M.Sc.-thesis_Matthias-Kellermann

cliff Intact Polar Lipid (IPL) Bacteri
ohopanepolyols (BHPs) Fatty Acid (
FA)…Phosphatidylcholine (PC) pho
sphatidyletholamine (PE) phosphat
idylserine (PS).phosphatidylserine (
PS) phosphatidylglycerol (PG) phos
phatidylinositol (PI) ceramide phos
phatidyletholamine (C-PE) ceramid
e-phosphonoetholamine (C-PnE) ce
rebroside (Cb)...(BHPs)…aminotriol
…aminotetrol…C16:1 7…C18:1 …C18:3
…C16:1 8…4 -methylcholesta-8(24)-
dien-3 -ol..B. childressi…B. brooksi
…B. cf.thermophilus…unknown.ster
ol…symbionts…methanotrophs…ro
Lipid Biomarkers of Thiotrophic and Methanotrophic
Symbionts in different Bathymodiolus mussel species:
Chemical and Isotopic Analysis
Master Thesis within the Master Program Geowissenschaften
at the Department of Geosciences at the University of Bremen, Germany
Matthias Kellermann
REVIEWER:
Prof. Dr. Kai-Uwe Hinrichs Prof. Dr. Jörn Peckmann
University of Bremen University of Bremen

Lipid Biomarkers of Thiotrophic and Methanotrophic
Symbionts in different Bathymodiolus mussel species:
Chemical and Isotopic Analysis
Master Thesis within the Master Program Geowissenschaften at the Department of
Geosciences at the University of Bremen, Germany
Matthias Kellermann
Reviewer:
Prof. Dr. Kai-Uwe Hinrichs Prof. Dr. Jörn Peckmann
University of Bremen, Bremen, Germany University of Bremen, Bremen, Germany
July – December 2007

i
TABLE OF CONTENTS
LIST OF FIGURES AND TABLES........................................................................................iii
LIST OF ABBREVIATIONS.................................................................................................. v
ABSTRACT......................................................................................................................viii
DANKSAGUNG.................................................................................................................. ix
1 INTRODUCTION ............................................................................................... 1
1.1 ENDOSYMBIOTIC BACTERIA IN SEEP MOLLUSKS ......................................... 1
1.2 GENERAL STRUCTURE OF A MUSSEL ........................................................... 3
1.3 DEFINITION OF BIOMARKERS ........................................................................ 3
1.4 MEMBRANE LIPIDS......................................................................................... 4
1.4.1 INTACT POLAR LIPIDS (IPLS)......................................................................... 7
1.4.2 BACTERIOHOPANEPOLYOLS (BHPS) ............................................................ 11
1.4.3 FATTY ACIDS AND CYCLIC TRITERPENOIDS - CONCENTRATIONS AND
COMPOUND-SPECIFIC ISOTOPE ANALYSIS................................................... 13
1.5 SITE DESCRIPTION........................................................................................ 14
1.5.1 B. BROOKSI & B. CHILDRESSI RECOVERED FROM THE GULF OF MEXICO
(GOM) .......................................................................................................... 14
1.5.2 B. CF. THERMOPHILUS RECOVERED FROM THE PACIFIC-ANTARCTIC
RIDGE (PAR) ............................................................................................... 15
1.6 OBJECTIVES AND RESEARCH QUESTIONS ................................................... 17
2 ANALYTICAL METHODS ............................................................................ 18
2.1 GENERAL LABORATORY PROCEDURE.......................................................... 18
2.2 TOTAL LIPID EXTRACTION AND EXTRACT PURIFICATION ......................... 19
2.2.1 SAMPLE PREPARATION AND EXTRACTION .................................................. 19
2.2.2 SAPONIFICATION ........................................................................................... 20
2.2.3 ASPHALTENE SEPARATION ........................................................................... 21
2.2.4 DERIVATIZATION .......................................................................................... 21
2.2.5 DMDS-ADDUCTS ......................................................................................... 23
2.3 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY COUPLED TO A
MASS SPECTROMETER................................................................................. 23

ii
2.3.1 BHPS ............................................................................................................ 25
2.4 GAS CHROMATOGRAPHY – MASS SPECTROMETRY (GC-MS) .................... 25
2.5 GAS CHROMATOGRAPHY ISOTOPE RATIO MASS SPECTROMETRY (GC-
IRMS)........................................................................................................... 26
3 RESULTS........................................................................................................... 28
3.1 INTACT POLAR LIPIDS (IPLS)....................................................................... 28
3.1.1 IPL-COMPOSITION AND -CONCENTRATIONS OF B. CHILDRESSI...................... 31
3.1.2 IPL-COMPOSITION AND -CONCENTRATIONS OF B. BROOKSI ......................... 32
3.1.3 IPL-COMPOSITION AND -CONCENTRATIONS OF B. CF. THERMOPHILUS ......... 33
3.2 BACTERIOHOPANEPOLYOLS (BHPS) ............................................................ 34
3.3 COMPOUND-SPECIFIC ISOTOPIC ANALYSIS (CSIA) .................................... 36
3.3.1 FATTY ACID FRACTION ................................................................................ 36
3.3.2 NEUTRAL FRACTION..................................................................................... 39
4 DISCUSSION..................................................................................................... 42
4.1 DISTRIBUTION OF IPLS AS INDICATOR FOR MICROBIAL ACTIVITY .......... 42
4.2 BHPS AS BIOMARKERS FOR BACTERIAL SOURCES ..................................... 45
4.3 SUMMARY OF THE COMPOUND-SPECIFIC ISOTOPE INVESTIGATION IN
B. CHILDRESSI, B. BROOKSI AND B. CF. THERMOPHILUS ................................ 46
4.3.1 CONCENTRATION AND
13
C TRENDS OF FATTY ACIDS AND STEROIDS
OF B. CHILDRESSI AND B. BROOKSI FROM THE ALAMINOS CANYON
(GOM) .......................................................................................................... 48
13
C TRENDS OF FATTY ACIDS AND STEROIDS
OF B. CF. THERMOPHILUS FROM THE PAR................................................... 53
4.4 MONOALKYL GLYCEROL ETHERS AS INDICATOR FOR BACTERIAL
ACTIVITY ..................................................................................................... 55
5 CONCLUSIONS AND OUTLOOK................................................................. 56
6 REFERENCES .................................................................................................. 58
APPENDIX.................................................................................................................... 65

iii
LIST OF FIGURES AND TABLES
FIGURES
Fig. 1: General structure of a mussel of the class bivalvia. ..................................... 3
Fig. 2: Schematic sketches of a phospholipid molecule, (top right),
phospholipid bilayer (top left) and a structure of the cytoplasmatic
membrane (central figure). .......................................................................... 5
Fig. 3: Lipid biomarkers from bacteria, eukarya and archaea. ................................ 6
Fig. 4: Role of hopanoids in cell membrane. .......................................................... 6
Fig. 5: Two classes of polar membrane lipids distinguished by their back bone
structure. Glycerol based lipids have two fatty acid chains and a polar
head group connected to a glycerol molecule. Sphingosine based lipids
are based on long-chain aliphatic aminodiols. ............................................ 8
Fig. 6: General chemical structure of sphingolipids. ............................................. 10
Fig. 7: Generalized structure of a BHP. ................................................................ 12
Fig. 8: Structure of a diagnostic fatty acid (C16:1 8). ............................................. 13
Fig. 9: Structure of a diagnostic triterpenoid. ........................................................ 13
Fig. 10: Map showing the northwestern part of the Gulf of Mexico. ...................... 15
Fig. 11: Map showing the position of the sampling site from the Pacific-
Antarctic Ridge. ........................................................................................ 16
Fig. 12: Picture of the recovered Bathymodiolus cf. thermophilus specimens. ....... 16
Fig. 13: Flow chart presenting an overview of the major laboratory processes. ..... 18
Fig. 14: Fragmentation pattern of the ms2 spectrum (positive and negative
ionization mode) clarifies the identity of phosphatidylethanolamine
(PE). ........................................................................................................... 29
Fig. 15: Base peak chromatograms showing the variation of the relative IPL
concentration through all mussel and tissue samples. ............................... 30
Fig. 16: Semi-quantitative estimates of all determined IPLs in the gill tissue of
B. childressi. .............................................................................................. 31
Fig. 17: Semi-quantitative estimates of all IPLs identified in B. brooksi - gill
(blue) and foot (red) tissue. ....................................................................... 32
Fig. 18: Semi-quantitative estimates of all IPLs identified in gill (blue) and foot
(red) tissue of B. cf. thermophilus. ............................................................ 34
Fig. 19: Mass chromatograms (m/z 714.5 + m/z 772.5) of BHP analysis of two
Bathymodiolus species. ............................................................................. 35

iv
Fig. 20: Five gas chromatograms showing the distribution of all determined
fatty acids in each mussel and tissue sample. ............................................ 38
Fig. 21: Five gas chromatograms showing a section (from 40 - 68 min) of the
neutral fraction of each mussel and tissue sample. ................................... 41
Fig. 22: Relative composition of IPLs subdivided in five samples. ........................ 43
Fig. 23: Weighted average isotopic values of three different Bathymodiolus
species. ...................................................................................................... 52
TABLES
Tab. 1: Membrane lipids and their source function. ................................................ 7
Tab. 2: List of samples including genus, location & dry weight. .......................... 19
Tab. 3: Phospholipid derived fatty acid composition and 13
C values for three
mussels of the genus Bathymodiolus. ........................................................ 36
Tab. 4: Concentration and isotopic composition of MAGEs, sterols and
unknown components for three diverse mussels of the genus
Bathymodiolus. .......................................................................................... 40

v
LIST OF ABBREVIATIONS
APCI Atmospheric pressure chemical ionization
BSTFA N,O-Bis(trimethylsilyl)trifluoroacetamide
C16-PAF 1-O-hexadecyl-2-acetoyl-sn-glycero-3-phosphocholine
(Platelet-activating Factor)
Da Dalton
DCM Dichloromethane
ESI Electrospray ionization
FA Fatty acid
FAME Fatty acid methyl ester
FISH Fluorescent in situ hybridization
GC gas chromatography
GC-irMS Gas chromatography – isotope ratio coupled to mass spectrometry
GC-MS Gas chromatography coupled to mass spectrometry
HPLC High performance liquid chromatography
HPLC-ESI-MSn
High performance liquid chromatography coupled to multistage
mass spectrometry with an electrospray ionization interface
IPL Intact polar lipid
IT Ion trap
MAGEs Monoalkylglycerolethers
MS Mass spectrometry
MSn
Multistage mass spectrometry
m/z Mass to charge ratio
MUFA Mono unsaturated fatty acid
PCR Polymerase chain reaction
PLFA Phospholipid-derived fatty acids

vi
PUFA Poly unsaturated fatty acid
rpm Rounds per minute
16S rRNA A large polynucleotide (~1500 bases) that functions as a part of
the small subunit of the ribosome of prokaryotes and from whose
sequence evolutionary relationship can be obtained (MADIGAN
AND MARINKO, 2006).
SFA Saturated fatty acid
sn Stereospecifically numbered
sp. Species
SPE Solid Phase Extraction
TLE Total lipid extract
TMS Trimethyl-silyl
VPDB Vienna Pedee Belemnite
g g-1
g g-1
dry weight

vii
Intact Polar Lipids:
MGDG Monogalactosyldiacylglycerol
PA Phosphatidic acid
PC Phosphatidylcholine
PDME Phosphatidyl-(N,N)-dimethylethanolamine
PE Phosphatidylethanolamine
PG Phosphatidylglycerol
PI Phosphatidylinositol
PS Phosphatidylserine
PME Phosphatidyl-(N)-methylethanolamine
PnE Phosphonoethanolamine
C-PnE Ceramide-phosphonoethanolamine
Cb Cerebroside (glycosylceramide)
C-PE Ceramide-phosphatidylethanolamine

viii
ABSTRACT
Three diverse mussel species of the genus Bathymodiolus were recovered from two
different chemosynthesis-based environments. B. childressi and B. brooksi were
collected from the Alaminos Canyon of the Gulf of Mexico (GoM), whereas B. cf.
thermophilus were obtained from the Pacific-Antarctic Ridge (PAR). The identification
and quantification of the mussel symbionts living within the gill tissue was achieved via
a complex lipid analysis. Intact polar lipids (IPLs) and bacteriohopanepolyols (BHPs)
were analyzed by HPLC-MS, whereas fatty acids and neutral lipids were analyzed by
GC-MS and GC-irMS to gain more information about the symbiotic relationship of
sulfide- and methane-oxidizing bacteria and the host organism. Compared to the
symbiont-free foot tissue, a higher diversity of distinct IPLs was detected in all three gill
tissues of the Bathymodiolus species which could indicate the presence of bacteria. The
results of the BHP analysis demonstrated the presence of methanotrophic bacteria in the
gill tissue of B. childressi and B. brooksi. The BHPs also indicate the presence of an
additional type-II methanotrophic symbiont abundant within the gill tissue of B.
childressi. High amounts of monounsaturated C16 FAs and the absence of typical
phytoplankton markers characterize all three Bathymodiolus species. Group-specific
biomarkers like type I-specific C16:1 fatty acids with double bond positions at 8 and
9, and 4 -methylsterols were detected in mussel species from the GoM. B. cf.
thermophilus contained primarily fatty acids with an unsaturation located at the 7
position (C16:1 7, C18:1 7, C19:1 7 and C20:1 7) which might be indicative for sulfide-
oxidizing bacteria. The compound-specific isotopic analysis for gill and foot tissue of
mussels from the GoM demonstrated different fractionation patterns between B.
childressi and B. brooksi, although they use the same methane source. This might
indicate different metabolic pathways of the symbionts within the gills or additional
filter-feeding capabilities of B. childressi. 13
C values from the Bathymodiolus species
taken from the GoM (between -45 and -60‰) are more negative than those found at the
PAR (between -23 and -37‰) reflecting different carbon substrates (CH4 and CO2,
respectively).

ix
DANKSAGUNG
Ich möchte die Gelegenheit nutzen, um meinen Dank all denjenigen auszusprechen, die mich bei der
Entstehung dieser Masterarbeit auf vielfältigste Weise unterstützt und damit einen großen Teil zum
Gelingen meines Vorhabens beigetragen haben.
Für das mir entgegengebrachte Vertrauen und die hervorragende Betreuung, möchte ich mich bei Herrn
Prof. Dr. Kai-Uwe Hinrichs bedanken. Mein Interesse am Gegenstand dieser Arbeit wurde nicht zuletzt
durch seine aufschlussreiche Heranführung an das Thema und die Ermöglichung des Erlernens
verschiedener, spannender Methoden geweckt.
Herrn Prof. Dr. Jörn Peckmann danke ich für die Durchsicht und Erstellung eines Zweitgutachtens.
Mein ganz besonderer Dank gilt Julius Lipp und Florence Schubotz, die mit ihrem Interesse und den
vielen konstruktiven Diskussionen für eine ganz besonders schöne Arbeitsatmosphäre gesorgt und mich
bis in die „letzten Meter“ in meinem Anliegen begleitet haben. Ihre stetige Ermutigung und
Reflexionshilfe, die mir immer wieder Kraft und Motivation gegeben haben, war insbesondere auch für
die Strukturierung meiner Ergebnisse von Bedeutung.
Dr. Marcus Elvert, Dr. Daniel Birgel, Xavier Prieto-Mollar und Tobias Ertefai möchte ich meinen Dank
für viele Anregungen und wertvolle Hinweise aussprechen, die mich speziell im fachlichen Bereich
gestützt und begleitet haben.
Generell möchte ich mich herzlich bei allen aus der Arbeitsgruppe „Organische Geochemie“ bedanken,
die auf die eine oder andere Weise zur Fertigstellung dieser Arbeit beigetragen haben. Ich habe die
gemeinsame Zeit und Gemeinschaft in dieser Gruppe sehr genossen.
Weiterhin gilt mein Dank Dr. Nicole Dubilier, für die Bereitstellung der Grundlage meiner Arbeit, die
Proben, und Luciana Raggi, für die hilfreiche Erläuterung im Bereich molekularer Techniken.
Ed C. Hathorne danke ich für seine Bereitschaft diese Arbeit Korrektur zu lesen.
Zu guter Letzt sei auch meinen Eltern ein großes Dankeschön ausgesprochen, die mich immer meinen
Weg haben gehen lassen und mich während meiner Studienzeit nicht nur finanziell unterstützt sondern
mich stets aufgefangen und ermuntert haben.

INTRODUCTION
1
1 INTRODUCTION
1.1 ENDOSYMBIOTIC BACTERIA IN SEEP MOLLUSKS
The venting of hydrogen, methane, and hydrogen sulfide charged fluids (geochemical
energy) at cold seeps and hot vents stimulates growth and metabolism of
chemosynthetic communities. The presence and abundance of benthic communities at
cold seeps and hot vents is largely based on chemosynthetic food chains. The oxidation
of reduced components (e.g. hydrogen, methane, hydrogen sulfate) by microorganisms
identifies the smallest member of the food chain in the deep ocean. LONSDALE (1977)
proposed for the first time, that chemoautotrophic production by bacteria might be
significant in the diet of bivalves which were first detected at the Galapagos Spreading
Center. His suggestion was later supported by carbon isotope data ( 13
C) which confirm
a dependence of vent mussels on in situ bacterial chemoautotrophic production as their
food source instead of a diet of organic material derived from the eutrophic zone. At
present it is well known that bacterial symbionts offer the host a source of nutrition
(carbon, nitrogen, and energy sources), which otherwise would be unavailable for
marine invertebrates. The symbiont provides nutrients for the host which can either be
released as metabolic byproducts or through digestion of symbiont tissue by the host.
The host, in turn, provides the symbiont with inorganic carbon, oxygen and compounds
from reduced environments (FISHER 1990; CAVANAUGH AND ROBINSON, 1996).
This study examines the symbiotic relationship of sulfide- and methane-oxidizing
bacteria found within the gill tissue of three different Bathymodiolus species.
Depending on the surrounding environment, different symbionts support their life with
energy and carbon. The deep-sea mussel of the genus Bathymodiolus is known to
dominate the biomass of many chemosynthesis-based cold seeps and hydrothermal vent
ecosystems worldwide (DUPERRON ET AL., 2005, VON COSEL, 2002; VAN DOVER, 2000;
VAN DOVER ET AL., 1996). The following three Bathymodiolus species are investigated:

INTRODUCTION
2
i. Bathymodioline species B. childressi (methane oxidizing symbionts), recovered
from the Gulf of Mexico (GoM), are known to harbor a monoculture of
methanotrophic bacteria which use methane as an electron donor and a carbon
source (DISTEL AND CAVANAUGH, 1994).
ii. Bathymodioline species, B. cf. thermophilus (sulfide-oxidizing
chemoautotrophic symbionts), recovered from the Pacific Antarctic Ridge
(PAR), are associated only with thiotrophic bacteria using sulfide and other
reduced sulfur compounds as electron donors and CO2 as a carbon source to
produce organic carbon (DISTEL ET AL., 1988).
iii. Bathymodioline species, B. brooksi (dual symbionts), recovered from the GoM,
are linked with thiotrophic and methanotrophic bacteria in the gill tissue
(CAVANAUGH ET AL., 1987).
B. childressi, B. brooksi and B. cf. thermophilus, recovered from two different
chemosynthesis-based environments, are known to harbor dense communities of a low
diversity of bacteria which are located within the gill tissue in the apical part of the
bacteriocytes (FISHER, 1990). Unlike B. cf. thermophilus and B. childressi with only one
symbiont, B. brooksi exhibits an intracellular dual symbiosis which allows this species
to exploit a wider range of chemical environments (DISTEL ET AL., 1995).
Mussels of the genus Bathymodiolus may also have working digestive tracts and can
therefore filter particular organic matter to supplement nutrients provided by
endosymbiotic chemoautotrophic bacteria (PAGE ET AL. 1990). Consequently, the
feeding strategies of the mussel can include two mechanisms, endosymbiotic and
suspension-feeding strategies, depending on resource availability during development
and growth (TRASK AND VAN DOVER, 1999; VAN DOVER, 1999; PAGE ET AL. 1990).
Most of the symbionts examined to date (B. childressi, B. brooksi and B. cf.
thermophilus included) have been classified as -proteobacteria (DISTEL ET AL., 1994;
DISTEL ET AL., 1988; DUPERRON ET AL., 2007).

INTRODUCTION
3
1.2 GENERAL STRUCTURE OF A MUSSEL
The anatomy of the mussel is shown in Fig. 1. Mussels of the class bivalvia comprise a
two part shell joined together dorsally by a hinge ligament. The bivalve‟s shell can be
opened and closed by two adductor muscles on the inner surface of the valves. Bivalves
contain within the shell a soft layer of tissue called mantle which encloses most of the
visceral organs. The gills are located inside of the mantle tissue and are used for
respiration and straining out food particles (filter-feeders). Mussels living at
hydrothermal vents or cold seeps can have prokaryotic cells (symbionts) living within
their gill tissue. Those mussels, living in “extreme environment”, are providing reduced
compounds and oxygen rich water via the action of the mussel‟s siphons (not shown in
Fig. 1). The muscular foot, located at their front end, can be used for movements.
1.3 DEFINITION OF BIOMARKERS
BROCKS AND SUMMONS (2003) described biomarkers as proxies for modern
environments as well as chemical fossils originating from formerly living organisms.
Therefore, biomarkers can be used for environmental and geological studies. The most
Fig. 1: General structure of a mussel of the class bivalvia. The two
valves of this mussel have been separated and the soft body
parts are laid open.
(http://www.ridge2000.org/seas/downloads/seas_cts_mussel_lab_procedure.pdf)

INTRODUCTION
4
useful biomarkers are organic molecules with high taxonomic specificity and a potential
for preservation (BROCKS AND SUMMONS, 2003). The application of biomarkers as
indicators of the origin of organic matter has been used to gain information about
biological sources (PETERS ET AL., 2005). Many detailed chemical structures (organic
molecules) and supplementary isotopic composition of individual organic compounds
are known and can be used to gain information on the predominant microbial players.
The analysis of isotopic signatures of individual biomarkers provides valuable
information on the composition of microbial communities and their influence on carbon
cycling (HAYES ET AL., 1990).
This following section presents an overview about biomarkers originating from
biological lipids which can be diagnostic for specific groups, especially if combined
with carbon isotope analyses.
1.4 MEMBRANE LIPIDS
Membrane lipids are the building blocks of all cell walls and therefore abundant in the
three domains of life, Eukarya, Bacteria, and Archaea. The general structure of
biological membranes is built by the so called phospholipid bilayer and therefore
presents an important class of complex lipids (Fig. 2). Phospholipids contain a
phosphorus element and two fatty acids bonded to the C3 alcohol (glycerol) (Fig. 3).
Lipids in general, are amphipathic macromolecules which have hydrophilic (water
loving) polar heads facing outwards to the aqueous external environment and the
hydrophobic (water hating) non-polar fatty acids/isoprenoid chains forming the core.
This property makes lipid membranes to ideal permeability barriers. A structure of a
cytoplasmic membrane is given in Fig. 2 (MADIGAN AND MARTINKO, 2006).

INTRODUCTION
5
Analyzing such membrane lipid biomarkers allows the discrimination between the
domains of life. For example, Bacteria, and Eukarya primarily synthesize membrane
lipids with fatty acid chains linked via an ester bond to a glycerol backbone, whereas
archaeal membrane lipids are characterized by isoprenoid alcohol chains connected via
ether linkage (Fig. 3). Furthermore, lipid analysis can be used for the identification and
quantification of different groups of organisms within a domain of life by recognizing
sources of specific lipids (MADIGAN AND MARTINKO, 2006).
Besides lipids, major cell membrane components are steroids and hopanoids. Hopanoids
and steroids are membrane rigidifiers. Eukaryotic organisms evolved sterols in their
membranes, whereas sterols are absent in prokaryotic membranes (with some notable
exceptions e.g. methanotrophic bacteria). Hopanoids are only found in bacteria and
regulate and rigidify membranes in the same way as sterols in eukarya. These
amphipathic lipids can be inserted between complex lipids (e.g. phospholipids) of the
bilayer membranes with their polar ends facing to the aqueous phase. Fig. 4 illustrates
the role of hopanoids in prokaryotic cell membranes.
Fig. 2: Schematic sketches of a phospholipid molecule, (top right),
phospholipid bilayer (top left) and a structure of the cytoplasmatic
membrane (central figure). Hydrophilic headgroups are facing
towards the outside, where they have contact with the aquaeous
environment while the hydrophobic non-polar fatty acid/isoprenoid
chains point inwards toward each other (MADIGAN AND MARTINKO,
2006).

INTRODUCTION
6
Fig. 3: Lipid biomarkers from bacteria, eukarya and archaea.
Archaeal lipids differ from bacterial and eukaryotal
lipids by building isoprenoid alkohols linked by ether
bonds, whereas bacteria and eukarya synthesize fatty
acid chains connected via ester bonds.
PETERS ET AL. (2005) described the importance of sterols and hopanoids as source of the
major saturated biomarkers in petroleum.
Tab. 1 provides a summary of all membrane lipids detected during this study. IPLs and
BHPs, as intact polar lipids, and fatty acids and sterols, as their apolar derivates, were
identified and assigned to their former living organisms (source function). The source
function was differentiated into three groups, eukaryotic, prokaryotic and a mixture
between those two. The detected membrane lipids in this study give some impression
about lipid diversity in prokaryotic and eukaryotic membranes, but the complexity of
lipids in membranes from different organisms is far more extensive (CULLIS ET AL.,
Fig. 4: Role of hopanoids in cell membrane.

INTRODUCTION
7
1996). A specification of most detected membrane lipids is presented in the next
chapters.
Tab. 1: Membrane lipids and their source function. IPLs, BHP, FAs and steroids are classified into eukaryotic or
prokaryotic sources. A third group labeled as mixture indicates prokaryotes and eukaryotes as possible
former living organism.
PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol;
PS, phosphatidylserine; PnE, phosphonoethanolamine; C-PnE, ceramide-phosphonoethanolamine; Cb,
cerebroside; C-PE, ceramide-phosphatidylethanolamine; SFA, saturated fatty acid; MUFA, monounsaturated
fatty acid; PUFA, polyunsaturated fatty acid; MAGEs, monoalkylglycerolethers;
1.4.1 INTACT POLAR LIPIDS (IPLS)
Traditionally, membrane lipids were analyzed by gas chromatography as phospholipid-
derived fatty acids (PLFAs) after transesterification (GUCKERT ET AL., 1985). Thereby,
only part of the information contained in the IPL is used. Due to recent developments in
analytical chemistry it is now possible to investigate membrane lipids in their intact
EUKARYOTIC PROKARYOTIC
IPLs
PC
PE
PS
PG
PI
C-PE
C-PnE
Cb
BHPs
aminotriol
aminotetrol
Fatty acid fraction
∑ SFA
∑ MUFA
∑ PUFA
specific fatty acids
C16:1 9
C16:1 8
C16:1 7
C18:1 8 Type-II
Neutral fraction
Sterols
Methylsterols
MAGEs
Compound
Class
Source Function
< MIXTURE >
e.g.Vokman et al., 1998; Bouvier et al., 1976;
Summons et al., 1994; Jahnke et. al., 1995;
Schouten, 2000; Elvert and Niemann, in press;
Hinrichs et al., 2000; Rütters et al. 2001
Type-I -
methanotrophs
GC/MS; GC-irMS
GC/MS; GC-irMS
References:
Analytical
technique
HPLC-ESI-MS
HPLC-APCI-MS
e.g. Abrajano et al., 1994; Niemann, 2005; Elvert
und Niemann, in press; Nichols et al., 1985;
Bowmann et al., 1991; Jahnke et al., 1995;
Guezennec and Fiala-Medioni. 1996; Fang et
al., 1993
e.g. Summons et al. , 1999; Talbot et al., 2001,
2003, 2007; Rohmer et al., 1984; Summons and
Jahnke, 1992)
e.g. Sohlenkamp et al. , 2003; Christie, 2007;
Mukhamedova and Glushenkova, 2000

INTRODUCTION
8
form using high-performance-liquid-chromatography coupled to electrospray ionization
mass spectrometry (HPLC-ESI-MS; FANG AND BARCELONA, 1998; RÜTTERS ET AL.,
2001, 2002). The advantage of direct IPL analysis is the complementary information on
the diversity of the polar head groups in addition to the fatty acid chains alone (diverse
chain length, number and position of double bonds). Therefore, a better taxonomic
differentiation can be achieved (FANG ET AL., 2000). Since IPLs are known to be
hydrolyzed within weeks after cell death (WHITE ET AL., 1979) their presence makes
them excellent biomarkers for viable biomass (WHITE ET AL., 1979; RÜTTERS ET AL.,
2002; STURT ET AL., 2004; BIDDLE ET AL., 2006).
In this lipid analysis, the major classes of complex polar membrane lipids were
subdivided into glycerol based lipids and sphingosine based lipids. Fig. 5 reflects all
IPLs relevant for this study. The detected lysophospholipids (e.g.
lysophosphatidylethanolamine (lyso-PE), lysophosphatidylcholine (lyso-PC),
lysophosphatidylserine (lyso-PS)) and two unidentified membrane lipids are not listed
in Fig. 5. Lysophospholipids contain only a single acyl chain (usually in position sn-1)
Fig. 5: Two classes of polar membrane lipids distinguished by their back bone
structure. Glycerol based lipids have two fatty acid chains and a polar
head group connected to a glycerol molecule. Sphingosine based lipids
are based on long-chain aliphatic aminodiols. The amino group of
sphingosine is linked to fatty acids by an amid bond and the polar head
group is esterified to the primary hydroxyl group.

INTRODUCTION
9
and are present in tissue only in trace amounts. Lysophospholipids could also be formed
during sampling, storage, or analytical processing (CHRISTIE, 2007).
1.4.1.1 GLYCEROL BASED LIPIDS
Glycerol based lipids are complex lipids containing a polar head group, often
phosphate-based, in position sn-3 and acyl groups in the sn-1 and sn-2 position (Fig. 3).
Phospholipids represent a structural role in the cytoplasmatic membrane and are
described as the most widely occurring membrane lipids in eukaryotes and prokaryotes
(SOHLENKAMP ET AL., 2003; CHRISTIE, 2007).
OCCURRENCE OF GLYCEROPHOSPHOLIPIDS IN PROKARYOTES
In most instances PE, PG, and cardiolipin (CL) have been found in bacteria as their
major membrane forming lipids. In addition to these three membrane lipids, a large
diversity can be found in bacteria, some as minor components but others as major
component in the bacterial membrane. Each family tends to have a distinct and
characteristic lipid composition (CHRISTIE, 2003).
PE is the main lipid fraction of microbial membranes, and also the second most
abundant phospholipid in animal and plant tissue (CHRISTIE, 2007). CHRISTIE (2007)
mentioned PE as a key building block of eukaryotic and prokaryotic membranes.
PG is found in almost all bacterial types and can be the main component of some
bacterial membranes. PG has also been proven to exist in small amounts in membranes
of eukaryotes (CHRISTIE, 2007).
CL is found in membranes of bacteria and should be named correctly as
„diphosphatidylglycerol‟. CL is a unique phospholipid with a diametric structure, four
acyl groups and potentially carrying two negative charges (CHRISTIE, 2007).

INTRODUCTION
10
PC plays a less important role in prokaryotic membranes. SOHLENKAMP ET AL. (2003)
explained a lack of PC in prokaryotes but also suggested that significant amounts (more
than 10%) can be present in rather diverse, mainly photosynthetic bacteria.
PS, as a major membrane lipid in eukaryotes, is only used as a biosynthetic intermediate
in PE biosynthesis and is rarely found in significant amounts in prokaryotic membranes.
OCCURRENCE OF GLYCEROPHOSPHOLIPIDS IN EUKARYOTES
In eukaryotic membranes the glycerol-based phospholipids are predominant, including
PS, PI, or the methylated derivates of PE, phosphatidyl-(N)-methylethanolamine (PME)
phosphatidyl-(N,N)-dimethylethanolamine (PDME), and PC, which can also occur in
some bacteria (SOHLENKAMP ET AL., 2003, and references therein; CULLIS ET AL.,1996).
PC is the major structural component of cellular membranes and the most abundant
phospholipid in eukaryotic cells, (MARTÍNEZ-MORALES ET AL., 2003). CHRISTIE (2007)
describes PC as the key building block of animal and plant membranes, which can have
an abundance of almost 50% of the total membrane.
PS is often found as a major membrane lipid of eukaryotes and also is present in some
microorganisms (CHRISTIE, 2007; SOHLENKAMP ET AL., 2003).
PI is described as a major and essential phospholipid of eukaryotic cells (SOHLENKAMP
ET AL., 2003; CHRISTIE, 2007).
1.4.1.2 SPHINGOSINE BASED LIPIDS
Long-chain bases are the characteristic
structural unit of the sphingolipid.
Sphingosine based lipids are linked via
an amide bond to a fatty acid to form a
ceramide (Fig. 6). The phosphate or
carbohydrate headgroups of the
Fig. 6: General chemical structure of sphingolipids.
Sphingosine linked via an amide bond to a fatty
acid builds a ceramide. The head group attaches
at the terminal hydroxyl group.

INTRODUCTION
11
sphingolipid are attached at the terminal hydroxyl group. CHRISTIE (2007) describes
sphingolipids as important and extremely versatile molecules which are quite distinct in
their physical and biological properties from the complex phospho- and glycerolipids.
CERAMIDE PHOSPHATIDYLETHANOLAMINE (C-PE)
C-PE is the sphingolipid analogue of PE and can be found in many bacteria (often
accompanied by ceramide phosphatidylglycerol). The fatty acid and long-chain bases
(sphingosine) vary with species (CHRISTIE, 2007).
CERAMIDE PHOSPHONOETHANOLAMINE (C-PNE)
C-PnE has a phophono-based headgroup attached to a sphingosine backbone. In
contrast to phosphatidyl-based headgroups which are derivates of phosphoric acid, the
PnE headgroup is based on phosphonic acid which lacks one oxygen atom. CHRISTIE
(2007) describes C-PnE as the most widespread phosphonolipid in nature. This class of
lipids occurs in protozoa and is also widely distributed among many species of marine
animals (e.g. anemone, mollusks, oysters and sponges) (MUKHAMEDOVA AND
GLUSHENKOVA, 2000). C-PnE was also detected in bacteria (CHRISTIE, 2007).
Cerebroside (Cb)
Chemically, cerebrosides are composed of a hexose and a ceramide moiety.
Cerebrosides are described as widespread in nature and have been found in nearly all
kinds of biological species (TAN AND CHEN, 2003).
1.4.2 BACTERIOHOPANEPOLYOLS (BHPS)
Hopanoids are classified into two groups, the biohopanoids and geohopanoids.
Bacteriohopanepolyols (BHPs; or biohopanoids) are pentacyclic triterpenoids produced
by a variety of bacteria which occur in immature sediments and biomass of some

INTRODUCTION
12
bacteria. These BHPs are thought to be cell membrane stabilizing components which
regulate and rigidify in the same way as sterols in eukaryotes (SUMMONS ET AL., 1999;
TALBOT ET AL., 2007). BHPs can be found in various immature modern to Cenozoic lake
sediments and in marine sediments (TALBOT ET AL., 2003). During diagenesis
biohopanoids are transformed to defunctionalized products called “geohopanoids”. A
generalized structure of a BHP is shown in Fig. 7:
OURISSON AND ALBRECHT (1992) described hopanoids as “the most abundant natural
products on Earth”, which can be found in records of recent (BHPs) and past (diagenetic
products) bacterial populations. Due to taxonomic variation of the BHP-producing
organism (ROHMER, 1993), they are important molecular markers (biomarkers) and can
be used to determine hopanoid-producing bacterial community structures in modern and
past environments. Bacteriohopanepolyols exhibit structural variation in the side chain
with many structures differing in terms of number, position and nature of the functional
groups. For example, tetrafunctionalized structures are the most commonly reported
BHPs. In addition, many more complex structures like amino sugar or amino acid
moieties can appear. Other structural variations include methylation at either C-2 or C-3
position of the triterpenoid ring system (TALBOT ET AL., 2007). These structural
variation allow, for example, a classification into cyanobacteria (C-2 methylated BHPs),
acetic acid bacteria, purple non sulfur bacteria, nitrogen fixing bacteria, in some
Fig. 7: Generalized structure of a BHP. Structural variation
can occur in the side chain (four, five or six functional
groups) and additional a methylation at either C2 or
C3 position. For example, C-2 methylated BHPs were
specific indicators of cyanobacteria (SUMMONS ET AL.,
1999) whereas methylation at C-3 are specific for
aerobic methanotrophs (NEUNLIST UND ROHMER,
1985a, b; TALBOT ET AL., 2003; BLUMENBERG ET AL.,
2007).

INTRODUCTION
13
gram-positive and -negative bacteria, methylotrophs and methanotrophs (methyl group
at C-3) (SUMMONS AND JAHNKE, 1992; ROHMER ET AL., 1984).
The capabilities to analyze BHPs by means of HPLC-APCI-MS are relatively new
(TALBOT ET AL., 2001) and applications to recent and particularly geological problems
are still rare. The large number of polar functional groups on the side chain of BHPs
precludes their analysis by conventional GC-MS techniques (TALBOT ET AL., 2001).
1.4.3 FATTY ACIDS AND CYCLIC TRITERPENOIDS - CONCENTRATIONS AND
COMPOUND-SPECIFIC ISOTOPE ANALYSIS
Fatty acids and cyclic triterpenoids are organic molecules which have a unique chemical
structure and/or isotopic signature that
allows a precise classification of
organisms. Fatty acids (FAs) are
aliphatic components of lipids,
particularly triacylglycerol and
phospholipids (CHRISTIE, 2007). FAs are
synthesized in nature by a group of
enzymes (fatty acid synthases) from
acetyl-CoA and malonyl-CoA
precursors (KANEDA, 1991). In general, they contain even numbers of carbon atoms in
straight chains (most abundant in the range of C14 to C24) with a carboxyl group at one
end. Odd-and branched-chain fatty acids can
also be synthesized. Fatty acids can include
double bonds (unsaturated fatty acids) or
other substituents which are normally
incorporated later by different enzyme
systems (KANEDA, 1991). The number and
position of double bonds can be characteristic
for the producing organism. For example,
polyunsaturated fatty acids (PUFAs) can be
Fig. 9: Structure of a diagnostic triterpenoid. Sterols
methylated at the C-4 position like 4 -
methylcholesta-8(14),24-dien-3 -ol are an
indicator for methanotrophic bacteria.
Fig. 8: Structure of a diagnostic fatty acid (C16:1 8). The
fatty acid has sixteen carbon atoms and a double
bond located between the eighth and ninth carbon
atom from the methyl end of the molecule.
C16:1 8 is characteristic for the appearance of
methanotrophic bacteria.

INTRODUCTION
14
found in plant tissue and are not often found in bacterial tissue whereas bacterial lipids
tend to contain higher amounts of C14 to C18 straight-chain saturated and monoenoic
fatty acids (CHRISTIE, 2003). Unsaturated fatty acids are a useful biomarker of bacterial
activity (e.g. POND ET AL., 1998; NICHOLS ET AL., 1985). An example of biomarker
studies of fatty acids and sterols/hopanols in mussels is given by JAHNKE ET AL. (1995).
These authors analyzed such biomarkers to confirm the presence of endosymbiotic
bacteria in a modern cold seep in the Gulf of Mexico. For example, Fig. 8 shows a
characteristic fatty acid for methanotrophic bacteria. Furthermore, they reported
substantial abundances of diagnostic triterpenoids which also indicate the appearance of
methanotrophic bacteria (Fig. 9). Steroids, as membrane lipids, are normally biomarkers
for eukaryotes but methylsterols such as 4 -methylcholesta-8(14),24-dien-3 -ol have
only been reported from aerobic methanotrophic bacteria (SCHOUTEN ET AL., 2000).
1.5 SITE DESCRIPTION
1.5.1 B. BROOKSI & B. CHILDRESSI RECOVERED FROM THE GULF OF MEXICO
(GOM)
Samples of Bathymodiolus brooksi and B. childressi specimens were collected in
October 2003 during the Deep Sea Cruise 11 Leg I at cold seeps in the Gulf of Mexico
near the Alaminos Canyon (26°21.32´N, 94°30.12´W, 2226m), using the deep-sea
submersible Alvin (Fig. 10).
Cold seeps are abundant in the northern Gulf of Mexico (LANOIL ET AL., 2001) and
provide rich energy sources (e.g. CH4 and H2S) for localized habitats on the sea floor
that are colonized by chemotrophic bacteria (LANOIL ET AL., 2001; SASSEN ET AL., 1993).
The GoM is a special site where both thermogenic (composed primary of hydrocarbon
gases derived from thermal degradation of organic matter) and biogenic (composed
primarily from biological methanogenesis) 13
C signatures have been recorded (LANOIL

INTRODUCTION
15
ET AL., 2001). For example, 13
C values of whole gill tissue from Bathymodiolus
heckerae at the West Florida Escarpment with -77.1‰ is more likely to show a biogenic
methane signal whereas the whole gill tissue from Bathymodiolus brooksi at Alaminos
Canyon with -46.0‰ reflects a thermogenic methane signal (DUPERRON ET AL., 2007).
1.5.2 B. CF. THERMOPHILUS RECOVERED FROM THE PACIFIC-ANTARCTIC RIDGE
(PAR)
Bathymodiolus cf. thermophilus was recovered from the Pacific-Antarctic Ridge near
the Foundation Chain by using a TV-controlled grab (GTVA). B. cf. thermophilus
samples were recovered from station 30-GTV at 37°47.443´S, 110°54.834´W in a water
depth of 2212 m in June 2001 during the Sonne cruise SO 157 (Fig. 11). Four
specimens of B. cf. thermophilus were taken close to an active vent (Fig. 12). The vent
is associated with cloudy bottom water with a temperature anomaly ( T) of +0.25°C.
The seafloor at the vent side was partly covered with active and fossil sulfide deposits
together with young glassy lava. Besides B. cf. thermophilus, more hydrothermal faunal
occurred on an area of 30 m x 30 m e.g. Neolepas cf. rapanui, chaetopterid tube worms,
dead vesicomyid clams, bythograeid crabs, actinians and macrourid fish (STECHER ET
AL., 2002). The TV-system also tape-recorded some open specimens of B. cf.
Fig. 10: Map showing the northwestern part of the Gulf of Mexico. Samples of B childressi and B. brooksi were
taken from the Alaminos Canyon (red point) (26°21.32‟N, 94°30.12‟W) in a water depth of 2226 m.

INTRODUCTION
16
thermophilus showing only moderately hypertrophic gills. STECHER ET AL. (2002)
describes the place of discovery as a changing environment from a symbiosis-
dominated community (vesicomyid clam assemblage) shifting to a filter feeding
assemblage (mussels of the genus Bathymodiolus – as an intermediate between
symbiosis-dominated and filter feeding community).
Fig. 11: Map showing the position of the sampling site from the Pacific-Antarctic Ridge. The black circle marks the
point where the Bathymodiolus cf. thermophilus specimens were recovered from (37°47.443´S,
110°54.834´W in a depth of 2212 m).
Fig. 12: Picture of the recovered
Bathymodiolus cf. thermophilus
specimens. Photo is courtesy of
Christian Borowski.

INTRODUCTION
17
1.6 OBJECTIVES AND RESEARCH QUESTIONS
This study investigated two seep mussels (B. childressi & B. brooksi) and one vent
mussel (B. cf. thermophilus) by analyzing two distinct tissue types (symbiont-containing
gill and symbiont-free foot tissue) with different lipid analysis methodologies. IPLs
mainly provided info on general compositional patterns; BHPs indicated the input for
bacterial sources and analysis of the apolar lipids demonstrated the presence of different
symbiotic bacteria living within the host organism. These powerful methods provide the
basis to solve the research questions which are summarized below.
To what extend can IPLs be used to distinguish between eukaryotal and
prokaryotal membrane lipids?
o How does the symbiont-containing gill tissue differ from the symbiont-
free-foot tissue?
o Are there IPLs which clearly differ between eukaryotic and prokaryotic
membranes? Can IPLs be used as a biomarker for bacterial input?
Do the methanotrophic and thiotrophic bacteria in B. childressi, B. brooksi and
B. cf. thermophilus produce BHPs?
o If they do, how specific are they?
Does the compound-specific carbon isotope analysis provide information about
group specific biomarkers for methanotrophic and thiotrophic bacteria?
o What are the biomarkers for the methane-oxidizing and sulfide-oxidizing
bacteria?
o Do the site specific differences between the samples from the GoM and
the PAR reflect in a different biomarker response? How do the sites
differ in terms of variations in specific lipid signals and by changing
isotopic composition?

ANALYTICAL METHODS
18
2 ANALYTICAL METHODS
2.1 GENERAL LABORATORY PROCEDURE
Fig. 13 shows all important steps during sample processing. Mussel tissues were
dissected directly after recovery. For each specimen, gill and foot tissue were frozen for
stable isotope and lipid analysis. The extraction of biomarkers from different mussels
and tissue types was carried out in an ultrasonic bath (Bligh & Dyer extraction). The
less polar -neutral and fatty acid fractions- were analyzed by gas chromatography-mass
spectrometry (GC-MS) and gas chromatography-isotope ratio monitoring-mass
spectrometry (GC-irMS), whereas biomarkers with a higher polarity like IPLs and
BHPs were detected by high performance liquid chromatography multistage mass
spectrometry (HPLC-MSn
). The following chapter describes laboratory processes in
more detail.
Fig. 13: Flow chart presenting an overview of the major laboratory processes.

ANALYTICAL METHODS
19
2.2 TOTAL LIPID EXTRACTION AND EXTRACT PURIFICATION
To prevent contamination of samples used for biomarker analysis, extensive precautions
were taken during sample handing. All glass materials used were either rinsed with
organic solvents (MeOH, DCM & n-hexane) or heated in the oven to 450°C for 16 h to
remove all organic material.
2.2.1 SAMPLE PREPARATION AND EXTRACTION
During storage, all samples were frozen at -20°C. Before extraction, the samples were
freeze dried and weighed to determine the dry weight (Tab. 2).
Before extraction, combusted sea sand was added to each sample in order to facilitate
physical disruption of cells and to achieve an equal amount of extracting material of
roughly 5 g (Tab. 2). The internal standard for intact polar lipids (MGDG) and four
standards for the measurements with the GC-MS (n-hexatriacontane, behenic acid
methyl ester, 1-nonadecanol, 2-methyl-octa-decanoic acid) were added at known
concentrations prior to the extraction. The use of internal standards allows for correction
of sample loss during the extraction and subsequent work-up steps, assuming that the
internal standards behave in the same way as other lipids in the sample.
The extraction protocol was originally described by BLIGH AND DYER (1959), however
the used extraction technique was a modified method following instructions in STURT ET
AL. (2004). After adding a mixture of MeOH, DCM and a 50 mM phosphate buffer at
Tab. 2: List of samples including genus, location & dry weight. Green bars visualize the amount
of dry weight.
Name of Sample Genus Location
M37 - Gill
Bathymodiolus childressi
(methanotrophs)
GoM 0.1366 4.8681 5.0047
0.0000
M31 - Gill 0.2436 4.8095 5.0531
M31 - Foot 0.0920 4.9724 5.06440.0000
157 - Foot 0.0787 4.9252 5.0039
157 - Gill 0.2128 4.8700 5.08280
Freeze dried Sample
Bathymodiolus brooksi
(thiotroph + methanotrophs)
GoM
Bathymodiolus sp.
(thiotroph )
PAR
∑
[g]
added comb.
Sand [g]
initial weight
[g]

ANALYTICAL METHODS
20
pH 7.4 (2:1:0.8 - v/v) to initiate lysis of bacterial cells, the extract was sonicated and
centrifuged at 2000 rpm for 10 minutes. The liquid phase was separated and the
extraction repeated five more times. For the last three extractions a 50 mM
trichloroacetic acid buffer was used instead of phosphate to facilitate the release of
archaeal compounds, like glyceroldialkylglyceroltetraether (GDGT, STURT ET AL.,
2004). The supernatants were combined in a separating funnel and deionized water
(Milli Q) and DCM added to accomplish a better separation of the organic phase and the
aqueous phase. After the two layers separated, the organic phase was drawn off and the
remaining aqueous rest was re-extracted three more times with DCM. Afterwards the
combined organic phases were washed three times with deionized water evaporated to
dryness under a stream of nitrogen at a temperature between 35-37°C, and stored at
-20°C. For IPL-measurements, the samples were dissolved in DCM/MeOH (1:1).
2.2.2 SAPONIFICATION
An aliquot (1/10 of the TLE) was transferred to a 5 ml conic screw cap glass vial and
evaporated to near dryness under a stream of nitrogen. The sample was then saponified
with 2 ml of a 6% methanolic KOH solution and placed into the oven to maintain the
reaction at 80°C for 3 hours. After cooling down, the sample was diluted with 2 ml of
0.05M KCL solution. The neutral lipids were released from the alkaline mixture (base
extraction) by extracting four times with 2 ml n-hexane. The remaining aquatic phase
was acidified by adding 25% HCL, one drop at a time, until the pH was close to 1. The
fatty acids (acid extraction) were extracted four times with 2 ml n-hexane. The neutral
lipids and the fatty acid lipids were dried under a stream of nitrogen and derivatized
with BF3 in MeOH and BSTFA, respectively.

ANALYTICAL METHODS
21
2.2.3 ASPHALTENE SEPARATION
A pasteur pipette packed with combusted glass wool was rinsed 5 times with 2 ml of
n-hexane. Half of the total lipid extract was separated and dried completely to ensure
that the sample contained no water. Afterwards, 500 µl of n-hexane was added to the
evaporated sample and sonicated for 10 minutes. The TLE dissolved in n-hexane was
rinsed onto the glass wool and stepwise eluted with approx. 2.5 ml n-hexane, the
fraction was labeled as maltenes. The maltene fraction was evaporated to near dryness
and stored at -20°C. The second fraction was rinsed from the glass wool with 4 ml
DCM (MeOH as a solvent was not used which probably influenced the results) and
labeled as asphaltenes. The asphaltene fraction was dried under a stream of nitrogen and
derivatized with acetic anhydrite before analyzing on the HPLC.
2.2.4 DERIVATIZATION
In gas chromatography it is often advantageous to derivatize polar functional groups
with suitable reagents. For example, prior to analysis on the GC, the fatty acid
components of lipids should be converted to low molecular weight non-polar
derivatives, such as methyl esters. Other polar functional groups should also be treated
in a similar manner to achieve a better gas chromatographic separation. Elution order
and fragmentation patterns in mass spectroscopy can be influenced by a specific
derivatization (CHRISTIE, 2007).
2.2.4.1 GC-MS (FAS AND STEROLS)
Fatty Acids were derivatized to fatty acid methyl esters (FAMEs). The neutral fraction
was analyzed as trimethylsilylethers (TMS derivatives). Both reactions take place at the
hydroxyl groups.

ANALYTICAL METHODS
22
Methylester derivatives:
An aliquot of the fatty acid fraction was transferred into a 5 ml conic screw cap glass
vial and evaporated under a stream of nitrogen. After adding 1 ml of a 20% BF3 in
methanol solution the samples were heated at 70°C for 1 hour. After cooling down 1 ml
water was added and the mixture extracted four times with 2 ml n-hexane. Each
derivatized extract was evaporated under a stream of nitrogen and re-dissolved in
1000 µl n-hexane for analysis on the GC-MS system.
Trimethylsilylether derivatives (TMS):
An aliquot of the neutral fraction was transferred into a 2 ml vial and evaporated under a
stream of nitrogen. 100 µl BSTFA (N,O-bis(trimethylsilyl)trifluoracetamide) and 100 µl
pyridine were added and the mixture heated to 70°C for 1 h. After cooling down, the
reaction mixture was evaporated under a stream of nitrogen and re-dissolved in 1000 µl
n-hexane for analysis on the GC-MS system.
2.2.4.2 LC/MS (BHPS)
50% of the asphaltene fraction was transferred into a 4 ml vial and then reduced to
dryness under a stream of nitrogen. The derivatization was done by adding 2 ml
pyridine and 2 ml acetic anhydride. After the initial warming at 50°C for 1 h the
samples were left to stand overnight at room temperature. The next day, acetic anhydrite
and pyridine were evaporated under a stream of nitrogen and re-dissolved in a mixture
of 1 ml methanol / propan-2-ol (60:40, v/v, 2 ml) for injection on the LC/MS system.

ANALYTICAL METHODS
23
2.2.5 DMDS-ADDUCTS
Monounsaturated fatty acid double bond positions were determined by mass
spectrometry (GC-MS) as dimethyldisulfide (DMDS) adducts (NICHOLS ET AL. 1986).
The reaction was catalyzed by iodine. This simple and rapid method provides
chromatographic separation and positive identification of adducts derived from cis/trans
isomers.
Briefly, the formation of the DMDS adducts was carried out in a 2 ml screw-cap
(teflon-lined) glass vial. The remainder of the fatty acid fraction was dissolved in 50 µl
n-hexane and treated with 100 µl DMDS and 20 µl of iodine solution (6% w/v in
diethylether). Before heating the mixture at 50°C for 48 h, the vial was flushed with
nitrogen and sealed. After 48 h, the sample was cooled and diluted with 500 µl n-hexane
and 500 µl of sodium thiosulfate (Na2S2O3 – 5%w/v in water). The sodium thiosulfate
removes the excess of iodine by formation of iodide ions. After the separation of the
organic phase, the aquaeous phase was extracted twice with 500 µl n-hexane. Combined
organic phases were evaporated under a stream of nitrogen and diluted with 200 µl
n-hexane prior to GC-MS analysis.
2.3 HIGH PERFORMANCE LIQUID CHROMATOGRAPHY COUPLED TO A
MASS SPECTROMETER
IPLs were analyzed on a ThermoFinnigan Surveyor HPLC coupled to an ion-trap mass
spectrometer (LCQ Deca XP Plus) equipped with an electrospray source (HPLC-ESI-
IT-MSn
). Electrospray ionization was used as a soft ionization technique in order to
retain all intact membrane lipid information. The used ion-trap mass spectrometer was
configured to run “data dependent ion tree” experiments where the base peak from each
scan was fragmented up to MS3
. All samples were analyzed in positive and negative
ionization mode to obtain complementary structural information (STURT ET AL., 2004).
The fragmentation pattern in the positive ionization mode is generally used to identify
the polar headgroup of the membrane lipid, whereas the negative mode offers

ANALYTICAL METHODS
24
information on the fatty acid side chains. More detailed information on the operation
mode and the data examination is given in SCHUBOTZ, (2005).
Technical information:
Analytical setup was adapted from STURT ET AL., (2004). In brief, IPLs were separated
according to headgroup polarity using a LiChrospher Diol-100 column
(125 mm x 2.1 mm, 5 µm; Alltech GmbH, München, Germany) fitted with a
7.5 x 2.1 mm guard column of the same packing material in a column oven at 30°C
using a ThermoFinnigan Surveyor HPLC system.
Two eluents (A+B) were used with a linear gradient method to achieve good separation.
The eluents are a mixture of different solvents. Eluent A contains hexane, isopropanol,
formic acid and ammonia (approximately 27% aqueous) in a ratio of 79:20:0.12:0.14.
Eluent B consists of isopropanol, water, formic acid and ammonia (approximately 27%)
at a ratio 90:10:0.12:0.04. The flow rate was set to 0.2 ml/min with the following linear
gradient: 100% A to 35% A; 65% B set for 45 min, then back to 100% A for 0.5 h to re-
equilibrate the column for the next run.
The samples in the Surveyor autosampler were kept at 10°C and the injection volume
was set to 10 µl using a sample loop of 20 µl in “no waste” injection mode.
MSn
measurements were carried out using a ThermoFinnigan LCQ Deca XP Plus ion-
trap mass spectrometer with an ESI interface. ESI parameters were as follows: Capillary
temperature 200°C, sheath gas flow 40 (arbitrary units), spray voltage ±5kV. Further
parameters were determined by manual tuning using a solution of 1,2-dipalmitoyl-sn-
glycero-3-phosphocholine introduced into the ESI source with LC flow of 100% A and
a flow rate of 0.2 ml/min.
Semi-quantitative concentrations due to response factors
All concentration values for IPLs in mg IPL/g tissue are semi-quantitative estimates
because response factors for each membrane lipid were not taken into account.
However, response factors of different IPL classes are relatively constant and differ
only by a factor of 2-3. Sample analysis was conducted in a continuous sequence which

ANALYTICAL METHODS
25
lowers the chance of changing response factors and therefore allows the relative
comparison of concentrations from different samples and tissue types.
2.3.1 BHPS
Atmospheric pressure chemical ionization liquid chromatography/multi-stage ion trap
mass spectrometry (APCI-LC/MSn
) with a procedure adapted from TALBOT ET AL.,
(2003) was used. A reversed-phase HPLC was accomplished using a reversed phase
column (Alltech Prevail C18, 150x2.1 mm, 3 µm, Alltech, München, Germany) and a
precolumn (7.5x2.1 mm) of the same packing material.
Three eluents (A+B+C) were used with a linear gradient to achieve good separation.
The eluents A, B, and C are different organic/inorganic solvents. Eluent A is MeOH,
eluent B is deionized water and eluent C is propan-2-ol. The flow rate was set to
1 ml/min with the following linear gradient: 90% A and 10% B (0-5 min); 59% A,
1% B and 40% C (at 45 min) then hold isocratic to 70 min. The samples (acetylated
total extracts) were dissolved in MeOH/propan-2-ol (60:40 v/v) prior to injection.
LC/MS settings were as follows: capillary temperature 150°C, APCI vaporizer
temperature 400°C, discharge current 5 µA, sheath gas flow 40 (arbitrary units). LCQ
instrument parameters were selected using the automated tune program on a direct
infusion of an acetylated standard of glucose.
2.4 GAS CHROMATOGRAPHY – MASS SPECTROMETRY (GC-MS)
The fatty acids and neutral lipids were examined using gas chromatography-mass
spectrometry (GC-MS) with a Thermo Electron Trace MS equipped with a 30 m
RTX-5MS fused silica column (0.32 mm i.d., 0.25 µm film thickness). The GC-MS
operated in the electron impact (EI+)-mode at 70 eV with a full mass range of
m/z 40-900 with 1.5 scans per second. The detector was set at 350 V, the interface
temperature was set to 300°C and the carrier gas was He at a constant flow rate of

ANALYTICAL METHODS
26
1.4 ml/min. Each sample was injected manually (injection volume: 1 µl). The GC oven
temperature program used two different methods depending on which fraction was
being analyzed.
The temperature program for the fatty acid fraction was 60°C held for 1 min then
increased to 150°C at a rate of 15°C min-1
, then to 320°C at 4°C min-1
and kept for
27.5 min at that temperature. For the neutral fraction a final temperature of 310°C was
used.
All compounds were identified and quantified by analyzing the mass spectral data and
comparison of the retention time of each compound.
2.5 GAS CHROMATOGRAPHY ISOTOPE RATIO MASS SPECTROMETRY
(GC-IRMS)
The carbon isotopic analysis of the fatty acid and neutral fraction was performed with a
Hewlett Packard 5890 series II gas chromatograph coupled via a Finnigan Combustion
Interface-II to a Finnigan MAT 252 mass spectrometer equipped with a 30 m RTX-5MS
fused silica column (0.25 mm i.d., 0.25 µm film thickness). Helium, as the carrier gas,
had a constant flow rate of 1.5 ml/min. The oxidation oven of the combustion interface
was set to 940°C and the temperature of the reduction oven was operated at 640°C.
Compared to the neutral fragments the fatty acids had a shorter temperature program.
The initial GC oven temperature for the fatty acids was 60°C, held for one minute,
increased to 150°C at 10°C min-1
, to 320°C at 4°C min-1
and held for 7 min. The final
temperature of the neutral fraction temperature program was longer as final temperature
was held for 32 min.
Each sample was injected manually with a sample volume of 1 µl and additionally
0.5 µl of injection standard. The injection standard cholestane ( 13
C: -25.7‰) was
introduced to examine reproducibility and precision of each measurement.

ANALYTICAL METHODS
27
All carbon isotope values are given in the notation (R = 13
C/12
C, RVPDB = 0.0112372;
values were given in permil relative to the Vienna Pee Dee Belemnite (VPDB)
standard):
By definition, VPDB has a 13
C value of 0‰, and therefore, negative values indicate
enrichment of the lighter isotope (12
C) compared to the standard.
Isotopic values of all measured fatty acids and neutral lipids were corrected for
additional carbon which was added during the derivatization. The neutral fraction was
analyzed as trimethylsilyl ether derivates (TMS-derivates), containing three additional
carbon atoms, whereas the fatty acids were converted via transesterification, containing
only one additional carbon atom. Isotopic values for all unknown components are not
corrected for the introduction of carbon during derivatization.

RESULTS
28
3 RESULTS
The lipid analysis was carried out on three different mussels of the genus
Bathymodiolus. Two of the three mussels were dissected into two distinct tissue types.
The B. childressi sample comprise only gill tissue, whereas B. brooksi and B. cf.
thermophilus contain gill and foot tissue. These five samples were treated and analyzed
with various methods. This chapter will present all major results from the complex lipid
structure (IPLs) and their cell membrane stabilizing components (BHPs and sterols) to
the composition of phospholipid derived fatty acids and their supplementary isotopic
composition.
3.1 INTACT POLAR LIPIDS (IPLS)
The identification of all biological membranes in all samples was based on typical
fragmentation patterns from IPLs in their mass spectra. The fragmentation of PE, as one
major membrane lipid, is shown, as an example in Fig. 14.
Using high performance liquid chromatography–electrospray ionization–ion trap–
multistage mass spectrometry (HPLC-ESI-IT-MSn
), different IPLs present in gill and
foot tissue in each of the three mussel species were detected. Fig. 15 shows five colored
chromatograms with all detected IPLs. Another possibility to visualize polar membrane
lipids is the use of density maps (Appendix IA-IE). Density maps provide a two-
dimensional view and are helpful during determination of different IPLs. A general
overview with semi-quantitative and relative concentrations of all IPLs in every mussel
and tissue type is presented in Appendix II.
The lipid composition in the different mussel and tissue types varies from major classes
of lipids found in biological membranes like phosphatidylcholine (PC),
phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylinositol (PI)
and phosphatidylserine (PS) and compounds containing only a single acyl chain
(lysophospholipids) like lyso-PE, lyso-PC, lyso-PS to membrane lipids which are not

RESULTS
29
usually found as primary part of the membrane. The structure and mass spectra of those
rare lipids like ceramide 2-aminoethylphosphonate (C-PoE), PE-ceramide (C-PE) and
glycosphingolipid are shown in the positive and negative mode in Appendix III, IV, V,
respectively. Two membrane lipids (M-154 and M-197) which could not be clearly
identified are described by their mass spectra in the positive ionization mode which is
shown in Appendix VI.
Fig. 14: Fragmentation pattern of the ms2 spectrum (positive and negative ionization mode) clarifies the
identity of phosphatidylethanolamine (PE). The mass spectral data represent a mixed signal of
two PE molecules (m/z 690 & m/z 688) with a different fatty acid composition (∑ C32:1 and
∑ C32:2). The positive ion mode shows two fragments with a characteristic neutral loss of
m/z 141. In the negative ionization mode, these two PE molecules produce a mixed signal.

RESULTS
30
Fig. 15: Base peak chromatograms showing the variation of the relative IPL concentration through all mussel and
tissue samples. Eleven known and two unknown IPLs were detected in five samples showing the lipid
composition and fatty acid distribution. M-197 & M-154, unknown components; PC, phosphatidylcholine;
PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine;
PnE, phosphonoethanolamine; C-PnE, ceramide-phosphonoethanolamine; Cb, cerebroside; C-PE,
ceramide-phosphatidylethanolamine; STD, injection standard (C16-PAF).

RESULTS
31
3.1.1 IPL-COMPOSITION AND -CONCENTRATIONS OF B. CHILDRESSI
Fig. 16 shows all detected IPLs with semi-quantitative concentrations. The IPL
composition in the gill tissue of B. childressi is dominated by PE. Next most common
membrane lipids are PC and C-PnE. The concentrations of the three main IPLs are 1.96,
1.41 and 0.69 mg/g tissue, respectively. PG, the unknown fragment M-197 and PI are
present in concentrations of 0.40, 0.32 and 0.16 mg/g tissue, respectively. Lyso-PE is
the most abundant lyso-lipid with 0.11 mg/g tissue followed by lyso-PC
(0.057 mg/g tissue) and lyso-PS (0.007 mg/g tissue). The sum of all membrane lipid
concentrations is 5.2 mg/g tissue; the corresponding foot tissue has not been analyzed.
Fig. 16: Semi-quantitative estimates of all determined IPLs in the gill tissue of B. childressi.
The concentrations of membrane lipids are given in mg/g tissue.

RESULTS
32
3.1.2 IPL-COMPOSITION AND -CONCENTRATIONS OF B. BROOKSI
Fig. 17 shows all semi quantitative concentrations of IPLs in B. brooksi for gill (blue)
and foot (red) tissue. The gill tissue has a total membrane lipid concentration of
2.4 mg/g tissue, whereas the total lipid concentration of the foot tissue is 2.2 mg/g tissue
(Appendix II). The gill tissue shows in general higher concentrations compared to the
foot tissue with exception of PC. PC is the major membrane lipid in the foot tissue and
at the same time the most abundant IPL (1.23 mg/g tissue) of these two samples. The
equivalent PC concentration in the gill tissue is 0.39 mg/g tissue which is approximately
1
/3 of the PC concentration in the foot tissue. PE, with 0.89 mg/g tissue, is the most
abundant IPL for the gill tissue and is slightly greater than PE for the foot tissue
(0.84 mg /g tissue), which is the second most abundant membrane lipid in this type of
tissue. C-PnE is with 0.64 mg/g tissue second abundant IPL for the gill tissue and is
Fig. 17: Semi-quantitative estimates of all IPLs identified in B. brooksi - gill (blue) and foot
(red) tissue. Concentrations of membrane lipids are given in mg/g tissue.

RESULTS
33
more abundant in the gill tissue than in the foot tissue (0.058 mg/g tissue). The
membrane lipid concentration of the unknown fragment M-197, lyso-PC, PI and lyso-
PE dominate (0.16, 0.114, 0.105 and 0.105 mg/g tissue) in the gill tissue over the IPL
concentration in the foot tissue (0.009, 0, 0.02 and 0.039 mg/g tissue). PG exists in
minor amounts in the gill tissue (0.02 mg/g tissue) and is not present in the foot sample.
The unknown fragment M-154 is only present in the gill tissue (0.0172 mg/g tissue) and
PS occurs in minor amounts in gill and foot tissue, with concentrations of 0.014 and
0.006 mg/g tissue, respectively.
3.1.3 IPL-COMPOSITION AND -CONCENTRATIONS OF B. CF. THERMOPHILUS
The concentration of all detected IPLs in gill and foot tissue of B. cf. thermophilus are
shown in Fig. 18. The total IPL concentration in the gill sample is 1.7 mg/g tissue and
therefore remarkably higher than the tissue concentration of the foot sample, which is
1.1 mg/g tissue. PE (0.87 mg/g tissue) is the most abundant lipid in the gill tissue
whereas PC (0.72 mg/g tissue) is the major membrane lipid in the foot tissue. PE in the
foot tissue (0.24 mg/g tissue) is almost four times less abundant than in the gill tissue.
The PC concentration in the gill tissue is 0.43 mg/g tissue. Lyso-PC is only abundant in
the gill tissue and comprises 0.22 mg/g tissue and is therefore the third most abundant
IPL in the gill tissue. The fourth highest concentrated IPL is only abundant in the gill
tissue at 0.10 mg/g tissue. PI and PS are only found in the gill sample with
concentrations of 0.044 and 0.024 mg/g tissue, respectively. Lyso-PE, with an
equivalent concentration of 0.06 mg/g tissue, is equally abundant in both gill and foot
tissue. C-PnE only occurs in foot tissue with a concentration of 0.031 mg/g tissue. The
determined amount of the unknown lipids M-197, M-154 and Cb in gill and foot tissue
is marginal.

RESULTS
34
Fig. 18: Semi-quantitative estimates of all IPLs identified in gill (blue) and foot (red) tissue of B.
cf. thermophilus. Concentrations of membrane lipids are given in mg/g tissue.
3.2 BACTERIOHOPANEPOLYOLS (BHPS)
All samples were acetylated and measured by a reversed-phase HPLC-APCI-MS
(TALBOT ET AL., 2001). The identification of BHPs was successful for B. childressi and
B. brooksi. The gill tissue of B. childressi contained two abundant BHPs, predominantly
35-aminobacteriohopane-31,32,33,34-tetrol and 35-aminobacteriohopane-32,33,34-triol
(Fig. 19). The gill tissue of B. brooksi contains an 35-aminobacteriohopane-32,33,34-
triol. Fig. 19 shows combined mass chromatograms (m/z 714 + m/z 772) of the two
mentioned BHPs.

RESULTS
35
Fig. 19: Mass chromatograms (m/z 714.5 + m/z 772.5) of BHP analysis of two
Bathymodiolus species (a) Gill tissue of B. childressi contains two BHPs.
Aminotetrol (35-aminobacteriohopane-31,32,33,34-tetrol) is eluting (RT:
28 min) two minutes before aminotriol (35-aminobacteriohopane-32,33,34-
triol) (RT: 30 min). (b) Gill tissue of B. brooksi contains only aminotriol
eluting at 29.6 min.

RESULTS
36
3.3 COMPOUND-SPECIFIC ISOTOPIC ANALYSIS (CSIA)
3.3.1 FATTY ACID FRACTION
All fatty acid fractions were treated with boron trifluoride (BF3) to produce fatty acid
methylesters (FAMEs) which were analyzed by GC-MS and GC-irMS. Tab. 3 shows
the concentration and the isotopic composition of all determined fatty acids in each
sample.
Tab. 3: Phospholipid derived fatty acid composition and 13
C values for three mussels
of the genus Bathymodiolus.
Total concentrations are given in mg g-1
(dry weight) and isotopic values are given in ‰. All 13
C
data are average values, measured between 2 and 4 times. The mean standard deviation of all
shown fatty acids accounts for 0.9 ‰. Fatty acids are listed in relative order of elution. Fatty acids
are identified by the total number of carbon atoms and the number of double bonds. The number
after the defines the position of the double bond from the methyl end of the molecule. The
relative concentration in each sample and tissue type is indicated through colored bars. GoM, Golf
of Mexico; AL, Alaminos Canyon; RT, retention time; ND, none detected; tr, trans geometry
TOTAL
CONCENTRATION
(mg g
-1
[dry wt])
13
C
(‰)
TOTAL
CONCENTRATION
(mg g
-1
[dry wt])
13
C
(‰)
TOTAL
CONCENTRATION
(mg g
-1
[dry wt])
13
C
(‰)
TOTAL
CONCENTRATION
(mg g
-1
[dry wt])
13
C
(‰)
TOTAL
CONCENTRATION
(mg g
-1
[dry wt])
13
C
(‰)
C16:1 9 ND - 2.46 -51.1 ND - ND - ND -
C16:1 8 7.44 -45.5 ND - ND - ND - ND -
C16:1 7 14.52 -45.5 11.10 -51.1 0.07 -50.6 7.28 -40.4 0.23 -35.8
C16:2 (RT: 20.9) 1.85 -44.6 ND - ND - ND - ND -
C16:0 5.43 -44.6 3.03 -52.3 0.08 -50.5 1.91 -36.6 0.15 -31.9
C17:0 ND - ND - ND - 0.15 -35.7 0.01 ND
C18:3 (RT: 24.3) 2.65 -47.3 2.24 -49.4 0.17 -51.6 0.82 -36.3 0.37 -37.7
C18:1 ND ND - 0.03 -32.2
C18:2 (RT: 24.5) 0.28 -47.5 0.19 -52.8 ND - 0.10 -36.1 ND -
C18:2 (RT: 24.7) 0.71 -46.2 0.20 -52.0 ND - 0.19 -34.3 ND -
C18:1 13 ND - ND - ND - 0.28 -34.4 ND -
C18:1 9 ND - ND - 0.02 -50.7 ND - ND -
C18:1 8 1.76 -38.9 0.29 -50.0 ND - ND - ND -
C18:1 7 2.34 -44.7 1.07 -50.7 0.03 -51.3 1.95 -35.7 0.12 -31.8
C18:1 7tr 2.24 -46.4 ND - ND - ND - ND -
C18:1 5 ND - ND - ND ND 0.11 -38.3 ND -
C18:2 (RT: 25.3) 1.12 -46.7 ND - ND - ND - ND -
C18:0 1.65 -45.2 0.64 -50.3 0.04 -48.4 0.75 -34.8 0.10 -32.6
C19:1 7 0.47 ND 1.29 -50.1 0.10 -50.6 1.27 -38.3 0.12 -40.3
C20:3 (RT: 28.4) 0.47 -45.8 0.08 -49.6 0.01 -48.7 ND - ND -
C20:3 (RT: 28.5) 1.53 -46.8 0.95 -48.7 0.11 -50.1 0.41 -34.8 0.21 -37.0
C20:2 (RT: 28.7) 1.45 -46.8 0.62 -50.3 0.06 -52.7 ND - 0.04 -36.7
C20:2 (RT: 28.7) 1.79 -46.8 1.58 -50.3 0.05 -52.8 2.25 -36.7 0.15 -36.7
C20:2 (RT: 28.9) 0.96 -46.9 0.29 -53.1 0.01 -52.8 ND - ND -
C20:2 (RT: 29.0) ND - ND - ND - 0.12 -35.4 ND -
C20:1 13 ND - 0.56 -50.4 ND - 0.86 -34.5 0.22 -32.9
C20:1 9 1.03 -45.7 ND - 0.05 -49.9 ND - ND -
C20:1 7 2.30 -45.0 0.95 -50.5 0.06 -51.1 2.86 -37.4 0.22 -36.0
C20:2 (RT: 29.6) 0.09 -46.0 ND - ND - ND - ND -
C21:2 (RT: 30.9) 0.12 ND ND - ND - 0.20 -39.0 ND -
C22:3 (RT: 32.4) 0.33 -47.0 ND - ND - 0.02 - 0.03 -35.8
C22:3 (RT: 32.6) 0.37 -47.0 ND - ND - ND - ND -
C22:2 (RT: 32.8) 1.75 -46.4 0.39 -48.9 0.05 -49.1 0.36 -36.3 0.12 -37.5
C22:2 (RT: 33.1) ND - 0.18 -48.9 ND - ND - ND -
∑ (mg g-1 [dry wt]): 54.06 28.12 0.91 21.88 2.11
weighted average: -45.5 -50.8 -50.8 -37.8 -35.7
∑ SFA: 7.08 -44.7 3.67 -51.9 0.12 -49.8 2.81 -36.1 0.26 -32.1
∑ MUFA: 32.11 -45.1 17.73 -50.9 0.33 -50.7 14.62 -38.5 0.93 -35.1
∑ PUFA: 15.47 -46.5 6.72 -49.9 0.45 -51.3 4.46 -36.4 0.92 -37.2
Gill tissue Gill tissue
FATTY ACID
FRACTION
B. childressi
(GoM - AL)
B. brooksi
(GoM - AL)
B. cf. thermophilus
(PAR)
B. cf. thermophilus
(PAR)
Gill tissue Foot tissue
B. brooksi
(GoM - AL)
Foot tissue

RESULTS
37
The fatty acid concentration is higher in the gill tissue compared to the foot tissue. The
gill tissue of B. childressi has a fatty acid concentration of 54.7 mg g-1
(dry weight) and
is ~two times higher than the other species (B. brooksi and B. cf. thermophilus) which
contain only 28.1 and 21.9 mg g-1
, respectively. The foot tissue of B. brooksi has
roughly 30 times less fatty acids compared to the associated gill tissue while the
difference for B. cf. thermophilus is only 10 times less. The arrangement of saturated
fatty acids (SFAs), monounsaturated fatty acids (MUFAs) and polyunsaturated fatty
acids (PUFAs) is distinct between gill tissue and foot tissue. The gill tissue consist
largely of MUFAs (e.g. C16:1 7) whereas the foot tissue contain large amounts of PUFAs
(e.g. C18:3) and MUFAs (e.g. C16:1, C20:1).
The compound specific carbon isotope analysis of the FAMEs is also shown in Tab. 3.
The total isotopic composition for B. childressi (gill tissue) and the gill and foot tissue
of B. brooksi were all highly depleted in 13
C. The 13
C values were -45.5, -51.4 and
-50.8 ‰, respectively. Gill and foot tissue of B. cf. thermophilus were less depleted in
13
C (-37.8 and -35.7‰, respectively).
Relative intensity and isotopic composition of all detected fatty acid in each mussel and
tissue type is presented in (Fig. 20).

RESULTS
38
Fig. 20: Five gas chromatograms showing the distribution of all determined fatty acids in each mussel and tissue
sample. The upper three chromatograms contain gill and foot tissue from mussels collected from the
Alaminos Canyon (AL) from the Gulf of Mexico. The lower two chromatograms describe one mollusk
from the Pacific Antarctic Ridge (PAR) dissected into gill and foot tissue. The foot samples from B. brooksi
and B. cf. thermophilus are ten times more concentrated compared to the gill tissue.

RESULTS
39
3.3.2 NEUTRAL FRACTION
The neutral fraction was measured as TMS-derivatives by GC-MS and GC-irMS. Tab. 4
shows the concentration and the isotopic composition of all determinate molecules in
the neutral fraction. The neutral fraction contains monoalkylglycerolethers (MAGEs)
with a variety of hydrocarbon chains differing in length and double bond position,
cyclic triterpenoids called sterols, and unknown compounds with unidentified structures
(APPENDIX VI). The concentration of MAGEs is generally higher in the gill tissue
compared to the foot tissue. The absolute concentrations of MAGEs from one mussel to
another differ strongly. The results of the compound specific isotopic analysis in the
MAGEs show that the gill tissue of B. childressi (-40.8 ‰) and the gill and foot tissue
of B. brooksi (-49.2 ‰ and -48.0 ‰, respectively) are highly depleted in 13
C. Gill and
foot tissue of B. cf. thermophilus are less depleted, they have values of -36.6 and
-33.7 ‰.
The composition of isoprenoid lipids (like sterols) differs between all three samples.
B. childressi (gill) sterol composition is dominated by cholesterol, followed by cholesta-
5,24-dienol, 5 (H)-cholestanol, cholesta-7-en-3-on, diplopterol and a 4 -methylsterol
named 4 -methylcholesta-8(14),24-dien-3 -ol. This 4 -methylsterol only comprises a
minor amount of the total sterol composition. The sterol compositions in gill and foot
tissue of B. brooksi differ only in concentration the relative proportion remains the
same. The sterol composition in gill and foot tissue of B. brooksi possess mainly the 4 -
methylcholesta-8(14),24-dien-3 -ol and contain only a minor fraction of cholesterol.
4 -methylcholesta-8(14),24-dien-3 -ol appears simultaneously with further 4 -
methylsterols named 4 -methylcholesta-8(14),24-en-3 -ol, 4 -methylcholesta-
8(14),24-trien-3 -ol, 4,4,14-trimethylcholesta-8(9),24-dien-3 -ol (also known as
lanosterol) and 4,4-dimethylcholesta-8(14),24-dien-3 -ol.
B. cf. thermophilus, recovered from the PAR, contains minor concentrations of
cholesterol and molecules with changing numbers and positions of double bonds but
methylsterols have not been detected.
Cholesterol, as the main sterol, in the gill tissue of B. childressi was depleted in 13
C.
The total weighted isotopic composition of B. childressi (gill tissue) and B. brooksi (gill

RESULTS
40
and foot tissue) cholesterol is 49.1, -58.9 and -60.0 ‰, respectively. B. cf. thermophilus
cholesterol has a total isotopic composition of -23.3 in the gill and -22.2 ‰ in the foot
tissue and is therefore less depleted in 13
C than the mussels from the GoM.
An unknown component was detected in every gill tissue of the three bivalves. B.
childressi and B. brooksi contain the same unknown molecule at 68.32 min (retention
time in the chromatogram) which has a 13
C value of -45.1 and -52.1 ‰, respectively.
The gill tissue of B. cf. thermophilus possesses a whole series of unknown compounds
which have an average 13
C value of -33.9 ‰. The mass spectra for the unknown
molecules in B. childressi and B. brooksi and the unknown series detected in
B cf. thermophilus are provided in the Appendix VIIA und VIIB.
Tab. 4: Concentration and isotopic composition of MAGEs, sterols and unknown components for three diverse
mussels of the genus Bathymodiolus.
Total concentration is given in mg g-1
(dry weight) and isotopic values are given in ‰. All 13
C data are
average values, measured between 2 and 4 times. The mean standard deviation of all shown neutral fraction
accounts for 1.1 ‰. The relative concentration in each sample and tissue type is indicated through colored
bars. GoM, Golf of Mexico; AL, Alaminos Canyon; RT, retention time; ND, none detected; MAGEs, sterols
and unknown components are listed in relative order of elution.
TOTAL
CONCENTRATION
(mg g
-1
[dry wt])
13
C
(‰)
TOTAL
CONCENTRATION
(mg g
-1
[dry wt])
13
C
(‰)
TOTAL
CONCENTRATION
(mg g
-1
[dry wt])
13
C
(‰)
TOTAL
CONCENTRATION
(mg g
-1
[dry wt])
13
C
(‰)
TOTAL
CONCENTRATION
(mg g
-1
[dry wt])
13
C
(‰)
C15:0 MAGE (RT: 30.68) ND - ND - ND - ND - ND -
C16:1 MAGE (RT: 32.59) ND - 0.10 -47.8 ND - ND - ND -
C16:1 MAGE (RT: 32.76) 0.12 -41.6 1.45 -50.1 0.017 -47.9 0.064 -38.4 0.064 -33.4
C16:1 MAGE (RT: 32.81) 0.05 -41.6 ND - ND - ND - ND -
C16:0 MAGE (RT: 33.10) 0.17 -40.6 0.29 -48.4 0.044 -47.9 0.035 -34.1 0.009 -33.4
C17:0 MAGE (RT: 34.89) ND - 0.01 ND ND - ND - ND -
C18:2 MAGE (RT: 35.92) 0.01 ND ND - ND - ND - ND -
C18:2 MAGE (RT: 36.02) 0.00 ND ND - ND - ND - ND -
C18:1 MAGE (RT: 36.35) ND - 0.04 -48.7 ND - 0.012 -34.9 0.009 -30.7
C18:1 MAGE (RT: 36.36) ND - 0.04 -48.7 ND - ND - ND -
C18:1 MAGE (RT: 36.55) ND - ND - ND - 0.002 -34.9 0.000 -
C18:0 MAGE (RT: 36.65) 0.24 -40.0 0.14 -48.7 0.004 -48.5 0.017 -34.9 0.004 -30.7
C20:1 MAGE (RT: 39.76) 0.19 -42.0 0.35 -47.2 ND - 0.073 -36.9 0.014 -38.3
C21:1 MAGE (RT: 41.40) ND - ND - ND - 0.005 ND ND -
∑ (mg g-1 [dry wt]): 0.96 2.44 0.07 0.209 0.101
5 (H)-Cholestanol ND - ND - ND - 0.005 -26.0 0.005 -24.6
unknown sterol ND - ND - ND - 0.006 -28.1 0.004 -27.4
Cholest-5,22-dien-3 -ol ND - ND - ND - ND - 0.006 -31.3
Cholesterol 5.88 -50.1 0.10 -46.8 0.124 -48.9 0.037 -22.4 0.285 -21.6
5 (H)-Cholestanol 0.47 -52.3 ND - ND - 0.013 -23.1 0.094 -22.2
Cholesta-5,24-dien-3 -ol 1.61 -47.9 ND - ND - ND - ND -
Cholesta-3-on ND - ND - ND- 0.007 -22.7 ND -
Cholesta-7-en-3-on ND - ND - ND - 0.005 ND ND -
Cholest-7-en-3 -ol 0.40 -47.8 ND - ND - ND - 0.036 -23.2
4 -methylcholesta-8(14),24-en-3 -ol ND - 0.24 -56.9 0.111 -60.5 ND - ND -
4 -methylcholesta-8(14),24-dien-3 -ol 0.78 -44.3 6.52 -59.5 2.769 -60.7 ND - ND -
4 -methylcholesta-8(14),24-trien-3 -ol ND - 0.31 ND 0.159 - ND - ND -
unknown sterol 0.79 -47.8 ND - ND - ND - ND -
4,4,14-trimethylcholesta-8(9),24-dien-3 -ol ND - 0.21 -53.5 0.006 - ND - ND -
4,4-dimethylcholesta-8(14),24-dien-3 -ol ND - 0.24 -53.0 0.086 -53.4 ND - ND -
Tetrahymanol ND - ND - ND - ND - 0.006 -29.7
Diplopterol 0.21 -45.2 ND - ND - ND - ND -
∑ (mg g-1 [dry wt]): 10.14 7.62 3.25 0.072 0.437
unknown (RT: 54.41) ND - ND - ND - 0.034 -37.1 ND -
unknown (RT: 68:32) 2.37 -45.1 1.35 -52.1 ND - ND - ND -
∑ (mg g-1 [dry wt]): 2.37 1.35 - 0.24 -
weighted average: -45.1 -52.1 - -33.9 -
Gill tissue Gill tissueNEUTRAL FRACTION
B. childressi
(GoM - AL)
B. cf. thermophilus
(PAR)
B. cf. thermophilus
(PAR)
Gill tissue Foot tissue
B. brooksi
(GoM - AL)
Foot tissue
B. brooksi
(GoM - AL)

RESULTS
41
Five chromatograms showing relative composition and isotopic data of the neutral
fraction is presented in Fig. 21.
Fig. 21: Five gas chromatograms showing a section (from 40 - 68 min) of the neutral fraction of each mussel and
tissue sample. The upper three chromatograms contain two gill and one foot tissue from two mussels
collected from the Alaminos Canyon (AL) from the Gulf of Mexico. The lower two chromatograms
describe one mollusk from the Pacific Antarctic Ridge (PAR) dissected into gill and foot tissue. The foot
tissue of B. brooksi is three times more concentrated compared to the corresponding gill tissue. Gill tissue
of B. cf. thermophilus is approximately 10 times more concentrated than the foot tissue and two times
higher than the gill samples from the GoM.

DISCUSSION
42
4 DISCUSSION
4.1 DISTRIBUTION OF IPLS AS INDICATOR FOR MICROBIAL
ACTIVITY
It is known that foot tissue is free of symbionts (JAHNKE ET AL., 1995) and is therefore
used as reference material to the symbiont-containing gill tissue. However, the gill
tissue reflects a mixed signal of eukaryotic and prokaryotic membrane lipids whereas
the foot tissue is of pure eukaryotic origin.
The pie charts in Fig. 22 show the relative IPL composition of each sample and allow
comparison between species and tissue types. The IPL composition of the symbiont
containing gill tissue compared to the IPL composition of the symbiont-free foot tissue
demonstrates a higher diversity of different IPLs in the gill tissue. This increase in
diversity in the gill tissue probably reflects the additional prokaryotic membrane lipids.
B. brooksi and B. cf. thermophilus were taken from different locations and are known to
harbor different symbionts (CAVANAUGH ET AL., 1987; DISTEL ET AL., 1988). However,
these two bivalves exhibit a very similar IPL composition in the foot tissue, dominated
by PC (> 55%) with some PE and minor amounts of 3 to 5 further IPLs, the foot tissues
reflect a pure eukaryotic signal (SOHLENKAMP ET AL., 2003; CHRISTIE, 2007).
In the symbiont-containing gill tissue PE is the dominative membrane lipid in all three
samples. However, the IPL compositions are clearly distinct between gills of different
species dominated by methanotrophs (GoM; DUPERRON ET AL. (2007)) and from the gill
sample only containing a single thiotrophic symbiont (PAR).

DISCUSSION
43
B. childressi and B. brooksi from the Alaminos Canyon have the same most abundant
membrane lipids at similar concentrations (Appendix I). The three main components of
those membranes are PE, C-PnE and PC. PC is reflecting the eukaryotic signal whereas
PE is known to be the main lipid fraction of microbial membranes but can still be
abundant in eukaryotic membranes (CHRISTIE, 2007). Ceramide-
phosphonoethanolamines (C-PnE) are thought to be part of marine mollusks
(MUKHAMEDOVA AND GLUSHENKOVA, 2000) but are also described to be abundant in a
Fig. 22: Relative composition of IPLs subdivided in five samples. Gill tissues are shown on the
left side and foot tissues on the right side. M-197 & M-154, unknown components; PC,
phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI,
phosphatidylinositol; PS, phosphatidylserine; PnE, phosphonoethanolamine; C-PnE,
ceramide-phosphonoethanolamine; Cb, cerebroside; C-PE, ceramide-phosphatidyl-
ethanolamine; lyso membrane lipids contain only a single acyl chain.

DISCUSSION
44
variety of bacteria (CHRISTIE, 2007). PG occurs in B. brooksi (1%) and B. childressi
(8%) in minor amounts but the membrane lipid is significant for bacterial activity as it
can be found in almost all bacterial membrane lipids (CHRISTIE, 2007).
The gill tissue of B. cf. thermophilus differs in many ways from the gill tissues of the
GoM samples. PE, also the main component, is followed by PC and lyso-PC. C-PnE is
not present but a new membrane lipid appears which only exists in this sample and
tissue, ceramide-phosphatidylethanolamine (C-PE). C-PE is described as an abundant
membrane lipid in many bacteria (CHRISTIE, 2007) and could be a significant marker for
thiotrophic bacteria.
Lyso-products are described to be formed during sampling, storage, or analytical
processing (CHRISTIE, 2007). However, the appearance of high lyso-PC concentrations
only in gill and foot tissue of B. cf. thermophilus at 13 and 7% indicates a major role in
the composition of the membranes and could possibly be linked to bacterial activity.
An unknown lipid with a neutral loss of 154 Da only occurs in minor amounts in the gill
tissue of mussels known to have thiotrophic symbionts and might therefore be
characteristic for the appearance of those specific prokaryotes. The unknown lipid
M-197 (characteristic neutral loss of 197 Da) suggests the appearance of
methanotrophic symbionts, since these membrane lipid only occur in the GoM samples.
PS and PI are indicators for eukaryotes (CHRISTIE, 2007) and are abundant in all three
gill tissues representing partly the host membrane composition.
This study provides IPLs which information can be used for the distinction between
eukaryotes and prokaryotes. The appearance of IPLs like PE, PG, C-PnE, C-PE and two
unknown membrane lipids (M-154 and M-197) is suggested to be part of prokaryotic
membranes but may to some extent also be present in eukaryotic membranes (CHRISTIE,
2007). Therefore, the foot tissue was used as reference to compare with the symbiont-
containing gill tissue. The distinct signals between gill and foot tissue should specify the
amount of symbiont membrane lipids abundant in the gill tissue. Unfortunately, no
information on structural membrane lipid differences between those two tissue types
(complex gill tissue and the muscular foot tissue) from bivalves could be obtained to
evaluate the general structural diversity. Therefore, the increase of detected IPLs in the
gill tissue might either be due to the appearance of symbionts, or a general structural

DISCUSSION
45
variation. Presumably, both possibilities generated the mixed signal. Hence, the exact
amount of bacterial derived lipids within the gill tissue remains unknown.
Nevertheless, the higher diversity of the membrane lipids indicates the potential of
using IPLs to distinguish between eukaryote- and prokaryote-derived cell membranes.
4.2 BHPS AS BIOMARKERS FOR BACTERIAL SOURCES
The appearance of BHPs in the gill tissue of mussels from the GoM adds further proof
for the presence of bacteria, since BHPs are known to be synthesized by many
prokaryotes, and BHPs have never been identified in eukaryotic organisms (SUMMONS
ET AL., 1999). The mass chromatograms (m/z 714 + m/z 772) of the gill tissue of B.
childressi and B. brooksi (Fig. 19) clearly indicate the presence of
bacteriohopanepolyols known to be produced by methanotrophic bacteria (TALBOT ET
AL., 2001; ROHMER ET AL., 1984).
The detected BHPs in the gill tissue of B. childressi are indicative of type-II
methanotrophic bacteria, since type-II methanotrophs are dominated by tetra- and penta-
functionalized compounds (TALBOT ET. AL., 2001). The distribution in that tissue and
sample was composed of aminotriol and -tetrol.
B. brooksi contains a single composition of tetra-functionalized bacteriohopanepolyols,
which could also indicate methanotrophic bacteria. However, these data provide less
information to distinguish between Type-I and Type-II methanotrophs compared to the
data obtained from B. childressi.
The subdivision of methanotrophs into Type-I, -II, or -X is conducted according to their
pathways of carbon assimilation and some other characteristics (SUMMONS ET AL.,
1994).
The indication of type-II methanotrophs in B. childressi do not correspond with the
results of the molecular approach carried out by DUPERRON ET AL. (2007). They
identified mussel symbionts in B. childressi and B. brooksi as -proteobacteria

DISCUSSION
46
containing exclusive Type-I methanotrophs. The fatty acid analysis (see chapter 4.3.1)
identified the dominant Type-I methanotroph in B. childressi but also suggests the
presence of Type-II methanotrophs. These findings may indicate a second type of
symbiont in B. childressi.
The symbiont-free foot tissue of B. brooksi did not contain any BHPs. Foot and gill
tissue of B. cf. thermophilus from the PAR, with a monoculture of thiotrophic bacteria
(DISTEL ET AL., 1988) did not show hopanoid producing bacteria. There has been no
evidence that thiotrophic bacteria produce BHPs reported to date.
4.3 SUMMARY OF THE COMPOUND-SPECIFIC ISOTOPE
INVESTIGATION IN B. CHILDRESSI, B. BROOKSI AND B. CF.
THERMOPHILUS
The total concentrations of phospholipid-derived fatty acids (PLFAs) in all
Bathymodiolus samples are in general many times higher in the gill tissue compared to
the corresponding foot tissue (Tab. 3). B. brooksi contain ~30 times, and B. cf.
thermophilus ~10 times, more fatty acids in the gill compared to the foot tissue.
However, IPL concentrations display much smaller gill and foot tissue differences
(Appendix II). This might be due to the fact that, in marine mollusks (CONWAY, 1991),
large amounts of fatty acids can also be esterified as triglycerides (three fatty acids
linked to one glycerol molecule). These would be not detected during IPL
measurements (triglycerides are less polar than complex lipids).
Among all three gill samples, the C16:1 FAs are much more abundant than the C16:0 FAs.
Compared to heterotrophic mytilids, C16:0 were roughly 3-times more abundant than
C16:1 fatty acids (ABRAJANO ET AL., 1994). High concentrations of unsaturated C16 FAs
in all gill tissues point to the presence of living symbiotic bacteria within that specific
tissue type (Tab. 3). A similar trend has been observed by JAHNKE ET AL., (1995) and
FANG ET AL., (1993).

DISCUSSION
47
Unsaturated fatty acids as group-specific biomarker provide valuable information.
These compounds have been used to distinguish between methanotrophic organisms
and other bacteria (NICHOLS ET AL., 1985; OURISSON ET AL., 1987). For example,
characteristic biomarkers for methanotrophic bacteria have been detected, such as
C16:1 8 (B. childressi) and C16:1 9 (B. brooksi) which are characteristic for Type-I
methanotrophs (NICHOLS ET AL., 1985; JAHNKE EL AL., 1995; NIEMANN, 2005, ELVERT
AND NIEMANN, in press). This was also concluded by DUPERRON ET AL. (2007), who
classified the dominant methanotrophic symbiont of B. childressi and B. brooksi as
-proteobacteria containing exclusively Type-I methanotrophs.
Type-II methanotrophs have been described to have C18 MUFAs containing a double
bond in the 8 position (NICHOLS ET AL., 1985; GUEZENNEC AND FIALA-MEDIONI,
1996). This specific C18:1 8 fatty acid was detected in the gill tissues of B. childressi and
also in B. brooksi. The fatty acid C16:1 7 has been used as a marker for the presence and
abundance of bacteria, and especially of thiotrophic bacteria (GUEZENNEC AND FIALA-
MEDIONI, 1996), and has been detected in high concentrations in B. cf. thermophilus.
The highest concentrations of polyunsaturated fatty acids, especially C18:3 and
polyunsaturated C20 FAs, can be observed in the foot tissue of B. brooksi and B. cf.
thermophilus. Polyunsaturated FAs like C20 and C22 are thought to be characteristic for
marine mytilid mussels (FANG ET AL., 1993).
Carbon isotopic compositions of gill and foot components (e.g. fatty acids and neutral
lipids) were measured for all three Bathymodiolus mussels. Different 13
C values for B.
childressi, B. brooksi and B. cf. thermophilus indicate different metabolic pathways
and/or different isotopic compositions of the carbon source (Tab. 3 & Tab. 4). 13
C
values from the Bathymodiolus species taken from the GoM (between -45 and -60‰)
and the PAR (between -23 and -37‰) reflect differences in substrates (CH4 vs. CO2).
The smaller isotopic shift between B. childressi (approximately -46‰) and B. brooksi
(approximately -52‰) might either be due to different symbiotic pathways of the
prokaryotes within the gills (FANG ET AL., 1993; POND ET AL., 1998) or it could reflect an
additionally heterotrophic food source (e.g. by filter-feeding) of B. childressi. However,
deep-sea invertebrates which incorporate sedimentary detritus as a primary carbon
source (nonevent environments) have 13
C values of about -17% (Fig. 23; VAN DOVER
AND FRY, 1989).

DISCUSSION
48
Similar carbon isotopic values for the symbiont-containing gill tissue and the symbiont-
free foot sample in B. brooksi and B. cf. thermophilus results from the eukaryotic
mussel (heterotroph), incorporating the isotope signature of their diet (symbionts)
(MACAVOY ET AL., 2003).
Exact isotopic values for the carbon sources from the GoM (CH4) and the PAR (CO2)
remain unknown since no isotopic analysis has been carried out during sampling.
13
C TRENDS OF FATTY ACIDS AND STEROIDS OF
B. CHILDRESSI AND B. BROOKSI FROM THE ALAMINOS CANYON (GOM)
The comparison of the fatty acid compositions of the gill tissues of B. childressi and B.
brooksi shows a distinct distribution which indicates the activity of different symbionts
(Tab. 3). In general, both mussels contain high concentrations of various C16:1 fatty
acids differing in the position of characteristic double bonds. This information about the
exact location of the double bond positions and the determination of fatty acids
concentrations was not determined by DUPERRON ET AL. (2007).
Type I and Type-X methanotrophs have been described to biosynthesize C16:1 fatty acid
containing double bonds at 8, 7, 6 and 5 (BOWMAN ET AL., 1991; JAHNKE ET AL.,
1986; NICHOLS, 1985) whereas Type-II methanotrophs contain unsaturated C18:1 fatty
acid with double bonds at position 8, 7 and 6 (BOWMAN ET AL., 1991; JAHNKE ET
AL., 1986; NICHOLS, 1985).
A high concentration of the characteristic Type-I methanotrophic C16:1 8 fatty acid
together with a minor concentration of the Type-II methanotrophic C18:1 8 and C18:1 7
fatty acid has been determined in B. childressi (Tab. 3). The appearance of the
characteristic Type-II fatty acids and additionally tetra- and penta- functionalized BHPs
(see chapter 4.2) indicates the presence of a previously unknown additional symbiont.
DUPERRON ET AL. (2007) could not exclude the presence of additional symbionts in B.
childressi.

DISCUSSION
49
B. brooksi also contained a characteristic C16:1 9 fatty acid, which is described to be
abundant in uncultured Type-I methanotrophs (NIEMANN, 2005). The presence of
C18:1 8 and C18:1 7 also indicates the presence of Type-II methanotrophic symbionts but
this has not been confirmed by BHP analysis.
However, the appearance of C18:1 fatty acids may also be due to the fatty acid elongation
mechanism via acetate that is known to occur in mytilid mussels (ZHUKOVA, 1991).
The neutral fraction of lipids extracted from B. childressi and B. brooksi displays a
distinct difference between the two species. Cholesterol is the main sterol in the gill
tissue of B. childressi whereas sterols methylated at C-4 are the most abundant
compounds in the gill tissue of B. brooksi.
High cholesterol (cholest-5-en-3 -ol) concentrations can indicate that this mussel
species was partly a filter-feeding organism, since cholesterol is generally considered to
be an indicator of zooplankton (VOLKMAN ET AL.1998). Alternatively B. childressi could
harbor a distinct unknown symbiont capable of synthesizing cholesterol.
The sterol composition of B. childressi is comparable with results from JAHNKE ET AL.
(1995). These authors analyzed tissues of a cold seep mytilid mussel which were
collected from the Louisiana slope of the Gulf of Mexico. The sterol patterns (high
concentration of cholest-5-en-3 -ol), the fatty acid composition, and the defunctionalized
hopanoids (C31- and C32-hopanol) show strong similarities. It seems to be the case that
the seep mussel analyzed by JAHNKE ET AL. (1995) is the same or a closely related
mussel to the B. childressi examined in this study.
The highly abundant 4-methyl-cholestadienol in B. brooksi is known to be characteristic
for some aerobic methane oxidizers (BOUVIER ET AL., 1976; SUMMONS ET AL., 1994;
JAHNKE ET AL., 1995; SCHOUTEN, 2000; ELVERT AND NIEMANN, in press). The
endosymbionts of B. brooksi might be closely related to M. capsulatus or M. hansonii,
since methylsterols are also highly enriched in both species (JAHNKE ET AL., 1992;
SCHOUTEN ET AL., 2000). Methylsterols are a reliable indicator for methanotrophs, since
they have never been detected in mytilid mussels free of methanotrophic symbionts
(CONWAY AND CAPUZZO, 1991; FANG ET AL., 1993).

M.Sc.-thesis_Matthias-Kellermann

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