25th International Meeting on Organic Geochemistry IMOG 2011

O-74
Tracing 13C-labeled inorganic carbon into intact polar lipids of
thermophilic anaerobic methanotrophs provides new insights
into pathways of archaeal lipid biosynthesis
Matthias Y. Kellermann 1 , Gunter Wegener 1,2 , Yu-Shih Lin 1 , Marcos Y. Yoshinaga 1 ,
Thomas Holler 2 , Marcus Elvert 1 , Kai-Uwe Hinrichs 1
1 MARUM and Dept. of Geosciences, University of Bremen, 28359 Bremen, Germany, 2 Max Planck Institute
for Marine Microbiology, 28359 Bremen, Germany (corresponding author:m.kellermann@uni-bremen.de)
Stable isotope probing using 13 C-labeled substrates is
widely applied in microbial biogeochemistry to trace
the incorporation of carbon into cell components. The
anaerobic oxidation of methane (AOM) has been
investigated by labeling of lipids [1,2] . However,
previous studies have combined the free and intact
lipid pools, thereby potentially mixing signals from
dead and living microbial biomass. Here we
conducted an in vitro stable isotope probing
experiment with a novel enrichment of thermophilic
ANME-1 archaea in order to identify substrate
specificities and patterns of lipid biosynthesis in
uncultured microorganisms.
A thermophilic active AOM community dominated by
ANME-1 consortia was enriched from hydrothermally
influenced Guaymas Basin sediments [3] . Sediment
aliquots were incubated with 13 C labeled bicarbonate
under a CH4 headspace. After extraction, we
separated individual intact polar lipids (IPLs) into
several fractions with normal- and reverse-phase
preparative HPLC. Selected archaeal IPLs were then
subjected to intramolecular isotopic analysis of their
isoprenoid chains (phytane and biphytane), sugar
headgroups [4] and the glycerol backbone [5] by GCirMS.
Pronounced differences in 13 C uptake were observed
among IPLs (e.g., 2Gly-AR and 2Gly-GDGT; Fig.1)
and within IPL components (i.e., sugars, glycerol and
isoprenoid chains; Fig.1). Weak 13 C incorporation of
2Gly-GDGT compared to other IPLs is likely due to
the succession of biosynthetic steps, with synthesis of
phosphatidylglycerol (PG)-GDGT preceding formation
of glycosidic GDGTs [6] . The stronger 13 C incorporation
of 2Gly- and PG-GDGT-PG suggests these
compounds are precursors of Gly-GDGTs.
The strong inter- and intramolecular variations in 13 C
uptake of IPLs open a new avenue for studying
membrane lipid biosynthesis of uncultured archaea
and identifying compounds diagnostic of various
stages of growth and activity. Time series incubations
of 24 day duration, different 13 C labeled substrates
and a comparison between bacterial and archaeal
IPLs will provide additional insights into the complex
biogeochemical processes involved in AOM.
Fig. 1. Differences in 13 C uptake of selected IPLs (e.g. PG-
AR, 2Gly-AR, 2Gly- and PG-GDGT-PG and 2Gly-GDGT),
including the hexose headgroups and glycerol are illustrated.
References
[1] Wegener et al. (2008) Envir. Microbiol. 10: 2287-2298.
[2] Blumenberg et al. (2005) Appl. Environ. Microbiol. 71: 4345-4351.
[3] Holler et al. (in review, ISME J.).
[4] Lin et al. (2010) Rapid Commun. Mass Spectrom. 24: 2817–2826.
[5] Lin et al. (unpublished protocol).
[6] Nemoto et al. (2003) Extremophiles 7: 235-243.
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