N. Chevalier1, I. Bouloubassi1, D. Birgel2, M.H Taphanel1

1 LOCEAN, UMR 7159, CNRS/Université P. et M. Curie, Paris, France2 University of Vienna, Department of Geodynamics and Sedimentology, Vienna, Austria

LIPID BIOMARKERS TRACE METHANE CONSUMPTION BY

MICROBIAL COMMUNITIES IN SEDIMENTS FROM THE

MARMARA SEA

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Mazurenko et al., 2003

Places where fluids (enriched in methane) are seeping out of the ocean floordue to the geology of the underlying sediments and/or due to the physiologicalfunctionning of the subsurface microbial community

COLD SEEPS

Widely distributed on ocean margins

Reservoirs of huge amounts ofmethane often in the form of hydrates

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SPECIFIC INTEREST FOR THE STUDY OF METHANE SEEPAGES

- Biology

Methane fuels peculiar forms of life ! Deep biosphere

- Economy

Methane is an appealing energy resource !

- Climate

Methane is a potent greenhouse gas !

Potential destabilisation of methane hydrates (through global warming ?) may release huge CH4 quantities (positive feedback!).This may have happened in the past ?

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• More than 90% of the methane produced/emitted in marine sediments is removed before it enters the hydrosphere and atmosphere

-> There is a methane barrier !

Microbial methane oxidation – key process

FACTS

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Two ways of microbial CH4 consumption

Process discovered in the 2000’sMajor regulator of methane fluxesSo far not cultivated archaeaPrecise metabolism?Other microbes as well?

Aerobic by methanotrophic bacteria at the sediment-water interface or in the water column

ΔG°= -204 kJ mol-1

CH4 + 2O2CO2 + 2H2O

Anaerobic by methanotrophic archaea and sulfate-reducing bacteria in deeper zones of the sediment

ΔG°= ~-17 kJ mol-1

CH4 + SO42-HCO3

- + HS- + H2O

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Cold seeps associated with the North Anatolian Fault zone in the Sea of Marmara

MARNAUT CRUISE – Nautile submersible

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EK60 sounder

Gas bubble emission

MARNAUT CRUISE – Nautile submersible : Field evidence

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‹#›

Reduced sediments, polychaetes, microbial mats

MARNAUT CRUISE – Nautile submersible : Field evidence

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Carbonate formations (crusts, chimneys)

MARNAUT CRUISE – Nautile submersible : Field evidence

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Origin of methane

• Thermogenic above leaky anticlines– Thrace basin source

• Dominantly biogenic in Cinarcik basin

Bourry et al., 200911

Chevalier, Bouloubassi . Geobiology (submitted)

165916601661

MARMARA SEA SEDIMENTS

We applied molecular and isotopic toolsto unravel microbial processes related with methane cycling

Push Cores

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Distinctive ‘taxonomic’ structural features > > community structure

Concentrations > > relative abundances of microbial groups

> > biomass of extant (active) organisms

Stable carbon isotopes > > carbon source utilized, carbon flow

O

OX

OH

X’OH

O

O X

Molecular – Isotopic Tools : lipid biomarkers

Constituents of microbial cell membranes

Which microbes are there ? What are they doing ?

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Core 1659 PC4 (S. Cinarcik Basin) – Archaeal lipids

AOM

δ13C, per mil

-140 -120 -100 -80 -60 -40 -20

De

pth

(cm

)

µg/g

0

2

4

6

8

10

12

14

16

0 5 10 15

Archaeol

sn-2-OHAr

ΣPMIs

Archaeal lipids highly depleted in 13C

Chevalier, Bouloubassi . Geobiology (submitted)

-> Anaerobic Oxidation of Methane (AOM) by ANME archaea

-> archaea assimilate methane-derived Carbon

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AOM/SR

Core 1659 PC4 (S. Cinarcik Basin) – Bacterial lipids D

ep

th(c

m)

δ13C, per milµg/g

-140 -120 -100 -80 -60 -40 -20

0

2

4

6

8

10

12

14

16

0 5 10 15

cyC17:0ω5,6

C16:1ω5

Me-C16

Highly 13C-depleted bacterial (sulphate reducers-SRB) lipids

CH4 + SO42- > HS- + HCO3

- + H2O

Chevalier, Bouloubassi . Geobiology (submitted)

-> SRB assimilate (at least partially) methane-derived carbon

-> AOM coupled to SR

SMTZ

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AOM-related microbial communities show specific lipid biomarker patterns

e.g., sn2-hydroxyarchaeaol/archaeol, isotopic offsets vs. CH4, C16:1w5 FA/ i-C15:0 FA, relative abundance of cyC17:0w5,6 FA, 10Me-C16:0 FA

Diagnostic biomarker indices can thus trace specific AOM-microbes

Identity of ANME Archaea and SR bacteria

Confirmation by 16S rRNA gene analyses

In core 1659 PC4 : dominant ANME-2 archaeaDesulfosarcina/Desulfococcus (DSS) SRB

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Chevalier, Bouloubassi . Geobiology (submitted)

1659

AOM

CH4 escapes ? – high flow ?Deeper SMTZ? – low flow ? √

MARMARA SEA SEDIMENTS

1660

AOM

1661

AOM

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CH4 + SO42- > HS- + HCO3

- + H2OAOM - SR

These authigenic carbonates sequester methane-derived carbon !

They are paleo-archives of previously active phases of seepage !

Fuels chemosynthetic fauna > typical cold seep benthic ecosystems

May trigger precipitation of diagenetic carbonates (through an increase in alkalinity of pore waters)

Ca2+, Mg2+, Fe2+ + HCO3- > ( Ca,Mg,Fe) CO3 + H+

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Chevalier, Bouloubassi et al., Marine Geology, 2011

MARMARA SEA CARBONATES

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Chevalier, Bouloubassi , Marine Geology, 2011

Authigeniccarbonates do precipitatedthrough microbialAOM

Dominant AOM assemblages consistof ANME-2 archaeaand associated DSS - SRB

MARMARA SEA CARBONATES

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Table 2

1659R1 1661R5 1664R2 1667R3

µg/g dw 13C (‰) µg/g dw 13

C (‰) µg/g dw 13C (‰) µg/g dw 13

C (‰)

Archaeol 8.7 −108 27.9 −107 14.5 −92 22.2 −111

Sn-2-hydroxyarchaeol 23.5 −113 85.8 −109 50.4 −95 58.1 −108

Extented-hydroxyarchaeol a

0.2 nd 0.4 nd 2.6 −92 2.4 nd

Crocetane nd nd 8.1 −103 3.2 −69 4.1 −109

Crocetene nd nd 1.0 −101 0.4 nd 0.6 −103

PMI:1b

nd nd − − − − 0.3 nd

PMI:2 nd nd 1.1 −88 3.4 −88 2.9 −108

PMI:3 nd nd 0.3 nd 2.1 −92 3.4 −111

PMI:4 nd nd 0.6 nd 1.8 −75 1.3 −100

PMI:5 nd nd − − − − 0.2 nd

3-O -phytanyl-glycerolether 0.5 −105 1.7 −110 1.2 −92 0.5 nd

−: not detected; nd: not determined.

a Stadnitskaia et al. (2008)

Concentrations and stable carbon isotopic composition (in ‰ V-PDB) of archaeal lipids extracted from carbonate

samples associated to cold seeps in the Marmara Sea.

In samples 1659R1 and 1664R2, an unresolved complex mixture (UCM) appeared in the hydrocarbon fraction as a hump

area between about n -C16 and n -C32. In sample 1659R1, the quantification of hydrocarbons was not possible because

of the too high amount of the UCM.

b Unsaturated pentamethylicosenes and the numeral refers to the number of double bonds

Table 3

1659R1 1661R5 1664R2 1667R3

µg/g dw 13C (‰) µg/g dw 13

C (‰) µg/g dw 13C (‰) µg/g dw 13

C (‰)

fatty acids

C14:0 FA 9.1 −78 5.4 −30 1.9 −48 19.8 −89

i -C15:0 FA 6.4 −90 6.0 −91 5.3 −80 8.0 −100

ai -C15:0 FA 3.5 nd 2.3 nd 2.4 −74 4.9 −95

C16:1ω5 FA 10.5 −85 1.1 nd 2.6 −68 7.7 −90

C16:0 FA 10.2 −58 17.6 −30 4.9 −36 8.0 −74

10Me-C16:0 FA 2.6 nd 3.5 nd 1.8 −82 9.7 −97

CyC17:0ω5,6 FA 22.8 −91 3.6 nd 1.5 −76 13.7 −89

monoalkyl glycerol ethers

C14:0 MAGE 0.9 −90 0.7 nd nd nd nd nd

C16:1 MAGE 3.1 −86 − − nd nd 0.6 nd

C16:0 MAGE 1.8 −90 nd nd 0.7 nd 0.3 nd

Me-C16:0 MAGE 0.9 −88 0.7 nd 0.6 nd 1.3 nd

CyC17:0 MAGE 1.5 −87 − − − − nd nd

−: not detected; nd: not determined.

Concentrations and stable carbon isotopic composition (in ‰ V-PDB) of fatty acids (FAs) and non-isoprenoid

monoalkyl glycerol ethers (MAGEs) extracted from carbonate samples associated to cold seeps in the Marmara Sea and

related to bacterial lipids from microbial sources.

Microbial biomass trapped within the mineral lattice

ARCHAEAL LIPIDS

BACTERIAL LIPIDS

In newly explored fluid emission zones in the Sea of Marmara :

13C-depleted archaeal and bacterial biomarkers evidence active

AOM coupled to SR within the upper 20 cm of sediments (S. ÇinarcikBasin)

Molecular evidence for AOM/SR is absent at sites from the N.Çinarcik Basin and the Central Basin, but pore water profiles suggest itlikely occurs in deeper sections

Diagnostic biomarker fingerprints point to ANME-2 archaea andsulphate-reducing bacteria from the Desulfosarcina/Desulfococcusclusters as predominant members of the AOM consortia

AOM is responsible for authigenic carbonate precipitation

SUMMARY

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Molecular isotopic approaches are powerfull tools to unravel:

- methane consumption processes- microbial communities involved

Molecular isotopic approaches are powerfull proxies to detect:

- Present day and past events of methane seepage

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Acknowledgements:

- Nicolas Chevalier’s Ph.D research

- ESONET (EU)

-MARNAUT Cruise staff (R/V L’Atalante, Nautile)

- IFREMER (France)

- Istanbul Technical University (Turkey), Maden Tetkikve Arama, Ankara (Turkey)

- Ministry of Education (France)

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