Geobiology 5, 413–421.

Treude, T., Orphan, V., Knittel, K., Gieseke, A., House, C.H., Boetius, A., 2007.

Consumption of methane and CO2 by methanotrophic microbial mats from gas

seeps of the anoxic Black Sea. Applied and Environmental Microbiology 73,

2271–2283.

Chapter II ________________________________________________________________________

61

II.2. SUPPLEMENTARY ONLINE MATERIAL

Supplementary Figure

Supplementary Fig. II.3. Structure of IPLs identified in this study

Chapter II ________________________________________________________________________

62

Chapter III ________________________________________________________________________

63

CHAPTER III

Factors controlling the distribution of anaerobic

methanotrophic communities in marine environments:

evidence from intact polar membrane lipids

Pamela E. Rossela, Marcus Elverta, Alban Rametteb,

Antje Boetiusb and Kai-Uwe Hinrichsa

Prepared for submission

aOrganic Geochemistry Group, Department of Geosciences, University of Bremen, 28334 Bremen,

Germany cMax-Planck-Institute for Marine Microbiology, 28359 Bremen, Germany

Chapter III ________________________________________________________________________

64

III.1. MANUSCRIPT

ABSTRACT

Three distinct types of anaerobic methanotrophic microbial consortia are globally

distributed in marine sediments. These communities are dominated by archaea of the

ANME-1, ANME-2 and ANME-3 clades and their bacterial partners. All three ANME

groups co-occur with sulfate reducing bacteria either of the Desulfosarcina-

Desulfococcus branch (ANME- 1/DSS and-2/DSS) or with Desulfobulbus spp (ANME-

3/DBB). Frequently one ANME group dominates, but the factors controlling their

distribution and abundance are not well constrained. We used a lipid-based approach to

investigate linkages between the composition of anaerobic methanotrophic communities

and environmental factors in a geographically diverse set of seep systems. Intact polar

lipids (IPLs) provided a better distinction of the composition of living communities than

their apolar (fossil) derivatives, probably due to the preservation of the apolar lipids

beyond the lifetime of the cells. Based on the analysis of a substantial set of different

microbial communities, assignments of IPLs to certain ANME community types were

found to be robust and taxonomically useful. In ANME-1/DSS communities glycosidic-

and phospho- glyceroldialkylglyceroltetraethers were abundant, while ANME-2/DSS and

ANME-3/DBB communities were dominated by a diverse range of glycosidic- and

phospho- archaeols in combination with bacterial phospholipids from sulfate reducing

bacteria. Beside these main IPL signatures, additional differences were related to the

habitat characteristics of these communities (e.g., lower amount of phosphorus-

containing IPLs were observed in communities inhabiting carbonate reefs compared to

sediments). Moreover, the habitats of ANME-1/DSS communities were characterized by

higher temperatures and lower oxygen content (or even anoxia) compared to ANME-

2/DSS and ANME-3/DBB habitats. In ANME-2 dominated environments, higher

oxygen availability from bottom waters and efficient supply of methane and sulfate were

the controlling factors.

Chapter III ________________________________________________________________________

65

Abbreviations: AR = archaeol, BL = betaine lipids, Crocetane = 2,6,11,15-tetramethylhexadecane,

2Gly = diglycosyl, DAG=diacylglycerol, DEG=dietherglycerol, GDGT = glyceroldialkylglyceroltetraether,

OH-AR = hydroxyarchaeol, OL = ornithine lipids, PC = phosphatidylcholine, PDME = phosphatidyl-

(N,N)-dimethylethanolamine, PE = phosphatidylethanolamine, PG = phosphatidylglycerol, PI =

phosphatidylinositol, PME = phosphatidyl-(N)-methylethanolamine, PMI =2,6,11,15,19-

pentamethylicosane, PS = phosphatidylserine, SRB = sulfate reducing bacteria, SRR = sulfate reduction

rate.

INTRODUCTION

Anaerobic oxidation of methane (AOM) is an important process in the carbon

cycle of marine environments and represents a major sink of the greenhouse gas methane

(Reeburgh, 1996). AOM has been documented in diffusive sedimentary environments

(Martens and Berner, 1974; Reeburgh, 1980; Iversen and Jørgensen, 1985) and at

advection-dominated cold seeps (Elvert et al., 1999; Hinrichs et al., 1999; Boetius et al.,

2000; Pancost et al., 2000; Michaelis et al, 2002). Cold seeps are broadly distributed

along active (Suess et al., 1985, 1998; Yun et al., 1999) and passive margins (Paull et al.,

1995) and are characterized by fluids expelled from deeper reservoirs which have high

contents of methane or other hydrocarbon gases. Furthermore, some hydrothermal vents

with high methane fluxes, such as the ones found in the Guaymas Basin, are also

supporting methanotrophic communities (Teske et al., 2002).

AOM is mediated by a syntrophic consortium of archaea and sulfate reducing

bacteria (SRB) (Boetius et al., 2000; Hinrichs et al., 2000; Orphan et al., 2001a) and

subsequent studies have shown evidence of at least three phylogenetically distinct

clusters of Euryarchaeota involved in the process. Two clusters of ANerobic

MEthanotrophs (i.e., ANME-1 and ANME-2) have been observed in close association

with SRB from the Desulfosarcina-Desulfococcus branch (DSS) (Hinrichs et al., 1999;

Boetius et al., 2000; Michaelis et al., 2002; Knittel et al., 2003), although ANME-1 has

also been frequently observed as monospecific aggregates or even single cells (Orphan et

al., 2002). Finally, members of the third cluster (i.e., ANME-3) occur together with

Desulfobulbus spp (DBB) (Niemann et al., 2006; Lösekann et al., 2007).

Chapter III ________________________________________________________________________

66

Several studies have provided evidence of diagnostic lipid biomarkers associated

with AOM-communities at hydrocarbon seeps (e.g., Elvert et al., 1999; Hinrichs et al.,

1999; Hinrichs et al., 2000; Pancost et al., 2000; Thiel et al., 2001; Michaelis et al., 2002;

Blumenberg et al., 2004; Elvert et al., 2005; Niemann et al., 2006; Rossel et al., 2008).

Distinct biomarker patterns have been attributed to ANME-1 and ANME-2 communities

(Blumenberg et al., 2004) with ANME-1 being characterized by a high abundance of

glyceroldialkylglyceroltetraethers (GDGTs), the absence of crocetane and a low

concentration of hydroxyarchaeol (OH-AR). In contrast, the main features of ANME-2

dominated communities are high amounts of crocetane and OH-AR, whereas GDGTs are

absent. ANME-3, on the other hand, are characterized by the presence of OH-AR,

polyunsaturated 2,6,10,15,19-pentamethylicosanes (PMI:4 and PMI:5), and the absence

of crocetane and GDGTs (Niemann et al., 2006).

Recently, the analysis of intact polar membrane lipids (IPLs) by high-performance

liquid chromatography/electrospray ionization mass spectrometry (HPLC-ESI-MS) has

largely extended the analytic window of lipid diversity in biogeochemistry and microbial

ecology (Rütters et al., 2002; Sturt et al., 2004). The IPL approach is based on ESI – ion

trap - MSn analysis that allows the simultaneous detection of characteristic IPLs from all

domains of life in a single analysis (Sturt et al., 2004; Ertefai et al., 2008; Rossel et al.,

2008). IPLs are reactive biomarkers considered to be indicative of living biomass (White

et al., 1979; Sturt et al., 2004; Biddle et al., 2006; Lipp et al., 2008) and sufficiently

specific for taxonomic distinction of the various ANME groups and their associated

bacterial partners (Rossel et al., 2008). ANME-1 is characterized by a high abundance of

diglycosyl-GDGT (2Gly-GDGT), while ANME-2 and ANME-3 produce mainly

archaeol-based IPLs, either with glycosidic and phospho headgroups or only phospho

headgroups, respectively. SRB members of AOM communities are characterized by

phosphatidylethanolamine (PE) and phosphatidyl-(N,N)-dimethylethanolamine (PDME)

headgroups, with the former occurring as dietherglycerol (DEG) phospholipids in

ANME-1/DSS dominated communities and as diacyl (DAG) or mixed acyl/ether (AEG)

phospholipids in ANME-2/DSS and ANME-3/DBB environments. The occurrence of

PDME is one distinctive feature of the bacteria associated with ANME-3. Relative

amounts of archaeal vs. bacterial IPLs differ systematically among the three major

Chapter III ________________________________________________________________________

67

community types, with the bacterial IPL contribution in ANME-1/DSS communities

being notably small (Rossel et al., 2008).

Most AOM studies focused on the diversity of community types but neglected the

importance of environmental factors selecting for each of these communities. Based on

field observations, it has been suggested that ANME-1/DSS dominate subsurface

sediments (Knittel et al., 2005) and microbial reef structures (Michaelis et al., 2002),

whereas ANME-2/DSS occurs in surface sediments above dissociating methane hydrates

(Elvert et al., 2005; Knittel et al., 2005) or in settings with high methane flux regimes

(Blumenberg et al., 2004) and ANME-3/DBB at mud volcanoes (Niemann et al., 2006;

Lösekann et al., 2007; Omoregie et al., 2008). Furthermore, based on results from in vitro

experiments, it has been suggested that ANME-2/DSS, contrary to ANME-1/DSS, is

better adapted to cold temperatures (Nauhaus et al., 2005). The influence of salinity and

pH on AOM activity has been evaluated but these factors seem not to be important

(Nauhaus et al., 2005). To better constrain the environmental factors influencing this

distribution we targeted AOM communities in a geographically diverse range of

hydrocarbon seeps. We performed the first comprehensive study of IPLs of AOM

communities, supplemented by a framework of community-related data (molecular

ecology and apolar lipid biomarkers) and information on geochemical conditions (e.g.,

concentrations of methane and sulfate, pH, salinity, etc.). IPL analysis provides a holistic

molecular view that integrates signals of all major microbial community members that

contribute substantially to the bulk living biomass. By contrast, other commonly used

culture-independent techniques such as fluorescence in situ hybridization (FISH) and

catalyzed reporter deposition-FISH (CARD-FISH) are highly selective and provide

information only on targeted organisms. Additionally, FISH techniques do not provide

information on the physiological status of microbes in relation to the environment

(Wagner et al., 2003). IPLs, on the other hand, may reflect environmental characteristics

because the structural composition of lipid membranes is influenced by growth

temperature (Khuller and Goldfine, 1974; Oliver and Colwell, 1973; Shimada et al.,

2008), pH (Minnikin and Abdolrahhimzadeh, 1974) and nutrient limitation (Van Mooy et

al., 2006). Therefore, our IPL analyses from globally distributed hydrocarbon seeps

Chapter III ________________________________________________________________________

68

represent a unique opportunity to evaluate both the distribution and the composition of

AOM communities and their relationship to the environmental conditions.

MATERIAL AND METHODS

Sample description

Table III.1. Samples analyzed in this study. Sampling location and the dominant AOM-phylotypes are indicated. Sample codes are based on the location, type of sample (sediment or mat) and field characteristics. Multiple samples of the same type are numbered sequentially.

Location Sample name Dominant

AOM community Lat. Long.

Water depth

(m) Station Research

Cruise

Arabian Sea (AS): AS-S-SOB orange unknown 24°54'N 63°01'E 551 GeoB12320 PC45, 2-3 cm,

Makran subduction zone

M74-3, 2007

AS-S-Thio unknown 24°51'N 63°01'E 1038 GeoB12313 PC4, 2-3 cm Makran subduction zone

M74-3, 2007

AS-S-Calyp unknown 24°51'N 63°01'E 1038 GeoB12313 PC15, 2-3 cm Makran subduction zone

M74-3, 2007

Black Sea (BS): BS-M-trunk-1a ANME-1/DSS [1] 44°47'N 31°59'E 189 P795, Dniepr area PO 317/3,

2004 BS-M-nodule-1a ANME-1/-2 mixed [1] 44°47'N 31°59'E 190 P822, Dniepr area PO 317/3,

2004 BS-M-interior ANME-1/DSS [2] 44°47'N 31°59'E 190 P822, Dniepr area PO 317/3,

2004 BS-M-trunk-2 ANME-1/DSS [2] 44°47'N 31°59'E 190 P822, Dniepr area PO 317/3,

2004 BS-M-trunk-3 unknown 44°01'N 36°41'E 2004 346, Shatsky Ridge R/V

Logachev TTR-15,

2005 BS-M-nodule-2 ANME-2a/DSS [2] 44°51'N 30°28'E 370 P780, Danube area PO 317/3,

2004 BS-M-nodule-3 ANME-2a/DSS [2] 43°57'N 30°17'E 295 P784, Danube area PO 317/3,

2004 BS-S unknown 44°48'N 31°55'E 235 Station 112, 0-2cm,

Crimean area R/V

Logachev TTR-11,

2001 Eastern

Mediterranean Sea (EMS):

EMS-S-SOB unknown 32°32'N 030°21’E 1698 770, PC 44, 2-4 cm Nile Delta

M70-2, 2006

Eel River Basin (ER):

ER-S-SOB ANME-1 [3] 40°48'N 124°36'W 500-520

PC 45, 3-6 cm Northern California continental slope

R/V Melville,

1998 Guaymas Basin

(GB):

GB-S-SOB orange ANME-1 [4] 27°1'N 111°24'W 2000 Core A, 0-2 cm Gulf of California

R/V Atlantis,

1998

Chapter III ________________________________________________________________________

69

Gullfaks oil field (GF):

GF-S-SOB white ANME-2a/2c mixed/DSS [5]

61°10'N 02°14’E 150 766, 0-10 cm North Sea

HE208, 2004

Gulf of Mexico (GOM):

GOM-S-SOB white ANME-1/DSS [6] 27°33'N 90°59'W 950 161, 0-10 cm Northern Gulf of Mexico

SO174, 2003

GOM-S-Campeche knolls

ANME-1/DSS [6] 21°54'N 93°26'W 2902 140, 8-10 cm Southern Gulf of Mexico

SO174, 2003

Håkon Mosby Mud Volcano

(HMMV):

HMMV-S-Beg-1a ANME-3/DBB [7] 72°00’N 14°44'E 1250 ATL 19, 1-2 cm South West Barents Sea

shelf

ATL, 2003

HMMV-S-Beg-2 ANME-3/DBB [7] 72°00’N 14°44'E 1250 Station 322, 0-2 cm South West Barents Sea

shelf

PS64, 2003

HMMV-S-Beg-3 ANME-3/DBB [7] 72°00’N 14°44'E 1250 Station 317, 0-1 cm South West Barents Sea

shelf

PS64, 2003

HMMV-S-Beg-4 ANME-3/DBB [7] 72°00’N 14°44'E 1250 Station 317, 2-3 cm South West Barents Sea

shelf

PS64, 2003

Hydrate Ridge (HR):

HR-S-Beg-1a ANME-2a/DSS [8, 9] 44°34'N 125°09'W 777 Station 19-2, 2-3 cm

Cascadia Margin SO148-1,

2000 HR-S-Beg-2 unknown 44°34'N 125°09'W 777 Station 19-2, 8-10 cm

Cascadia Margin SO148-1,

2000 HR-S-Beg-3 unknown 44°34'N 125°09'W 777 Station 165, 0-3 cm

Cascadia Margin SO165-2,

2002 HR-S-Calyp-1b ANME-2c/DSS [8, 9] 44°34'N 125°09'W 787 Station 38, 2-6 cm

Cascadia Margin SO148-1,

2000 HR-S-Calyp-2b ANME-2c/DSS [10] 44°34'N 125°09'W 787 Station 44D, 4-6 cm

Cascadia Margin SO148-1,

2000 HR-S-Calyp-3 unknown 44°34'N 125°09'W 787 Station 44D, 16-19 cm

Cascadia Margin SO148-1,

2000 a Samples previously reported by Rossel et al. (2008): BS-M-trunk-1(Black Sea mat 795), BS-M-nodule-1(Black Sea mat 822 reef top), HR-S-Beg-1(HR sediment station 19-2, 2-3 cm) and HMMV-S-Beg-1(HMMV sediment station 19, 1-2cm). b Phylogenetic information of HR-S-Calyp-2 indicates that this sample contains 80% of ANME-2c/DSS aggregates (2E10 of total cells from which 7E9 are archaeal cells) and an average of 15% of single ANME-1 cells (6.5E9 total cells), with cell diameters of 0.5nm and 0.6nm for ANME-2 and ANME-1, respectively (Knittel et al., 2003, 2005). Based on the cell shapes (coccus vs. rods) we calculate 1.7 and 10.3 fg of lipid for ANME-2 and ANME-1 cells, respectively (Lipp et al., 2008), which suggest that 60% of the lipids in this sample are associated with ANME-2c and 40% to ANME-1. The same estimate is probably valid for HR-S-Calyp-2, a sample collected in parallel to HR-S-Calyp-1 (Elvert et al., 2005). Abbreviations: Beg = Beggiatoa; Calyp = Calyptogena, SOB = sulfide oxidizing bacteria, Thio = Thioploca observed at the surface sediment, M = mat, S = sediment. References: [1] Rossel et al., 2008; [2] Arnds et al., unpublished data; [3] Orphan et al., 2002; [4] Teske et al., 2002; [5] Wegener et al., 2008; [6] Orcutt PhD thesis 2007; [7] Lösekann et al., 2007; [8] Knittel et al., 2003; [9] Knittel et al., 2005 and [10] Elvert et al., 2005.

The survey of IPLs associated with AOM communities included a broad range of

methane-rich sediments from nine major hydrocarbon seep settings: Arabian Sea, Black

Sea, Eastern Mediterranean Sea, Eel River Basin, Guaymas Basin, Gulf of Mexico,

Gullfaks oil field, Håkon Mosby Mud Volcano, and Hydrate Ridge (Fig. III.4,

Chapter III ________________________________________________________________________

70

supplementary material). IPL analyses were performed at locations at which AOM and

the community members had been previously reported as well as at sites for which no

prior taxonomic characterization was available (see ref.s in Table III.1).

AOM community composition

The taxonomic identification of the different community types studied here

(ANME-1/DSS, ANME-2/DSS and ANME-3/DBB) was based on culture independent

techniques such as 16S ribosomal RNA clone libraries, FISH and CARD-FISH analyses

as described elsewhere (see ref.s in Table III.1).

Lipid analysis

Samples were extracted using an automated microwave assisted extraction system

(MARS-X, CEM, USA) following a modified Bligh and Dyer protocol (Sturt et al., 2004)

except for HR-S-Beg-1, GB-S-SOB orange and ER-S-SOB samples, which were

extracted via ultrasonication at Woods Hole Oceanographic Institution (Teske et al.,

2002; Orphan et al., 2002; Sturt et al., 2004). IPLs were analyzed in total lipid extracts

(TLEs) for the majority of samples, except samples HR-S-Beg-1, GOM-S-SOB white,

GOM-S-Campeche knolls, GB-S-SOB orange and ER-S-SOB, from which the polar

fractions were analyzed after liquid chromatographic separation of the TLE on silica

(White et al., 1998). The utilization of different extraction and separation methods were

tested in some of the samples providing no difference in the lipid distribution (data not

shown). IPL analyses were performed on a HPLC–ESI–MSn system with the instrumental

parameters described previously (Sturt et al., 2004; Biddle et al., 2006). IPL

identification was based on mass spectral interpretation (cf. Sturt et al., 2004; Ertefai et

al., 2008; Rossel et al., 2008; Schubotz et al., submitted) and by comparison with IPL

inventories of cultures of different archaea and bacteria (e.g., Koga et al., 1998; Koga and

Morii, 2005; Hinrichs et al., unpublished data). Due to the limited availability of

commercial standards for the accurate determination of absolute concentrations, IPL

diversity was evaluated based on their relative abundances (cf. Rossel et al., 2008) under

the assumption of uniform response factors. While this procedure is inadequate to

accurately reflect the “real” relative IPL distribution, it is suitable for the differentiation

Chapter III ________________________________________________________________________

71

of a large set of samples. Additionally, unidentified IPLs that were present in at least

three samples and with relative concentrations of more than 2% in at least one sample

were included in the data set. These criteria were used to avoid the inclusion of rare IPLs

signatures potentially not related to methanotrophic habitats and those with unclear

molecular structure. The IPL inventories of the samples HR-S-Beg-1, HMMV-S-Beg-1,

BS-M-trunk-1, and BS-M-nodule-1 were previously reported (Rossel et al., 2008) and

have now been complemented by a few additional compounds such as

phosphatidylglycerol-GDGTs, ornithine (OL) and betaine (BL) lipids due to recent

progress in IPL identification.

In this study we integrated data from apolar lipid biomarkers associated with

AOM, that is, degradation products of IPLs formed either in the sediment or during

sample manipulation and analysis (e.g., free fatty acids, the archaeal core lipids archaeol

(AR) and OH-AR and bacterial glycerol-ether lipids) as well as hydrocarbons (crocetane,

PMI, and their unsaturated derivatives). We presumed that most compounds in this pool

have longer turnover times than IPLs and are thus likely to integrate longer episodes in

the evolution of the respective seep ecosystem. Therefore, the inclusion of these data

may provide additional clues on intrinsic properties of a seep system.

Some of the respective data were acquired in previous studies focusing on the

distribution of apolar lipids (Elvert et al., 2005; Niemann et al., 2006; Wegener et al.,

2008). For microbial mats from the Black Sea, sediments from the Arabian Sea, and

sample HMMV-S-Beg-2, apolar lipids and IPLs of the same TLEs were analyzed.

Chromatographic separation, identification, and quantification were performed according

to previously reported methods (Hinrichs et al., 2000; Elvert et al., 2005).

Environmental data

Biogeochemical parameters from all locations are summarized in Table III.2.

Environmental data were mainly represented by variables associated with regional scale

characteristics (fluid flow, temperature, salinity as well as bottom water oxygen and

phosphate concentration), while other variables focused on small-scale variations at the

respective sampling location (total organic carbon, sulfate reduction rate,

Chapter III ______________________________________________________________________

72

Table III.2. Environmental data selected for redundancy analysis (RDA).

Oxygen

(μM) SRR

(μmol cm-3 d-1) TOC

(wt%) Methane

(mM) Sulfate (mM) Temp (°C) pH

Arabian Sea (AS):

AS-S-SOB orange 15 [1] <0.1 [2] 2.0 [3, 4] 1.25 [5] 33 [2] 12.5 [1] 7.4 [2]

AS-S-SOB Thio 15 [1] <0.1 [2] 2.0.[3, 4] 0.08 [5] 32 [2] 8.0 [1] 7.4 [2]

AS-S-SOB Calyp 15 [1] <0.1 [2] 2.0 [3, 4] 0.08 [5] 32 [2] 8.0 [1] 7.4 [2]

Black Sea (BS):

BS-M-trunk-1 <10 [6] 39.6 [7]* 35 [8] 3.8 [7, 9] 3.0 [9, 10] 8.5 [11] 7.7 [12]

BS-M-nodule-1 <10 [6] 113 [7]* 35 [8] 3.8 [7, 9] 3.0 [9, 10] 8.5 [11] 7.7 [12]

BS-M-interior <10 [6] 36 [7]* 35 [8] 3.8 [7, 9] 3.0 [9, 10] 8.5 [11] 7.7 [12]

BS-M-trunk-2 <10 [6] 39.6 [7]* 35 [8] 3.8 [7, 9] 3.0 [9, 10] 8.5 [11] 7.7 [12]

BS-M-trunk-3 <10 [6] 39.6 [7]* 35 [8] 3.8 [7, 9] 3.0 [9, 10] 8.5 [11] 7.7 [12]

BS-M-nodule-2 <10 [6] 113 [7]* 15 [8] 3.8 [7, 9] 3.0 [9, 10] 8.5 [11] 7.7 [12]

BS-M-nodule-3 <10 [6] 113 [7]* 15 [8] 3.8 [7, 9] 3.0 [9, 10] 8.5 [11] 7.7 [12]

BS-S <10 [6] <0.1 [13] 3.2 [14] 0.1 [15] 18 [13, 15] 8.5 [11] 7.5 [9]

Eastern Mediterranean Sea (EMS):

EMS-S-SOB 200 [16] 0.2 [17, 18] 0.6 [17] 0.3 [17, 19] 24 [18] 14 [17] 8.0 [19]

Eel River Basin (ER):

ER-S-SOB 90 [20] <0.1 [21] 1.0 [20] 0.16 [22] 12 [22] 5.5 [20] 8.4 [23]

Guaymas Basin (GB):

GB-S-SOB orange 28 [24] 0.3 [24] 3.3 [25] 14 [26] 26 [24] 15 [27] 7.5 [28]

Gullfaks oil field (GF):

GF-S-SOB white 275 [29] 0.5 [30]* 0.2 [30] 25 [30] 28 [30] 8.0 [31] 7.4 [32]

Gulf of Mexico (GOM):

GOM-S-SOB-white 200 [33] 0.6 [34] 11 [34] 38 [34] 14 [34] 8.0 [34] 8.0 [35]

GOM-S-Campeche knolls 200 [33] <0.1 [34] 13 [34] 1.5 [34] 3.0 [34] 8.0 [34] 8.0 [35] Haakon Mosby Mud Volcano

(HMMV):

HMMV-S-Beg-1 300 [36] 0.6 [37] 1.0 [38] 2.5 [37] 14 [39] -1.0 [36,39] 8.0 [36]

HMMV-S-Beg-2 300 [36] 1.5 [37] 1.0 [38] 2.5 [37] 19 [37] -1.0 [36,39] 8.0 [36]

HMMV-S-Beg-3 300 [36] 0.5 [37] 1.0 [38] 2.5 [37] 15 [37] -1.0 [36,39] 8.0 [36]

HMMV-S-Beg-4 300 [36] 0.2 [37] 1.0 [38] 2.5 [37] 11 [37] -1.0 [36, 39] 8.0 [36]

Hydrate Ridge (HR):

HR-S-Beg-1 70 [40] 1.0 [41, 42] 2.6 [41] 50 [43, 44] 16 [42, 43] 3.0 [43] 8.3 [11]

HR-S-Beg-2 70 [40] 0.4 [41, 42] 1.6 [41] 50 [43, 44] 2.0 [42] 3.0[43] 8.3 [11]

HR-S-Beg-3 70 [40] 1.0 [42] 2.2 [41] 50 [43, 44] 16 [42, 43] 3.0 [43] 8.3 [11]

HR-S-Calyp-1 70 [40] 1.2 [41] 1.9 [41] 5.5 [43, 44] 18 [42] 3.0 [43] 8.3 [11]

HR-S-Calyp-2 70 [40] 1.2 [41] 1.8 [41] 5.5 [43, 44] 18 [42] 3.0 [43] 8.3 [11]

HR-S-Calyp-3 70 [40] 0.3 [45] 1.6 [41] 5.5 [43, 44] 2.0 [42] 3.0 [43] 8.3 [11] *SRR from the Black Sea mats and Gullfaks sediments were transformed from μmol gdw-3 d-1 to μmol cm-3 d-1 considering 0.12 gdw and 1.2 gdw for 1cm3 mat and sediment, respectively. References: [1] Bohrmann and cruise participants, 2008; [2] Schmaljohann et al., 2001; [3] Cowie et al., 1999; [4] Grandel et al., 2000; [5] Yoshinaga unpublished data; [6] Shaffer, 1986; [7] Arnds et al., unpublished data; [8] Roberts et al., 2008; [9] Krüger et al., 2008; [10] Treude et al., 2005; [11] Nauhaus et al., 2004; [12] Lichtschlag, Wenzhöfer, DeBeer unpublished data [13] Jørgensen et al., 2001; [14] Wakeham et al., 1995; [15] Knab PhD thesis 2007; [16] Yilmaz and Tugrul 1998; [17] Omoregie et al, 2008; [18] Felden unpublished data; [19] Heijs et al., 2007; [20] Levin et al., 2003; [21] Ziebis and Haese, 2005; [22] Orphan et al., 2004; [23] Day MSc dissertation 2003; [24] Weber and Jørgensen 2002; [25] Schouten et al., 2003; [26] Teske et al., 2002; [27] Jorgensen et al., 1992; [28] Gieskes et al., 1982; [29] Lohse et al., 1996; [30] Wegener et al., 2008; [31] Shovitri MSc dissertation 2007; [32] Dando et al., 1994; [33] Yan et al., 2006; [34] Orcutt PhD thesis 2007; [35] Aharon and Fu, 2000; [36] De Beer et al., 2006; [37] Niemann PhD thesis 2005; [38] Milkov et al., 2004; [39] Niemann et al., 2006; [40] Suess et al.,1999; [41] Elvert et al., 2005; [42] Treude et al., 2003; [43] Knittel et al., 2005 [44] Torres et al., 2002; [45] Boetius and Suess 2004.

Chapter III ______________________________________________________________________

73

pH, and concentrations of methane, sulfate and sulfide). Only the environmental

variables that explained the IPL variability according to the redundancy analysis (RDA)

are presented in Table III.2.

Statistical analyses

IPL patterns were subjected to a Hellinger transformation prior to applying

linear multivariate methods (Legendre and Gallagher, 2001). Principal component

analysis (PCA) was performed with a focus on inter-species distances and principal

axes were calculated for samples with available molecular characterization (Table

III.1). The remaining samples, whose ANME community types were unknown, were

then projected as passive samples in the ordination plot by using their IPL patterns. In

order to relate variation in IPL patterns to variation in contextual parameters, RDA was

performed on quantitative variables that were standardized to unit variance and zero

mean, and qualitative variables (i.e., fluid flow, sample type [sediment vs. mat]) were

converted to dummy variables (Ramette, 2007). A forward selection procedure was

performed to retain only the spatial terms that significantly explained variation in the

lipid data. The selected terms were then analyzed in concert with the other contextual

parameters. Significances in the RDA models were assessed by 1000 data permutations

using CANOCO (version 4.5. Microcomputer Power, Ithaca, NY).

The overall distribution and total variability of lipids in the different settings

was evaluated first by the relative abundance of different IPL types (Fig. III.1)

combined with three PCA. In the first PCA (Fig. III.2), archaeal IPLs were represented

as full molecules, whereas bacterial IPLs were distinguished by the headgroup and

bond type between the alkyl moieties and the glycerol backbone (DEG or DAG; AEG

was not possible to distinguish, therefore alkyl chains are given as DAG). This

approach used for bacterial IPLs intends to avoid underestimation of bacterial IPLs,

which would occur from the separation of each IPL depending on the variability in the

fatty acid chains (e.g., PE-DAG has 25 different fatty acid combinations represented by

diverse chain lengths and degrees of unsaturation). Furthermore, betaine lipids (BL)

were separated in two groups according to the presence of odd (BL-odd) and even (BL-

Chapter III ______________________________________________________________________

74

even) fatty acid chains in order to evaluate the possible contribution of specific sources

distinct from algae (Schubotz et al., submitted).

In the second PCA (Fig. III.5, supplementary material), possible differences in

the side chain distribution of fatty acids in bacterial IPLs were evaluated. Four samples

were excluded in this analysis because no bacterial IPLs were detected: HR-S-Calyp-1

to -3 and GOM-S-Campeche knolls. Similarly, the overall distribution of apolar lipids

was evaluated by a third PCA (Fig. III.6, supplementary material).

RESULTS AND DISCUSSION

Diversity of IPLs at hydrocarbon seeps

A total of 46 different IPLs (35 known, nine with tentative names and two

unknowns) were evaluated in detail (Table III.3). 34 IPLs (25 known and nine

tentatively identified) were assigned to archaeal sources (Arabian numbers) and 10 to

bacterial sources (Roman numbers, except VIII which is derived from aquatic algae).

Additionally, two unknown IPLs (a and b) were likely derived from bacteria and

archaea inhabiting carbonate reefs and sediments, respectively. These assignments

were based on characteristics in the mass spectra, which indicate the presence of a

series of acyl moieties in compound a and a lipid structure analogical to a glycosidic-

AR in compound b (Table III.3). Archaeal IPL diversity included several glycosidic-

GDGTs (IPLs # 1 to 7) and glycosidic-ARs (IPLs # 18 to 22) as well as phospho-

GDGTs (IPLs # 8 to 17) and phospho-ARs (IPLs # 23 to 34) (Table III.3).

Chapter III ______________________________________________________________________

75

Table III.3. Lipid code and source assignment of IPLs detected in this study. Lipid Code Lipid name Source assignement

1 2Gly-GDGT Archaea, ANME-1 (Rossel et al., 2008) and deep subsurface (Biddle et al., 2006; Lipp et al., 2008; Sturt et al., 2004), Sulfolobus shibatae (Sturt et al., 2004); Methanobacterium thermoautotrophicum (Koga et al., 1993).

2 3Gly-GDGT Archaea, ANME-1 (this study).

3 4Gly-GDGT Archaea, ANME-1 (this study).

4 2Gly-GDGT+14 Archaea, ANME-1 (this study) and in deep subsurface (Fredricks and Hinrichs, 2007).

5 2Gly-GDGT+18 Archaea, ANME-1(this study) and in deep biosphere sediments (Lipp and Hinrichs, unpublished data) and Nitrosopumilus maritimus (Schouten et al., 2008).

6 2Gly-GDGT+28 Archaea, ANME-1 (this study).

7 2Gly-GDGT+145 Archaea, ANME-1 (this study).

8 Tentative 2Gly-GDGT-PE

Archaea, ANME-1 (this study), Methanobacterium thermoautotrophicum (Koga et al., 1993).

9 MAPT-GDGT-PG Archaea, ANME-1 (this study), aminopentatetrol -GDGTs with two and three methyl groups on the amino group have been describe in Methanomicrobiales (Koga and Morii, 2005; Koga and Nakano, 2008).

10 Gly-GDGT-PG Archaea, ANME-1 (this study).

11 2Gly-GDGT-PG Archaea, ANME-1 (this study), Methanospirillum hungatei (Koga et al., 1993).

12 PG-GDGT Archaea, ANME-1 (this study).

13 2PG-GDGT Archaea, ANME-1 (this study).

14 Tentative PE-GDGT-PG Archaea, ANME-1 (this study).

15 Tentative APT-GDGT-PG

Archaea, ANME-1 (this study), aminopentatetrol-GDGTs without methyl group on the amino group have been describe in Methanomicrobiales (Koga and Morii, 2005; Koga and Nakano, 2008).

16 Tentative APT-GDGT-238

Archaea, ANME-1 (this study), aminopentatetrol-GDGTs without methyl group on the amino group have been describe in Methanomicrobiales (Koga and Morii, 2005; Koga and Nakano, 2008).

17 Tentative 2P-GDGT+155 Archaea, ANME-1 (this study).

18 Gly-MAR Archaea, ANME-2 (this study), Methanocaldococcus jannaschii (Sturt et al., 2004).

19 2Gly-AR Archaea, ANME-2 (Rossel et al., 2008; this study), Methanocaldococcus jannaschii (Sturt et al., 2004), deep subsurface (Biddle et al., 2006, Fredricks and Hinrichs, 2007, Lipp et al., 2008).

20 Gly-OH-AR Archaea, ANME-2 (this study); Methanothrix soehngenii (koga et al., 1993).

21 2Gly-OH-AR Archaea, ANME-2 (Rossel et al., 2008; this study), Methanothrix soehngenii (koga et al., 1993).

22 2Gly-MAR Archaea, ANME-2 (this study), Methanocaldococcus jannaschii (Koga et al., 1993; Sturt et al., 2004).

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23 PE-OH-AR Archaea, ANME-2 (Rossel et al., 2008; this study), Methanothrix soehngenii (koga et al., 1993), Methanosarcina barkeri (Koga and Morii et al., 2005).

24 PG-AR Archaea, ANME-2 (Rossel et al., 2008; this study).

25 PG-OH-AR Archaea, ANME-2 (Rossel et al., 2008; this study), Methanosarcina barkeri (Koga and Morii et al., 2005), Halophiles (Kates, 1978).

26 Tentative APT-OH-AR Archaea, ANME-2 (this study).

27 PI-OH-AR Archaea, ANME-2 and ANME-3 (Rossel et al., 2008; this study).

28 PS-AR Archaea, ANME-2 and ANME-3 (Rossel et al., 2008; this study), Methanobacterium thermoautotrophicum (Koga et al., 1993), Methanocaldococcus jannaschii (Sturt et al., 2004).

29 PS-OH-AR Archaea, ANME-2 and ANME-3 (Rossel et al., 2008; this study), Methanosarcina barkeri (Koga et al., 1993).

30 PS-2OH-AR Archaea, ANME-2 and ANME-3 (Rossel et al., 2008; this study).

31 Tentative P-AR+223 Archaea, ANME-2 (Rossel et al., 2008; this study).

32 Gly-PG-AR Archaea, ANME-1 (this study).

33 Tentative Gly-PS-AR Archaea, possibly ANME-2 (this study).

34 Tentative Gly-P-OH-AR, extended

Archaea, possibly ANME-2 (this study), archaeols with C25 chain have been previously reported in extreme Halophiles (Koga et al., 1993; 2008) and in cold seep sediments from Eastern Mediterranean Sea (Stadnitskaia et al., 2008).

I PC-DAG* Methanotrophic bacteria (Makula, 1978; Goldfine, 1984; Fang et al., 2000), photosynthethic bacteria and green algae (Imhoff and Bias-Imhoff, 1995; Thompson, 1996).

II PG-DAG*

Methanotrophic bacteria (Makula, 1978; Goldfine, 1984; Fang et al., 2000), Desulfosarcina variabilis (Rütters et al., 2001; Sturt et al., 2004), photosynthethic bacteria and green algae (Imhoff and Bias-Imhoff, 1995; Thompson, 1996).

III PE -DAG* Methanotrophic bacteria (Makula, 1978; Goldfine, 1984; Fang et al., 2000), Desulfosarcina variabilis (Rütters et al., 2001; Sturt et al., 2004).

IV PE-DEG Methanotrophic bacteria (Makula, 1978; Goldfine, 1984; Fang et al., 2000).

V PME-DAG* Methanotrophic bacteria (Makula, 1978; Goldfine, 1984; Fang et al., 2000), sulfide oxidizer (Barridge and Shively, 1968).

VI PDME-DAG* Methanotrophic bacteria (Makula, 1978; Goldfine, 1984; Fang et al., 2000), sulfide oxidizer (Barridge and Shively, 1968).

VII OL Bacteria gram-negative performing SR, sulfur and iron oxidation (Makula and Finerty, 1975; Knoche and Shively, 1972; Imhoff and Bias-Imhoff, 1995).

VIII BL-even Photosynthetic eukaryote (Sato, 1992; Dembitsky, 1996; Kato, 1996).

IX BL-Odd Bacteria from anoxic waters (Schubotz et al., submitted; this study).

X Surfactin Bacillus sp (Vater, 1986).

Chapter III ______________________________________________________________________

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Unknown IPLs Distinctive features a Unknown a+ Retention time: 27 min (-0.73 Retention index relative to C-16 PAF)

m/z of quasi-molecular ion: 706.3, 734.3 neutral loss in ms2 positive mode: 194 and then 18

b Unknown b+ Retention time: 43-45 min(+1.3 Retention index relative to C-16 PAF) m/z of quasi-molecular ion: 1148.0 ms2 in positive mode yield the m/z fragments: 873, 993

* Distinction of AEG and DAG not possible under HPLC-MS conditions applied, alkyl chain in Fig. III.3 of supplementary material provided for DAG. + Unknown IPL a was solely detected in microbial mats from the Black Sea. The occurrence of this IPL was specifically observed in ANME-2a/DSS and in the mat displayed between ANME-1 and ANME-2a grouping (BS-M-interior). This unknown was characterized by two major quasi-molecular ions (706.3 and 734.3, Table III.3), both with daughter fragments ions indicative of a loss of 193. Negative ion mode information showed the presence of fatty acids C16:1 and C17:1, which suggest that these lipids are bacterial-derived. The unknown IPL b was displayed, although with a small arrow, between ANME-2a and ANME-2c dominated sediments. The occurrence of this lipid was higher in sediments dominated by ANME-2a/DSS (except in GOM-S-SOB white where it makes up to 21%). This IPL was characterized by one quasi-molecular ion (1148.1, Table III.3) which shows a loss of a 155 (which could indicate the presence of PME) followed by a loss 120 Da in MS2. Negative ion mode information was rather noisy and did not allow a clear identification of the molecule. However, the occurrence of the 993.4 fragment during MS2, caused by the loss of 155, is a possible indication of the presence of 2Gly-MAR. Abbreviations: APT = phosphoaminopentatetrol, AR = archaeol, BL = betaine lipids, 2Gly = diglycosyl, 3Gly = triglycosyl, 4Gly = tetraglycosyl, OH-AR = hydroxyarchaeol, 2OH-AR = dihydroxyarchaeol, GDGT = glyceroldialkylglyceroltetraether, MAPT = phospho methylaminopentatetrol, MAR = macrocyclic archaeol, OL = ornithine lipids, P = phospho headgroup, PC = phosphatidylcholine, PDME = phosphatidyl-(N,N)-dimethylethanolamine, PE = phosphatidylethanolamine, PG = phosphatidylglycerol, PI = phosphatidylinositol, PME = phosphatidyl-(N)-methylethanolamine, PS = phosphatidylserine.

In order to evaluate the general trend of IPLs, the samples were separated in two

groups using the criteria of a contribution to the total archaeal IPLs higher than 50% of

GDGT-based IPLs (Fig. III.1a) or AR-based IPLs (Fig. III.1b), with the former

considered to be diagnostic of ANME-1 and the latter of ANME-2 and ANME-3

dominated communities (Rossel et al., 2008). In the first group the glycosidic-GDGTs

(IPLs # 1 to 7, Table III.4, supplementary material), with 2Gly-GDGT being the

dominant IPL, contributed over 75% in the microbial mats from the Black Sea, whereas

in sediments the contribution varied between 57 and 100%. However, this general

trend of a high glycosidic-GDGTs content was not followed by three samples (HR-S-

Calyp-1 and HR-S-Calyp-2 and AS-S-SOB orange, the first two taxonomically

affiliated with ANME-2c, Table III.1), which contained less than 40% of glycosidic-

GDGTs but more than 56% of phospho-GDGTs, with 2Gly-GDGT-PG and 2PG-

GDGT being the most abundant IPLs. Glycosidic-GDGTs have been previously

reported in isolates of Sulfolobus shibatae (Sturt et al., 2004), whereas the presence of

GDGTs with only phospho or mixed phospho and glycosidic headgroups has been

documented in Methanobacterium thermoautotrophicum (Koga et al., 1993).

Chapter III ______________________________________________________________________

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Fig. III.1. Grouping of samples according to the dominance of GDGT- (a) and AR-based IPLs (b), considering the former as diagnostic for ANME-1 and the latter for ANME-2 and ANME-3 communities. Major lipids (BL-even and the unknowns a and b were not included) were grouped according to IPL classes and normalized to archaeal (a and b) and bacterial (c and d) IPLs. Contribution of archaeal IPLs relative to the total are shown (e). Abbreviations: Gly = glycosidic headgroups, P = phospho headgroups (including mixed glycosidic and phospho headgroup), GDGT = glyceroldialkylglyceroltetraether, AR = archaeol-based IPLs (archaeol and hydroxyarchaeol), PE = phosphoethanolamine, DAG = diacyl, DEG = diether. Notice that some of the samples in the first group (c) did not contain any bacterial IPLs.

The second group of samples was strongly dominated by AR-based IPLs with

both phospho and glycosidic headgroups (Fig. III.1b), compounds that have been found

in cultures of methanogens such as Methanocaldococcus jannaschii (Koga et al., 1993;

Sturt et al., 2004). All samples from Håkon Mosby Mud Volcano and AS-S-Calyp

contained exclusively phospho-ARs, with PG-OH-AR and PS-OH-AR being the major

IPLs in the first setting and PI-OH-AR the dominant in the second one. The microbial

mats from the Black Sea, which contained low amounts of glycosidic-GDGTs (< 34%;

Chapter III ______________________________________________________________________

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Table III.1), presented high abundance of glycosidic-ARs (up to 57%), with 2Gly-AR

and 2Gly-MAR (macrocyclic) being the major IPLs. Besides the generally high

contribution of phospho-ARs in the sediments (> 56%), in the samples HR-S-Beg-3

and EMS-S-SOB also high amounts of GDGTs (~40% in each sample) were observed.

The bacterial IPL distribution in the two groups of samples was variable. In the

samples dominated by GDGT-based IPLs the bacterial IPLs presented a low

contribution or even absence (less than 28%), whereas in the group of samples

dominated by AR-based IPLs the abundance was as high as 66% (Figs. III.1c, d and e,

Table III.4, supplementary material). The most abundant bacterial IPL was PE-DAG

with contributions between 18 and 100% (Figs. III.1c and d) but revealed no clear

pattern in relation to ANME community types or sample characteristics. In contrast,

PE-DEG was present in relatively higher amounts (up to 38%) in the microbial mats

from the Black Sea dominated by glycosidic-GDGTs (Fig. III.1c). Additionally, high

contribution of PME and PDME in samples from the Håkon Mosby Mud Volcano was

a distinctive feature of these sediments dominated by ANME-3/DBB. PE has been

previously reported to be the major phospholipid in SRB such as Desulfosarcina

variabilis (Rütters et al., 2001; Sturt et al., 2004) and its occurrence together with PME

and PDME in the anoxic water column and surface sediments from the Black Sea has

also been suggested to derive from SRB (Schubotz et al., submitted). Nevertheless,

PME and PDME are also produced by methanotrophic bacteria such as Methylosinas

trichosporium and Methylobacterium organophilum (Makula, 1978; Goldfine, 1984;

Fang et al., 2000), as well as by sulfide oxidizers (Barridge and Shively, 1968). It

should be noted that two of these samples from the Håkon Mosby Mud Volcano, which

included the uppermost sediment surface (HMMV-S-Beg-2 and -3, Table III.1),

contained very low amounts of archaeal IPLs (7 to 9%, Fig. III.1e). This suggest an

additional contribution to the bacterial IPLs from either aerobic methanotrophic

bacteria, which have been shown to be present in surface sediments in this habitat

(Lösekann et al., 2007; Elvert and Niemann, 2008) or from sulfide oxidizers, both of

which contain PME and PDME in their membranes (Barridge and Shively, 1968;

Makula, 1978; Fang et al., 2000). PC and PG, which have been observed in

methanotrophic bacteria (Makula, 1978; Golfine, 1984; Fang et al., 2000) as well as in

Chapter III ______________________________________________________________________

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green algae (Thompson, 1996), were highly variable but generally higher in sediment

samples (up to 85%) dominated by AR-based IPLs. The bacterial-derived BL-odd and

OL with non-phospho headgroups were mainly present in the mat samples from the

Black Sea but were also found (contribution up to 37%) in AS-S-SOB orange sample

dominated by GDGTs (Fig III.1c). BL-odd is probably derived from bacteria as

suggested by the exclusive presence in deep anoxic waters of the Black Sea (Schubotz

et al., submitted), whereas OL have been previously observed in SRB (Makula and

Finerty, 1975), sulfur-oxidizing and iron-oxidizing bacteria (Knoche and Shively,

1972). It has been suggested that OL play a functional role in the iron oxidation

metabolism in Thiobacillus ferrooxidans (Ghosh and Misha, 1987). Interestingly,

Reitner et al., (2005) observed that DSS in ANME-2 dominated mats from the Black

Sea presented intracellular iron sulfide precipitates, which suggest an active iron cycle

in these mats. Surfactin, a common lipopeptide previously found in Bacillus sp. (Vater,

1986), was only present in three of the mat samples from the Black Sea (BS-M-trunk-3,

BS-M-nodule-1 and -2), two of them dominated by AR-based IPLs in which surfactin

was as high as 55%. Because previous clone libraries from microbial mats do not

provide evidence for the presence of Bacillus sp (Knittel et al., 2005), we suggest that

other unidentified bacteria are the main producers of this compound.

Patterns of IPL – sample associations indicated by PCA

In order to examine systematic relationships between lipid distributions, AOM

community type and sample characteristics, we examined the data set with three PCAs:

(1) all IPLs, (2) all bacterial IPLs with individual acyl and alkyl moieties distinguished,

and (3) apolar lipids. In the first PCA (Fig. III.2) the total number of IPLs was reduced

from 46 to 41 due to the removal of some IPLs with low variation and/or frequency

among the samples (IPLs # 2, 3, 8 were found in Black Sea at the Shatsky Ridge, IPL

#33 was present at Eastern Mediterranean Sea and IPLs # 2 and 26 were observed in

Arabian Sea at the orange and Thioploca sites, respectively). The remaining 41 IPLs

explained most of the data variability (59% based on two principal components).

Chapter III ______________________________________________________________________

81

Fig.III.2. PCA plot displaying the overall distribution of IPLs among the various samples analyzed. Samples are shown in color codes according to the phylogenetic affiliation. The mixture of ANME-2a and -2c is displayed with both orange and brown circles combined (GF-S-SOB white). Eigenvectors of archaeal, bacterial, algal and unknown IPLs are displayed in red, blue, green and black, respectively. Their direction and length represents the main behavior of the lipid and the rate of change in two dimensional space, respectively (Ramette, 2007). IPL names are given according to Table III.3. As an example, mats dominated by ANME-2a/DSS (dots with white cross) were characterized by the archaeal IPLs # 22, 21, 24 and bacterial IPLs IV, X, VII. Notice that for the construction of this plot archaeal IPLs were represented as full molecules, whereas bacterial IPLs were distinguished by the headgroup and bond type between the acyl/alkyl moieties and the glycerol backbone (DEG or DAG; AEG was not possible to be distinguished). This approach was used to avoid underestimation of bacterial IPLs. Additionally, betaine lipids (BL) were separated in two groups according to the presence of odd (BL-odd) and even (BL-even) fatty acid chains.

IPLs of ANME-1/DSS dominated systems

A distinct group was formed by sediments and microbial mats samples

dominated ANME-1/DSS (BS-M-trunk-1, BS-M-interior, BS-M-trunk-2, GOM-S-

Campeche knolls, ER-S-SOB, GB-S-SOB-orange, and GOM-S-SOB white; Fig. III.2,

black circles) and taxonomically uncharacterized samples (BS-M-trunk-3, HR-S-Calyp-

Chapter III ______________________________________________________________________

82

3, BS-S, and AS-S-SOB orange; Fig. III.2, grey circles). The main feature of the

samples from this group was the high contribution of 2Gly-GDGT, which corroborates

our earlier findings (Rossel et al., 2008). In addition to glycosidic-GDGTs, diverse

types of GDGTs with mixed glycosidic and phospho headgroups (Gly-GDGT-PG,

2Gly-GDGT-PG) and phospho headgroups (PG-GDGT, 2PG-GDGT) were observed.

Together with 2Gly-GDGTs, 2Gly-GDGT-PG, 2PG-GDGT, other GDGTs with

unknown headgroups were observed (Table III.3). Only two AR-based IPLs were

observed in the ANME-1 grouping, however, with less than 1% contribution: Gly-PG-

AR and a tentatively identified extended Gly-P-OH-AR. In the ANME-1 group, which

includes sediments and microbial mats, the contribution of bacterial IPLs was very low

between 0 and 7% (Table III.4, supplementary material), in agreement with our

previous observations in ANME-1/DSS dominated mats (Rossel et al., 2008).

However, we observed two sediment samples with higher contributions of bacterial

IPLs: one from the Eel River Basin (ER-S-SOB) and one from the Black Sea (BS-S)

which contained 28 and 14% of the total IPLs, respectively. The low contribution or

even absence of bacterial IPLs in ANME-1/DSS dominated sediments and microbial

mats is in agreement with the observation of Orphan et al. (2002), who reported that

ANME-1 frequently occurs as a monospecific aggregates or as single cells.

IPLs of ANME-2/DSS dominated systems

Samples dominated by ANME-2 were separated into three main groups (Fig.

III.2; brown and orange circles). The first group was represented by microbial mat

nodules observed on the outside of carbonate reefs from the Black Sea (BS-M-nodule-1

to -3, brown circles with a white cross), which are dominated by ANME-2a/DSS

(Arnds et al., unpublished data). Characteristic features of these nodules were IPLs

based on AR and OH-AR with both glycosidic and phospho headgroups (Gly-OH-AR,

Gly-MAR, 2Gly-AR, 2Gly-OH-AR, 2Gly-MAR, 2-Gly-OH-AR, tentative-P-AR, PG-

AR and PE-OH-AR). These archaeal IPLs were accompanied by the presence of

bacterial-derived PE-DAG, PE-DEG, OL, BL-odd and surfactin. The general presence

of PE-DEG in microbial mats, independent of ANME type, suggests that it may not be

Chapter III ______________________________________________________________________

83

derived from a bacterial partner that is exclusively associated with one particular

ANME community type.

The three mat nodules dominated by the ANME-2a/DSS group differed by the

higher contribution of phospho-ARs in the first nodule (42%, BS-M-nodule-1), whereas

in the samples BS-M-nodule-2 to -3 both phospho and glycosidic headgroups were

similarly abundant. Additionally, the high contribution of surfactin of up to 27% in

these two nodules (Table III.4, supplementary material) suggests that this compound

has a functional role in ANME-2a/DSS mats. Surfactin is a lipopeptide composed of a

hydrophilic part (seven amino acids) and hydrophobic tail (hydroxylated fatty acids

with 13, 14 or 15 carbon atoms), and it is the most efficient microbial biosurfactant

known (Vater, 1986). Surfactin is mainly produced during the maximum growth phase

of the bacterial cell cycle, and so far has been mainly observed in several Bacillus

subtilis strains (Vater, 1986). It has surface-, interface- and membrane-active

properties and has been shown to improve mechanisms of cell adhesion (Ahimou et al.,

2000). This suggests that surfactin may facilitate the formation of zones of ANME-

2a/DSS aggregates in the methanotrophic mats.

The second cluster linked to the ANME-2 group comprised sediment samples

dominated by the ANME-2a subgroup. Some of the samples have been taxonomically

characterized (HR-S-Beg-1, Knittel et al., 2005; GF-S-SOB white, Wegener et al 2008)

whereas others are uncharacterized (AS-S-Thio, AS-S-Calyp, HR-S-Beg-3, HR-S-Beg-

2 and EMS-S-SOB; grey circles). The main feature of this group was the high

abundance of phospho-ARs (PS-2OH-AR, PS-AR, PS-OH-AR, PG-OH-AR and PI-

OH-AR) and bacterial IPLs, (PE-DAG, PG-DAG, PC-DAG) and the presence of BL-

even, considered to originate from aquatic plants (cf. Ertefai et al., 2008; Table III.4,

supplementary material). The archaeal IPL pattern was strongly dominated by OH-AR,

while glycosidic-AR was not abundant in this group (< 3%). The IPL composition of

sample EMS-S-SOB from the Eastern Mediterranean Sea differed from the rest due to a

high abundance of phospho-GDGTs and the tentatively identified Gly-P-OH-AR

extended (22 and 16%, respectively; Table III.4, supplementary material), with the

latter being the intact counterpart of the apolar OH-AR previously reported in this

setting by Stadniskaia et al. (2008). The occurrence of phospho-GDGTs and Gly-P-

Chapter III ______________________________________________________________________

84

OH-AR suggest the presence of a mixed ANME-1 and ANME-2a community in this

setting.

The third group of samples linked to ANME-2 was represented by two

Calyptogena-influenced sediment samples dominated by ANME-2c (Knittel et al.,

2005), HR-S-Calyp-1 and HR-S-Calyp-2 (Fig. III.2, orange circles). This group is

distinguished from the previous ANME-2 groups due to the high contribution of

GDGTs with phospho and mixed phospho and glycosidic headgroups. The main

GDGTs were 2Gly-GDGT-PG and 2PG-GDGT, followed by the tentatively identified

IPLs PE-GDGT-PG, APT-GDGT-PG and MAPT-GDGT-PG and additional unknown

intact GDGTs (# 16 and 17, Table III.4, supplementary material). By contrast, the IPLs

typically associated with ANME-2 (PG-OH-AR, PI-OH-AR, PS-OH-AR) were less

abundant (Table III.4, supplementary material). The presence of GDGTs in ANME-2c

was previously suggested by Elvert et al. (2005), who reported a maximum of free

GDGTs with one and two cyclopentane rings in sample HR-S-Calyp-2, which was

characterized by maximum rates of sulfate reduction and high numbers of ANME-

2c/DSS aggregates. However, relative cellular abundance is not necessarily a reliable

predictor for the corresponding IPL ratios since cellular size and surface area of

ANME-2c and ANME-1 cells differ significantly. Even though the ANME-1 cell

concentration is relatively small, due to their significantly larger total surface size

compared to ANME-2c cells, the expected IPL concentration account for up to 40% of

total archaeal IPL (Table III.1). And notably, in the sample from Gullfaks (GF-S-SOB

white), which is a mixture of ANME-2a and ANME-2c cells (Wegener et al., 2008), we

did not detect any GDGT-based IPLs. This suggests that the GDGTs observed in the

samples HR-S-Calyp-1 and -2 could be associated with the presence of ANME-1 cells.

Nevertheless, the GDGT composition of the sample is significantly different to those

from other ANME-1 dominated systems, which is specifically expressed in the low

abundance of glycosidic-GDGTs (Fig. III.1a, Table III.4, supplementary material).

Therefore, we suggest that the GDGTs in the samples HR-S-Calyp-1 and -2 originate

from ANME-1 that, unlike at other settings, produce phospholipids, although we cannot

exclude that ANME-2c, unlike ANME-2a, has the capability to produce GDGTs.

However, the absence of GDGTs at Gullfaks combined with the dominance of ANME-

Chapter III ______________________________________________________________________

85

1b genes in the clone library of this sample (Knittel et al., 2005) rather supports the

former alternative. Furthermore, we found no indications of bacterial phospho-IPLs,

which is contrary to the finding of SRBs by Elvert et al. (2005) and Knittel et al.

(2005), moving this group into closer relation to ANME-1 rather than to ANME-2.

IPLs of ANME-3/DBB dominated systems

ANME-3 dominated samples (HMMV-S-Beg-1 to -4; green circles) were

closely clustered with the group of sediments characterized by ANME-2a. The

respective ANME-3 samples were characterized by very similar phospho-ARs as those

commonly associated with ANME-2a and by the absence of GDGTs and glycosidic-

ARs, consistent with previous observations (Rossel et al., 2008). However, ANME-3

dominated samples were distinguished by high contributions of bacterial PMEs and

PDMEs (Table III.4, supplementary material), thus providing a possible indication of

DBB species (Rossel et al., 2008). Nevertheless, due to lack of information about IPLs

from DBB isolates and the previously reported production of similar lipids by aerobic

methanotrophs and sulfide oxidizers (Barridge and Shively, 1968; Makula, 1978; Fang

et al., 2000), it is possible, especially in surface sediments, that a fraction of the lipid

contribution may derive from these bacteria. The low contribution of bacterial IPLs in

sediments and microbial mats dominated by ANME-1/DSS compared to ANME-

2a/DSS and -3/DBB suggests that the latter two communities inhabit environments

suitable for a wide variety of microbes.

Diversity of bacterial IPLs in each ANME system

The bacterial communities inhabiting AOM environments were evaluated in

more detail by a second PCA including not only the characteristic headgroups of

bacterial IPLs (PE, PME, PDME, PC, PG, OL and BL), but also variations in the acyl

and alkyl chains (chain length and saturation degree) (Fig. III.5, supplementary

material). The most striking feature of this PCA was the strong separation of the

ANME-3/DBB group from Håkon Mosby Mud Volcano, whereas the other

methanotrophic communities were dominantly separated due to sample characteristics

into mats vs. sediments. The ANME-3/DBB group was characterized by phospho-IPLs

Chapter III ______________________________________________________________________

86

with the DAG bond type (PE C32:3, PME C32:2, PDME C34:2, PDME C32:2, PDME C32:1,

PC C32:2) as well as OL and BL (OL C32:1, OL C32:2, BL C32:2 and BL C34:2). All of

these were positively correlated. The association of PDME with combined C32:2 and

C34:2 acyl moieties with ANME-3/DBB is in agreement with previous observations

(Rossel et al., 2008). These acyl moieties are consistent with combinations of the fatty

acids such as C16:1�5c and C17:1�6c associated with ANME-3/DBB, but also with the

fatty acid C16:1�8 attributed to aerobic methanotrophs observed in surface sediments

from the same location (Niemann et al., 2006).

Distribution of apolar lipids and their taxonomic significance

Strongly 13C-depleted apolar lipids such as crocetane, PMI of archaeal origin

(e.g., Elvert et al., 1999, Thiel et al., 1999), various fatty acids and mono- and di-O-

alkyl glycerol ethers, putatively produced by SRB and the derivatives of polar DEG and

AEG lipids (Hinrichs et al., 2000; Pancost et al., 2000; Elvert et al., 2003), AR and sn-

2-OH-AR (e.g., Hinrichs et al., 1999, 2000) as well as biphytanes obtained by ether-

cleavage reactions of free GDGTs (e.g., Pancost et al., 2001; Schouten et al., 2001;

Thiel et al., 2001) have been routinely used to identify AOM. These and other apolar

lipids were analyzed by PCA in order to identify taxonomic relationships with

individual ANME groups (Fig. III.6, Table III.5, supplementary material). The sample

set had to be reduced from 27 down to 17 due to the lack of available contextual data.

As evident in supplementary Fig. III.6, apolar lipids alone did not separate the

three dominating AOM communities. Interestingly, most of the selected lipids (i.e.,

2OH-AR, DAGE C30:0, C23:1, AR, Crocetane, PMI, PMI:4, Crocetane:1, FA ai-C15:0, FA

cyC17: 0�5,6, C31:x and DAGE C32:2a) were mainly associated with the samples previously

grouped with ANME-1/DSS and ANME-2c/DSS based on IPLs. In opposite direction

to the majority of the lipids, were sn-2-OH-AR and MAGE C16:1�5c, (frequently

observed in ANME-2/DSS dominated systems, e.g., Blumenberg et al., 2004; Elvert et

al., 2005), here related to microbial mats containing both ANME-1 and ANME-2

populations, thus not providing a clear separation. Similarly, no clear relationship was

identified between ANME-3/DBB dominated samples and apolar lipids.

Chapter III ______________________________________________________________________

87

Possible explanations for the poor taxonomic differentiation in this data set are

the lack of appropriate data on GDGT abundances, the major ANME-1 core lipid, and,

importantly, the longer turnover times of apolar lipids compared to IPLs. As a

corollary, the mismatch between these two lipid-based lines suggests that community

compositions are probably not uniform through the time interval integrated by a typical

sample (largely on the order of 100 to 1000 yrs), suggestive of community changes in

the course of the geological evolution of highly dynamic seep systems.

Discrepancies between FISH and IPL data

We observed a number of discrepancies between the data sets of IPLs and FISH.

For example, we interpret the IPL distributions of two Calyptogena-influenced

sediment samples (HR-S-Calyp-1 and -2) as evidence of a substantial contribution of

ANME-1, while FISH analysis suggest a strong predominance of ANME-2c (Knittel et

al., 2003, 2005) (Table III.1). This discrepancy can partly be explained by the large

difference of cell surface areas of ANME-1 and ANME-2 (cf. Table III.1). If we take

these differences into account and calculate the surface area of ANME-1 vs. ANME-2

cells, we would predict that ANME-1 lipids are almost equally abundant as ANME-2

lipids. However, ANME-1 derived IPLs are much higher concentrated than their

ANME-2 counterparts. Potential explanations for these discrepancies can be sought in

both methodologies. For example, the large predominance of ANME-1 lipids in

samples HR-S-Calyp-1 and -2 could also be due to a fossil component in the IPL signal

although this would require that the system has evolved from an ANME-1 to an

ANME-2 dominated community. One explanation could be a period of starvation

which has been proposed to induce a dramatic decrease in phospholipid content (Oliver

and Stringer, 1984).

On the other hand, FISH counts may underestimate certain members of the

archaeal community due to the low permeability of their cell membranes (Wagner et

al., 2003). Based on the membrane lipid structure, we can argue that ANME-1 cells are

probably more rigid than ANME-2 cells (cf. Valentine, 2007), which may negatively

impact their detectability by FISH. Moreover, the selective FISH approach will not

detect archaea outside of the window of interest that may contribute IPLs similar to

Chapter III ______________________________________________________________________

88

those of ANME-1, i.e., the ubiquitous Marine Benthic Group B, which has been

detected previously in clone libraries of the Black Sea (Knittel et al., 2005) and is

presumed to produce 2Gly-GDGT as the major lipid (cf. Biddle et al., 2006; Lipp et al.,

2008). Additionally, there are several samples for which FISH suggests a relatively

high proportion of bacteria while IPL analysis did not detect bacterial IPLs at all (HR-

S-Calyp-1, -2 and GOM-S-Campeche knolls). This could be a result of generally lower

detectability of archaeal cells via FISH, thus resulting in an overestimation of bacteria.

Other factors such as current and past physiological state of a cell can likewise

influence the RNA content and therefore the detection by FISH techniques (Oda et al.,

2000), but probably also the lipid content of cells.

Environmental factors controlling the distribution and composition of AOM

communities

The linkage of environmental conditions and AOM community type was

evaluated by RDA based on the distribution of IPLs (Fig. III.3). Environmental

variables which were previously inferred to influence AOM community distribution are

fluid flow (Girguis et al., 2005), temperature (Nauhaus et al., 2005), oxygen, sulfate

and methane availability (Knittel et al., 2005). Furthermore, the influence of salinity

and pH over AOM activity has been investigated, although both do not appear to be

important (Nauhaus et al., 2005). From a total of twelve variables, 7 were shown to be

related to the IPL distribution (in priority order based on forward selection): oxygen

concentrations in the bottom water (O2), sulfate (SO42-) and methane concentrations

(CH4), sulfate reduction rate (SRR), total organic carbon concentration (TOC),

temperature and pH (Table III.2). From these variables, SRR and TOC were positively

correlated with each other and negatively correlated with SO42-. Variables additionally

included in the statistical analysis, which finally did not contribute to the variability of

the IPLs, were fluid flow (included as qualitative data), water depth, salinity, sulfide

and phosphate concentrations.

Chapter III ______________________________________________________________________

89

Fig. III.3. RDA plot showing the distribution of samples and IPLs in function of environmental variables that explain most of the variability. Environmental variables are shown in red arrows. TOC = total organic carbon, SRR= sulfate reduction rate, O2 = oxygen concentration in the bottom water, CH4 = methane concentration, SO4

2- = sulfate concentration. Color code is as in Figure III.2, except that molecularly uncharacterized samples (grey circles in Figure III.2) are colored according to the phylogenetic grouping in which they were displayed based on the IPL distribution. IPL names are given according to the abbreviation in Table III.3. As an example, mats dominated by ANME-2a/DSS (dots with white cross) were characterized by high sulfate reduction rates and diagnostic IPLs # 21 and 22.

The RDA separated all microbial mat samples from the Black Sea, independent

of the dominant AOM community type, from all of the sediment samples (Fig. III.3).

The main variables associated with this separation were temperature, TOC and SRR.

Microbial mats dominated by ANME-1/DSS (BS-M-interior, BS-M-trunk-1 to -3) were

characterized by higher TOC content and lower SRR compared to the ANME-2a/DSS

dominated mats (BS-M-nodule-1 to -3). Furthermore, temperature affected ANME-

1/DSS dominated AOM communities (mats and sediments), as illustrated by a very

Chapter III ______________________________________________________________________

90

similar direction of its vector in relation to that of 2Gly-GDGT, the IPL diagnostic of

ANME-1. A relationship between ANME-1/DSS and temperature has been previously

suggested by Nauhaus et al. (2005) based on results from in vitro experiments that

indicated higher AOM activity of ANME-1/DSS from microbial mats between 16°C

and 24°C compared to ANME-2a/DSS from sediments, for which a temperature

optimum between 10°C and 15°C was observed. Sediment samples of the ANME-

1/DSS type, by contrast, were more widely distributed during RDA and just weakly

affected by temperature and pH. The respective plot region was characterized by 2Gly-

GDGT-PG and 2PG-GDGT (# 11 and 13 in Fig. III.3, respectively), which were IPLs

also displayed in the Calyptogena-influenced sediments (HR-S-Calyp-1 and -2) and in

uncharacterized sediments from the Eastern Mediterranean Sea (EMS-S-SOB). The

wide distribution of ANME-1/DSS dominated samples relative to pH is in agreement

with previous observations that were not suggestive of a direct relationship (Nauhaus et

al., 2005).

O2 influenced the data distribution in an opposite direction as TOC and

contributed to the separation of mats and sediments. Macrofauna is less abundant in

areas where oxygen is scarce (Levin et al., 2002). Thus, grazing on microbial

communities by macrofauna in anoxic water bodies is absent, allowing the increase of

biomass and therefore TOC. Another environment with low oxygen concentrations

was represented by a sample from the oxygen minimum zone of the Arabian Sea (AS-

S-SOB orange - grouped with ANME-1/DSS dominated samples). However here, TOC

was not as high as in the carbonate reefs from the Black Sea, where typical values of

~25mg of TOC mL-1 of mat were reported (Michaelis et al., 2002).

Displayed opposite to SRR, pH varied similarly in both sediments and in the

Black Sea mats (between 7.4 and 8.3) and was expressed with a rather short vector.

Broader pH values between 6.8 and 8.1 are suggested to be optimum for ANME-1/DSS

activity, whereas for ANME-2/DSS communities the reported optimum is at 7.4

(Nauhaus et al., 2005). The overlapping of the pH values from ANME-1/DSS and

ANME-2/DSS dominated habitats suggested that the communities are not strongly

influenced by pH. Additionally, the metabolic activities of sulfide oxidizing bacterial

communities contribute to an effective supply of SO42-, a variable influencing the group

Chapter III ______________________________________________________________________

91

of sediment samples, particularly those dominated by ANME-2a/DSS. The occurrence

of both SO42-and CH4 (although expressed in a shorter vector compared to most of the

other variables) in the sediment samples dominated by ANME-2a/DSS suggests that a

high supply of these two reactants is an important criterion selective for ANME-

2a/DSS.

ANME-2a/DSS and ANME-3/DBB dominated sediments and some of the

molecularly uncharacterized samples (AS-S-Calyp, AS-S-Thio and HR-S-Beg-1) were

related to O2, SO42- and CH4. The most prominent IPLs found in this grouping were

PG-OH-AR and PI-OH-AR (# 25 and 27 in Fig. III.3, respectively), whereas for the

ANME-3 group, PME, PE-DAG, PDME and BL-even were observed. Among these,

the bacterial IPLs were inversely related to temperature. This relationship is explained

by the fact the ANME-3 type communities were only observed at Håkon Mosby Mud

Volcano, at bottom temperatures of around –1°C.

Based on IPL distribution, it was possible to distinguish microbial mats at

carbonate reefs from sediment samples (Figs. III.1 and III.2). This distinction was

corroborated by the inclusion of environmental variables for the purpose of the RDA

(Fig. III.3). With respect to IPL distribution, the major distinctive feature of these two

habitats is the importance of glycosidic vs. phospho-IPLs. Microbial mats in the Black

Sea affiliated with both ANME-1/DSS and ANME-2/DSS groups contained more than

75% glycosidic IPLs derived from archaea compared to the same consortia inhabiting

sediments which showed lower relative amounts (Figs. III.1a and b). This trend was

also accompanied by higher contributions of bacterial IPLs with non-phospho

headgroups such as OL, BL and surfactin in mats compared to sediments (Fig. III.1d).

The low abundance of phospho-IPLs in samples from reef-like structures in the Black

Sea could be related to phosphate availability (cf. van Mooy et al., 2006). Dissolved

phosphate in sediment pore water has been shown to be strongly adsorbed on calcium

carbonate (Cole et al., 1953; de Kanel and Morse, 1978). During AOM, precipitation

of calcium carbonate is highly stimulated by the increase in alkalinity (e.g., Barnes and

Goldberg, 1976; Ritger et al., 1987; Michaelis et al., 2002). In case of the chimney-like

structures of the Black Sea, Mg-calcite minerals rich in iron sulfide precipitates co-

occur with aragonite phases producing the characteristic highly cavernous stable fabric

Chapter III ______________________________________________________________________

92

(Reitner et al., 2005). Peckmann et al. (2001) suggested that, contrary to Mg-calcite

precipitation, aragonite precipitation in these chimney systems occurs under low

phosphate and high sulfate concentration. Hence, it is conceivable that the chimneys

act as a sink for dissolved phosphate, thus limiting phosphate availability for the

microbial communities inhabiting these carbonate structures. In analogy to marine

planktonic communities and cyanobacteria (van Mooy et al., 2006), AOM communities

may adapt their lipid membrane composition towards IPLs with higher proportions of

glycosidic lipids in case of archaeal, and BL and OL in case of bacterial community

members. For example, Pseudomonas fluorescens has been shown to substitute

phospholipids with OL in response to phosphate limitation (Minnikin and

Abdolrahimzadeh, 1974). Hence, the difference in IPL distribution of mats and

sediments may result from phosphate limitation rather than taxonomic control.

CONCLUSIONS

We distinguished the major microbial communities involved in AOM based on

the distribution of IPLs. IPL distribution allowed the identification of the major ANME

groups with and without phylogenetic information. In line with previous observations

(Rossel et al., 2008), one key feature of ANME-1/DSS dominated systems was the

higher abundance of intact GDGTs compared to ANME-2a/DSS and ANME-3/DBB in

which higher abundance of AR-based IPLs and bacterial lipids were characteristic.

Furthermore, within the main IPL types present in each community, additional

differences related to the habitat characteristics were also influencing the IPL

composition. For example, limitation of dissolved phosphate in AOM mats in

carbonate reef environments of the Black Sea is likely responsible for the generally low

amount of phospho-IPLs in both ANME-1/DSS and ANME-2a/DSS dominated mats in

the Black Sea when compared to sediments inhabited by the same communities.

We constrained several factors selecting for one of the three major ANME

community types. The dominance of ANME-1/DSS was associated with higher

temperatures and anoxia. In sediments dominated by ANME-2a/DSS, higher

concentrations of oxygen in the bottom water, methane, and, most importantly, sulfate

Chapter III ______________________________________________________________________

93

were key environmental parameters involved in selection of this community.

Effective supply of sulfate in sediments in which ANME-2a/DSS inhabits are possibly

facilitated by the production of sulfate coupled to removal of sulfide by sulfide

oxidizing bacterial mats.

The diversity of bacterial IPLs was high and strongly differed among the

settings analyzed. These differences reflect the diversity of bacteria in AOM

environments. Bacterial IPLs were generally less abundant and diverse in ANME-

1/DSS dominated systems compared to ANME-2a/DSS and ANME-3/DBB. The

taxonomic resolution of apolar lipids, i.e., compounds commonly targeted in lipid-

based studies of AOM environments, was insufficient for a distinction of the major

ANME community types.

ACKNOWLEDGMENTS

We thank the captain, crew, and shipboard scientist from the R/V SONNE SO

148-1, SO 165-2, SO 174, R/V L’Atalante 2003, R/V Polarsten PS64, R/V Poseidon

PO 317/3, Meteor M74-3, M70-2, R/V Atlantis, 1998, R/V Melville, 1998, R/V

Heincke HE208, R/V Logachev TTR-15 and TTR-11 for the support during sample

collection. Helge Niemann, Beth Orcutt, Victoria Orphan, Andreas Teske, Tina Treude,

and Gunter Wegener are gratefully acknowledged for providing several of the samples

analyzed here. We also thank Julius Lipp and Xavier Prieto for technical support on the

LC-ESI-MS and the GC-MS. We thank Julia Arnds and Katrin Knittel for phylogenetic

information and also Janine Felden, Helge Niemann, Florence Schubotz, Beth Orcutt,

Ana Lichtschlag, Frank Wenzhöfer and Dirk DeBeer for the unpublished data supplied.

This study was part of the program MUMM II (grant 03G0608C), funded by the

Bundesministerium für Bildung und Forschung (BMBF, Germany) and the Deutsche

Forschungsgemeinschaft (DFG, Germany). Further support was provided by the Center

for Marine Environmental Sciences (MARUM) at the University of Bremen funded by

the DFG-Research Center/Excellent Cluster “The Ocean in the Earth System.

Chapter III ______________________________________________________________________

94

REFERENCES

Aharon, P., Fu, B., 2000. Microbial sulfate reduction and sulfur and oxygen isotope

fractionations at oil and gas seeps in deepwater Gulf of Mexico. Geochimica et

Cosmochimica Acta 64, 233-246.

Ahimou, F., Jacques, P., Deleu, M., 2000. Surfactin and iturin A effect on Bacillus

subtilis surface hydrophobicity. Enzyme and Microbial Technology 27, 749-754.

Barnes, R., Goldberg, E., 1976. Methane production and consumption in anoxic marine

sediments. Geology 4, 297-300.

Barridge, J. K., Shively, J. M., 1968. Phospholipids of the Thiobacilli. Journal of

Bacteriology 95, 2182-2185.

Benning, C., Huang, Z. -H., Gage, D. A., 1995. Accumulation of a novel glycolipid and

a betaine lipid in cells of Rhodobacter sphaeroides grown under phosphate

limitation. Archives of Biochemistry and Biophysics 317, 103-111.

Biddle, J. F., Lipp, J. S., Lever, M. A., Lloyd, K. G., Sörensen, K. B., Anderson, R.,

Fredricks, H. F., Elvert, M., Kelly, T. J., Schrag, D. P., Sogin, M. L., Brenchley, J.

E., Teske, A. House, C. H., Hinrichs, K. –U., 2006. Heterotrophic archaea

dominate sedimentary subsurface ecosystems off Peru. Proceedings of the

National Academy of Science U.S.A. 103, 3846-3851.

Blumenberg, M., Seifert, R., Reitner, J., Pape, T., Michaelis, W., 2004. Membrane lipid

patterns typify distinct anaerobic methanotrophic consortia. Proceedings of the

National Academy of Science U.S.A. 101, 11111-11116.

Boetius, A., Ravenschlag, K., Schubert, C. J., Rickert, D., Widdel, F., Gieseke, A.,

Amann, R., Jørgensen, B. B., Witte, U., Pfannkuche, O., 2000. A marine

microbial consortium apparently mediating anaerobic oxidation of methane.

Nature 407, 623-626.

Boetius, A., Suess, E., 2004. Hydrate Ridge: a natural laboratory for the study of

microbial life fueled by methane from near-surface gas hydrates. Chemical

Geology 205, 291-310.

Bohrman, G., cruise participants., 2008. Report and preliminary results of R/V Meteor

cruise M74/3, Fujairah-Male, 30 October-28 November, 2007. Cold seeps of

Chapter III ______________________________________________________________________

95

Makran subduction zone (continental margin of Pakistan). Berichte, Fachbereich

Geowissenschaften, Universität Bremen, No. 266, 161 pages. Bremen.

Cole, C. V., Olsen, S. R., Scott, C. O., 1953. The nature of phosphate sorption by

calcium carbonate. Soil Science Society of America Journal 17, 352-356.

Cowie, G. L., Calvert, S. E., Pedersen, T. F., Schultz, H., Von Rad, U., 1999. Organic

content and preservational controls in surficial shelf and slope sediments from the

Arabian Sea (Pakistan margin). Marine Geology 161, 23-38.

Curatolo, E., 1987. The physical properties of glycolipids. Biochimica et Biophysica

Acta 906, 111-136.

Dando, P. R., Bussmann, I., Niven, S. J., O´Hara, S. C. M., Schmaljohann, R., Taylor,

L. J., 1994. A methane seep area in the Skagerrak, the habitat of the pogonophore

Siboglinum poseidoni and the bivalve mollusk Thyasira sarci. Marine Ecology

Progress Series 107, 157-167.

Day, A. S., 2003. Documenting modern and ancient methane release from cold seeps

using deep-sea benthic foraminifera, M.Sc. Thesis. University of Florida.

De Beer, D., Sauter, E., Niemann, H., Kaul, N., Foucher, J. -P. Witte, U., Schlüter, M.,

Boetius, A., 2006. In situ fluxes and zonation of microbial activity in surface of

the Håkon Mosby Mud Volcano. Limnology and Oceanography 51, 1315-1331.

De Kanel, J., Morse, J. W., 1978. The chemistry of orthophosphate uptake from

seawater on to calcite and aragonite. Geochimica et Cosmochimica Acta 42, 1335-

1340.

DeLong, E. F., Wickham, G. S., Pace, N. R., 1989. Phylogenetic stains: Ribosomal-

based probes for the identification of single cells. Science 243, 1360-1363.

Dembitsky, V. M., 1996. Betaine ether-linked glycerolipids: chemistry and biology.

Progress in Lipid Research 35, 1-51.

Elvert, M., Suess E., Whiticar, M. J., 1999. Anaerobic methane oxidation associated

with marine gas hydrates: superlight C-isotopes from saturated and unsaturated

C20 and C25 irregular isoprenoids. Naturwissenschaften 86, 295-300.

Elvert, M., Hopmans, E. C., Treude, T., Boetius, A., Suess E., 2005. Spatial variations

of methanotrophic consortia at cold methane seeps: implications from a high-

resolution molecular and isotopic approach. Geobiology 3, 195-209.

Chapter III ______________________________________________________________________

96

Elvert, M., Niemann, H., 2008. Occurrence of unusual steroids and hopanoids derived

from aerobic methanotrophs at an active marine mud volcano. Organic

Geochemistry 39, 167-177.

Ertefai, T. F., Fisher, M. C., Fredricks, H. F., Lipp, J. S., Pearson, A., Birgel, D., Udert,

K. M., Cavanaugh, C. M., Gschwend, P. M., Hinrichs, K. -U., 2008. Vertical

distribution of microbial lipids and functional genes in chemically distinct layers

of a highly polluted meromictic lake. Organic Geochemistry 39, 1572-1588.

Fang, J. S., Barcelona, M. J., Semrau, J. D., 2000. Characterization of methanotrophic

bacteria on the basis of intact phospholipid profiles. FEMS Microbiology Letters

189, 67-72.

Fredricks, H. F., Hinrichs, K. -U. 2007. Data report: intact membrane lipids as

indicators of subsurface life in Cretaceous and Paleogene sediments from site

1257 and 1258. Proceedings of the Ocean Drilling Program, Scientific Results

207.

Ghosh, M., Misha, A. K., 1987. Occurrence, identification and possible significance of

ornithine lipid in Thiobacillus ferrooxidans. Biochemical and Biophysical

Research Communications 142, 925-931.

Gieskes, J. M., Elderfield, H., Lawrence, J. R., Johnson, J., Meyers, B., Campbell, A.,

1982. Geochemistry of interstitial waters and sediments, Leg 64, Gulf of

California. Initial Reports, Deep Sea Drilling Project 64, 675-694. Washington,

DC: US Government Printing Office.

Goldfine, H., 1984. Bacterial membrane and lipid packing theory. Journal of Lipid

Research 25, 1501-1507.

Grandel, S., Rickert, D., Schlüter, M., Wallmann, K., 2000. Pore-water distribution and

quantification of diffusive benthic fluxes of silicic acid, nitrate and phosphate in

surface sediments of the deep Arabian Sea. Deep-Sea Research II 47, 2707-2734.

Guirguis, P. R., Cozen, A. E., DeLong, E. F., 2005. Growth and population dynamics of

anaerobic methane oxidizing archaea and sulfate.reducing bacteria in a

continuous-flow bioreactor. Applied and Environmental Microbiology 71, 3725-

3733.

Chapter III ______________________________________________________________________

97

Heijs, S. H., Haese, R. R., Van der Wielen, P. W. J. J., Forney, L. J., Van Elsas, J. D.,

2007. Use of 16S rRNA gene based clone libraries to assess microbial

communities involved in anaerobic methane oxidation in a Mediterranean cold

seep. Microbial Ecology 53, 384-398.

Hinrichs, K. -U., Hayes, J. S., Sylva, S. P., Brewer, P. G., DeLong, E. F., 1999.

Methane-consuming archaebacteria in marine sediments. Nature 398, 802-805.

Hinrichs, K. -U, Summons, R. E, Orphan, V., Sylva, S. P., Hayes, J. M., 2000.

Molecular and isotopic analyses of anaerobic methane-oxidizing communities in

marine sediments. Organic Geochemistry 31, 1685-1701.

Hofman, M., Eichenberger, W., 1996. Biosynthesis of diacylglyceryl-N,N,N-

trimethylhomoserine in Rhodobacter sphaeroides and evidence for lipid-linked N

methylation. Journal of Bacteriology 178, 6140-6144.

Imhoff, J. F., Bias-Imhoff, U., 1995. Lipids, Quinones and Fatty Acids of Anoxygenic

Phototrophic Bacteria. In Anoxygenic Photosynthetic Bacteria (eds. Blankenship,

R. E., Madigan, M. T., Bauer, C. E.), 179-205. Kluwer Academic Publishers,

Netherlands.

Ishikawa, M., Ichikuni, M., 1981. Coprecipitation of phosphate with calcite.

Geochemical Journal 15, 283-288.

Iversen, N., Jørgensen, B. B., 1985. Anaerobic methane oxidation rates at the sulfate-

methane transition in marine sediments from Kattegat and Skagerrak (Denmark).

Limnology and Oceanography 30, 944-955.

Jørgensen, B. B., Isaksen, M. F., Jannasch, H. W., 1992. Bacterial sulfate reduction

above 100°C in deep-sea hydrothermal vent sediments. Science 258, 1756-1757.

Jørgensen, B. B., Weber, A., Zopfi, J., 2001. Sulfate reduction and anaerobic methane

oxidation in Black Sea sediments. Deep-Sea Research I 48, 2097-2120.

Kato, M., Sakai, M., Adachi, K., Ikemoto, H., Sano, H., 1996. Distribution of betaine

lipids in marine algae. Phytochemistry 42, 1341-1345.

Khuller, G. K., Goldfine, H., 1974. Phospholipids of Clostridium butyricum. V. effects

of growth temperature on fatty acid, and alk-1-enyl ether group, and phospholipid

composition. Journal of Lipid Research 15, 500-507.

Chapter III ______________________________________________________________________

98

Knab, N. J., 2007. Controls of Anaerobic oxidation of methane in ocean margin

sediments. Ph.D. Thesis. University of Bremen.

Knittel, K., Boetius, A., Lemke, A., Eilers, H., Lochte, K., Pfannkuche, O., Linke, P.,

2003. Activity, distribution, and diversity of sulfate reducers and other bacteria in

sediments above gas hydrate (Cascadia Margin, Oregon). Geomicrobiology

Journal 20, 269-294.

Knittel, K., Lösekann, T., Boetius, A., Kort, R., Amann, R., 2005. Diversity and

Distribution of Methanotrophic Archaea at Cold Seeps. Applied and

Environmental Microbiology 71, 467-479.

Knoche, H. W., Shively, J. M., 1972. The structure of an ornithine –containing lipid

from Thiobacillus thioxidans. The Journal of Biological Chemistry 247, 170-178.

Koga, Y., Nishihara, M., Morii, H., Akagawa-Matsushita, M., 1993. Ether polar lipids

of methanogenic bacteria: structures, comparative aspects, and biosyntheses.

1996. Microbiological Reviews 57, 164-182.

Koga, Y., Morii, H., Akagawa-Matsushita, M., Ohga, I., 1998. Correlation of polar

lipid composition with 16S rRNA phylogeny in methanogens. Further analysis of

lipid component parts. Bioscience Biotechnology and Biochemistry 62, 230-236.

Koga, Y., Morii, H., 2005. Recent advances in structural research on ether lipids from

archaea including comparative and physiological aspects. Bioscience

Biotechnology and Biochemistry 69, 2019-2034.

Krüger, M., Wolters, H., Gehre, M., Joye, S. B., Richnow, H. -H., 2008. Tracing the

slow growth of anaerobic methane-oxidizing communities by 15N-labelling

techniques. FEMS Microbiology Ecology 63, 401-411.

Legendre, P., Gallagher, E. D., 2001. Ecologically meaningful transformations for

ordination of species data. Oecologia 129, 271-280.

Levin, L. A., 2002. Deep-ocean life where oxygen is scarce. American Scientist 90,

436-444.

Levin, L. A., Ziebis, W., Mendoza, G. F., Growney, V. A., Tryon, M. D., Brown, K.

M., Mahn, C., Gieskes, J. M., Rathburn, A. E., 2003. Spatial heterogeneity of

macrofauna at northern California methane seep: influence of sulfide

concentration and fluid flow. Marine Ecology Progress Series 265, 123-139.

Chapter III ______________________________________________________________________

99

Lipp, J. S., Morono, Y, Inagaki, F., Hinrichs, K. -U., 2008. Significant contribution of

Archaea to the extant biomass in marine subsurface sediments. Nature 454, 991-

994.

Lohse, L., Epping, E. H. G., Helder, W., Van Raaphorst, W., 1996. Oxygen pore water

profiles in continental shelf sediments of the North Sea: turbulent versus

molecular diffusion. Marine Ecology Progress Series 145, 63-75.

López -Lara, I. M., Sohlenkamp, C., Geiger, O., 2003. Membrane lipids in plant-

associated bacteria: their biosyntheses and possible functions. Molecular Plant-

Microbe Interactions Overview 16, 567-579.

Lösekann, T., Knittel, K., Nadalig, T., Fuchs, B., Niemann, H., Boetius, A., Amann, R.,

2007. Diversity and abundance of aerobic and anaerobic methane oxidizers at the

Haakon Mosby Mud Volcano, Barents Sea. Applied and Environmental

Microbiology 73, 3348–3362.

Makula, R. A., 1978. Phospholipid composition of methane-utilizing bacteria. Journal

of Bacteriology 134, 771-777.

Makula, R. A., Finnerty, W. R., 1975. Isolation and characterization of an ornithine-

containing lipid from Desulfovibrio gigas. Journal of Bacteriology 123, 523-529.

Martens, C. S., Berner, R. A., 1974. Methane production in the interstitial waters of

sulfate-depleted marine sediments. Science 185, 1167-1169.

Michaelis, W., Seifert, R., Nauhaus, K., Treude, T., Thiel, V., Blumenberg, M., Knittel,

K., Gieseke A., Peterknecht, F., Pape, T., Boetius A., Amann, R., Jørgensen, B.

B., Widdel, F., Peckmann, J., Pimenkov, N., Gulin, M. B., 2002. Microbial reefs

in the Black Sea fueled by anaerobic methane oxidation. Science 297, 1013-1015.

Milkov, A. V., Vogt, P. R., Crane, K., Lein, Y. A., Sassen, R., Cherkashev, G. A.,

2004. Geological, geochemical, and microbiological processes at the hydrate-

bearing Håkon Mosby Mud Volcano: a review. Chemical Geology 205, 347-366.

Mills, H. J., Martinez, R. J., Story, S., Sobecky, P. A., 2004. Identification of members

of the metabolically active microbial populations associated with Beggiatoa

species mat communities from Gulf of Mexico cold-seep sediments. Applied and

Environmental Microbiology 70, 5547-5458.

Chapter III ______________________________________________________________________

100

Minnikin, D. E., Abdolrahimzadeh, H., 1974. Effect of pH on the proportions of polar

lipids, in chemostat cultures of Bacillus subtilis. Journal of Bacteriology 120,

999-1003.

Mucci, A., 1986. Growth kinetics and composition of magnesian calcite overgrowths

precipitated from seawater: quantitative influence of orthophosphate ions.

Geochimica et Cosmochimica Acta 50, 2255-2265.

Nauhaus, K., Treude, T., Boetius, A., Krüger, M., 2005. Environmental regulation of

the anaerobic oxidation of methane a comparison of ANME-1 and ANME-II

communities. Environmental Microbiology 7, 98-106.

Niemann, H., 2005. Rates and signatures of methane turnover in sediments of

continental margins, Ph.D. Thesis. University of Bremen.

Niemann, H., Lösekann T., de Beer, D., Elvert, M., Nadalig, T., Knittel, K., Amann, R.,

Sauter, E., Schlüter, M., Klages, M., Foucher, J. -P., Boetius, A., 2006. Novel

microbial communities of the Haakon Mosby mud volcano and their role as a

methane sink. Nature 443, 854-858.

Oda, Y., Slagman, S. -J., Meijer, W. G., Forney, L. J., Gottschal, J. C., 2000. Influence

of growth rate and starvation on fluorescence in situ hybridization of

Rhodopseudomonas palustris. FEMS Microbiology Ecology 32, 205-213.

Oliver, J. D., Colwell, R. R., 1973. Extractable lipid of gram-negative marine bacteria:

phospholipid composition. Journal of Bacteriology 114, 897-908.

Oliver, J. D., Stringer, W. F., 1984. Lipid composition of a psychrophilic marine Vibrio

sp. during starvation-induced morphogenesis. Applied and Environmental

Microbiology 47, 461-466.

Omoregie, E. O., Mastalerz, V., De Lange, G., Straub, K. L., Kappler, A., Røy, H.,

Stadniskaia, A., Foucher, J. -P., Boetius, A., 2008. Biogeochemistry and

community composition of iron- and sulfur-precipitating microbial mats at the

Chefren Mud Volcano (Nile Deep Sea Fan, Eastern Mediterranean). Applied

Environmental Microbiology 74, 3198-3215.

Orcutt, B. N., 2007. Anaerobic oxidation of methane in cold seeps and gas hydrates:

responsible microorganisms, rates of activity, and interactions with other

processes, Ph.D. Thesis. University of Georgia, Athens.

Chapter III ______________________________________________________________________

101

Orphan, V. J., House, C. H., Hinrichs, K. -U., McKeegan, K. D., DeLong, E. F., 2001a.

Methane-consuming archaea revealed by directly coupled isotopic and

phylogenetic analysis. Science 20, 484-487.

Orphan, V. J., House, C. H., Hinrichs, K. -U., McKeegan, K. D., DeLong, E. F., 2001b.

Comparative analysis of methane-oxidizing archaea and sulfate-reducing bacteria

in anoxic marine sediments. Applied and Environmental Microbiology 67, 1922-

1934.

Orphan, V. J., House, C. H., Hinrichs, K. -U., McKeegan, K. D., DeLong, E. F., 2002.

Multiple archaeal groups mediate methane oxidation in anoxic cold seep

sediments. Proceedings of the National Academy of Science U.S.A. 99, 7663-

7668.

Orphan, V. J., Ussler, III. W., Naehr, T. H., House, C. H., Hinrichs, K. -U. Paull, C. K.,

2004. Geological, geochemical, and microbiological heterogeneity of the seafloor

around methane vents in the Eel River Basin, offshore California. Marine

Geology 205, 265-289.

Pancost, R. D., Sinninghe Damsté, J. S, Lint, S. D., van der Maarel, M. J. E. C.,

Gottschal J. C., Shipboard Scientific Party., 2000. Biomarker evidence for

widespread anaerobic methane oxidation in Mediterranean sediments by a

consortium of methanogenic archaea and bacteria. Applied Environmental

Microbiology 66, 1126-1132.

Pancost, R. D., Bouloubassi, I., Aloisi, G., Sinninghe Damsté, J. S., Party M. S. S.,

2001. Three series of non-isoprenoidal dialkyl glycerol diethers in cold-seep

carbonate crust. Organic Geochemistry 32,695-707.

Paull, C. K., Ussler III, W., Browski, W. S., Spiess, F. N., 1995. Methane- Rich plumes

on the Carolina continental rise: associations with gas hydrates. Geology 23, 89-

92.

Peckmann, J., Reimer, A., Luth, U., Hansen, B. T., Heinicke, C., Hoefs, J., Reitner. J.,

2001. Methane-derived carbonates and authigenic pyrite from northwestern Black

Sea. Marine Geology 177, 129-150.

Ramette, A., 2007. Multivariate analysis in microbial ecology. FEMS Microbiology

Ecology 62, 142-160.

Chapter III ______________________________________________________________________

102

Reeburgh, W. S., 1980. Anaerobic methane oxidation: rate depth distributions in Skan

Bay sediments. Earth and Planetary Science Letters 47, 345-352.

Reeburgh, W. S., 1996. “Soft Spots” in the global methane budget. In: Microbial

growth on C1 compounds (eds. Lidstrom, M. E., Tabita, F. R.), 334-342, Kluwer

Academic Publishers, Dordrecht.

Reitner, J., Peckmann, J., Reimer, A., Schumann, G., Thiel, V., 2005. Methane-derived

carbonate build-ups and associated microbial communities at cold seeps on the

lower Crimean shelf (Black Sea). Facies 51, 66-79.

Ritger, S., Carson, B., Suess, E., 1987. Methane-derived authigenic carbonates formed

by subduction-induced pore-water expulsion along Oregon/Washington margin.

Geological Society of America Bulletin 98, 147-156.

Roberts, Z. E., Meldrum, F. C., Pancost, R. D., 2008. The archaeal lipid composition of

partially lithified cold seep mats. Organic Geochemistry 39, 1000-1006.

Rossel, P. E., Lipp, J. S., Fredricks, H. F., Arnds, J., Boetius, A., Elvert, M., Hinrichs,

K. -U., 2008. Intact polar lipids of anaerobic methanotrophic archaea and

associated bacteria. Organic Geochemistry 39, 992-999.

Rütters, H., Sass, H., Cypionka, H., Rullkötter, J., 2001. Monoalkylether phospholipids

in the sulfate-reducing bacteria Desulfosarcina variabilis and Desulforhabdus

amnigenus. Archives of Microbiology 176, 435-442.

Rütters, H., Sass, H., Cypionka, H., Rullkötter, J., 2002. Phospholipid analysis as a tool

to study complex microbial communities in marine sediments. Journal of

Microbiological Methods 48, 149-160.

Sato, N., 1992. Betaine lipids. The botanical Magazine Tokyo 105, 185-197.

Schmaljohann, R., Drews, M., Walter, S., Linke, P., Von Rad, U., Imhoff, J, F., 2001.

Oxygen-minimum zone sediments in the northeastern Arabian Sea off Pakistan: a

habitat for the bacterium Thioploca. Marine Ecology Progress Series 211, 27-42.

Schouten, S., Wakeham, S. G., Sinninghe Damsté, J. S., 2001. Evidence for anaerobic

methane oxidation by archaea in euxinix waters of the Black Sea. Organic

Geochemistry 32,1277-1281.

Chapter III ______________________________________________________________________

103

Schouten, S., Wakeham, S. G., Hopmans, E. C., Sinninghe Damsté, J. S., 2003.

Biogeochemical evidence that thermophilic archaea mediate the anaerobic

oxidation of methane. Applied and Environmental Microbiology 69, 1680-1686.

Schouten, S., Hopmans, E. C., Bass, M., Boumann, H., Standfest, S., Könneke, M.,

Stahl, S. A., Sinninghe Damsté, J. S., 2008. Intact membrane lipids of

“Candidatus Nitrosopumilus maritimus”, a cultivated representative of the

cosmopolitan mesophilic group I Crenarchaeota. Applied and Environmental

Microbiology 74, 2433-2440.

Shaffer, G., 1986. Phosphate pumps and shuttles in the Black Sea. Nature 321, 515-

517.

Shimada, H., Nemoto, N., Shida, T., Yamagishi, A., 2008. Effects of pH and

temperature on the composition of polar lipids in Thermoplasma acidophilum

HO-62. Journal of Bacteriology 190, 5504-5411.

Shovitri, M., 2007. Biogeochemistry and molecular ecology of sandy sediment from

North Sea gas seep, M.Sc. Thesis. University of Bremen.

Stadnitskaia, A., Bouloubassi, I., Elvert, M., Hinrichs, K. -U., Sinninghe Damsté, J. S.,

2008. Extended hydroxyarchaeaol, a novel lipid biomarker for anaerobic

methanotrophy in cold seepage habitats. Organic Geochemistry 39, 1007-1014.

Sturt, H. F., Summons, R. E., Smith, K. J., Elvert, M., Hinrichs, K. -U., 2004. Intact

polar membrane lipids in prokaryotes and sediments deciphered by high-

performance liquid chromatography/electrospray ionization multistage mass

spectrometry-new biomarkers for biogeochemistry and microbial ecology. Rapid

Communications in Mass Spectrometry 18, 617-628.

Suess, E., Carson B., Ritger S. D., Moore, J. C., Jones, M. L., Kulm, L. D., Cochrane,

G. R., 1985. Biological communities at vent sites along the subduction zone off

Oregon. Biological Society of Washington Bulletin 6, 475-484.

Suess, E., Bohrmann, G., Huene, R., Linke, P., Wallmann, K., Lammers, S., Sahling,

H., 1998. Fluid venting in the eastern Aleutian subduction zone. Journal of

Geophysical Research 103, 2597-2614.

Suess, E., Torres, M. E., Bohrmann, G., Collier, R. W., Greinert, J., Linke, P., Rehder,

G., Trehu, A., Wallmann, K., Winckler, G., Zuleger, E., 1999. Gas Hydrate

Chapter III ______________________________________________________________________

104

destabilization: enhanced dewatering, benthic material turnover and öarge

methane plumes at the Cascadia convergent margin. Earth and Planetary Science

Letters 170, 1-15.

Teske, A., Hinrichs, K. -U., Edgecomb, V., de Vera Gomez, A., Kysela, D., Sylva, S.

P., Sogin, M. L., Jannasch, H. W., 2002. Microbial diversity of hydrothermal

sediments in the Guaymas Basin: Evidence for anaerobic methanotrophic

communities. Applied and Environmental Microbiology 68, 1994-2007.

Thiel, V., Peckmann, J., Siefert, R., Wehrung, P., Reitner, J., Michaelis, W., 1999.

Highly isotopically depleted isoprenoids: Molecular markers for ancient methane

venting. Geochimica et Cosmochimica Acta 73, 97-112.

Thiel, V., Peckmann, J., Richnow, H. H., Luth, U., Reitner, J., Michaelis, W., 2001.

Molecular signals for anaerobic methane oxidation in Black Sea seep carbonates

and a microbial mat. Marine Chemistry 73, 97-112.

Thompson, G. A., 1996. Lipids and membrane function in green algae. Biochimica et

Biophysica Acta 1302, 17-45.

Torres, M., McManus, J., Hammond, D. E., de Angelis, M. A., Heeschen, K., Colbert,

S. L., Tyron, M. D., Brown, K. M., Suess, E., 2002. Fluid and chemical fluxes in

and out of sediments hosting methane hydrate deposits on Hydrate Ridge, OR, I:

hydrological provinces. Earth and Planetary Science Letters 201, 525-540.

Treude, T., Boetius, A., Knittel, K., Wallmann, K., Jørgensen, B. B., 2003. Anaerobic

oxidation of methane above has hydrates at Hydrate Ridge, NE Pacific Ocean.

Marine Ecology Progress Series 264, 1-14.

Treude, T., Knittel, K., Blumenberg, M., Seifert, R., Boetius, A., 2005. Subsurface

microbial methanotrophic mats in the Black Sea. Applied and Environmental

Microbiology 71, 6375-6378.

Valentine, D. L., 2007. Adaptations to energy stress dictate the ecology and evolution

of the Archaea. Nature Reviews Microbiology 4, 316-323.

Van Mooy, B. A. S., Rocap, G., Fredricks H. F., Evans, C. T., Devol, A. H., 2006.

Sulfolipids dramatically decrease phosphorus demand by picocyanobacteria in

oligotrophic marine environments. Proceedings of the National Academy of

Science U.S.A. 103, 8607-8612.

Chapter III ______________________________________________________________________

105

Vater, J., 1986. Lipopeptides, an attractive class of microbial surfactants. Progress in

Colloid and Polymer Science 72, 12-18.

Wagner, M., Horn, M., Daims, H., 2003. Fluorescence in situ hybridization for the

identification and characterization of prokaryotes. Current opinion in

Microbiology 6, 302-309.

Wakeham, S. G., Sinninghe Damsté, J. S., Kohnen, M. E. L., de Leeuw, J. W., 1995.

Organic sulfur compounds formed during early diagenesis in Black Sea

sediments. Geochimica et Cosmochimica Acta 59, 521-533.

Weber, A., Jørgensen, B. B., 2002. Bacterial sulfate reduction in hydrothermal

sediments of the Guaymas Basin, Gulf of California, Mexico. Deep-Sea Research

I 49, 827-841.

Wegener, G., Shovitri, M., Knittel, K., Niemann, H., Hovland, M., Boetius, A., 2008.

Biogeochemical processes and microbial diversity of the Gullfaks and

Tommeliten methane seeps (Northern North Sea). Biogeosciences 5, 1127-1144.

White, D. C., Davis, W. M., Nickels, J. S., Kind, J. D., Bobbie, R. J., 1979. Oecologica

40, 51-62.

White, D. C., Ringelberg D. B., 1998. Signature lipid biomarker analysis. In:

Techniques in microbial ecology (eds. Burlage, R. S., Atlas, R, Stahl, D., Geesey,

G., Sayler, G.), 255-259, Oxford University Press, New York.

Yan, T., Ye, Q., Whou, J., Zhang, C. L., 2006. Diversity of functional genes for

methanotrophs in sediments associated with gas hydrates and hydrocarbon seeps

in the Gulf of Mexico. FEMS Microbiology Ecology 57, 251-259.

Yilmaz, A., Tugrul, S., 1998. The effect of cold-and warm-core eddied on the

distribution and stoichiometry of dissolved nutrients in the northeastern

Mediterranean. Journal of Marine Systems 16, 253-268.

Yun, J. W., Orange, D. L., Field, M. E., 1999. Subsurface gas offshore of Northern

California and its link to submarine geomorphology. Marine Geology 154, 357-

368.

Ziebis, W., Haese, R. R., 2005. Interactions between fluid flow, geochemistry, and

biogeochemical processes at methane seeps. In: Macro and microorganisms in

Chapter III ______________________________________________________________________

106

marine sediments (eds. Kristensen, E., Kostka, J., Haese, R. R.), 267-298, AGU

Coastal and Estuarine Studies (Vol 60 Coastal and Estuarine Studies).

III.2. SUPPLEMENTARY MATERIAL

Supplementary Figures and Tables

Supplementary Fig. III.4. Location of samples included in the global survey.

Chapter III ______________________________________________________________________

107

Supplementary Fig. III.5. PCA plot of the overall distribution of bacterial IPLs distinguishing their bond types (DEG = diether, DAG = diacyl), headgroups, sum of carbon atoms and number of unsaturations. Distinction between DAG and AEG was not possible, thus alkyl chains are provided for DAG. Bacterial IPLs with DEG are displayed with the names, all other are shown as DAG (AEG was not possible to distinguish, thus alkyl chains are provided for DAG). Color code of samples is according to Figure III.2 of the manuscript.

Chapter III ______________________________________________________________________

108

Supplementary Fig. III.6. PCA plot of the overall distribution of apolar lipids among the samples analyzed. Color code of samples is according to Figure III.2 of the manuscript. Abbreviations: ai = anteiso, AR = archaeol, Crocetane = 2,6,11,15-tetramethylhexadecane, Crocetene:1/2 = 2,6,11,15-tetramethylhexadecane with one or two double bond(s), DAGE = sn-1,2-di-O-alkyl glycerol ether, OH-AR = hydroxyarchaeol, 2OH-AR = dihydroxyarchaeol, FA = fatty acid, MAGE = sn-1 mono-O-alkyl glycerol ether, PMI =2,6,11,15, 19-pentamethylicosane, PMI:4 =2,6,11,15, 19-pentamethylicosene with four double bonds.

Cha

pter

III

____

____

____

____

____

____

____

____

____

____

____

____

____

____

____

____

____

____

____

____

____

____

____

____

____

____

____

109

Su

pple

men

tary

Tab

le II

I.4. R

elat

ive

abun

danc

e of

IPLs

in p

erce

ntag

e.

A

rabi

an S

ea

Bla

ck S

ea

Eas

tern

Med

iterr

anea

n Se

a E

el R

iver

B

asin

G

uaym

as

Bas

in

Gul

lfaks

G

ulf o

f Mex

ico

Håk

on M

osby

mud

V

olca

no

Hyd

rate

Rid

ge

IPL

AS-S-SOB orange

AS-S-thio

AS-S-calyp

BS-M-trunk-1

BS-M-nodule-1

BS-M-interior

BS-M-trunk-2

BS-M-trunk-3

BS-M-nodule-2

BS-M-nodule-3

BS-S

EMS-S-SOB

ER-S-SOB

GB-S-SOB orange

GF-S-SOB white

GOM-S-SOB white

GOM-S- Campeche

knolls

HMMV-S-Beg-1

HMMV-S-Beg-2

HMMV-S-Beg-3

HMMV-S-Beg-4

HR-S-Beg-1

HR-S-Beg-2

HR-S-Beg-3

HR-S-Calyp-1

HR-S-Calyp-2

HR-S-Calyp-3

2Gly

-GD

GT

(1)

29.7

74

.3

24.4

67

.2

82.1

79

.1

3.0

3.8

61.6

64.0

60

.9

41

.1

80.5

1.

4

5.6

18.1

7.

0 10

0.0

3Gly

-GD

GT

(2)

5.0

3.1

4G

ly-G

DG

T (3

)

1.6

2G

ly-G

DG

T+1

4 (4

)

14.5

2Gly

-GD

GT

+18

(5)

0.

6

1.2

1.1

2Gly

-GD

GT

+28

(6)

9.

1

2Gly

-GD

GT

+145

(7)

0.

5

1.

9 1.

5

0.6

4.

1

2Gly

-GD

GT

-PE

(8)

0.

3

MA

PT-G

DG

T-P

G (9

)

7.

9

G

ly-G

DG

T-P

G (1

0)

4.8

0.

2 1.

0

1.

3 3.

1

2Gly

-GD

GT

-PG

(11)

13

.6

0.3

1.1

3.7

14.2

8.

0 1.

2 8.

7

5.9

2.

7 14

.9

20.6

PG-G

DG

T (1

2)

20

.2

1.

7 6.

7 2.

5

2PG

-GD

GT

(13)

29

.5

0.3

0.

4 3.

7 0.

2

3.

7 14

.12

3.

3

3.3

20.2

48

.2

PE

-GD

GT

-PG

(14)

0.1

4.

8

APT

-GD

GT

-PG

(15)

13

.5

APT

-GD

GT

-238

(16)

4.

7

2P

-GD

GT

+155

(17)

14.3

GL

Y_M

AR

(18)

0.3

1.

4

2.

5 1.

1

2Gly

-AR

(19)

0.7

0.

3 3.

0 3.

2 0.

6 0.

9 14

.5

9.3

3.3

0.5

2.3

2.

4 1.

1 11

.2

2.8

2.2

1.6

G

ly-O

H-A

R (2

0)

0.4

0.

7

2G

ly-O

H-A

R (2

1)

0.9

0.7

0.1

3.

7 1.

9

2Gly

-MA

R (2

2)

0.

1 1.

7 1.

5 0.

4 0.

2 7.

9 4.

1

0.6

PE

-OH

-AR

(23)

0.

8 0.

2

0.

6 0.

6 3.

7

3.

7

0.6

PG

-AR

(24)

6.3

25.6

5.

9 0.

2

12.7

12

.5

4.

8

0.

6 2.

9

PG-O

H-A

R (2

5)

10

.2

4.5

0.2

0.6

16

.5

16.9

6.

7 19

.9

2.6

1.1

13.2

5.

8 29

.7

6.5

7.1

0.7

T

enta

tive

A

PT-O

H-A

R (2

6)

6.

5

PI-O

H-A

R (2

7)

2.5

11.9

18

.8

0.

1

7.

6 0.

4 0.

4 12

.5

1.4

1.6

2.8

0.3

0.5

1.3

5.1

13.0

2.

4 5.

9 0.

6

PS-A

R (2

8)

2.

2

0.

2

0.

6

0.5

0.8

0.3

1.0

0.8

PS

-OH

-AR

(29)

1.

0 29

.4

1.3

0.7

0.2

2.

2 0.

5

0.3

0.9

0.3

4.

9 0.

9 1.

1 4.

7 3.

4 25

.4

10.2

6.

0 2.

7

PS-2

OH

-AR

(30)

3.7

0.1

0.3

1.

6 0.

1 0.

3 0.

9 0.

6 3.

1 0.

6

Ten

t P-A

R+2

23 (3

1)

0.

9

14

.0

6.0

4.1

8.1

0.

5

0.4

G

LY

-PG

-AR

(32)

0.

1 0.

1

0.6

T

ent.G

ly-P

S-A

R (3

3)

6.

9

0.3

T

en. G

ly-P

-AR

ext

ende

d (3

4)

15

.7

0.5

0.4

0.

5

PC-D

AG

(I)

0.6

12.8

0.2

0.

1

1.

2

1.2

0.9

3.0

1.6

3.0

PG

-DA

G (I

I)

0.

1

23.2

21

.6

9.

7 3.

6

4.8

1.2

6.

2 20

.8

12

.5

PE

-DA

G (I

II)

2.9

10.8

27

.6

0.4

8.9

3.3

0.8

2.4

15.9

7.

8 8.

8 19

.2

6.6

2.9

47.6

1.

4

9.2

18.6

8.

0 23

.0

28.0

10

.2

36.5

PE-D

EG

(IV

) 1.

0 2.

7

0.4

3.9

0.9

0.3

2.2

4.1

2.5

4.7

1.9

0.5

3.7

3.0

10.6

PME

-DA

G- (

V)

0.8

6.

9

1.

4

8.

4

3.

0 5.

4 10

.2

3.7

0.3

PD

ME

.-DA

G (V

I)

11

.0

14.5

21

.4

14.5

O

L (V

II)

7.0

1.6

0.

1 4.

8 8.

0

1.4

2.

4 1.

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Chapter IV ________________________________________________________________________

111

CHAPTER IV

Experimental approach to evaluate stability and reactivity of

intact polar membrane lipids of archaea and bacteria

in marine sediments

Pamela E. Rossela, Julius S. Lippa, Verena Heuera and Kai-Uwe Hinrichsa

aOrganic Geochemistry Group, Department of Geosciences, University of Bremen, 28334 Bremen,

Germany

Keywords: intact polar membrane lipids, biomarker, archaea, bacteria, sediment,

degradation

Chapter IV ________________________________________________________________________

112

IV.1. MANUSCRIPT

ABSTRACT

A 465-days-long incubation experiment was performed in order to asses the

stability and reactivity of archaeal and bacterial membrane lipids in anoxic marine

sediments. Subsurface sediments with low organic carbon content were spiked with both

archaeal (diglycosyl glycerodialkylglyceroltetraether, from a freeze dried and ground

microbial mat) and bacterial membrane lipid (C16-phosphatidylcholine, available as a

commercial standard), and incubated under oxygen-free conditions at 5 °C and 40 °C.

Incubations were performed using both “sterile” (previously autoclaved sediment) and

“alive or active” conditions to evaluate differences between biotic and abiotic

degradation. An overall decay for both membrane lipids, although at different rates, was

observed under sterile conditions at 5°C and 40°C, contrary to previous observations

suggesting only the occurrence of biotically mediated degradation. The degradation of

lipids under sterile conditions can be accounted by: 1) the presence of active microbial

enzymes likely derived from the microbial mat powder added to the sediments; 2) the

presence of active resistant spores even after sterilization of the sediment; 3) partial

decrease due to adsorption onto minerals, 4) partial degradation of the membrane lipids

takes place abiotically. Additionally, temperature appeared to be an important factor in

IPL degradation. In the incubations with active sediment the archaeal IPL increased at

5ºC and 40ºC, whereas the bacterial IPL only increased at 5ºC. This suggests that

microbes were growing during the experiments, although this could not be evaluated due

to the fact that both pools, degraded and newly produced IPLs, were indistinguishable.

Further improvements in future experiments are needed to better distinguish between

degraded and in situ produced IPLs as well as to evaluate the abundance of microbial

cells, the production of degradation products and the effect of adsorption processes over

time. However, our results provide an important baseline for guiding such experiments.

Chapter IV ________________________________________________________________________

113

INTRODUCTION

Intact polar lipids (IPLs) are ubiquitous in all cell membranes of living organisms.

Due to the instability of the bond between the head group and the glycerol backbone,

IPLs are assumed to be highly unstable after cell’s decay and are therefore used as

biomarkers for living biomass (White et al, 1979; Sturt et al 2004; Lipp et al., 2008).

During degradation, the cleavage of the polar head group from the intact molecule occurs,

leaving behind their apolar derivatives such as archaeol, hydroxyarchaeol or varying fatty

acid side chains. These derivatives have been commonly used in the study of modern

prokaryotic ecosystems such as those associated with anaerobic oxidation of methane

(AOM; e.g., Hinrichs et al., 2000; Blumenberg et al., 2004; Elvert et al., 2005). However,

the use of apolar lipids may be influenced by fossil biomass and therefore it does not

necessarily provide a direct evidence of active communities.

Even though IPLs are currently used as marker for living biomass, their stability

has not been studied systematically and the understanding of their reactivity is poorly

constrained. Therefore, the potential contribution of IPLs to a fossil sedimentary pool

remains unknown. In this study, we evaluated the stability and reactivity of archaeal and

bacterial IPLs, i.e., the hydrolytic cleavage of the glycosidic or phosphate-ester bond

between the polar head group and the core lipid, in a long-term experiment. A better

understanding of IPL degradation is essential because it severely affects the interpretation

of lipid biomarker signals in natural environments.

MATERIAL AND METHODS

Anoxic subsurface sediments with low organic carbon content (~0.7 wt%) were

obtained from IODP Leg 311 (Cascadia Margin; collected from 34 and 53 meter below

the sea floor). Sediments were mixed in a bottle with autoclaved artificial seawater (in

duplicate) in a 1:1 proportion to obtain 1 L of slurry per bottle, and were later incubated

at 5°C over a week to allow the formation of microbial films (Fig. IV.1). Artificial

seawater was prepared using sodium chloride (26.4 g L-1), magnesium chloride (5.7 g L-

1), potassium chloride (0.682 g L-1), potassium bromide (0.099 g L-1) and nutrients

Chapter IV ________________________________________________________________________

114

(ammonium chloride and potassium dihydrogen phosphate), the latter recommended for

culture media of sulfate reducing bacteria (SRB, Widdel and Bak, 1992). One slurry

bottle was kept at 5°C (active sediment incubation). The second bottle was autoclaved

twice (sterile conditions) with two days in between each autoclave cycle; afterwards,

water was removed and replaced for freshly autoclaved artificial seawater to avoid

contamination by potential microbe-derived spores growing in the sediment (Fig IV.1). In

parallel to the preparation of slurries, ~ 30 μg of bacterial C16-Phosphocoline (C16-PC)

and archaeal diglycosyl glyceroldialkylglyceroltetraether (2Gly-GDGTs) were introduced

in a series of Hungate tubes and stored at -80°C to avoid degradation (Fig IV.1). The

bacterial IPL correspond to a commercially available standard, whereas the archaeal IPL

is the dominant lipid in a microbial mat associated with an AOM system from the Black

Sea (R/V Logachev Cruise 2005). The occurrence of the ester lipid C16-PC has been

previously described in SRB such as Desulforhabdus amnigenus (Rütter et al., 2001),

methanotrophic bacteria (Makula, 1978) as well as photosynthetic eukaryotes

(Thompson, 1996). On the other hand, the ether lipid 2Gly-GDGT has been reported only

in methanogenic and thermogenic archaea (de Rosa et al., 1986) as well as in the

methanotrophic archaea ANME-1 (Rossel et al., 2008).

After both slurries were prepared, Hungate tubes with the IPL mixture were filled

up to completing a volume of 10 mL, and were sealed with butyl rubber stoppers

(previously sterilized) under anaerobic conditions using a glove box (Fig. IV.1). Only

anaerobic degradation was evaluated in this study because it is a most accurate

representation of the environmental conditions in which AOM take place.

Experiments were performed in order to monitor abiotic- and/or biologically-

mediated decay of IPLs. Incubations were performed under anaerobic conditions in

darkness for 465 days at 5°C and 40°C with irregular sampling intervals (Table IV.1).

Abiotic degradation of IPLs is known to occur under exposure to oxygen, light, or high

temperatures (Peterson and Cummings, 2006). Degradation of lipids and bulk AOM

biomass was monitored by analysis of IPLs and apolar GDGT cores concentrations, as

well as the concentrations and the carbon isotopic composition of the metabolite acetate

at irregular intervals (Table IV.1). For acetate analysis, an aliquot of ~1 mL was collected

Chapter IV ________________________________________________________________________

115

from the supernatant water and stored at -20°C, whereas for lipid, sediments were stored

at -80°C prior to extraction and analysis (Fig. IV.1).

Fig. IV.1. Diagram of the experimental design. 2Gly-GDGT = Diglycosyl dialkylglcerotetraether, C16-PC = C16-phosphocholine.

Acetate was analyzed by isotope ratio monitoring - liquid chromatography mass

spectrometry (irm-LC/MS) according to Heuer et al. (2006). Total lipid extracts (TLEs)

were obtained from freeze dried samples, previously stored at -80°C, with a microwave

assisted extraction systems (MARS-X, CEM, USA) for 15 min at 70°C using a modified

Bligh and Dyer method (Sturt et al., 2004). TLEs were evaporated to dryness under

nitrogen stream and stored at -80°C until IPL analysis was performed by high-

performance liquid chromatography/electrospray ionization mass spectrometry (HPLC-

ESI-MS) according to Sturt et al. (2004). For GDGT core analysis, selected TLE used for

IPL were analyzed by high-performance liquid chromatography/atmospheric pressure

chemical ionization-mass spectrometry (HPLC-APCI-MS) as described elsewhere

(Schouten et al., 2007; Lipp and Hinrichs, submitted). The quantification of the IPLs

Chapter IV ________________________________________________________________________

116

(using phosphatidyl-(N,N)-dimethylethanolamine as internal standard) and GDGTs are

expressed as the percentage relative to the initial amount (T0).

Table IV.1. Frequency of analysis performed in the experiments Days IPL at 5°C IPL at 40°C GDGT cores at 5°C Acetate at 40°C sterile active sterile active active active 0 + + + + + + 1 + + --- --- + --- 3 + + --- --- --- --- 6 + + --- --- --- --- 9 + + + + --- + 15 + + --- --- --- --- 21 + + --- --- --- --- 29 + + + + --- --- 40 --- + + + --- --- 95 + + + + + + 465 + + + + + +

RESULTS AND DISCUSSION

Sterile incubation

Generally, and despite the analytical error, a more rapid cleavage of the archaeal

IPL (glycosidic ether bond) compared to the bacterial IPL (phosphate ester bond) is

observed (Figs. IV.2 A and B). After 465 days of incubation, 14 and 16% of the initial

2Gly-GDGT was still present at 5°C and 40°C, respectively, whereas for C16-PC 46 and

~1% was present, respectively (Figs. IV.2 A and B). This result differs to previous

observations by Harvey et al. (1986) who reported a 20 fold higher degradation for the

bacterial phospholipid (phosphoethanolamine) than for a glycosidic-archaeol. However,

these authors performed incubations under oxic conditions, and suggested that the high

turnover of the phospholipid is expected due to its high solubility, making it more

accessible for enzymatic attack. Interestingly, by the end of the 40°C incubation the

decrease of bacterial IPLs was more dramatic than for the archaeal IPL. Although

stimulation of organic matter degradation rates due to higher temperatures is likely to

occur, a similar trend for both lipids is expected.

The abundance of polar lipids was highly variable through time, especially for the

bacterial IPL during the initial 50 days of the incubation. A possible mechanism

explaining such variability is adsorption of IPLs onto minerals, which seems to affect

Chapter IV ________________________________________________________________________

117

more strongly the bacterial IPL (larger error bars). The adsorption of organic matter onto

minerals facies may occur soon after deposition, providing physical protection which

decreases its availability for microbial degradation (Mayer, 1994; Keil, 1994; Hedges and

Keil, 1995). Experimental studies have reported that apparently recalcitrant organic

matter becomes more labile and it is rapidly degraded when separated from its mineral

matrix (Keil, 1994). Unfortunately, no controls to address the effect of adsorption on

IPLs were performed during our experiments. Therefore, we can not judge the

importance of this effect.

Time (days)0 50 100 150 450 500

0

10

20

30

40

50

60

70

80

90

100

110

120

130

140

150

Archaeal IPL (2Gly-GDGT) at 5°CArchaeal IPL (2Gly-GDGT) at 40°C

Time (days)0 50 100 150 450 500

0

10

20

30

40

50

60

70

80

90

100

110

120

130

140

150

Bacterial IPL (C16-PC) at 5°CBacterial IPL (C16-PC) at 40°C

A B

%IP

Ls re

lativ

e to

T0

Fig. IV.2. Degradation of archaeal 2Gly-GDGT (A) and bacterial C16-PC (B) at 5°C and 40°C in sterile sediments. Values are expressed as the percentage of the original IPL concentration at the beginning of the experiment.

The rapid decrease at the beginning of the experiment followed by a slow

turnover observed in the sterile experiment is in agreement with other observations of

IPLs degradation in natural sediments (White et al., 1979; Harvey et al., 1986). However,

contrary to previous observations by Harvey et al. (1986), who did not observed

abiotically-mediated degradation in the sterile sediments (previously autoclaved and

treated with formaldehyde), we observed a decrease of lipid abundance over time in the

sterile incubations. Degradation in sterile conditions may be caused by several reasons: 1)

it is possible that enzymes from the mat powder used as archaeal lipid standard were still

active; 2) that the used sterilization procedure (only autoclaved steps) did not efficiently

kill some resistant spores; 3) that the decrease of IPLs is due to adsorption; or 3) that an

Chapter IV ________________________________________________________________________

118

important fraction of IPLs is really degraded abiotically, contrary to the observations of

Harvey et al. (1986).

Whereas Harvey et al. (1986) performed anoxic incubations in a glove box during

the curse of the experiment, we only prepared and sealed our samples under anoxic

conditions, but were not incubated under oxygen-free environment. This opens the

possibility that the samples were exposed to oxygen during part of the experiment and

thus oxic degradation may occur. Harvey et al. (1986) observed degradation rates 40%

lower under anoxic than under oxic conditions.

Active sediment incubations

Experiments with active sediments generally showed a decrease during the first

100 days of incubation, especially at 40°C (down to 11 and 1% for the archaeal and

bacterial IPLs, respectively; Figs. IV.3 A and B). After 100 days, a subsequent increase in

the abundance of 2Gly-GDGT up to 64 and 75% at 5°C and 40°C, respectively, was

observed, while C16-PC increased only during the 5°C incubation (Figs. IV.3 A and B).

The unexpected finding of high abundances of 2Gly-GDGT at higher temperatures can be

best explained by the growth of archaeal cells in the incubated sediments used for this

experiment. Subsurface sediments have been found to contain abundant archaea

producing 2Gly-GDGT (Biddle et al., 2006; Lipp et al., 2008). Moreover, due to the

extensive hydrogen bonding capacity, glycolipids-based membranes are more stable at

higher temperatures than phospholipid-based membranes (Curatolo, 1987). The growth of

2Gly-GDGT-producing archaea in the 40°C experiment is also in agreement with

previous observations by Nauhaus et al. (2005). Based on in vitro experiments, these

authors found evidence that ANME-1, the main producer of GDGTs in AOM

environments, showed higher activity than ANME-2 at higher temperatures.

The observed decrease of IPLs over the first 100 days in the active sediment

incubation at 40°C was accompanied by a rapid release of strongly 13C-depleted acetate

(Fig. IV.3B), whereas at 5°C acetate was below detection limit. The �13C value of the

acetate pool shifted from -26‰ to -73‰ in only nine days, strongly suggesting that fresh

AOM biomass was quickly turned over into acetate. After 465 days of incubation, acetate

concentrations were up to 590 μM, and exhibited a �13C value of -90‰. This strong

Chapter IV ________________________________________________________________________

119

depletion towards the end of the experiment is similar to values reported by Heuer et al.

(2006) in pore water analysis of a methane seep in the Black Sea (-85‰). These authors

suggested that such depletion in acetate was probably due to the role of acetate as an

intermediate in AOM, or that acetate may be also produced from 13C depleted organic or

inorganic molecules. Although acetate production was observed, the simultaneous

increase of IPLs in the active sediment incubations did not allow the clear assignation of

biologically mediated IPL degradation since the degraded and produced IPL pools were

indistinguishable in our study.

Time (days)0 50 100 150 450 500

% o

f IP

Ls re

lativ

e to

T0

0

20

40

60

80

100

120

140

160

180

200

220

240Archaeal IPL (2Gly-GDGT) at 40°CArchaeal IPL (2Gly-GDGT) at 5°C

A

D

Time (days)0 50 100 150 450 500

0

20

40

60

80

100

120

140

160

180

200

220

240

Acet

ate

(μM

)

0

50

100

150

200

250

300

350

400

450

500

550

600

650

Bacterial IPL (C-16-PC) at 5°CBacterial IPL (C-16-PC) at 40°CAcetate μM

B

-73‰

-72‰

-90‰

-26‰

Fig. IV.3. Degradation of archaeal 2Gly-GDGT (A) and bacterial C16-PC (B), at 5°C and 40°C in active sediments over time. Acetate production and isotopic values at 40°C are displayed in figure B.

The decreasing trend of IPL abundances during the first 100 days under sterile

and active conditions points to the fact that IPL degradation occurs quiet rapidly, with a

loss of ~80% for 2Gly-GDGT and ~50% for C16-PC at 5°C. However, these results are

notoriously higher than those reported by Harvey et al. (1986), who observed remaining

amounts of glycosidic archaeol between 60 and 80% in the aerobic and anaerobic

incubation, respectively. Additionally, they found 30% of the phospholipid remaining in

the oxic experiment (anoxic incubations were not performed). In our study, which is more

than a year longer than the one by Harvey and coworkers, 14 and 16% of archaeal IPL,

and 46 and 1% of bacterial IPL were still present at 5°C and 40°C at the end of the sterile

experiment, respectively. The higher turnover of IPLs in these incubations compared to

the experiment reported by Harvey et al. (1986) could be related to the pre-incubation

Chapter IV ________________________________________________________________________

120

periods used in both studies. The short incubation time used by Harvey et al. (1986)

previous to the lipid addition (48 h), compared to one week used in this study, may not be

enough time for the formation of microbial films and for the growth of an abundant active

microbial population, which may result in lower degradation rates of IPLs. Unfortunately,

we did not measure the increase of microbial cells over the time of the experiment;

therefore the possibility of a higher degradation due higher abundance of microbial cells

could not be tested.

In order to evaluate the production of GDGT cores caused by the degradation of

2Gly-GDGT, selected samples from the active sediment incubation at 5°C were analyzed

(0, 1, 95 and 465 days, Table IV.1). The obtained results show that the concentration of

the GDGT cores with 0, 4 and 5 cyclopentane rings (GDGT-0, -4, and -5) decreased

significantly during the first day of the experiment (from 100% to 55%, 23% and 13%,

respectively; Fig. IV.4). GDGT cores with 2 and 3 cyclopentane rings (GDGT-2 and -3),

on the other hand, displayed only a moderate decrease of ~10% (Fig. IV.4). Distinctly,

GDGT core 1 cyclopentane ring (GDGT-1) increased relative to T0.

Time (days)

0 1 2 3 4 5 6 7 8 9 10 100 200 300 400 500

% o

f GD

GT

core

s re

lativ

e to

T0

0

20

40

60

80

100

120

140GDGT-0 GDGT-1 GDGT-2 GDGT-3 GDGT-4 GDGT-5

Fig. IV.4. Changes in GDGT core abundance during the active sediment incubation at 5°C. 0 to 5 stands for number of rings in the GDGT core

The increase of GDGT-1 over time suggests a preferential degradation of the

2Gly-GDGT with 1 ring, which is the third most abundant core observed in the intact

Chapter IV ________________________________________________________________________

121

molecule from the mat used in this experiment. After a period of rather stable

concentrations during the first 100 days, relative abundances of GDGT-1, -3, and -4 also

increased, suggesting a higher degradation of intact GDGTs after this time of incubation.

However, IPL analyses showed the opposite trend, with degradation in the first 100 days

followed by a production after 100 days of incubation (Fig. IV.3A). Therefore, our results

from the active experiment, at 5°C incubation cannot be fully interpreted. Degradation of

intact GDGTs during the active experiments is not in accordance with the results from the

analyses of GDGT cores, calling for further and improved experiments which may allow

the distinction between the in situ produced and degraded IPL pools over time. A possible

solution for differentiating both pools could be the utilization of 13C labeled membrane

lipids. This approach would additionally improve the quantification of lipids over long-

time experiments, as well as the possibility to measure the production of gases and

degradation products specifically enriched in 13C.

CONCLUDING REMARKS

The results from our degradation experiments of IPLs suggest a rapid decrease of

membrane lipids (i.e., 2Gly-GDGT and C16-PC) under sterile conditions at 5°C and 40°C.

This decrease may be caused by several reasons: 1) the presence of active enzymes

derived from the added microbial mat powder with the archaeal lipid; 2) the loss of

anoxic conditions; 3) adsorption processes; 4) the presence of resistant spores even after

autoclaving the sediment; 4) the degradation of membrane lipids in marine sediments can

be partially abiotically mediated.

It was also observed that temperature, a factor not taken into account in previous

studies, affects degradation of both membrane lipids differently. During the incubation at

40°C, degradation of bacterial IPL was more dramatic than for the archaeal IPL.

Furthermore, an increase of membrane lipids after 465 days in the active sediment

incubations suggested that microbial communities were growing in situ.

Unfortunately, degraded and newly produced IPL pools were indistinguishable in

the active experiment; therefore the potential growth of microbes can not be proved.

Thus, an improved experimental design is required for future attempts. For these, not

Chapter IV ________________________________________________________________________

122

only the degraded pool and the fresh IPLs should be carefully determined, but also

problems such as possible loss of oxygen conditions, abundance of microbial cells,

degradation products and adsorption process should be evaluated over the curse of a long

term experiment.

ACKNOWLEDGMENTS

We thank the crew and shipboard scientist of IODP expedition 311 for support

during sample collection. Augusta Dibbel is gratefully acknowledged for laboratory

assistance and also Thomas Holler and Cristian Deusner from the Max Planck Institute

for Marine Microbiology in Bremen for assisting with the use of the glove box. This

study was part of the program MUMM II (grant 03G0608C), funded by the

Bundesministerium für Bildung und Forschung (BMBF, Germany) and the Deutsche

Forschungsgemeinschaft (DFG, Germany). Further support was provided from the Center

of Marine Environmental Sciences (MARUM) at the University of Bremen funded by the

DFG.

REFERENCES

Biddle, J. F., Lipp, J. S., Lever, M. A., Lloyd, K. G., Sörensen, K. B., Anderson, R.,

Fredricks, H. F., Elvert, M., Kelly, T. J., Schrag, D. P., Sogin, M. L., Brenchley, J.

E., Teske, A. House, C. H., Hinrichs, K. -U., 2006. Heterotrophic archaea dominate

sedimentary subsurface ecosystems off Peru. Proceedings of the National Academy

of Science U.S.A. 103, 3846-3851.

Blumenberg, M., Seifert, R., Reitner, J., Pape, T., Michaelis, W., 2004. Membrane lipid

patterns typify distinct anaerobic methanotrophic consortia. Proceedings of the

National Academy of Science U.S.A. 101, 11111-11116.

Curatolo, E., 1987. The physical properties of glycolipids. Biochimica et Biophysica

Acta 906, 111-136.

Chapter IV ________________________________________________________________________

123

De Rosa, M., Gambacorta, A., Gliozzi, A., 1986. Structure, biosíntesis, and

physicochemical properties of archaebacetrial lipids. Microbiological Reviews 50,

70-80.

Elvert, M., Hopmans, E.C., Treude, T., Boetius, A., Suess E., 2005. Spatial variations of

methanotrophic consortia at cold methane seeps: implications from a high-

resolution molecular and isotopic approach. Geobiology 3, 195–209.

Harvey, H. R., Fallon, R. D., Patton, J. S., 1986. The effect of organic matter and oxygen

on the degradation of bacterial membrane lipids in marine sediments. Geochimica et

Cosmochimica Acta 50, 795-804.

Hedges, J. I., Keil, R. G., 1995. Sedimentary organic matter preservation: an assesment

and speculative synthesis. Marine Chemistry 49, 81-115.

Heuer, V., Elvert, M., Tille, S., Krummen, M., Mollar, X. P., Hmelo, L. R., Hinrichs, K. -

U., 2006. Online �13C analysis of volatile fatty acids in sediment/porewater system

by liquid chromatography-isotope ratio mass spectrometry. Limnology and

Oceanography: Methods 4, 346-357.

Hinrichs, K. -U, Summons, R. E, Orphan, V., Sylva, S. P., Hayes, J. M., 2000. Molecular

and isotopic analyses of anaerobic methane-oxidizing communities in marine

sediments. Organic Geochemistry 31, 1685-1701.

Keil, R. G., Montlucon, D, B., Prahl, F. G., Hedges, J. I., 1994. Sorptive preservation of

labile organic matterin marine sediments. Nature 370, 549-552.

Lipp, J. S., Morono, Y, Inagaki, F., Hinrichs, K. -U., 2008. Significant contribution of

Archaea to the extant biomass in marine subsurface sediments. Nature 454, 991-

994.

Makula, R. A., 1978. Phospholipid composition of methane-utilizing bacteria. Journal of

Bacteriology 134, 771-777.

Mayer, L., 1994. Surface area controlof organic carbon accumulation in continental shelf

sediments. Geochimica et Cosmochimica Acta 58, 1271-1284.

Nauhaus, K., Treude, T., Boetius, A., Krüger, M., 2005. Environmental regulation of the

anaerobic oxidation of methane a comparison of ANME-1 and ANME-II

communities. Environmental Microbiology 7, 98-106.

Chapter IV ________________________________________________________________________

124

Peterson, B. L., Cummings, B. S., 2006. A review of chromatographic methods for the

assessment of phospholipids in biological samples. Biomedical Chromatography

20, 227-243.

Rossel, P. E., Lipp, J. S., Fredricks, H. F., Arnds, J., Boetius, A., Elvert, M., Hinrichs, K.

-U., 2008. Intact polar lipids of anaerobic methanotrophic archaea and associated

bacteria. Organic Geochemistry 39, 992-999.

Rütters, H., Sass, H., Cypionka, H., Rullkötter, J., 2001. Monoalkylether phospholipids in

the sulfate-reducing bacteria Desulfosarcina variabilis and Desulforhabdus

amnigenus. Archives of Microbiology 176, 435-442.

Schouten, S., Hughet, C., Hopmans, E. C., Kienhuis, M. V. M., Sinninghe Damsté, J. S.,

2007. Analyticyl methodology for TEX86 paleothermometry by high-performance

liquid chromatography/atmospheric pressure chemical ionization-mass

spectrometry. Analytical Chemistry 79, 2940-2944.

Sturt, H. F., Summons, R. E., Smith, K. J., Elvert, M., Hinrichs, K. -U., 2004. Intact polar

membrane lipids in prokaryotes and sediments deciphered by high-performance

liquid chromatography/electrospray ionization multistage mass spectrometry-new

biomarkers for biogeochemistry and microbial ecology. Rapid Communications in

Mass Spectrometry 18, 617-628.

Thompson, G. A., 1996. Lipids and membrane function in green algae. Biochimica et

Biophysica Acta 1302, 17-45.

White, D. C., Davis, W. M., Nickels, J. S., Kind, J. D., Bobbie, R. J., 1979. Oecologica

40, 51-62.

Widdel, F., Bak, F., 1992. Gram-negative mesophilic sulfate-reducing bacteria. In: The

prokaryotes, a handbook on the biology of bacteria: ecophysiology, isolation,

identification, applications (eds. Balows A, Trüper, H. G., Dworkin, M., Harder,

W., Schleifer, K. H.), 3352-3378, 2nd edn. Springer, Berlin Heidelberg New York.

Chapter V ________________________________________________________________________

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CHAPTER V

Diversity of intact polar membrane lipids

in marine seep environments

Pamela E. Rossela, Marcus Elvert and Kai-Uwe Hinrichsa

aOrganic Geochemistry Group, Department of Geosciences, University of Bremen, 28334 Bremen,

Germany

Keywords: intact polar membrane lipids, archaea, bacteria, seep, phospholipids,

glycolipids, non-phospholipids

Chapter V ________________________________________________________________________

126

ABSTRACT

Determination of the microbial community structure in natural habitats has been

the focus of many microbiological studies. However, most of the techniques applied are

inadequate because of their selectivity. Current approaches successfully applied to

characterize microbial communities include the analysis of intact polar membrane lipids

(IPLs). In this study structural information of IPLs from a variety of methane-bearing

environments is presented. This report provides a comprehensive spectral analysis of

IPLs from both archaea and bacteria occurring in seep environments. Analysis of lipid

extracts by high-performance liquid chromatography/electrospray ionization mass

spectrometry (HPLC-ESI-MS) provide information of the diversity in archaeal core lipids

including diphytanyl glyceroltetraether (GDGTs) and diphytanyl glyceroldiethers

(archaeols), with the latter presenting hydroxylation and cyclization in the lipid core.

Both core lipids were linked to a variety of glycosidic, phospho or mixed glycosidic and

phospho headgroups. Within the phospho headgroups, phosphatidylglycerol (PG) and

phosphatidylethanolamine (PE) were found attached to both GDGTs and archaeols,

whereas phosphatidylinositol (PI) and phosphatidylserine (PS) were only occurring with

archaeol. Additionally, bacteria produced both phospho and non-phospho lipids. Among

these, the major ones were PE and its methyl derivatives phosphatidyl-(N)-

methylethanolamine (PME) and phosphytidyl-(N,N)-dimethylethanolamine (PDME).

Bacteria derived non-phospho lipids included ornithine lipids, surfactin and betaine

lipids, with the latter characterized by odd fatty acid chains.

The results of membrane lipid analysis from a wide variety of seep environments

presented in this study confirmed the high diversity of microbes inhabiting these systems

and represent a base for further IPL studies from habitats in which anaerobic oxidation of

methane takes place.

Chapter V ________________________________________________________________________

127

INTRODUCTION

Determination of the microbial community structure in natural habitats has been

the focus of many microbiological studies. However, most of the applied methods,

including fluorescence in situ hybridization and cultures techniques, are inadequate for

several reasons: 1) they are selective methods (Wagner et al., 2003), 2) only a fraction of

the viable microbes is cultivable in laboratory conditions (MacCarthy and Murray, 1996

in fang 1998) and 3) limited information is gained about interaction between different

microbes (Findlay, 1996; Findlay et al., 1990). Current approaches to characterize

microbial communities include the analysis of intact polar membrane lipids (IPLs) by

high-performance liquid chromatography/electrospray ionization mass spectrometry

(HPLC-ESI-MS), technique that has been successfully applied in complex mixtures

(Sturt et al., 2004; Rütters et al., 2002; Biddle et al., 2006; Ertefai et al., 2008; Lipp et al.,

2008; Rossel et al., 2008; Schubotz et al., unpublished).

Methane-bearing environments represent a good opportunity to study microbial

communities using IPLs. These environments are considered as oases in which the

advection of fluids rich in methane and hydrogen sulfide support abundant

chemosynthetic life (Campbell, 2006). Communities inhabiting these systems include

sulfide oxidizing bacterial mats, diverse benthic macrofauna with methanotrophic

symbionts (Sahling et al., 2002; Levin, 2005) and the consortium of anaerobic

methanotrophic archaea (ANME) and sulfate reducing bacteria (SRB), which perform the

anaerobic oxidation methane (AOM, e.g., Hinrichs et al., 1999; Boetius et al., 2000;

Lösekann et al., 2007).

Among the diversity of IPLs observed in seep environments, several GDGTs and

archaeols with glycosidic and phospho headgroups from archaea and phospholipids from

bacteria have been observed (Rossel et al., 2008). Some of these lipids have already been

reported in other habitats such as anoxic water columns (ocean and lakes) (Schubotz et

al., unpublished; Ertefai et al., 2008) and in the deep subsurface (Biddle et al., 2006; Lipp

et al., 2008).

Chapter V ________________________________________________________________________

128

In order to evaluate the structural diversity of IPLs in seep systems using HPLC-

ESI-MS, a comprehensive spectral interpretation of both archaeal and bacterial IPLs from

these natural environments is provided.

MATERIAL AND METHODS

IPL analysis

Total lipid extracts of the samples were obtained with an automated microwave-

assisted extraction system (MARS-X, CEM, USA) for 15 minutes at 70°C or via

ultrasonication, using a modified Bligh and Dyer method (Sturt et al., 2004)

IPL analysis was performed with an HPLC system equipped with an ion-trap

mass spectrometer (ThermoFinnigan LCQ Deca XP) with an electrospray ionization

source (ESI) using protocols described previously by Sturt et al. (2004) and Biddle et al.

(2006). Briefly, a LiChospher Diol column (125mm x 2 mm, 5μm; Alltech Associates

INC., Deerfield, Il, USA) was used isothermally at 30°C in a ThermoFinnigan Surveyor

HPLC system. The following linear gradient was applied with a flow of 0.2 mL min-1:

100% A to 35% A: 65% B over 45 min, hold for 20min, then back to 100% A for 1 h to

equilibrate the system for the next injection, where A = 72:20:0.12:0.04 of hexane/2-

propanol/formic acid/14.8 M NH3aq and B = 88:10:0.12:0.04 of 2-propanol/water/formic

acid/14.8 M NH3aq..

Structural assignments were based on characteristic fragmentation patterns (cf.

Sturt et al., 2004) and by comparison with IPL inventories of cultured archaea and

bacteria (e.g., Koga et al., 1998; Koga and Morii, 2005; Hinrichs et al., unpublished

data). Individual IPLs were extracted by using the quasi-molecular ions obtained from the

full scan (m/z 500–2000), from which the MS2 daughter ion spectra information were

obtained. A general overview of the diversity of IPLs is given in Table V.1. The IPLs

identified are described according to their lipid class (i.e., glycolipids, phospholipids and

non-phospho lipids).

Chapter V ________________________________________________________________________

129

RESULTS AND DISCUSSION

IPL identification

1. Glycolipids

Carbohydrate-containing lipids are abundant molecules within thermophilic

archaea and bacteria (Langworthy, 1982). In some cases, these lipids are the major

components of the cytoplasmic membrane, especially in microbes without cell walls or

inhabiting hostile environments (Curatolo, 1987a). Most of the proposed functions for

glycolipids are based on their physical properties, which generally suggest that these

molecules participate in the stabilization, shape, extracellular recognition and ion bonding

in the membrane (Curatolo, 1987b).

Among the samples analyzed, the observed glycolipids were only associated with

archaea. Glycolipids have been suggested to be widely distributed among gram-positive

bacteria but rarely in gram-negative bacteria (López-Lara et al., 2003), which may

suggest low abundance of the former in seep environments. Within the archaeal

glycolipids, several archaeol-based IPLs (archaeol and hydroxyarchaeols) and GDGT-

based IPLs are described below.

1.1. Archaeol-based IPLs

Glycosidic archaeol-based IPLs ranged from archaeols (sn-2,3-diphytanyl

glycerol) containing 1 and 2 glycosidic headgroups (2Gly-AR, Fig. V.1a) to archaeols

with varying chain length, hydroxylation (extended archaeol, Fig. V.1b) and cyclization

(macrocyclic archaeol, Fig. V.1c). The occurrence of glycosidic archaeols has been

shown to be characteristic of the order Methanosarcinales (Koga et al., 1998).

Furthermore, the decrease in the proportion of archaeol relative to macrocyclic archaeol

as a response to the increase in the growth temperature has been observed in

Methanococcus jannaschii, a microbe isolated from hydrothermal vent systems (Sprott et

al., 1991).

During the fragmentation of archaeols, the MS2 daughter ion spectra exhibit a

main fragment indicative of the archaeol core (653 Da), together with another diagnostic

fragment (373 Da), which corresponds to one phytanyl chain with the glycerol positively

Chapter V ________________________________________________________________________

130

charged. Differently, the MS2 daughter ion spectra of hydroxylarchaeols show as major

fragment the loss of 296 Da, which represents the phytanyl chain with the hydroxyl

group. This pattern was consistently observed in all hydroxyarchaeols present in the

samples from this study.

Fig. V.1. MS2 positive ion spectra of several archaeol-based IPLs with glycosidic or mixed glycosidic and phospho headgroups. a) diglycosidic-archaeol (2Gly-AR), b) tentative glycosidic phospho-hydroxyarchaeol (Gly-P-OH-AR) and c) glycosidic-macrocyclic archaeol (Gly-MAR).

1.2. GDGT-based IPLs

Despite the occurrence of 1 to 8 cyclopentane rings depending of the growth

conditions (De Rosa et al., 1986), the C40 phytanyl chains of glycolipid tetraethers

(GDGTs) do not vary in length or saturation degree. However, GDGTs exhibit a wide

structural variety as is shown in this study. Glycosidic-GDGTs have been observed in

cultures of Sulfolobus schibatae and Methanobacterium thermoautotrophicum, the latter

also presenting GDGTs with mixed glycosidic and phospho headgroups (Koga et al.,

1993; Sturt et al., 2004).

The dominance of glycosidic headgroups in GDGTs has been reported in the deep

subsurface (Biddle et al., 2006; Lipp et al., 2008). Similarly, diglycosyl-GDGT (2Gly-

Chapter V ________________________________________________________________________

131

GDGT, Fig. V.2a) and 2Gly-GDGT with 18 Da more (Fig. V.2b) (H342-GDGT, Lipp et

al., 2008) have also been observed in seeps. The MS2 daughter ion spectrum of 2Gly-

GDGT shows the GDGT core as the most prominent fragment (Fig. V.2a). On the other

hand, the MS2 of 2Gly-GDGT with the additional 18 Da shows that this GDGT seems to

initially lose the diglycosyl headgroup with an ammonium adduct (1314 Da although

small fragment was observed), followed by a loss of 18 Da more (1314 Da to 1296 Da)

(Fig. V.2b). At this moment it is unclear if the additional 18 Da are contained in the

GDGT core or in the headgroup as previously suggested (GDGT core is the only

fragment observed in MS2, Lipp et al., 2008; Lipp and Hinrichs, submitted).

Nevertheless, the retention time and quasi-molecular ion information suggest that this

GDGT is the same as the one reported in deep subsurface environments. Furthermore,

although this lipid is present in seep environments, it is not the most abundant GDGT.

Other glycosidic GDGTs observed in seep environments included GDGTs with

up to 4 sugars (Fig. V.2c) and mixed glycosidic and phospho headgroups (Fig. V.2d and

e), as well as another 2Gly-GDGT with an additional unknown head group of 145 Da

(Fig. V.2e).

The MS2 daughter ion spectra of GDGTs show that GDGT containing only

glycosidic headgroups have the GDGT core as the most prominent fragment. However,

GDGTs containing both glycosidic and phospho headgroups lose first the glycosidic

headgroup and therefore the major fragment in MS2 is the GDGT core with the phospho

headgroup. Furthermore, during the fragmentation of some GDGTs with mixed

glycosidic and phospho headgroups it is also possible to observe two fragments in the

MS2: one indicative of the GDGT core with the phospho headgroup and another with the

GDGT core alone. Although we can not confirm the position of the glycosidic and

phospho headgroups, it has been previously suggested that in methanogens these two

headgroups are located in opposite ends of the GDGT core (Kates, 1997).

Chapter V ________________________________________________________________________

132

Fig. V.2. MS2 positive ion spectra of several glyceroldialkylglyceroltetraether (GDGT) based IPLs with glycosidic or mixed glycosidic and phospho headgroups. a) 2Gly-GDGT, b) 2Gly-GDGT+18, c) tetraglycosidic-GDGT (4Gly-GDGT), d) 2Gly-GDGT-phosphatidylglycerol (2Gly-GDGT-PG) and e) 2Gly-GDGT+ unknown head group of 145 Da (2Gly-GDGT+145).

Chapter V ________________________________________________________________________

133

2. Phospholipids

According to the fluid mosaic model, the primary function of phospholipids is to

define the permeability of the cell membrane (Madigan et al., 2003). Phospholipids are

also involved in solute transport, cell signaling as well as cell to cell recognition

(Madigan, et al., 2003). Additionally, phospholipids regulate the membrane structure by

modifying the headgroups, unsaturation degree and chain length of the acyl chains

(Hasegawa et al., 1980; Langworthy, 1982).

Phospholipids are ubiquitous in bacteria, however, the occurrence of archaeol and

GDGT with phospho headgroups among archaea is also widely distributed (Kates, 1997).

2.1 Phospholipids derived from archaea

Several phospholipids were observed in AOM environments. Among them,

archaeal phospholipids included phosphatidylglycerol (PG), phosphatidylinositol (PI),

phosphatidylserine (PS) and phosphatidylethanolamine (PE) linked to archaeol (Fig. V.3a

to 3e), whereas the common headgroups linked to GDGT were only PG and PE (Fig.

V.4a and b). The occurrence of PG, PI, PS and PE with GDGT and archaeol has been

frequently observed in methanogens (Kates, 1997; Sprott, 1992). Nevertheles, PG-

archaeol, which has been reported in Methanosarcina mazei (Sprott, 1992), is also a

characteristic IPL of Halophiles (Kates, 1997).

In agreement with the fragmentation pattern of hydroxyarchaeol described for

glycosidic-archaeol based IPL (section 1.1), the loss of the phytanyl chain with the

hydroxyl group (loss of 296 Da) was also observed in the MS2 daughter ion spectra of

the hydroxyarchaeols with phospho headgroups. Furthermore, the occurrence of 373 Da

fragment in MS2 previously described was also observed here. Nevertheless, the most

common fragment for phospho-hydroxyarchaeols in MS2 was 453 Da, which

corresponds to one phytanyl chain with the phosphate group. Different from glycosidic-

archaeols, the MS2 daughter ion spectra from archaeols with phospho headgroups show

the archaeol core with the phosphate group (733 Da). In addition to the previously

described phospho-archaeol based IPLs, the occurrence in ANME-2 dominated sediments

and carbonate mats of another archaeol with an unknown phospho headgroup of 223 Da

was observed.

Chapter V ________________________________________________________________________

134

Fig. V.3. MS2 positive ion spectra of diverse archaeol-based IPLs with phospho headgroups. a) PG-OH-AR, b) PI-OH-AR, c) PS-AR, c) PS-OH-AR, d) PE-OH-AR and e) archaeol with unknown phospho headgroup of 223 Da (P-AR+223).

Chapter V ________________________________________________________________________

135

Differently from the glycosidic GDGTs, the MS2 daughter ion spectra of the

GDGTs with phospho headgroups usually show two or three fragments: 1) fragment

indicative of the GDGT core with one of the two phospho headgroups, 2) the GDGT core

with only the phosphate group and 3) the GDGT core alone (Fig. V.4.).

Fig. V.4. MS2 positive ion spectra of diverse GDGT-based IPLs with phospho headgroups. a) PE-GDGT-PG and b) 2PG-GDGT.

2.2 Phospholipids derived from bacteria

The most common bacterial phospholipids observed were PE and its methyl

derivatives phosphatidyl-(N)-methylethanolamine (PME) and phosphytidyl-(N,N)-

dimethylethanolamine (PDME) (Fig. V.5a to V.5c). PE has been found to be the most

dominant IPL in SRB such as Desulfosarcina variabilis and Desulforhabdus amnigenus

(Rütter et al., 2001; Sturt et al., 2004). PME and PDME have been reported in

methanotrophic bacteria such as Methylosinas trichosporium and Methylobacterium

organophilum (Makula, 1978; Goldfine, 1984; Fang et al., 2000) as well as in sulfide

oxidizers (Barridge and Shively, 1968). The MS2 daughter ion spectra of the

diacylglycerol phospholipids PE, PME and PDME show the loss of their phospho

headgroups (141, 155 and 169 Da, respectively). This is the same fragmentation pattern

Chapter V ________________________________________________________________________

136

of phospholipids previously described in literature (e.g., Rütter et al., 2001; Sturt et al.,

2004).

Fig. V.5. MS2 positive ion spectra of the major bacterial phospholipids observed in seep environments. As examples a) PE-DAG C32:2, b) PME C32:2, c) PDME C34:2. DAG = diacylglycerol

3. Phosphorus-free membrane lipids

The production of phosphorus-free membrane lipids has been suggested to occur

in organism exposed to physiological stress conditions such as limitation of nitrogen or

phosphate (López-Lara et al., 2003). In our data set three main types of bacterial IPLs

which do not contain carbohydrates or phospho headgroups were identified. The

occurrence of some of them has been related to phosphate limitation conditions during

the growth (e.g., ornithine and betaine lipids), whereas others seem to provide surface

active properties to the membrane containing these molecules (e.g., surfactin, Vater,

1986).

3.1. Ornithine lipids

Ornithine lipids (OL) contain one amidified 3-hydroxy fatty acid to which another

fatty acid residue is attached (López-Lara et al., 2003). OL are widely spread among

Chapter V ________________________________________________________________________

137

gram-negative bacteria (Imhoff and Bias-Inhoff, 1995) involved in sulfate reduction

(Desulfovibrio gigas), sulphur oxidation (Thiobacillus thiooxidans) and iron metabolism

(Rhodomicrobium vannielii) (Makula and Finnerty, 1975; Knoche and Shively, 1972).

High abundance of OL seems to substitute phospholipids such as PE, PG and DPG, in

Pseudomonas fluorescens in response to change towards phospho-limited conditions

(Minnikin and Abdolrahimzadeh, 1974). Moreover, it has also been suggested that OL

partially control the iron oxidation metabolism in Thiobacillus ferrooxidans (Ghosh and

Misha, 1987).

The MS2 daughter ion spectra of OL usually show three fragments, with the first

indicative of the ornithine with one fatty acid, and the other two corresponding to the two

consecutive losses of 18 Da, indicative of loss of two molecules of water according to the

OL fragmentation pattern reported by Aygun-Sunar, et al. (2006) (Fig V.6.).

Fig. V.6. MS2 positive ion spectrum of C34:1 OL as an example for ornithine lipids.

3.2. Betaine lipids with odd fatty acid chains

Betaine ether linked glycerolipids (BL) are membrane components widely

distributed among higher plants, algae, protozoa and some fungi (Dembitsky, 1996).

Their structure in aquatic algae is frequently characterized by the presence of C14, C16,

C18, C20 and C22 fatty acids (Sato et al., 1992; Dembitsky, 1996). Their synthesis in

bacteria has been observed in the anoxygenic photosynthetic bacterium Rhodobacter

sphaeroides (Benning et al., 1995; Hoffman and Eichenberger, 1996) and in plant-

associated bacteria such as Sinorhizobium meliloti (Lopez-Lara et al., 2003). It has been

documented that phosphate-deprived cells of Rhodobacter s. growing in phosphate

Chapter V ________________________________________________________________________

138

concentrations <0.1mM can decrease their membrane phospholipid content from 90% to

22% (Benning et al., 1995).

Characteristic fatty acids of BL from bacteria have not been reported, but detailed

description of the BL from algae, which are composed by fatty acids with even carbon

numbers, suggest that the BL containing odd fatty acids chains (BL-odd) may be bacterial

derived. This has been previously suggested by Schubotz et al. (submitted), who

observed an increase of BL content with C15 and C17 fatty acids in the anoxic water from

the Black Sea.

In general the MS2 daughter ion spectra of BL show four fragments; the first and

second fragments correspond to the fatty acid chain in the sn1 position with and without

hydroxyl group, whereas the third and fourth fragments correspond the fatty acids in the

sn2 position with and without hydroxyl group (Fig V.7).

Fig. V.7. MS2 positive ion spectrum of C31:1 BL-odd as an example for betaine lipids.

3.3. Surfactins

Biosurfactants are molecules of interest in biotechnology and are grouped in five

different classes: glycolipids, phospholipids, lipopeptides/lipoproteins, polymeric

surfactants and particulate surfactants (Muthusamy et al., 2008). Among these classes,

surfactins, which are macrocyclic heptapeptides linked to a long-chain �-hydroxy fatty

acid (Hue et al., 2001), are considered as one of the most powerful biosurfactants (Vater,

1986). The cyclic form of surfactins results from the link between the hydroxyl group of

the fatty acid with the C-terminal carbonyl to form a lactone ring (Fig. V.8) (Hue et al.,

2001). Glutamic acid, leucine, valine and aspartic acid are the common amino acids

Chapter V ________________________________________________________________________

139

forming the ring. Surfactin structure has been shown to vary in both amino acid

composition and acyl chain length, the latter found with 12 and 15 carbon atoms (Hue et

al., 2001).

Several are the properties assigned to surfactins, including surface active (Vater et

al., 1986), antibiotics (Georgiou et al., 1992) and antifungal (Thimon et al., 1992), among

others. The surface active properties of surfactin have been suggested to increase

significantly when glutamic and aspartic acids are present (Georgiou et al., 1992).

The production of these molecules is affected by several factors. The carbon

source present in the system (usually hydrocarbons or carbohydrates) can influence not

only the surfactant production but also their structure, especially the hydrophobic tail

(Georgiou et al., 1992). Furthermore, temperature, pH and oxygen seem to affect

surfactant production as well (Kim et al., 1990; Gerson and Zajic, 1978). It has also been

reported that surfactin production is enhanced by the increase in iron and manganese

concentrations in the growth media (Cooper et al., 1981).

The MS2 daughter ion spectra of surfactins (Fig. V.8) were characterized by a

prominent 685 Da fragment, which corresponds to the loss of the protonated peptide (six

out of seven aminoacids with H+). The main quasi-molecular ion present is 1036.5, which

corresponds to a surfactin with glutamic acid, leucine, leucine, valine, aspartic acid,

leucine and leucine amino acids and a hydroxy fatty acid iso-C15. Other quasi-molecular

ions observed within the surfactin peak were 1008.5 and 1022.5, which indicate the

change of the hydroxy fatty acid from 15 to 13 and 14 carbon atoms, respectively.

Fig. V.8. MS2 positive ion spectrum of surfactin with glutamic acid, leucine, leucine, valine, aspartic acid, leucine and leucine amino acids and a hydroxyfatty acid iso-C15.

Chapter V ________________________________________________________________________

140

4. Unknown IPLs

Two unknown IPLs frequently observed in the analyzed samples were IPL a and

b, the first represented by the two quasi molecular ions 734.3 and 706.4 m/z, and the

second by the quasi molecular ion 1148.0 m/z. The MS2 daughter ion spectrum of IPL a

in positive mode (Fig. V.9a and b) shows three main fragments (losses of 193.5 Da, 18

Da and 46 Da). Information obtained from negative ion mode for 706.4 m/z indicate that

the molecule has 18 Da less when is negatively charged (687.5 m/z), which is also

indicated in the MS2 by a loss of 175.2 Da instead of 193.5 Da (Fig. V.9c). Additionally,

the occurrence of two other fragments in the MS2 of the negative ion mode, indicates the

presence of the fatty acids C17:1 and C16:1. The occurrence of these lipids in carbonate

mats from the Black Sea, together with presence of fatty acids in their lipid structure,

suggest that these lipids are bacterial derived.

The MS2 daughter ion spectrum of IPL b (Fig. V.9e) shows several fragments.

The first is 993.6 Da, which could be analogical to the diglycosyl archaeol core after loss

of 155 Da (possible analog to PME). However, the major fragment in the MS2 daughter

ion spectrum was 873.4 Da, which results from a consecutive loss of 120 Da.

Unfortunately, negative ion mode for this lipid was always very noisy and did not

provide additional information about its molecular structure. Unknown b was frequently

observed in ANME-2 dominated sediments.

Chapter V ________________________________________________________________________

141

Fig. V.9. MS2 positive ion spectra of two unknowns frequently observed in seep environments. a) unknown IPL a with quasi molecular ion 734.3 m/z b) unknown IPL a with quasi molecular ion 706.4 m/z c) MS2 daughter ion spectrum in negative mode for unknown IPL a 706.5, which is 687.5 m/z due to a loss of 18 Da and d) unknown IPL b with quasi molecular ion 1148.0 m/z.

Cha

pter

V

_

142

Tab

le V

.1. I

ntac

t pol

ar m

embr

ane

lipid

div

ersi

ty in

seep

env

iron

men

ts

Lip

id n

ame

RT

R

ange

of q

uasi

m

olec

ular

ions

N

eutr

al lo

ssa

diag

nost

ic

ion

in M

S2

neut

ral l

oss o

r di

agno

stic

fr

agm

ent i

n M

S2 r

epre

sent

s O

bser

ved

in:

2Gly

-AR

-0

.68

994.

6 [M

+ N

H4]+1

34

1 65

3 Lo

ss

of

digl

ycos

yl

head

gr

oup

with

an

NH

4 add

uct

Arc

haea

, AN

ME-

2 (R

osse

l et

al.,

200

8), M

etha

noca

ldoc

occu

s ja

nnas

chii

(Stu

rt et

al.,

200

4), d

eep

subs

urfa

ce (B

iddl

e et

al.,

200

6; L

ipp

et a

l., 2

008)

Te

ntat

ive

Gly

-P-O

H-A

R

exte

nded

-0.9

5 98

1.7

[M+H

]+1

296

685

Loss

of

phyt

anyl

cha

in w

ith a

n hy

drox

yl g

roup

A

rcha

ea, p

ossi

bly

AN

ME-

2 (th

is s

tudy

), ar

chae

ols

with

C25

cha

in h

ave

been

pre

viou

sly

repo

rted

in

extre

me

Hal

ophi

les (

Kog

a et

al.,

199

3; 2

008)

and

in c

old

seep

sedi

men

ts fr

om E

aste

rn M

edite

rran

ean

Sea

(Sta

dnits

kaia

et a

l., 2

008)

G

ly-M

AR

-0

.75

831.

2 [M

+NH

4]+1

180

651

Loss

of

glyc

osyl

hea

d gr

oup

with

an

NH

4 add

uct

Arc

haea

, AN

ME-

2 (th

is st

udy)

, Met

hano

cald

ococ

cus j

anna

schi

i (St

urt e

t al.,

200

4)

PG-O

H-A

R

0.54

82

3.4

[M+H

]+1

296

527,

453

Lo

ss o

f ph

ytan

yl c

hain

with

an

hydr

oxyl

gro

up

Arc

haea

, A

NM

E-2

(Ros

sel

et a

l., 2

008)

, M

etha

nosa

rcin

a ba

rker

i (K

oga

and

Mor

ii et

al.,

200

5),

Hal

ophi

les (

Kat

es, 1

997)

PI

-OH

-AR

1.

00

911.

5 [M

+H]+1

29

6 61

5, 4

53

Loss

of

phyt

anyl

cha

in w

ith a

n hy

drox

yl g

roup

A

rcha

ea, A

NM

E-2

and

AN

ME-

3 (R

osse

l et a

l., 2

008)

PS-O

H-A

R

-0.9

0 83

6.4

[M+H

]+1

296

540,

453

Lo

ss o

f ph

ytan

yl c

hain

with

an

hydr

oxyl

gro

up

Arc

haea

, AN

ME-

2 an

d A

NM

E-3

(Ros

sel e

t al.,

200

8), M

etha

nosa

rcin

a ba

rker

i (K

oga

et a

l., 1

993)

PS-A

R

-0.8

7 82

0.4

[M+H

]+1

87

733,

453

Lo

ss o

f ser

ine

Arc

haea

, A

NM

E-2

and

AN

ME-

3 (R

osse

l et

al.,

200

8),

Met

hano

bact

eriu

m t

herm

oaut

otro

phic

um

(Kog

a et

al.,

199

3), M

etha

noca

ldoc

occu

s jan

nasc

hii (

Stur

t et a

l., 2

004)

PE

-OH

-AR

-0

.72

792.

4 [M

+H]+1

29

6 49

6, 4

53

Loss

of

phyt

anyl

cha

in w

ith a

n hy

drox

yl g

roup

A

rcha

ea,

AN

ME-

2 (R

osse

l et

al

., 20

08),

Met

hano

thri

x so

ehng

enii

(Kog

a et

al

., 19

93);

Met

hano

sarc

ina

bark

eri (

Kog

a an

d M

orii

et a

l., 2

005)

2G

ly-G

DG

T -0

.72

1632

.1-1

645.

1b [M

+NH

4]+1

341

GD

GT

core

Lo

ss

of

digl

ycos

yl

head

gr

oup

with

an

NH

4 add

uct

Arc

haea

, AN

ME-

1 (R

osse

l et a

l., 2

008)

and

dee

p su

bsur

face

(B

iddl

e et

al.,

200

6; L

ipp

et a

l., 2

008;

St

urt e

t al.,

200

4), S

ulfo

lobu

s sh

ibat

ae (

Stur

t et a

l., 2

004)

, Met

hano

bact

eriu

m th

erm

oaut

otro

phic

um

(Kog

a et

al.,

199

3)

2Gly

-GD

GT+

18

-0.7

7 16

50.1

-166

1.1b

[M+N

H4]+1

34

1 or

360

13

14,

GD

GT

core

Lo

ss

of

digl

ycos

yl

head

gr

oup

with

an

NH

4 ad

duct

or

unkn

own

head

gro

up o

f 34

2 D

a w

ith a

n N

H4 a

dduc

t (36

0 D

a)

Arc

haea

in

deep

bio

sphe

re s

edim

ents

(Li

pp a

nd H

inric

hs,

unp

ublis

hed

data

) an

d N

itros

opum

ilus

mar

itim

us (S

chou

ten

et a

l., 2

008)

4Gly

-GD

GT

+1.1

0 19

58.1

-196

9.1b

[M+N

H4]+1

66

7 G

DG

T co

re

Loss

of

tetra

glyc

osyl

hea

d gr

oup

with

an

NH

4 add

uct

Arc

haea

, AN

ME-

1 (th

is st

udy)

2Gly

-GD

GT-

PG

+1.0

6 17

87.1

-179

8.1b

[M+N

H4]+1

34

1 G

DG

T co

re+P

G

Loss

of

di

glyc

osyl

he

ad

grou

p w

ith a

n N

H4 a

dduc

t A

rcha

ea, A

NM

E-1

(this

stud

y), M

etha

nosp

irill

um h

unga

tei (

Kog

a et

al.,

199

3)

2Gly

-GD

GT+

145

-0.6

9 17

78.1

-178

9.7b

[M+N

H4]+1

34

1 G

DG

T co

re+1

45

Loss

of

di

glyc

osyl

he

ad

grou

p w

ith a

n N

H4 a

dduc

t A

rcha

ea, A

NM

E-1

(this

stud

y)

PE-G

DG

T-PG

+1

.06

1569

.1-1

579.

7b [M

+H]+1

15

4, 4

3 G

DG

T+PE

Lo

ss o

f PG

with

the

pho

spha

te

grou

p w

ithou

t on

e ox

ygen

, fo

llow

ed

by

the

lost

of

et

hano

lam

ine

Arc

haea

, AN

ME-

1(th

is st

udy)

2PG

-GD

GT

+1.0

9 16

00.1

-161

0.1b

[M+H

]+1

154,

74

GD

GT+

PG

Loss

of

PG w

ith t

he p

hosp

hate

gr

oup

with

out

one

oxyg

en,

follo

wed

by

the

lost

of g

lyce

rol

Arc

haea

, AN

ME-

1 (th

is st

udy)

PE (D

AG

) -0

.76

608.

6-74

4.5c

[M+H

]+1

141

Fatty

ac

ids

+gly

cero

l Lo

ss o

f PE

Met

hano

troph

ic b

acte

ria (M

akul

a, 1

978;

Gol

dfin

e, 1

984;

Fan

g et

al.,

200

0), D

esul

fosa

rcin

a va

riabi

lis

(Rüt

ters

et a

l., 2

001;

Stu

rt et

al.,

200

4)

PME

(DA

G)

-075

70

2.4-

802.

5c [M

+H]+1

15

5 Fa

tty

acid

s +g

lyce

rol

Loss

of P

ME

Met

hano

troph

ic b

acte

ria (

Mak

ula,

197

8; G

oldf

ine,

198

4; F

ang

et a

l., 2

000)

, su

lfide

oxi

dize

r (B

arrid

ge a

nd S

hive

ly, 1

968)

PD

ME

(DA

G)

-0.7

5 71

6.6-

746.

6c [M

+H]+1

16

9 Fa

tty

acid

s +g

lyce

rol

Loss

of P

DM

E M

etha

notro

phic

bac

teria

(M

akul

a, 1

978;

Gol

dfin

e, 1

984;

Fan

g et

al.,

200

0),

sulfi

de o

xidi

zer

(Bar

ridge

and

Shi

vely

, 196

8)

OL

-0.8

2 59

7.5-

721.

5c [M

+H]+1

--

----

----

--

Orn

ithin

e+

fatty

aci

d Lo

ss o

f on

e fa

tty a

cid

chai

n an

d re

mai

n th

e hy

drox

yl

fatty

ac

id

with

the

orni

thin

e he

adgr

oup

Bac

teria

gra

m-n

egat

ive

perfo

rmin

g su

lfur

redu

ctio

n, s

ulfu

r an

d iro

n ox

idat

ion

(Mak

ula

and

Fine

rty,

1975

; Kno

che

and

Shiv

ely,

197

2; Im

hoff

and

Bia

s-Im

hoff

, 199

5)

BL

-0.6

8 71

6.6-

746.

6c [M

+H]+1

--

----

----

--

236

Indi

cativ

e of

th

e be

tain

e he

adgr

oup

BL

with

C14

, C16

, C18

fat

ty a

cids

der

ived

fro

m a

quat

ic a

lgae

(D

embi

tsky

, 199

6), B

L w

ith o

dd f

atty

ac

ids s

uch

as C

15 a

nd, C

17 s

eem

to d

eriv

e fr

om b

acte

ria d

ue to

thei

r occ

urre

nce

in a

noxi

c w

ater

s fr

om

the

Bla

ck S

ea (S

chub

otz

et a

l., su

bmitt

ed; t

his s

tudy

) Su

rfac

tin

-0.5

9 10

08.5

-103

6.0d

[M+H

]+1

----

----

----

68

5 Lo

ss

of

the

prot

onat

ed

pept

ide

(six

out

of

seve

n am

inoa

cids

with

H

+ )

Baci

llus

sp (

Vat

er, 1

986)

, unk

now

n ba

cter

ia d

ue to

thei

r oc

curr

ence

in th

e bl

ack

nodu

les

from

the

Bla

ck S

ea m

ats (

this

stud

y)

Unk

now

n a

-0.7

3 70

6.3

and

734.

3 19

4 an

d th

en 1

8 --

----

----

--

----

----

----

O

bser

ved

in c

arbo

nate

mat

s fro

m th

e B

lack

Sea

U

nkno

wn

b +1

.3

1148

.0

993.

6 an

d 87

3 --

----

----

--

----

----

----

Fr

eque

ntly

obs

erve

d in

AN

ME-

2 do

min

ated

sedi

men

ts

RT=

rete

ntio

n in

dex

rela

tive

to C

16-P

AF

inte

rnal

sta

ndar

d, a

The

neut

ral l

oss

resu

lts fr

om th

e lo

ss o

f the

hea

dgro

up p

lus

the

nece

ssar

y [H

]+ to c

harg

e th

e co

re o

f the

IPL

in M

S2, b

Ran

ge o

f mas

ses

cons

ider

a G

DG

T co

re w

ith 0

to 5

cy

clop

enta

ne r

ings

; c Inc

lude

fat

ty a

cids

of

diff

eren

t len

gth

and

satu

ratio

ns; d I

nclu

de s

urfa

ctin

mol

ecul

es w

ith g

luta

mic

aci

d, le

ucin

e, le

ucin

e, v

alin

e, a

spar

tic a

cid,

leuc

ine

and

leuc

ine

and

the

hydr

oxyl

fat

ty a

cids

with

13,

14

and

15

carb

on a

tom

s. A

bbre

viat

ions

: AR

= a

rcha

eol,

BL

= be

tain

e lip

ids,

2Gly

= d

igly

cosy

l, D

AG

=dia

cylg

lyce

rol,

GD

GT

= gl

ycer

oldi

alky

lgly

cero

ltetra

ethe

r, O

H-A

R =

hyd

roxy

arch

aeol

,, O

L =

orni

thin

e lip

ids,

PDM

E =

phos

phat

idyl

-(N

,N)-

dim

ethy

leth

anol

amin

e, P

E =

phos

phat

idyl

etha

nola

min

e, P

G =

pho

spha

tidyl

gylc

erol

, PI =

pho

spha

tidyl

inos

itol,

PME

= ph

osph

atid

yl-(

N)-

met

hyle

than

olam

ine,

PS

= ph

osph

atid

ylse

rine.

Chapter V ________________________________________________________________________

143

CONCLUDING REMARKS

Using HPLC-ESI-MS a total of 25 different IPL structures observed in seep

environments were discussed. The interpretation of mass spectra provided useful

structural information of archaeal lipids including GDGTs and archaeols linked to a

variety of glycosidic and phospho headgroups such as diglycosyl, tetraglycosyl, PG, PE

PI and PS. Bacterial IPLs commonly observed included the phospholipids PE, PME and

PDME as well as non-phospho lipids such as ornithine lipids, surfactin and betaine lipids,

with the latter characterized by odd fatty acid chains.

These results show the potential of intact polar membrane lipid analysis in the

evaluation of microbial diversity in a variety of methane-bearing environments and

provide a base for further IPL studies in natural environments such as those in which

anaerobic oxidation of methane takes place.

REFERENCES

Aygun-Sunar, S., Mandaci, S., Koch, H. -G., Murray, I. V. J., Goldfine, H., Daldal, F.,

2006. Ornithine lipid required for optimal steady-state amounts of c-typer

cytochromes in Rhodobacter capsulatus. Molecular Microbiology 61, 418-435.

Barridge, J. K., Shively, J. M., 1968. Phospholipids of the Thiobacilli. Journal of

Bacteriology 95, 2182-2185.

Benning, C., Huang, Z. –H., Gage, D. A., 1995. Accumulation of a novel glycolipid and a

betaine lipid in cells of Rhodobacter sphaeroides grown under phosphate limitation.

Archives of Biochemistry and Biophysics 317, 103-111.

Biddle, J. F., Lipp, J. S., Lever, M. A., Lloyd, K. G., Sörensen, K. B., Anderson, R.,

Fredricks, H. F., Elvert, M., Kelly, T J., Schrag, D. P., Sogin, M. L., Brenchley, J.

E., Teske, A. House, C. H., Hinrichs, K. -U., 2006. Heterotrophic archaea dominate

sedimentary subsurface ecosystems off Peru. Proceedings of the National Academy

of Science U.S.A. 103, 3846-3851.

Boetius, A., Ravenschlag, K., Schubert, C. J., Rickert, D., Widdel, F., Gieseke, A.,

Amann, R., Jørgensen, B. B., Witte, U., Pfannkuche, O., 2000. A marine microbial

Chapter V ________________________________________________________________________

144

consortium apparently mediating anaerobic oxidation of methane. Nature 407, 623-

626.

Campbell, K. A., 2006. Hydrocarbon seep and hydrothermal vent paleoenvironments and

paleontology: past developments and future research directions. Palaeogeography,

Palaeoclimatology, Palaeoecology 232, 362-407.

Cooper, D. G., MacDonald, C. R., Duff, S. J. B., Kosaric, N., 1981. Enhanced production

of surfactin from Bacillus subtilis by continuos product removal and metal cation

additions. Applied and Environmental Microbiology 42, 408-412. 1981

Curatolo, E., 1987a. Glycolipid function. Biochimica et Biophysica Acta 906, 137-160.

Curatolo, E., 1987a. The physical properties of glycolipids. Biochimica et Biophysica

Acta 906, 111-136.

De Rosa, M., Gambacorta, A., Gliozzi, A., 1986. Structure, biosynthesis, and

physicochemical Properties of Archaeabacterial Lipids. Microbiological Reviews

50, 70-80.

Dembitsky, V. M., 1996. Betaine ether-linked glycerolipids: chemistry and biology.

Progress in Lipid Research 35: 1-51.

Ertefai, T. F., Fisher, M. C., Fredricks, H. F., Lipp, J. S., Pearson, A., Birgel, D., Udert,

K. M., Cavanaugh, C. M., Gschwend, P. M., Hinrichs, K. -U., 2008. Vertical

distribution of microbial lipids and functional genes in chemically distinct layers of

a highly polluted meromictic lake. Organic Geochemistry 39, 1572-1588.

Fang, J. S., Barcelona, M. J., Semrau, J. D., 2000. Characterization of methanotrophic

bacteria on the basis of intact phospholipid profiles. FEMS Microbial Letters 189,

67-72.

Findlay, R. H., 1996. The use of phospholipids fatty acids to determine microbial

community structure. Molecular Microbial Ecology Manual 4, 1-17.

Findlay, R. H., Trexler, M. B., White, D. C., 1990. Response of a benthic microbial

community to biotic disturbance. Marine Ecology Progress Series 62, 135-148.

Georgio, G., Lin, S. -C., Sharma, M., 1992. Surface-active compound from

microorganisms. Nature biotechnology 10, 60-64.

Chapter V ________________________________________________________________________

145

Gerson, D. F., Zajic, J. E., 1978. Surfactant production from hydrocarbons by

Corynebacterium lepus, sp nov. And Pseudomonas asphaltenicus, sp nov.

Developments in Industruial Microbiology 19, 597-599.

Ghosh, M., Misha, A. K., 1987. Occurrence, identification and possible significance of

ornithine lipid in Thiobacillus ferrooxidans. Biochemical and Biophysical Research

Communications 142, 925-931.

Goldfine, H., 1984. Bacterial membrane and lipid packing theory. Journal of Lipid

Research 25, 1501-1507.

Hasegawa, Y., Kawada, N., Nosoh, Y., 1980. Change in Chemicals composition of

membrane of Bacilus caldotenax after shifting the growth temperature. Archives of

Microbiology 126, 103-108.

Hinrichs, K. -U., Hayes, J. S., Sylva, S. P., Brewer, P. G., DeLong, E. F., 1999. Methane-

consuming archaebacteria in marine sediments. Nature 398, 802-805.

Hofman, M., Eichenberger, W., 1996. Biosynthesis of diacylglyceryl-N,N,N-

trimethylhomoserine in Rhodobacter sphaeroides and evidence for lipid-linked N

methylation. Journal of Bacteriology 178, 6140-6144.

Hue, N., Serni, L., Laprévote, O., 2001. Structural investigation of cyclic peptidolipids

from Bacillus subtilis by high-energy tandem mass spectrometry. Rapid

Communications in Mass Spectrometry 15, 203-209.

Imhoff, J. F., Bias-Imhoff, U., 1995. Lipids, Quinones and Fatty Acids of Anoxygenic

Phototrophic Bacteria. In Anoxygenic Photosynthetic Bacteria (eds. Blankenship, R.

E., Madigan, M. T., Bauer, C. E.), 179-205. 1995 Kluwer Academic Publishers.

Printed in The Netherlands.

Kates, M., 1997. Diether and tetraether phospholipids and glycolipids as molecular

markers for archaeabacteria (archaea). In: Molecular Markers in Environmental

Geochemistry (ed. Eganhause, R. P.), 35–48, Oxford University Press.

Kim, J. -S., Powalla, M., Lang, S., Wagner, F., Lundsdorf, H., Wray, F., 1990. Microbial

glycolipid production under nitrogen limitation and resting cell conditions. Journal

of Biotechnology 13, 257-266.

Knoche, H. W., Shively, J. M., 1972. The structure of an ornithine –containing lipid from

Thiobacillus thioxidans. The Journal of Biological Chemistry 247, 170-178.

Chapter V ________________________________________________________________________

146

Koga, Y., Morii, H., 2005. Recent advances in structural research on ether lipids from

archaea including comparative and physiological aspects. Bioscience Biotechnology

and Biochemistry 69, 2019-2034.

Koga, Y., Morii, H., Akagawa-Matsushita, M., Ohga, I., 1998. Correlation of polar lipid

composition with 16S rRNA phylogeny in methanogens. Further analysis of lipid

component parts. Bioscience Biotechnology and Biochemistry 62, 230-236.

Koga, Y., Nishihara, M., Morii, H., Akagawa-Matsushita, M., 1993. Ether polar lipids of

methanogenic bacteria: structures, comparative aspects, and biosyntheses. 1996.

Microbiological Reviews 57, 164-182.

Langworthy, T. A., 1982. Lipids of bacteria in extreme environments. In: Current Topics

in Membranes and Transport, Membrane lipids of prokaryotes 17 (eds. Razin, S.,

Rottem, S.), 45-77. Academic press.

Levin, L., 2005. Ecology of cold seep sediments: interactions of fauna with fluid flow,

chemistry and microbes. In: Oceanography and Marine Biology: An annual Review

43 (eds. Gibson, R. N., Atkinson, R. J. A., Gordon, J. D. M), 1-46. Taylor and

Francis.

Lipp, J. S., Morono, Y, Inagaki, F., Hinrichs, K.-U., 2008. Significant contribution of

Archaea to the extant biomass in marine subsurface sediments. Nature 454, 991-

994.

López –Lara, I. M., Sohlenkamp, C., Geiger, O., 2003. Membrane lipids in plant-

associated bacteria: their biosyntheses and possible functions. Molecular Plant-

Microbe Interactions Overview 16, 567-579.

Lösekann, T., Knittel, K., Nadalig, T., Fuchs, B., Niemann, H., Boetius, A., Amann, R.,

2007. Diversity and abundance of aerobic and anaerobic methane oxidizers at the

Haakon Mosby Mud Volcano, Barents Sea. Applied and Environmental

Microbiology 73, 3348–3362.

MacCarthy, C. M., Murray, L., 1996. Viability and metabolic features of bacteria

indigenous to a contaminated deep aquifer. Microbial Ecology 32, 305-321.

Madigan, M. T., Martinko, J. M., Parker, J., 2003. Brock - Biology of Microorganisms.

10th Edition, Pearson education Inc. Upper Saddle River, New Jersey.

Chapter V ________________________________________________________________________

147

Makula, R. A., 1978. Phospholipid composition of methane-utilizing bacteria. Journal of

Bacteriology 134, 771-777.

Makula, R. A., Finnerty, W. R., 1975. Isolation and characterization of an ornithine-

containing lipid from Desulfovibrio gigas. Journal of Bacteriology 123, 523-529.

Minnikin, D. E., Abdolrahimzadeh, H., 1974. Effect of pH on the proportions of polar

lipids, in chemostat cultures of Bacillus subtilis. Journal of Bacteriology 120, 999-

1003.

Muthusamy, K., Gopalakrishnan, S., Ravi, T. K., Sivachidambaram, P., 2008.

Biosurfactants: properties, commercial production and application. Current Science

94, 736-746.

Rossel, P. E., Lipp, J. S., Fredricks, H. F., Arnds, J., Boetius, A., Elvert, M., Hinrichs, K.

-U., 2008. Intact polar lipids of anaerobic methanotrophic archaea and associated

bacteria. Organic Geochemistry 39, 992-999.

Rütters, H., Sass, H., Cypionka, H., Rullkötter, J., 2001. Monoalkylether phospholipids in

the sulfate-reducing bacteria Desulfosarcina variabilis and Desulforhabdus

amnigenus. Archives of Microbiology 176, 435-442.

Rütters, H., Sass, H., Cypionka, H., Rullkötter, J., 2002. Phospholipid analysis as a tool

to study complex microbial communities in marine sediments. Journal of

Microbiological Methods 48, 149-160.

Sahling, H., Rickert, D., Lee, R. W., Linke, P., Suess, E., 2002. Macrofaunal community

structure and sulfide flux at gas hydrate deposits from the Cascadia convergent

margin, NE Pacific. Marine Ecological Progress Series 231, 121-138.

Sato, N., 1992. Betaine lipids. The botanical Magazine Tokyo 105, 185-197.

Sprott, D. G., 1992. Structures of archaebacterial membrane lipids. Journal of

Bioenergetics and Biomembranes 24, 555-565.

Sprott, D. G., Meloche, M., Richards, J. C., 1991. Proportions of diether, macrocyclic

diether and tetraether lipids in Methanococcus Jannaschii grown at different

temperatures. Journal of Bacteriology 173, 3907-3910.

Stadnitskaia, A., Bouloubassi, I., Elvert, M., Hinrichs, K. -U., Sinninghe Damsté, J. S.,

2008. Extended hydroxyarchaeaol, a novel lipid biomarker for anaerobic

methanotrophy in cold seepage habitats. Organic Geochemistry 39, 1007-1014.

Chapter V ________________________________________________________________________

148

Sturt, H. F., Summons, R. E., Smith, K. J., Elvert, M., Hinrichs, K. -U., 2004. Intact polar

membrane lipids in prokaryotes and sediments deciphered by high-performance

liquid chromatography/electrospray ionization multistage mass spectrometry-new

biomarkers for biogeochemistry and microbial ecology. Rapid Communications in

Mass Spectrometry 18, 617-628.

Thimon, L., Peypoux, F., Maget-Dana, R., Roux, B., Michel, G., 1992. Interactions of

surfactin, a biosurfactant from Bacillus subtilis, with inorganic cations.

Biotechnology Letters 14, 713-718.

Vater, J., 1986. Lipopeptides, an attractive class of microbial surfactants. Progress in

Colloid and Polymer Science 72, 12-18.

Wagner, M., Horn, M., Daims, H., 2003. Fluorescence in situ hybridization for the

identification and characterization of prokaryotes. Current opinion in Microbiology

6, 302-309.

Chapter VI ________________________________________________________________________

149

CHAPTER VI

Concluding remarks and perspectives

Chapter VI ________________________________________________________________________

150

VI.1. Conclusions

This dissertation focused on the study of different microbial communities

involved in the process of AOM. This work began with the identification of several intact

polar lipids (IPLs) from few samples phylogenetically dominated by each of three

anaerobic methanotrophic consortia (ANME-1, -2 and 3 and sulfate reducer bacterial

partners). After the identification of several diagnostic IPLs characteristic of the each

AOM-community, these lipids were analyzed in a variety of globally distributed cold

seep systems. Among these hot spots of AOM, different habitats were analyzed such as

anoxic water bodies, mud volcanoes, oil fields, gas hydrate environments and

hydrothermal vents. In the course of this work, it was possible to address several open

questions regarding AOM-research: (1) identification of communities involved in AOM

based on few diagnostic of IPLs, (2) microbial-derived IPL diversity in AOM hot spots

and (3) environmental factors influencing the dominance and distribution of AOM-

communities.

This work is the first to demonstrate that IPLs, which are biomarkers associated to

living biomass, enable not only the distinction of the three main groups of AOM-

mediating microbes from a wide variety of methane-bearing habitats (Chapter II and III)

but, more importantly, provides additional insights on the environmental factors

influencing the distribution of these communities (Chapter III).

The three phylogenetically distinct clusters of Euryarchaeota called ANME-1, -2

and -3 (e.g., Hinrichs et al., 1999; Boetius et al., 2000; Lösekann et al., 2007) which have

been observed in association with sulfate-reducing bacteria (SRB) of the

Desulfosarcina/Desulfococcus group (Boetius et al., 2000; Orphan et al., 2001; Michaelis

et al., 2002, ‘‘ANME-1/DSS and -2/DSS aggregates”) or Desulfobulbus spp (Lösekann et

al., 2007, ‘‘ANME-3/DBB aggregates”) exhibit a characteristic IPL composition.

ANME-1, which is not directly affiliated with any of the major orders of methanogens

(Hinrichs et al., 1999; Orphan et al., 2001; Knittel et al., 2005) is characterized by the

production of glyceroldialkylglyceroltetraether (GDGTs) with glycosidic and phospho as

well as mixed glycosidic and phospho headgroups. The main glycosidic-GDGT in

Chapter VI ________________________________________________________________________

151

ANME-1 system, is diglycosyl-GDGT (2Gly-GDGT, Rossel et al., 2008; Chapter II and

Chapter III), a lipid also frequently observed in deep subsurface (Biddle et al., 2006; Lipp

et al., 2008), as well among several species within the order Methanomicrobiales (Koga

et al., 1998). In addition to glycosidic-GDGTs, GDGTs with mixed glycosidic and

phospho or only phospho headgroups were dominated by 2Gly-GDGT-PG and 2PG-

GDGT (Chapter III), which have been also previously reported in Methanobacterium

thermoautotrophicum (Koga et al., 1993). Interestingly, contribution of 2Gly-GDGT,

2Gly-GDGT-PG and 2PG-GDGT varied depending of the ANME-1 habitat. Beside the

general dominance of 2Gly-GDGT, the contribution of 2Gly-GDGT-PG and 2PG-GDGT

was much higher in sediment than in carbonate reefs dominated by ANME-1.

Different from ANME-1, diagnostic IPLs of ANME-2 were archaeols with both

glycosidic and phospho headgroups, which also occur in Methanocaldococcus jannaschii,

Methanococcus voltae and Methanothirx soehngenii (Koga et al., 1993; Sturt et al.,

2004). Within the glycosidic archaeols the main IPLs were 2Gly-archaeol (2Gly-AR),

2Gly-MAR (2Gly-macrocyclic archaeol), 2Gly-hydroxyarchaeol (2Gly-OH-AR),

whereas the major phospho-archaeols were PG-OH-AR, phosphatidylserine-OH-AR (PS-

OH-AR) and phosphatidylinositol-OH-AR (PI-OH-AR) (Chapter III). Similar to ANME-

1 systems, archaeal IPLs containing phospho headgroups were more abundant in

sediments than in carbonate reefs.

ANME-3, contrary to ANME-2 and ANME-1 contained neither glycosidic-

archaeols nor GDGT-based IPLs. However, the phospho-archaeols composition was very

similar to ANME-2, although with a generally less contribution of PI-OH-AR (Chapter

III). The phylogenetic affiliation of ANME-2 and ANME-3 with the order

Methanosarcinales, was consistent with the dominance of archaeol and hydroxyarchaeol

with both glycosidic and phospho headgroups (Kates, 1997; Koga et al., 1998).

Among the major bacterial IPLs, relative high abundance of

phosphatidylethanolamine (PE), phosphatidyl-(N)-methylethanolamine (PME) and

phosphatidyl-(N,N)-dimethylethanolamine (PDME) with diacylglycerol (DAG) bond

type, were found in ANME-2/DSS and ANME-3/DBB dominated settings (Rossel et al.,

2008; chapter II and chapter III). PE is the major phospholipid type of SRB such as

Desulfosarcina variabilis (Rütters et al., 2001) and its occurrence together with PME and

Chapter VI ________________________________________________________________________

152

PDME in anoxic waters and surface sediments from the Black Sea has been also

suggested to derive from SRB (Schubotz et al., submitted). However PME and PDME

have been also described in some methanotrophic bacteria (Makula, 1978; Fang et al.,

2000) as well as sulfide oxidizers (Barridge and Shively, 1968). The presence of PME

and PDME seems to be a general feature of ANME-3/DBB dominated systems, although

it needs to be taken into account, that a fraction of these two IPLs may derived either

from aerobic methanotrophic bacteria or from sulfide oxidizers, both which contain

similar membrane lipids (Barridge and Shively, 1968; Makula, 1978; Fang et al., 2000).

Other bacterial IPLs, which contributed mainly to ANME-2/DSS dominated mats,

were the non-phospho IPLs ornithine lipids (OL), surfactin and betaine lipids (BL), with

the latter characterized by odd fatty acid chains (BL-odd) (Chapter III). OL have been

reported in SRB, and sulfur and iron oxidizing bacteria (Knoche and Shively, 1972;

Makula and Finerty, 1975), whereas surfactin is a lipopeptide with surface active

properties common of Bacillus sp. (Vater et al., 1986) that may also be produced by an

unknown bacteria in the mats from the Black Sea. On the other hand, BL-odd, contrary to

BL with even fatty acid chains, have been suggested to derive from bacteria, do to their

occurrence in deep anoxic water of the Black Sea (Schubotz et al., submitted).

Based on IPL distribution, it was possible to observe a clear separation within the

chimney-like structures and the sediment habitats. ANME-1 and ANME-2/DSS

inhabiting carbonate reefs contained high abundance of glycosidic-IPLs and IPL with

non-phospho headgroups. Both archaeal (2Gly-GDGT, 2Gly-AR, 2Gly-MAR, 2Gly-OH-

AR) and bacterial IPL (OL, surfactin and BL odd) composition point to the low

abundance of phospho-IPLs in carbonate mats compared to sediments. Dissolved

phosphate in sediment pore water has been shown to be strongly adsorbed on calcium

carbonate (Cole et al., 1953; de Kanel and Morse, 1978). Therefore, limitation of

dissolved phosphate in AOM carbonate mats from the Black Sea is likely responsible for

the generally low abundance of IPLs with phospho headgroups in both ANME-1/DSS

and ANME-2a/DSS dominated mats (Chapter III).

Beside the general differences in IPL composition of ANME-1, -2 and -3

communities, additional variations in the IPL pattern in relation to several environmental

Chapter VI ________________________________________________________________________

153

variables provided new insights into the ecological niches dominated by these

communities (Chapter III). ANME-1/DSS, in which the diagnostic IPL was 2Gly-GDGT,

dominates habitats with higher temperature and lower oxygen content in bottom waters

compared to the systems in which ANME-2/DSS and ANME-3/DBB inhabit. This

relationship between ANME-1/DSS and temperature is in agreement with the detected

higher AOM-activity of ANME-1/DSS at higher temperatures (up to 24°C) compared to

ANME-2/DSS (up to 15°C) based on in vitro experiments (Nauhaus et al., 2005).

Furthermore, the dominance of ANME-1/DSS in low oxygen bottom waters is in

agreement with previous field observations, which suggest that ANME-1/DSS may be

more sensitive to oxygen than ANME-2/DSS (Knittel et al., 2005). Based on IPL

diversity, ANME-2/DSS systems were separated in two groups: the carbonate reefs and

the sediments. ANME-2/DSS dominated sediments were characterized not only by lower

temperature and higher oxygen content in bottom waters, but also by higher methane and

sulfate concentrations. These environmental variables were accompanied by the presence

of PG-OH-AR and PI-OH-AR. On the other hand, ANME-2/DSS dominated carbonate

mats were associated with higher sulfate reduction rates (SRR) and to the occurrence of

2Gly-OH-AR and 2Gly-MAR. These differences between carbonate reefs and sediments

dominated by ANME-2/DSS could be explained by the presence of sulfide oxidizing

bacteria (SOB) in the sediments, which efficiently remove sulfide and produce sulfate.

The environmental characteristics, as well as the archaeal IPL composition of

ANME-3 and ANME-2 from sediments, suggest that these two communities dominate in

similar environments, although due to the fact that the lowest temperatures were observed

at ANME-3/DBB dominated sediments from Håkon Mosby Mud Volcano, it is possible

that temperature may also select for either ANME-2/DSS or ANME-3/DBB.

IPL data in general was in good agreement with the phylogenetic information

based on FISH methods. Nevertheless, in a few cases both methods have some

discrepancies due to several potential reasons. It was observed that in sediments

dominated by ANME-2c/DSS according to FISH counting, the contribution of ANME-1

derived GDGT-based IPLs was higher than the ANME-2/DSS IPL signal. The high

contribution of GDGT-based IPLs was probably due to the presence of extremely large

ANME-1 cells in this setting. Additionally, FISH methods could also underestimate

Chapter VI ________________________________________________________________________

154

archaeal abundance, especially ANME-1, due to the low permeability of their membranes

compared to the bacterial phospholipid (Wagner et al., 2003).

The evaluation of apolar lipids distribution provided a poor taxonomic separation

between the three AOM-communities (Chapter III). This was probably due to the lack of

GDGTs in our data set, which is the main core lipid of ANME-1, but also to the

presumed longer turnover times of apolar lipids than of IPLs. Apolar signals may

integrate longer periods in the geologic evolution of the studied seep systems, in which

community changes are likely to occur resulting in a mixed signal from current and past

microbial communities.

Furthermore, IPL behavior on marine sediment systems was evaluated using an

experimental approach (Chapter IV). Incubations were performed using slurries of

sediments with (sterile condition) and without sterilization (active condition), in which

membrane lipid of archaea (2Gly-GDGT) and bacteria (C16-PC) were spiked. Both sterile

and active conditions were incubated at 5°C and 40°C. According to our results both

archaeal and bacterial IPLs were degraded under sterile conditions. However, after 465

days of incubation under active conditions, an increase of both IPLs was observed

(although the bacterial IPL only increased at 5°C). This suggests that the microbial

communities were growing. Unfortunately, degradation of IPLs in the active conditions

could not be proved because the IPLs produced and degraded were indistinguishable.

Therefore, an improved experimental approach is necessary.

We demonstrated that few IPLs enable the distinction of AOM-communities,

although the diversity of IPLs identified in methane-bearing habitats is very high (chapter

III and V). Among the archaeal IPLs identified, GDGT-based and archaeol-based IPLs

with glycosidic, mixed phospho and glycosidic or pure phospho headgroups were

observed. Bacterial IPLs were also diverse having not only different phospho headgroups

but also containing non-phospho IPLs. Structural information and fragmentation patterns

of diverse IPL classes are provided in this thesis (Chapter V) as base for further IPL

identification in AOM systems.

The results obtained during this thesis provide a clear distinction between the

major microbial communities involved in AOM in marine sediments (ANME-1, -2 and -3

Chapter VI ________________________________________________________________________

155

and SRB partners) based on IPL distribution. Additionally these results demonstrate that

IPLs varied not only according to the community type but also in relation to the habitat

characteristics. Furthermore, IPL distribution was also related to several environmental

factors selecting for one of the three major AOM-community types. Thus, allowing to

define the ecological niches dominated by each of these groups.

VI.2. Future perspectives

This thesis contributes to a better understanding of the microbial communities

involved in the process of AOM and the environmental factors controlling their

dominance in a variety of seeps globally distributed. However, several open questions

regarding the process of AOM and the potential applications of IPLs for future research

can still be addressed:

� Few diagnostic IPLs enable the distinction between the three major communities

performing AOM. However the diversity of IPLs in hot spots of AOM is quite

high and includes some IPLs which are abundant in just a few settings. This

suggest that either the same microbial communities produce different IPLs

depending of the environment or other ANMEs, so far not identified, are present.

In this settings will be necessary to characterized in detail the microbial

community present.

� The high concentration of IPLs in AOM systems provides an excellent

opportunity to elucidate structural diversity of IPLs derived from both archaea and

bacteria living in marine systems. Some of these IPLs, still with tentative

structures, can be purified to confirm their structures.

� During this work, we have learned that IPLs are strongly influenced by the habitat

conditions in which microbial communities dominate. In this study the role of

phosphate limitation was discussed, although many other factors may influence

the composition of IPLs in microbial membranes. These effects can be studied

either by covering a variety of extreme environments or by culture experiments in

Chapter VI ________________________________________________________________________

156

which environmental factors such as nutrients, pH, pressure, temperature, carbon

source, starvation are controlled and properly monitored.

� Environmental factors selecting between ANME-1/DSS and ANME-2/DSS were

clearly defined. However ANME-2/DSS and ANME-3/DBB presented similar

IPL compositions as well as the habitat characteristics, which do not allow a good

separation between these two groups. It is necessary to study in more detail

ANME-3/DBB from other dominated settings, to confirm the presence of similar

diagnostic IPLs as well as the environmental factors influencing their distribution.

� An improvement of the experimental design used in this study to evaluate stability

of IPLs in sediments is needed. This information affects the interpretation and

validation of IPLs as biomarkers for currently active communities. In this new

experimental approach, the distinction between degraded and produced IPLs, the

effects of adsorption in IPLs behavior over time, the abundance of microbial cells

as well as degradation products should be considered.

REFERENCES

Barridge, J. K., Shively, J. M., 1968. Phospholipids of the Thiobacilli. Journal of

Bacteriology 95, 2182-2185.

Biddle, J. F., Lipp, J. S., Lever, M. A., Lloyd, K. G., Sörensen, K. B., Anderson, R.,

Fredricks, H. F., Elvert, M., Kelly, T J., Schrag, D. P., Sogin, M. L., Brenchley, J.

E., Teske, A. House, C. H., Hinrichs, K. –U., 2006. Heterotrophic archaea dominate

sedimentary subsurface ecosystems off Peru. Proceedings of the National Academy

of Science U.S.A. 103, 3846-3851.

Boetius, A., Ravenschlag, K., Schubert, C. J., Rickert, D., Widdel, F., Gieseke, A.,

Amann, R., Jørgensen, B. B., Witte, U., Pfannkuche, O., 2000. A marine microbial

consortium apparently mediating anaerobic oxidation of methane. Nature 407, 623-

626.

Cole, C.V., Olsen, S. R., Scott, C. O., 1953. The nature of phosphate sorption by calcium

carbonate. Soil Science Society of America Journal 17, 352-356.

Chapter VI ________________________________________________________________________

157

De Kanel, J., Morse, J. W., 1978. The chemistry of orthophosphate uptake from seawater

on to calcite and aragonite. Geochimica et Cosmochimica Acta 42, 1335-1340.

Elvert, M., Niemann, H., 2008. Occurrence of unusual steroids and hopanoids derived

from aerobic methanotrophs at an active marine mud volcano. Organic

Geochemistry 39, 167–177.

Fang, J. S., Barcelona, M. J., Semrau, J. D., 2000. Characterization of methanotrophic

bacteria on the basis of intact phospholipid profiles. FEMS Microbial Letters 189,

67-72.

Hinrichs, K.-U., Hayes, J. S., Sylva, S. P., Brewer, P. G., DeLong, E. F., 1999. Methane-

consuming archaebacteria in marine sediments. Nature 398, 802-805.

Kates, M., 1997. Diether and tetraether phospholipids and glycolipids as molecular

markers for archaeabacteria (archaea). In: Molecular Markers in Environmental

Geochemistry (ed. Eganhause, R.P.), pp. 35–48. Oxford University Press.

Knittel, K., Lösekann, T., Boetius, A., Kort, R., Amann, R., 2005. Diversity and

Distribution of Methanotrophic Archaea at Cold Seeps. Applied and Environmental

Microbiology 71, 467-479.

Knoche, H. W., Shively, J. M., 1972. The structure of an ornithine –containing lipid from

Thiobacillus thioxidans. The Journal of Biological Chemistry 247, 170-178.

Koga, Y., Nishihara, M., Morii, H., Akagawa-Matsushita, M., 1993. Ether polar lipids of

methanogenic bacteria: structures, comparative aspects, and biosyntheses. 1996.

Microbiological Reviews 57, 164-182.

Koga, Y., Morii, H., Akagawa-Matsushita, M., Ohga, I., 1998. Correlation of polar lipid

composition with 16S rRNA phylogeny in methanogens. Further analysis of lipid

component parts. Bioscience Biotechnology and Biochemistry 62, 230-236.

Lipp, J. S., Morono, Y, Inagaki, F., Hinrichs, K.-U., 2008. Significant contribution of

Archaea to the extant biomass in marine subsurface sediments. Nature 454, 991-

994.

Lösekann, T., Knittel, K., Nadalig, T., Fuchs, B., Niemann, H., Boetius, A., Amann, R.,

2007. Diversity and abundance of aerobic and anaerobic methane oxidizers at the

Haakon Mosby Mud Volcano, Barents Sea. Applied and Environmental

Microbiology 73, 3348–3362.

Chapter VI ________________________________________________________________________

158

Makula, R. A., Finnerty, W. R., 1975. Isolation and characterization of an ornithine-

containing lipid from Desulfovibrio gigas. Journal of Bacteriology 123, 523-529.

Makula, R. A., 1978. Phospholipid composition of methane-utilizing bacteria. Journal of

Bacteriology 134, 771-777.

Nauhaus, K., Treude, T., Boetius, A., Krüger, M., 2005. Environmental regulation of the

anaerobic oxidation of methane a comparison of ANME-1 and ANME-II

communities. Environmental Microbiology 7, 98-106.

Niemann, H., Lösekann T., de Beer, D., Elvert, M., Nadalig, T., Knittel, K., Amann, R.,

Sauter, E., Schlüter, M., Klages, M., Foucher, J. -P., Boetius, A., 2006. Novel

microbial communities of the Haakon Mosby mud volcano and their role as a

methane sink. Nature 443, 854-858.

Orphan, V. J., House, C. H., Hinrichs, K. –U., McKeegan, K. D., DeLong, E. F., 2001.

Comparative analysis of methane-oxidizing archaea and sulfate-reducing bacteria in

anoxic marine sediments. Applied and Environmental Microbiology 67, 1922-1934.

Rossel, P. E., Lipp, J. S., Fredricks, H. F., Arnds, J., Boetius, A., Elvert, M., Hinrichs, K.

–U., 2008. Intact polar lipids of anaerobic methanotrophic archaea and associated

bacteria. Organic Geochemistry 39, 992-999.

Rütters, H., Sass, H., Cypionka, H., Rullkötter, J., 2001. Monoalkylether phospholipids in

the sulfate-reducing bacteria Desulfosarcina variabilis and Desulforhabdus

amnigenus. Archives of Microbiology 176, 435-442.

Sturt, H. F., Summons, R. E., Smith, K. J., Elvert, M., Hinrichs, K.-U., 2004. Intact polar

membrane lipids in prokaryotes and sediments deciphered by high-performance

liquid chromatography/electrospray ionization multistage mass spectrometry-new

biomarkers for biogeochemistry and microbial ecology. Rapid Communications in

Mass Spectrometry 18, 617-628.

Wagner, M., Horn, M., Daims, H., 2003. Fluorescence in situ hybridization for the

identification and characterization of prokaryotes. Current opinion in Microbiology

6, 302-309.

Chapter VI ________________________________________________________________________

159

VI.3. Presentations and other activities

August 2008 Gordon Research Conference in Organic Geochemistry, Plymouth, USA. “Intact polar membrane lipids associated with microbial communities performing AOM from globally distributed hydrocarbon seeps. (Poster)

February 2008 Anaerobic Oxidation of Methane Exchange Meeting

together with groups from Universities of Wageningen and Nijmegen (The Netherlands), Aselage, Germany. “Intact polar membrane lipid analyses of anaerobic methanotrophic archaea and associated bacteria”. (Talk)

October 2007 International Conference and 97th Annual Meeting of the

Geologische Vereinigung e.V. University of Bremen, Bremen, Germany. “Diversity of polar lipids in anaerobic communities and their stability in marine sediments”. (Talk)

September 2007 International Meeting on Organic Geochemistry

Conference, Torquay, UK. “Polar and apolar lipids of anaerobic methanotrophic communities from marine seep environments and their relation to environmental conditions” (Poster)

August 2006 Gordon Research Conference in Organic Geochemistry,

Plymouth, USA. “Diversity of polar lipids in anaerobic methanotrophic communities”. (Poster)

November 2005 2nd Northern German Organic Geochemistry Meeting.

University of Oldenburg, Oldenburg, Germany. “Diversity of polar lipids in anaerobic methanotrophic communities”. (Talk)

Participation in Fieldtrips November 2007 M74/3 on board of R/V Meteor (Fujairah to Maldives,

Indian Ocean) in the framework of the “Methane seeps and sediment transport on the Makran accretionary prism and biogeochemical investigations of the oxygen minimum zone.

Academic Supervision July-August 2006 Supervision of summer student Augusta Dibbell,

Massachusetts Institute of Technology-USA.

Chapter VI ________________________________________________________________________

160

Courses September 2007 Organic Facies modelling. European Graduate College in

Marine Sciences (ECOLMAS) January 2007 Methane Biogeochemistry and Geophysics & Remote

Sensing and Ocean-Land Interaction. Austral Summer Institute (ASI-VII)

May-June 2006 Advanced Organic Biogeochemistry. European Graduate

College in Marine Sciences (ECOLMAS) March 2006 Signal and time series analysis. European Graduate College

in Marine Sciences (ECOLMAS)