10ο Πανελλήνιο Συμπόσιο Ωκεανογραφίας & Αλιείας 7-11 Μαΐου 2012, Αθήνα Βιοποικιλότητα, απειλούμενα είδη στα υδάτινα συστήματα. Βιο-γεω-χημικές διεργασίες - Τροφικά πλέγματα

1. LIPID BIOMARKERS TRACE METHANE CONSUMPTION BY
MICROBIAL COMMUNITIES IN SEDIMENTS FROM THE
MARMARA SEA
N. Chevalier1, I. Bouloubassi1, D. Birgel2, M.H Taphanel1
1 LOCEAN, UMR 7159, CNRS/Université P. et M. Curie, Paris, France
2 University of Vienna, Department of Geodynamics and Sedimentology, Vienna, Austria
1

2. COLD SEEPS
Places where fluids (enriched in methane) are seeping out of the ocean floor
due to the geology of the underlying sediments and/or due to the physiological
functionning of the subsurface microbial community
Widely distributed on ocean margins
Reservoirs of huge amounts of
methane often in the form of hydrates
Mazurenko et al., 2003
2

3. SPECIFIC INTEREST FOR THE STUDY OF METHANE SEEPAGES
- 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 ?
- Biology
Methane fuels peculiar forms of life ! Deep biosphere
- Economy
Methane is an appealing energy resource !
3

4. FACTS
• 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
4

5. Two ways of microbial CH4 consumption
Aerobic by methanotrophic bacteria at the
sediment-water interface or in the water column
CH4 + 2O2CO2 + 2H2O
ΔG°= -204 kJ mol-1
Anaerobic by methanotrophic archaea and sulfate-
reducing bacteria in deeper zones of the sediment
Process discovered in the 2000’s
CH4 + SO42- HCO3- + HS- + H2O Major regulator of methane fluxes
So far not cultivated archaea
ΔG°= ~-17 kJ mol-1 Precise metabolism?
Other microbes as well?
5

6. Cold seeps associated with the North Anatolian Fault zone
in the Sea of Marmara
MARNAUT CRUISE – Nautile submersible
6

7. MARNAUT CRUISE – Nautile submersible : Field evidence
Gas bubble emission
EK60 sounder
7

8. ‹#›

9. MARNAUT CRUISE – Nautile submersible : Field evidence
Reduced sediments, polychaetes, microbial mats
9

10. MARNAUT CRUISE – Nautile submersible : Field evidence
Carbonate formations (crusts, chimneys)
10

11. Origin of methane
• Thermogenic above leaky anticlines
– Thrace basin source
• Dominantly biogenic in Cinarcik basin
11
Bourry et al., 2009

12. MARMARA SEA SEDIMENTS
Push Cores
1661 1660 1659
We applied molecular and isotopic tools
to unravel microbial processes related with methane cycling
12
Chevalier, Bouloubassi . Geobiology (submitted)

13. Molecular – Isotopic Tools : lipid biomarkers
Constituents of microbial cell membranes
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
Which microbes are there ? What are they doing ?
OH O
X’
O X
O
OX OH 13

14. Core 1659 PC4 (S. Cinarcik Basin) – Archaeal lipids
µg/g δ13C, per mil
0 5 10 15 -140 -120 -100 -80 -60 -40 -20
0
2 Archaeol
4 sn-2-OHAr
6 ΣPMIs
Depth(cm)
8
10 AOM
12
14
16
Archaeal lipids highly depleted in 13C
-> archaea assimilate methane-derived Carbon
-> Anaerobic Oxidation of Methane (AOM) by ANME archaea
14
Chevalier, Bouloubassi . Geobiology (submitted)

15. Core 1659 PC4 (S. Cinarcik Basin) – Bacterial lipids
µg/g δ13C, per mil
0 5 10 15 -140 -120 -100 -80 -60 -40 -20
0
2 cyC17:0ω5,6
4 C16:1ω5
6 Me-C16
Depth(cm)
8
10
AOM/SR
SMTZ
12
14
16
Highly 13C-depleted bacterial (sulphate reducers-SRB) lipids
-> SRB assimilate (at least partially) methane-derived carbon
-> AOM coupled to SR
CH4 + SO42- > HS- + HCO3- + H2O
15
Chevalier, Bouloubassi . Geobiology (submitted)

16. Identity of ANME Archaea and SR bacteria
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
In core 1659 PC4 : dominant ANME-2 archaea
Desulfosarcina/Desulfococcus (DSS) SRB
Confirmation by 16S rRNA gene analyses
16

17. MARMARA SEA SEDIMENTS
AOM AOM AOM
1661 1660 1659
CH4 escapes ? – high flow ?
Deeper SMTZ? – low flow ? √
17
Chevalier, Bouloubassi . Geobiology (submitted)

18. Fuels chemosynthetic fauna > typical cold seep benthic ecosystems
AOM - SR CH4 + SO42- > HS- + HCO3- + H2O
May trigger precipitation of diagenetic carbonates
(through an increase in alkalinity of pore waters)
Ca2+, Mg2+, Fe2+ + HCO3- > ( Ca,Mg,Fe) CO3 + H+
These authigenic carbonates sequester methane-derived carbon !
They are paleo-archives of previously active phases of seepage !
18

19. MARMARA SEA CARBONATES
19
Chevalier, Bouloubassi et al., Marine Geology, 2011

20. MARMARA SEA CARBONATES
Table 2 ARCHAEAL LIPIDS
Concentrations and stable carbon isotopic composition (in ‰ V-PDB) of archaeal lipids extracted from carbonate
samples associated to cold seeps in the Marmara Sea. Authigenic
1659R1 1661R5 1664R2 1667R3
µg/g dw  13 C (‰) µg/g dw  13 C (‰) µg/g dw  13 C (‰) µg/g dw  13 C (‰) carbonates do
Archaeol 8.7 −108 27.9 −107 14.5 −92 22.2 −111 precipitated
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 through microbial
Crocetane nd nd 8.1 −103 3.2 −69 4.1 −109 AOM
Crocetene nd nd 1.0 −101 0.4 nd 0.6 −103
PMI:1b nd nd − − − − 0.3 nd
Table 3
PMI:2 nd BACTERIAL LIPIDS
nd 1.1 −88 3.4 −88 2.9 −108
Concentrations and stable carbon isotopic composition (in ‰ V-PDB) of fatty acids (FAs) and non-isoprenoid
PMI:3 nd nd 0.3 nd 2.1 −92 3.4 −111
monoalkyl glycerol ethers (MAGEs) extracted from carbonate samples associated to cold seeps in the Marmara Sea and
PMI:4 nd nd
related to bacterial lipids from microbial sources. 0.6 nd 1.8 −75 1.3 −100 Dominant AOM
− − − −
PMI:5 nd
1659R1
3-O -phytanyl-glycerolether µg/g dw  C (‰)
0.5
nd
13−105
1661R5
µg/g dw  −110
1.7 13
1664R2
µg/g dw  −92(‰)
1.2 13
0.2
1667R3 nd
µg/g dw nd (‰)
0.5 13
assemblages consist
C (‰) C C
−: not detected; nd: not determined.
fatty acids of ANME-2 archaea
C14:0 FA 1659R1 and 1664R2, an unresolved complex mixture−30
In samples 9.1 −78 5.4 (UCM) appeared in the hydrocarbon fraction as a hump
1.9 −48 19.8 −89
area between about n -C16 and n -C32 . In sample 1659R1, the quantification of hydrocarbons was not possible because
i -C15:0 FA 6.4 −90 6.0 −91 5.3 −80 8.0 −100 and associated DSS
ai -C too high amount of the3.5
of the FA
15:0
UCM. nd 2.3 nd 2.4 −74 4.9 −95 - SRB
a
Stadnitskaia et al. (2008) 10.5
C16:1ω5 FA −85 1.1 nd 2.6 −68 7.7 −90
b
C16:0 FA 10.2 −58 17.6 −30 4.9 −36
Unsaturated pentamethylicosenes and the numeral refers to the number of double bonds 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
Microbial biomass trapped within the mineral lattice
C14:0 MAGE
C16:1 MAGE
0.9
3.1
−90
−86
0.7
−
nd
−
nd
nd
nd
nd
nd
0.6
nd
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
20
−: not detected; nd: not determined. Chevalier, Bouloubassi , Marine Geology, 2011

21. SUMMARY
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. Çinarcik
Basin)
 Molecular evidence for AOM/SR is absent at sites from the N.
Çinarcik Basin and the Central Basin, but pore water profiles suggest it
likely occurs in deeper sections
 Diagnostic biomarker fingerprints point to ANME-2 archaea and
sulphate-reducing bacteria from the Desulfosarcina/Desulfococcus
clusters as predominant members of the AOM consortia
 AOM is responsible for authigenic carbonate precipitation
21

22. 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
22

23. 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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