Exploring the Lipidomes of Shallow-Water and Deep-Sea Hydrothermal Systems

Exploring the lipidomes of shallow-water and deep-sea hydrothermal systems

Dissertation zur Erlangung des Doktorgrades der Naturwissenschaften - Dr. rer. nat. -

Am Fachbereich Geowissenschaften der Universität Bremen

vorgelegt von

Miriam Sollich

Bremen Mai 2018

1. Gutachter: Dr. Solveig I. Bühring 2. Gutachter: Associate Prof. Dr. Eoghan P. Reeves

Tag des Promotionskolloquiums:16. Februar 2018

Den Wissenschaftlern geht es wie den Chaoten. Es ist alles da, man muss es nur suchen. - Franz Kern -

CONTENTS

Abstract Zusammenfassung Acknowledgements List of Abbreviations

Chapter I 1 Introduction and Methods

Chapter II 37 Scope and Outline

Chapter III 43 Heat stress dictates the microbial lipid composition along a thermal gradient in marine sediments

Chapter IV 91 Shallow-water hydrothermal systems offer ideal conditions to study archaeal lipid membrane adaptations to environmental extremes

Chapter V 113 Transfer of chemosynthetic fixed carbon and its ecological significance revealed by lipid analysis of fluids at diffuse flow deep-sea vents (East Pacific Rise 9°50’N)

Chapter VI 143 Concluding Remarks and Future Perspectives

ABSTRACT

Shallow-water and deep-sea hydrothermal systems are environments where seawater percolates downward through fractures in the oceanic crust, and becomes progressively heated and chemically altered. Finally, the entrained water is expelled into the overlying water column as a hydrothermal fluid. Hydrothermal circulation occurs at all active plate boundaries like mid-ocean ridges, submarine volcanic arcs and backarc basins. They represent one of the most extreme and dynamic ecosystems on the planet with steep physico-chemical gradients. Nevertheless, these environments are characterized by exceptional high biomass representing hotspots of life in the mostly hostile and desolated deep sea. Although shallow-water and deep-sea hydrothermal systems share basic physical processes regarding heat transfer and water circulation they differ in various physico-chemical parameters. The cut-off between “shallow” and “deep” was defined to ~200 m water depth, which loosely correlates with the extent of the photic zone and results in striking differences of species community structures. This dissertation is focused on the microbial ecology in hydrothermal systems, more specifically on the lipidome and encoded lipid structure-functions of microbial membranes. The first study examines sediments from the shallow-water hydrothermal sediments off the coast of Milos along a temperature gradient from 18 to 101 °C [Chapter III]. The rationale was to test the concept that membrane lipids dictate the thermodynamic ecology of bacteria and archaea, by minimizing loss of energy by ion diffusion across the membrane. A detailed investigation of archaeal and bacterial polar lipids revealed a membrane quandary: not only the expected low permeability, but also increased fluidity of membranes are required as a unified property of microbial membranes for energy conservation under heat stress. For instance, bacterial fatty acids were composed of longer chain lengths in concert with a higher degree of unsaturation while archaea modified their tetraethers by incorporation of additional methyl groups at elevated sediment temperatures. These configurations toward a more fluidized membrane at elevated temperatures were observed with a concomitantly high abundance of archaeal glycolipids and bacterial sphingolipids. It is suggested that glycolipids and sphingolipids could reduce membrane permeability through strong intermolecular hydrogen bonding. Results of this chapter provide a new angle for interpreting membrane lipid structure-function enabling archaea and bacteria to survive and grow in hydrothermal systems. In chapter III, the modifications observed for abundant archaeal glycosidic tetraethers (e.g., increase in “H-shaped” structures and number of methyl groups and

cyclopentane rings) were correlated with elevated sediment temperatures. This result was reproduced in another shallow-water hydrothermal system sampled along a thermal gradient off Dominica in the second study of this thesis [Chapter IV]. Furthermore, statistical multivariate analyses helped to identify additional geochemical key players which strongly impacted the lipid distribution in hydrothermally-influenced sediments. H-shaped structures clearly correlated with temperature, while H-shaped methylated GDGT showed a strong correlation with increasing hydrogen sulfide concentrations and salinity. The last study of this thesis investigated, for the first time, the lipid diversity in diffuse flow fluids and adjacent bottom water from the East Pacific Rise [Chapter V]. Lipids in these samples were dominated by triacylglycerols, wax esters and alkyldiacylglycerols (> 90% of total lipids), rarely found in bacteria – especially in Epsilonproteobacteria, which are the most prevalent chemosynthetic taxa in these systems. Moreover, polyunsaturated fatty acids (PUFA, both ω-3 and ω-6) represented up to 20% of total fatty acids linked to major phospholipids such as phosphatidyl-choline and -ethanolamine, an uncommon feature among bacterial membranes. Thus the high abundance of storage lipids and PUFA linked to phospholipids indicate eukaryotes rather than bacteria as the major source of lipids in diffuse hydrothermal fluids and adjacent bottom water. Stable carbon isotopic analysis of fatty acids, which are mainly derived from storage lipids, revealed an intimate energy flux from chemosynthetic fixed carbon to higher trophic levels in diffuse flow systems. Chapter V highlights the trophic transfer in these diffuse flow systems and addresses the origin of PUFA in deep-sea hydrothermal vents.

ZUSAMMENFASSUNG

Flachwasser- und Tiefseehydrothermalsysteme sind Gebiete in denen Meerwasser durch Spalten und Risse tief in die Ozeankruste eindringt. Auf dem Weg tiefer in die Ozeankruste wird das Meerwasser kontinuierlich erwärmt und auch die chemische Zusammensetzung verändert sich bevor es zurück an die Oberfläche gelangt und dort als hydrothermales Fluid an das Meerwasser abgegeben wird. Hydrothermale Zirkulation findet an allen aktiven Plattenrändern statt zu denen Mittelozeanische Rücken, Backarc- Becken und unterseeische vulkanische Inselbögen zählen. Hydrothermalsysteme weisen ausgeprägte physikalische und chemische Gradienten auf und stellen eines der extremsten und dynamischsten Ökosysteme dieser Welt dar. Trotzdem sind diese Gebiete durch eine atemberaubende Vielfalt an Lebewesen charakterisiert und stellen sogenannte Oasen in der sonst öden und lebensfeindlichen Tiefsee dar. Obwohl Flachwasser- und Tiefseehydrothermalsysteme auf den gleichen physikalischen Prozessen des Wärmetransfers und der Wasserzirkulation beruhen, unterscheiden sie sich stark in einigen physikalischen und chemischen Parametern. Die Grenze zwischen „Flach“ und „Tief“ verläuft bei ~200 m Wassertiefe, ungefähr bis zu der Tiefe in der maximal das Sonnenlicht vordingen kann. Dies wiederum hat erhebliche Auswirkungen auf die Artenzusammensetzung, die sehr unterschiedlich in beiden Systemen ist. Diese Dissertation fokussierte sich auf die mikrobielle Ökologie von Hydrothermalsystemen, insbesondere des Lipidoms und den in mikrobiellen Membranen enthaltenden Informationen über die Struktur und Funktion von Lipiden. Die erste Studie untersuchte Sedimente eines Flachwasserhydrothermalsystems entlang eines Temperaturgradienten von 18 bis 101 °C vor der Küste von Milos. Es sollte das Konzept getestet werden, ob Membranlipide die thermodynamische Ökologie von Bakterien und Archaeen dadurch bestimmen, dass sie den Energieverlust mittels Ionendiffusion durch die Membran minimieren. Eine detaillierte Untersuchung archaeeller und bakterieller Polarlipide brachte jedoch eine konträre Situation zum Vorschein: neben der bereits erwarteten geringen Permeabilität, konnte eine zeitgleiche erhöhte Fluidität der Membran als einheitlicher Code mikrobieller Membranen zur Energiekonservierung unter Temperaturstress beobachtet werden. Zum Beispiel bestanden bakterielle Fettsäuren aus längeren Ketten bei zeitgleichem höheren Unsättigungsgrad, während Archaeen ihre Tetraether dadurch modifizierten, indem sie zusätzliche Methylgruppen bei höherer Temperatur einbauten. Diese Konfigurationen in Richtung einer erhöhten Fluidität bei höheren Temperaturen wurde bei zeitgleicher

erhöhter Abundanz glykosidischer achaeeller Lipide und bakterieller Sphingolipide beobachtet. Es wird diskutiert ob Glykolipide und Sphingolipide die Membranpermeabilität dadurch verringern, indem sie die Ausbildung intermolekularer Wasserstoffbrückenbindungen intensivieren. Diese Ergebnisse eröffnen völlig neue Interpretationsmöglichkeiten bezüglich der Untersuchung gewisser Abhängigkeiten von Struktur und Funktionalität von Membranlipiden, die Archaeen und Bakterien überhaupt erst ermöglichen in Hydrothermalgebieten erfolgreich zu gedeihen. Die in Kapitel III beobachteten Modifikationen archaeeller glykosidischer Tetraether (Abundanz der „H-shape“ Struktur und Anzahl zusätzlicher Methylgruppen und Cyclopentanringe), korrelierten positiv mit ansteigender Sedimenttemperatur. Diese Ergebnisse konnten in einem anderen Flachwasserhydrothermalsystem entlang eines Temperaturgradienten vor der Küste Dominicas in einer zweiten Studie reproduziert werden [Kapitel IV]. Darüber hinaus konnten multivariate Analysemethoden weitere geochemische Schlüsselparameter identifizieren, die die Lipidverteilung in hydrothermalen Sedimenten stark beeinflussten. H-shape Strukturen zeigten eine deutliche Korrelation mit Temperatur, während H-shape methylierte GDGT vor allem mit ansteigenden Schwefelwasserstoffkonzentrationen und Salinität korrelierten. In der letzten Studie dieser Dissertation wurde zum ersten Mal die Lipiddiversität in diffusen Fluidsystemen sowie angrenzendem Tiefseemeerwasser des Ostpazifischen Rückens untersucht [Kapitel V]. Der überwiegende Anteil der Lipide (> 90%) bestand aus Triacylglycerolen, Wachsestern und Alkyldiacylglycerolen, die extrem selten in Bakterien – besonders in Epsilonproteobakterien – zu finden sind, die jedoch den überwiegenden Anteil der chemosynthetisch lebenden Arten in diesem System darstellen. Darüber hinaus stellten mehrfach ungesättigte Fettsäuren (PUFA, sowohl ω-3 als auch ω-6) einen Anteil von bis zu 20% dar, deren Fettsäuren an Phospholipide wie z.B. Phosphatidylcholin und Phosphatidylethanolamin gekettet waren, eine Besonderheit die bisher nur sehr selten in bakteriellen Membranen beobachtet werden konnte. Die besondere Häufigkeit detektierter Speicherfette und PUFA verlinkt mit Phospholipiden deutet daher eher auf eine eukaryotische als eine bakterielle Lipidquelle in Fluidproben als auch in den angrenzenden Tiefseemeerwasserproben hin. Stabile Kohlenstoffisotopenanalysen der Fettsäuren, die überwiegend von den Speicherfetten abstammten, zeigten einen engen Energiefluss von chemosynthetisch fixiertem Kohlenstoff zu höheren trophischen Stufen in diffusen Fluidsystemen. Kapitel V hebt den

trophischen Transfer in diffusen Fluidsystemen hervor und behandelt insbesondere das Thema des Ursprungs der PUFA in Hydrothermalsystemen.

ACKNOWLEDGEMENTS

First of all, I would like to express my sincere thanks to my long term supervisor Dr. Solveig Bühring for giving me the opportunity to do my PhD thesis within her group. You introduced me into the fascinating world of hydrothermal systems and nurtured my research interest through exciting and challenging field trips and you brought hydrothermal systems literally closer to me than everybody else. Your patience and encouraging words motivated me throughout my thesis. I also would like to thank Associate Prof. Eoghan Reeves for being my second reviewer and also for being my “first address” whenever I had questions regarding all the different physico-chemical processes around hydrothermal systems. Without you some of them would probably have stayed a mystery to me. Special thanks to Prof. Kai-Uwe Hinrichs, for introducing me into the world of lipid biomarkers. For having an open door even during busy times, for joining many hours of thesis committee meetings and for giving me always constructive and objective feedback. Thanks for supporting me all these years. I am indebted to Prof. Ray Valentine, Dr. Marcos Y. Yoshinaga and Dr. Matthias Kellermann from the MLCCS for creative and inspiring video chat sessions. It was always informative and entertaining at the same time and you guys teached me a very important lesson: to think outside the box from time to time. Furthermore I would like to thank my other thesis committee members Drs. Petra Pop Ristova and Stefan Sievert for stimulating discussions. Thanks Stefan for joining my thesis committee meetings personally despite the long journey and for inviting me to the scientific expedition AT26-10 to the East Pacific Rise, which was an unforgettable experience. Many thanks to my outstanding colleagues from the Organic Geochemistry Group who helped me generously in many different ways and together with the Hydrothermal Geomicrobiology Group created the best working atmosphere I could wish for. I particularly want to thank Drs. Florence Schubotz, Marcus Elvert, Lars Wörmer and Julius Lipp for their great expertise and knowledge about polar lipids, fatty acids and isotopes and for answering countless questions throughout the years. Xavier Prieto, Jenny Wendt, Jessica Arndt and Raika Himmelsbach are acknowledged for their great technical support and for keeping the labs running. Kevin Becker, Felix Elling, Nadine Goldenstein and Bernhard Viehweger are acknowledged for extensive discussions and inspirations, essential for this thesis.

Many thanks to Gonzalo Gómez-Sáez, Evert Kramer and Heidi Taubner who always cheered me up in the most positive way.

Danke auch an meine großartigen Freunde, in und außerhalb von Bremen, die mich zu jeder Zeit tatkräftig unterstützt, motiviert und wenn nötig abgelenkt haben. Im Besonderen möchte ich Marcos danken. Ich kenne dich seit deinem ersten Tag in Bremen und ich bin dankbar dafür, dass sich unsere Wege gekreuzt haben. Danke für all die unzähligen Stunden in denen wir Daten, Ideen und Theorien diskutiert haben und dafür, dass du mir alles über Lipidbiomarker beigebracht hast, was du weißt. Ich weiß, dass ich deine Geduld des Öfteren auf eine harte Probe gestellt habe und ich werde es wohl kaum in Worte fassen können wie wichtig du als Mentor, Motivator, Personal Trainer und guter Freund für das Ergebnis dieser Arbeit gewesen bist! Berni, es gibt so viele kleine und große Dinge für die ich dir danken möchte. Deine Freundschaft hat mich stets begleitet und die vielen gemeinsamen Abende mit Wein und Mensch ärger dich nicht® waren immer eine willkommene Auszeit zum oft stressigen Alltag. Auch wenn wir nicht für ewig nebeneinander wohnen können, so hoffe ich doch, dass wir uns nicht gänzlich aus den Augen verlieren werden. Als letztes möchte ich meiner Familie danken. Opa, ohne das du dir dessen vermutlich überhaupt bewusst bist, verdanke ich dir wohl die eigentliche Fertigstellung dieser Arbeit. Dein Interesse und deine ehrliche Begeisterung für meine Doktorarbeit haben mich in der Endphase zu einer solchen Disziplin verleitet, ohne die ich wohl noch immer schreiben würde. Mama und Papa, ihr seid die Basis, zu der ich jeder Zeit zurückkehren kann. Ihr habt jeden Höhepunkt meiner wissenschaftlichen Laufbahn mit mir gefeiert und jeden Tiefpunkt für mich erträglicher gemacht. Ohne euch, und eure grenzenlose Unterstützung, Vertrauen und Liebe wäre diese Arbeit nie zustande gekommen! Danke auch für meine großartige Schwester Nina. Egal wann, ich konnte immer zu dir aufs „Eiland“ flüchten. Das turbulente Leben in deinem Haus und dein liebevoller, groß- schwesterlicher Beistand haben mich des Öfteren auf andere Gedanken gebracht und mein Leben wieder in geordnetere Bahnen gelenkt!

LIST OF ABBREVIATIONS

1G monoglycosyl 2G diglycosyl 1Me monomethylated 2Me dimethylated ADG alkyldiacylglycerols AEG acyletherglycerol Amp amphipod AR archaeol ARA arachidonic acid ATP adenosine triphosphate Beg beggiatoa BL betaine lipid BSTFA bis-(trimethylsilyl)trifluoroacetamide RS reference sample Cb crab CBB Calvin-Benson-Bassham Cil ciliate CNEXO Centre National pour l’Exploitation des Océans (french) CL cardiolipin Cop copepod Cren crenarchaeol CS Crab Spa DAG diacylglycerol DCM dichloromethane DEG dietherglycerol DFG Deutsche Forschungsgemeinschaft DGTS 1,2-dipalmitoyl-sn-glycero-3-O-4’-(N,N,N-trimethyl)-homoserine DHA docosahexaenoic acid DNA deoxyribonucleic acid DT thermal diffusivity dw dry weight EPA eicosapentaenoic acid EPR East Pacific Rise Epsilon Epsilonproteobacteria ESI electrospray ionization FA fatty acids FAMES fatty acid methyl esters FID flame ionization detector GC gas chromatography GC-FID gas chromatography coupled to flame ionization detector GC-irMS gas chromatography coupled to isotope ratio mass spectrometer GC-MS gas chromatography coupled to mass spectrometer GDGT glycerol dibiphytanyl glycerol tetraether (C40-C40 isoprenoidal chains) Gt gastropod HPLC high performance liquid chromatography H-shaped covalent bond between isoprenoidal chains of a GDGT HUFA highly unsaturated fatty acid ICP-OES inductively coupled plasma-optical emission spectroscopy IPL intact polar lipid

LC liquid chromatography Lp limpet LVP large volume pump MARUM Center for Marine Environmental Sciences MDS molecular dynamics simulations MeOH methanol MIX methylation-index Ms mussel MS mass spectrometer MS1 primary order mass spectrometry stage MS2 secondary order mass spectrometry stage m/z mass to charge ratio n.d. no data nMe multiple methylated n.s. not significant NSF National Science Foundation OH hydroxylated OL ornithine lipid PA phosphatidic acid PC phosphatidylcholine PCA principal component analysis PDME phosphatidyl-(N,N)-dimethylethanolamine PE phosphatidylethanolamine PG phosphatidylglycerol PI phosphatidylinositol PME phosphatidyl-(N)-methylethanolamine Pq polychaete PUFA polyunsaturated fatty acid qToF-MS quadrupole time-of-flight mass spectrometer r ratio of volumetric heat capacity RDA redundancy analysis RI ring-index RNA ribonucleic acid rTCA reverse tricarboxylic acid cycle S Spathi Sh shrimp sP-Uk unknown phosphatidyl sphingolipid SQ sulfoquinovosyl T temperature TAG triacylglycerols TB Teddy Bear Tbw bottom water temperature TCA tricarboxylic acid TLE total lipid extract TMS trimethylsilyl Ts source temperature Tw tubeworm UK unknown Uns unsaturated v darcy velocity VPDB Vienna Pee Dee Belemnite standard

WE wax esters z depth

Chapter I

CHAPTER I – INTRODUCTION AND METHODS

I.1. Submarine hydrothermal systems

I.1.1. Discovery of the first deep-sea hydrothermal vents

In 1969 a historical hallmark for humankind was to land successfully on the moon’s surface for the first time. It is still astonishing that mankind was first able to fly to outer space before they were able to explore the deep seafloor of our oceans personally. In year 1971 Xavier Le Pichon, head of the French Centre National pour l’Exploitation des Océans (CNEXO), had envisioned a journey to the mid-ocean ridge by human-occupied submersible. Finally, on February 17, 1977 researchers Jack Corliss and Jerry van Andel together with Alvin pilot Jack Donnelly were puzzled and astonished when they reached the ocean seafloor in the depth of the Galápagos Rift.

“Isn’t the deep ocean supposed to be like a desert?” geologist Jack Corliss, one of the three divers in the submersible, asked his grad student Debra Stakes on the phone. “Yes,” his student answered, to which a startled Corliss replied: “Well, there’s all these animals down here.”1

The famous discovery of the first deep-sea hydrothermal vents in 1977 (Ballard, 1977; Lonsdale, 1977; Corliss et al., 1979) started a new era of deep-sea research. Astonishing discoveries of bizarre creatures with exotic physiologies and mysterious microbial metabolic pathways were made. These findings accounted for a change of paradigm in the current opinion at time that no life is possible in the absence of solar energy.

I.1.2. The definition of hydrothermal

The term hydrothermal derives from the two Greek words “water” and “heat”. The word was invented by the British geologist Sir Roderick Murchison (1792–1871) to describe the action of water at elevated temperature and under high pressure. This event,

1https://www.the-scientist.com/foundations/life-on-the-ocean-floor-1977-40523 , April 24th, 2018 1

Introduction and Methods in turn, is associated with changes in the Earth’s crust and leads to the formation of various rocks and minerals (Byrappa and Yoshimura 2001; Yoshimura and Byrappa, 2008). The term was also used by many scientists to summarize the action of hot waters and mineralization associated with magmatism (Gilbert, 1875; Morey and Niggli, 1913; Holmes, 1928; Stearns et al., 1935; Machel and Lonnee, 2002). Several definitions for the word hydrothermal emerged over the past years according to the scientific perspective. For example, Byrappa and Yoshimura defined hydrothermal as any homogeneous or heterogeneous chemical reaction in the presence of a solvent (whether aqueous or non-aqueous) above the room temperature and at pressure greater than 1 atm in a closed system (Byrappa and Yoshimura, 2001). Throughout the following chapters, the word hydrothermal will be used in the context of hydrothermal activity, which refers to the movement of heated water beneath the seafloor. The most common source for hydrothermal activity is a volcanic magma chamber, which heats up subsurface seawater causing the upward movement of fluids that are released at the seafloor.

I.1.3. The geosphere-biosphere interface – Submarine hydrothermal vent systems

Submarine hydrothermal activity is a consequence of Earth’s plate tectonics. It occurs at tectonically active plate boundaries like mid-ocean ridges, submarine volcanic arcs and backarc basins (Hannington et al., 2011). Water circulation and heat transfer during hydrothermal activity may occur in depths of 1 to perhaps more than 8 km underneath the seafloor (Sinton and Detrick, 1992; Wolfe et al., 1995; Kelley et al., 2002; Davis and Elderfield, 2004; Carbotte et al., 2012). Therefore, hydrothermal systems and the related hydrothermal processes have been theoretically divided into three major areas: the recharge zone, the reaction zone and the upflow zone (the latter one is also known as discharge zone) (Figure I.1.). The recharge zone is characterized by percolation of cold, oxygenated seawater into the oceanic crust through cracks and fissures. When the seawater penetrates depths of 2 to 8 km beneath the ocean seafloor it undergoes substantial metamorphosis. Along the downward journey it becomes progressively heated concomitantly with several interactions with the surrounding host rock and consequently rock and fluid compositions get profoundly altered (Kelley et al., 2002) (Figure I.1.). At the base of the reaction zone, near the magma chamber, fluids reach their end-member composition. The highest

Chapter I temperatures of the deep circulating fluids are speculated to be in the range of 500 °C to 750 °C (Gillis and Roberts, 1999; Manning et al., 2000; Coogan et al., 2006). Here, the fluids usually get enriched in magmatic volatiles like H2O, CO2, SO2, H2S, H2, He and undergo further high-temperature reactions with the surrounding rock (Craig and Lupton, 1981; Gamo et al., 1997; German and Von Damm, 2003; Hannington et al., 2005; Lupton et al., 2008; Reeves et al., 2011). The extremely heated fluids are buoyant and hence tend to rush toward the seafloor while passing the upflow zone (Figure I.1.). At the crust- hydrosphere interface (the discharge zone), fluids may be released either in focused or in “diffuse” form. Focused flow is characterized by hot (up to ~400 °C), acidic (pH 2 to 6), reduced, metal-rich fluids with respect to seawater (Von Damm, 1995). Focused flow occurs when hot fluids are sufficiently channeled (e.g. chalcopyrite channels in sulfide chimneys) and get discharged directly from the subsurface reservoir (Alt, 1995). The final chemical composition of hydrothermal fluids depends on the nature of the heat source, the composition of the source rock as well as the temperature and time span of the water- rock interaction. Relative to bottom waters, hydrothermal fluids are typically depleted in

2− Mg, O2 and SO4 while enriched in other chemical species like CH4, H2, reduced sulfides and metals (Allen and Seyfried, 2003; Tivey, 2007). However, depending on the underlying geology in concert with the tectonic setting, the chemistry of individual hydrothermal fluids can vary significantly in time and space. Once a high-temperature venting system is established, the precipitation of minerals and metals at the high-temperature fluid – cold seawater interface leads to formation of characteristic chimneys and sulfide deposits (Juniper and Tebo, 1995). Mineral precipitation at this interface also results in formation of characteristic dense smoke (black and white smoker chimneys). Black smoker chimneys expel fluids with temperatures that might exceed 400 °C (Haase et al., 2007; Koschinsky et al., 2008), and are characterized by intense precipitation of sulfidic mineral phases (including anhydrite, barite, pyrite, chalcopyrite and sphalerite). The temperatures of hydrothermal fluids emanating from white smoker chimneys are usually between 100 and 300 °C. Their typically white appearance is caused by the precipitation of white particles like silica, anhydrite and barite (Hannington et al., 1995).

Introduction and Methods

Figure I.1. | Schematic diagram illustrating processes of hydrothermal activity. The recharge zone is characterized by seawater intrusion into the oceanic crust. Along the downward journey (reaction zone), the seawater gets progressively heated and substantially chemically altered. Because of the heat, hydrothermal fluids get more buyant and rush back to the seafloor (upflow zone) before they get emitted into the cool, oxygenated seawater. Figure adapted from Karson et al. (2015).

Another possibility of hydrothermal fluids in submarine hydrothermal systems is the so-called diffuse fluid flow. In this case, high-temperature fluids get intensively diluted with seawater during the upflow process. Discharging fluids exhibit relatively mild temperatures (< 0.2 to ~100 °C) and billow out through the seafloor as “diffuse fluids” (Bemis et al., 2012). Diffuse fluid flow systems exhibit a great variety of forms, but in general, they occur over extensive areas in the surrounding of black and white smokers (Van Dover, 2000). Sometimes diffuse venting can be observed as shimmering water but this is often obscured by overgrowing fauna and microbial mats. The relatively slow and inconspicuous upward flow of diffuse hydrothermal venting is likely the main reason why geochemical studies have been centered on high-temperature vents with the spectacular appearance of black smokers (McCollom and Shock, 1997). However, based on heat-output data, it has been estimated that diffuse venting exceed by the order of one magnitude the heat and mass flux of high-temperature venting in some areas (Rona and Trivett, 1992; Schultz et al., 1992). The global budget of diffuse venting systems is estimated at 50 to 90% of the total fluid flow in the oceans (Rona and Trivett, 1992; Schultz et al., 1992; Baker et al., 1993; Ramondenc et al., 2006; Veirs et al., 2006; Bemis et al., 2012).

Chapter I

From a biological perspective, diffuse flow systems are characterized by relatively low temperatures relative to high-temperature systems (100 to > 400 °C, respectively), with the former ones well within the so far determined limit of life at 122 °C (Kashefi and Lovley, 2003; Takai et al., 2008a). Because of the low temperature they contribute very little to mineral deposition, but instead these diffuse fluid flow systems can sustain quite productive populations of thermophilic microorganisms and dense invertebrate communities at deep-sea vent areas (Van Dover, 2000).

I.1.4. Importance of hydrothermal systems to the global element cycle

Hydrothermal activity is a process of global implication, including global heat loss, weathering processes and geochemical cycling of elements. Therefore, it has profound impacts on the biogeochemistry of deep-ocean waters and sediments. Recent computer simulated models have estimated that hydrothermal fluid flow circulates the entire volume of the ocean through the ocean crust and out at mid-ocean ridges every 70,000 to 200,000 years (Johnson and Pruis, 2003; Mottl, 2003; Wheat et al., 2003). Hydrothermal systems are important sinks for sulfate and magnesium and significant sources of manganese, iron, lithium, rubidium and cesium (German and Angel, 1995; Elderfield and Schultz, 1996). For example, iron that is usually a trace element in the ocean has been measured in hydrothermal fluids in the millimolar range (Van Dover, 2000). Moreover, Sander and Koschinsky (2011) estimated that the amount of iron in hydrothermal fluids may account for 9% of the total dissolved iron budget of the deep ocean. Finally, the global potential for chemosynthetic primary production at deep-sea hydrothermal vents is estimated at 1013 g year−1, which amounts to ca. 0.02% of the global primary production from the surface oceans (McCollom and Shock, 1997). Therefore, hydrothermal circulation has far-reaching implications for global element cycles.

I.1.5. Hydrothermal vents and the origin of life

The discovery of hydrothermal vent systems more than 40 years ago dramatically changed our view of the biogeochemical history of the Earth. High-temperature sulfide chimneys typical of deep-sea vent systems harbor both chemical and thermal gradients that are similar to conditions of the early Earth. The latter have likely existed as soon as liquid water accumulated on the Earth more than 4.2 billion years ago (Martin et al., 2008). The idea that many features of hydrothermal systems constituted suitable conditions for the origin and early evolution of life is particularly interesting (Baross and Hoffman, 1985). 5

Introduction and Methods

Several lines of evidence suggest the chemically reducing conditions at hydrothermal environments as conducive to the conversion of inorganic compounds (e.g.

CO2, H2, NH4, H2S) or simple organics to prebiotic organic compounds (lipids, amino acids, sugars, nucleotides). For instance, concepts such as the RNA world (as the early form preceding the DNA- and protein-based life; Joyce, 2002) are consistent with the prebiotic conditions of the early Earth. McCollom and coworkers (1999) observed the formation of lipids homologues after incubating formic acid or oxalic acid in water at 175 °C for 60 to 72 hours in stainless steel vessels or tubes. This is equally relevant to the spontaneous formation of micelles (membrane-like vesicles) from lipids during cooling of hydrothermal fluids (Monnard et al., 2002). From these studies derive the idea of self- assembled lipid vesicles as the origin of primitive membranes (Deamer, 2017) which might have been essential for maintenance of ion gradients for early bioenergetics (Lane and Martin, 2012). Thus it is likely that present-day microorganisms from vents and hydrothermal systems carry physiological features that resemble the early life forms of the Earth.

I.1.6. Submarine hydrothermal activity – the global distribution

The total strike length of the global ocean plate boundaries is ~89,000 km, including mid ocean ridges (64,000 km), submarine volcanic arcs and backarc basins (25,000 km) (Bird, 2003; de Ronde et al., 2003; Hannington et al., 2011). Baker and colleagues (1996) suggested that 16% of the total ridge length is hydrothermally active at any given time and based on simulation models, Baker and German (2004) hypothesized that there are ~1000 active hydrothermal vent fields worldwide. Until today around 688 vent fields are registered in the InterRidge vents database2 (Beaulieu, 2015), occurring over a wide range of water depths from the very shallow coastline to the abyssal in depths of more than 5.5 km (Figure I.2.). Within this number, 286 vent fields are confirmed to be active, 345 are inferred to be active and 57 are determined as inactive (Beaulieu, 2015). A closer look on the active confirmed and inferred fields revealed, that 570 vent fields belong to the category of “deep-sea” hydrothermal vent fields while only 61 belong into the category of “shallow” (Figure I.2.). Due to innovative deep-sea technologies and analytical methods the amount of discovered vent fields has approximately been tripled within the last decade. Nevertheless, it is very likely that much more sites will be discovered in future ocean surveys and expeditions, when the remaining ~80% of the global ridge crest

2Retrieved from https://vents-data.interridge.org , February 3rd, 2018 6

Chapter I will undergo systematic exploration for the presence and location of high-temperature venting (Baker and German, 2004). In 2015, Beaulieu and colleagues predicted that there are likely ~1000 undiscovered vent fields, most of them probably located in the regions of the poorly explored ultraslow and slow-spreading mid-ocean ridges (Beaulieu et al., 2015).

Figure I.2. | Global distribution of seafloor hydrothermal vent fields. Version 3.3 of the InterRidge vents database (Beaulieu, 2015) used for this figure contains information on 688 sites of seafloor hydrothermal activity (confirmed and unconfirmed sites of deep-sea and shallow-water hydrothermal vent systems) and inactive sites. About 570 vent fields are declared as deep-sea hydrothermal fields, while 61 are declared as shallow-water hydrothermal fields. This figure was made with Generic Mapping Tools (GMT) (Wessel et al., 2013) and included the bathymetric map of Amante and Eakins (2009).

I.1.7. The importance of chemosynthesis at hydrothermal systems

The discovery of areas characterized by excessive faunal communities in the vicinity of deep-sea vents represented a paradigm shift and a reconsideration of the concept that there is no life independent of solar energy. It seemed unlikely that these enormous blooms of life on the deep ocean seafloor could be supported by just the flux of particulate organic matter derived from the surface layers of the ocean (Van Dover, 2000; Tarasov et al., 2005). Although the process of chemosynthesis was known since the 19th century (Winogradsky, 1887), the global significance of this process finally became appreciated by the time when deep-sea vents were first discovered. From then on, deep-sea vents represented a window of opportunity for microbiologists to study the versatile metabolic pathways utilized by microbes, which are the basis of the food chain in an area remote of solar energy.

Introduction and Methods

All living organisms, independently of environment and life style, have three basic requirements to maintain their metabolic functions: i) a source of energy, ii) a carbon source and iii) a source of electrons (Van Dover, 2000). The energy source can be either light (photo-) or chemical nature (chemo-); the carbon source may be inorganic (auto-) or organic (hetero-) and electron donors may be inorganic (litho-) or organic (organo-) (Van Dover, 2000). Therefore, the typically shortened term “photosynthesis” (Eq. 1) may be more precisely defined as photoautolithotrophy.

Eq. 1

Hydrothermal ecosystems are generally characterized by extreme geochemical conditions with sharp physical and chemical gradients, which provide a variety of habitats and microniches for highly specialized and metabolically diverse microorganisms (Fisher et al., 2007; Dubilier et al., 2008). Mixing of hydrothermal fluids with oxidized seawater

2− − – enriched in abundant oxidants like O2, SO4 , NO3 and CO2 – yields a variety of redox

− 0 2− 2+ couples (e.g., H2, H2S, HS , S , S2O3 and Fe ) scavenged by versatile microbes for energy generation (Jannasch and Mottl, 1985; Jannasch, 1995; Orcutt et al., 2011; Sievert and Vetriani, 2012). Table I.1. shows the diversity of electron donors and electron acceptors for energy generation under aerobic and anaerobic conditions which will be further discussed in the next section.

Chapter I

Table I.1. | Examples of thermodynamically favorable redox reactions exploited by microorganisms living in marine hydrothermal environments.

Conditions Electron donor Electron acceptor Metabolic process Aerobic H2O NADP Oxygenic photosynthesis Anaerobic HsA O2, NADP Anoxygenic aerobic photosynthesis Aerobic H2 O2 Hydrogen oxidation − HS O2 Sulfide oxidation 0 S O2 Sulfur oxidation 2− S2O3 O2 Thiosulfate oxidation 2+ Fe O2 Iron oxidation 4+ NH O2 Nitrification CH4 + other C-1 O2 Methane oxidation compounds 2+ Mn O2 Manganese oxidation Organics O2 Heterotrophic metabolism − Anaerobic H2 NO3 Denitrification 0 H2 S Sulfur reduction 2− H2 SO4 Sulfate reduction H2 CO2 Methanogenesis 2− Organics SO4 Heterotrophic sulfate reduction Organics S0 Heterotrophic sulfur reduction Organics Organics Fermentationa Complied and adapted from Flores and Reysenbach (2011) and from Hügler and Sievert, 2011

aExample reaction calculated from acetate + ethanol  butyrate + water

I.1.8. Energy sources and frequently identified microorganisms in hydrothermal ecosystems

A rich diversity of bacteria and archaea associated with hydrothermal systems has been detected using culture-dependent and culture-independent methods (Jeanthon, 2000; Takai et al., 2001; Schrenk et al., 2003; Kormas et al., 2006; Reysenbach et al., 2006; Takai et al., 2008b; Takai et al., 2009; Kato et al., 2010; Flores et al., 2011; Flores et al., 2012; Olins et al., 2013). In deep-sea vents, bacteria dominate over archaea, with contributions ranging from 35 to 99% of the total microbial population, depending on the type of method and the samples position within a mineral deposit (Flores and Reysenbach, 2011 and references therein). It is widely believed that a sizable fraction of biomass at hydrothermal vents is directly linked to chemolithoautotrophy (Jannasch and Mottl, 1985) and the global primary production has been estimated to be about 1013 g biomass per year, representing ~0.02% of the global primary production by photosynthesis (McCollom and Shock, 1997; McCollom, 2000). In deep-sea vent areas of high-temperature fluids, anaerobic reactions tend to be the dominant process – although transition to aerobic processes may occur over short spatial

Introduction and Methods scales due to diffusive and advective fluid mixing (Kormas et al., 2006). Among anaerobic reactions, generally mediated by thermophilic or thermotolerant species, methanogenic archaea that utilize H2 coupled to CO2 reduction are potential autotrophic candidates (McCollom and Shock, 1997), although many known thermophilic archaea are heterotrophic (Baross and Deming, 1995). The microbial communities in high- temperature basalt-hosted systems tend to be dominated by sulfur- and H2-respiring Epsilonproteobacteria and in thermophilic to hyperthermophilic methanogens and sulfate-reducing Euryarchaeota (Campbell et al., 2006; Longnecker and Reysenbach,

2001; Nunoura et al., 2010, Opatkiewicz et al., 2009; Reysenbach et al., 2000). Similar patterns in microbial ecology were found at diffuse hydrothermal vents (25 °C) in the flanks of Juan de Fuca Ridge (Baby Bare, Cowen et al., 2003; Huber et al., 2006; Nakagawa et al., 2006; Orcutt et al., 2011) and 9°N East Pacific Rise (McNichol et al., 2016). In low-temperature, Fe-rich, diffusely venting environments, such as at Loihi Seamount and around Suiyo Seamount, the bacterial communities appear to be dominated by Fe-oxidizing microbes belonging to the Zetaproteobacteria (Moyer et al., 1995; Emerson and Moyer, 1997, 2002; Emerson et al., 2007; Davis et al., 2009; Kato et al., 2009; Rassa et al., 2009), which are also frequently observed in massive inactive sulfides (Kato and Yamagishi, 2015). A complete overview of the microbial ecology of these systems is beyond the scope of this thesis, and readers are referred to comprehensive reviews available in the literature (Jeanthon, 2000; Orcutt et al., 2011; Sievert and Vetriani, 2012; Price and Giovannelli, 2017.) In contrast to abundant publications devoted to the microbial ecology of deep-sea vents, much less is available for submarine shallow-water hydrothermal systems (Price and Giovannelli, 2017). Given that these ecosystems are the main focus of chapters III and IV of this thesis, the fundamental differences between shallow and deep hydrothermal systems will be introduced next.

I.1.9. Deep-sea versus shallow-water hydrothermal systems

Although the discovery of the first deep-sea hydrothermal vents in 1977 represented a milestone in deep-sea research, historically this event was not the first report of a hydrothermal system. Venting related to volcanic activity was already known since the 1800’s when the Panarea shallow-water hydrothermal vent was described (InterRidge vents database, Beaulieu, 2015). Unlike their deep-sea counterparts, shallow-water hydrothermal systems had received much less scientific attention (Price and Giovannelli,

Chapter I

2017). The classification of “deep” and “shallow” is based on findings of Tarasov and colleagues who recognized a strong shift of the hydrothermal vent community composition at approx. 200 m water depth (Tarasov et al., 2005) (Figure I.3.). Shallow-water hydrothermal systems are located in close proximity to the coast of either the continents or islands, and therefore commonly related to coastal volcanism. Moreover, these submarine ecosystems are roughly within the maximum extent of the photic zone. Therefore, the main difference between these ecosystems is photosynthesis in shallow-water hydrothermal systems. Primary production occurs both in the water column above the hydrothermal vent and on the seafloor where pronounced bacterial and/or algal mats are commonly observed (Tarasov et al., 2005; Tarasov, 2006). It is speculated that shallow-water hydrothermal systems may support an increase of photosynthetic rates because fluids are directly discharged into the photic zone (Sorokin et al., 1993, 1994, 1998, 2003; Kleint et al., 2017). Although chemosynthesis in shallow- water hydrothermal systems can account for 1 to 65% of the total primary production (Tarasov et al., 2005; Gomez-Saez et al., 2017), the amount of photosynthetically produced organic matter, taking the primary production within the overlying water column into account, seems to be comparatively greater (Tarasov et al., 2005). Another characteristic feature of shallow-water hydrothermal systems is the presence of volatile species in free gas and/or dissolved phase forms that result in intense bubbling through the seafloor (Tarasov et al., 2005; Price and Giovannelli, 2017) (Figure I.3.). Escaping gas bubbles have been documented from different locations worldwide (Glasby, 1971; Horibe et al., 1980; Benjamínsson, 1988; Tarasov and Zhirmunsky, 1989; Pichler et al., 1990; Dando et al., 1995; Calanchi et al., 1995; Forrest et al., 2005; McCarthy et al., 2005; Kilias et al., 2013). Although volatile gases are also very abundant in deep-sea hydrothermal systems, the higher hydrostatic pressure usually prevents the formation of a free gas phase (Tarasov et al., 2005). Furthermore, the close proximity to the coastline implies that these ecosystems are subjected to terrigenous input of minerals and organic matter through weathering and run- off. Input of meteoric groundwater, which affects the chemical composition of the hydrothermal fluids (e.g. elevated metal concentration, low pH), has been frequently documented for shallow sites (Pichler et al., 1999; Prol-Ledesma et al., 2004; McCarthy et al., 2005; Couto et al., 2015). The influence of terrigenous input and meteoric water over the productivity of these hydrothermal systems is speculated to be important, but it has not been thoroughly evaluated (Price and Giovannelli, 2017). Although periodicity of

Introduction and Methods hydrothermal systems is common at all water depths, the effects of tides, wave action and storms have probably a greater impact on microbial communities from shallow areas than in the deep sea (Price and Giovannelli, 2017).

Figure I.3. | A) Schematic overview of deep-sea and shallow-water hydrothermal systems. B) Deep-sea vent (black smoker) occurring in the absence of sunlight, showing high macrofaunal biomass with diffuse and focused hydrothermal fluid flow. C) Shallow-water hydrothermal system dominated by microbial and algal mats and characterized by a free gas phase and diffuse fluid flow. This figure has been modified after Kellermann (2012).

A stunning visual difference between shallow-water and deep-sea hydrothermal sites is the common absence of sulfide structures and macrofauna in the former (Figure I.3.). Although large sulfide deposits can only be found under the high hydrostatic pressure of the deep sea, a few sulfide structures are documented for shallow-water hydrothermal systems (e.g. off the coast of Taiwan; Wang et al., 2015). Similarly, only a few species of macrofauna are known to be directly related to shallow-water venting, including vestimentifera worms of Kagoshima Bay, Japan (Hashimoto et al., 1993; Miura et al., 1997), crabs off Kuishan Island, Taiwan (Ng et al., 2014; Yang et al., 2016) and nematodes of the Bay of Pleanty, New Zealand (Kamenev et al., 1993). It is believed that

Chapter I the broad diversity in food sources (e.g. chemotrophic- and planktonic-derived) presumably favors opportunistic species, preventing endemic and/or symbiont-bearing metazoan communities at shallow-water hydrothermal systems (Tarasov et al., 2005; Dubilier et al., 2008). The microbial communities inhabiting shallow-water hydrothermal sites consist largely of meso-, thermo- and hyper-thermophilic archaea and bacteria (e.g. Alfredsson et al., 1988; Dando et al., 1999; Sievert et al., 2000; Tarasov, 2006; Hirayama et al., 2007). Contrasting with their deep-sea counterparts, photosynthetic communities (e.g. cyanobacteria, diatoms and anoxygenic phototrophs; Kamenev et al., 1993; Thiermann et al., 1997; Dando et al., 1999; Hirayama et al., 2007; Roeselers et al., 2007; Giovannelli et al., 2013; Maugeri et al., 2013; Dávila-Ramos et al., 2014; Lentini et al., 2014) and heterotrophs (Hoaki et al., 1995; Gugliandolo and Maugeri, 1998; Sievert et al., 2000; Tarasov, 2006; Pop Ristova et al., 2017) are also observed in high abundance likely due to the presence of sunlight and high input of allochthonous organic matter, respectively, in these systems (Tarasov et al., 2005). It is interesting, however, that many biogeochemical processes occurring in subsurface/near surface of shallow-water hydrothermal sites, to a certain extent, resemble those at deep-sea vents (reviewed in Tarasov et al., 2005 and Price and Giovannelli, 2017). Reflecting these similarities, the two major groups of bacteria appear to be uniformly dominant and related to the sulfur metabolism in both submarine hydrothermal systems: Epsilonproteobacteria and Gammaproteobacteria and/or Firmicutes (Price and Giovannelli, 2017 and references therein). Another similar aspect between both ecosystems is that the distribution of microbial taxa seems to be largely controlled by temperature and availability of electron acceptors and donors (Sievert et al., 2000; Price et al., 2007; Giovannelli et al., 2013; Yücel et al., 2013). In this thesis, chapters III and IV involve the study of microbial lipid distribution relative to sediment temperature at submarine shallow-water hydrothermal systems.

I.2. Methods

I.2.1. Lipid ecology applied to hydrothermal systems

This thesis focuses on the interplay between microbial ecology and marine hydrothermal systems. The rationale is that environmental conditions at these locations influence the chemosynthetic population and associated communities. For instance, gradients in temperature, hydrogen sulfide concentrations and pH, among others, may

Introduction and Methods directly affect the composition of microbial communities thriving in hydrothermal environments. In addition, vent fluids directly drive substrate and electron acceptor concentrations, which will in turn determine the major pathways of carbon fixation. In this context, lipids may thus be used as biomarkers for living microbes, inform about cell membrane adaptation to the physico-chemical conditions and elucidate food web relationships from primary producers to higher trophic levels. Below, a general introduction to lipids and a brief history of lipid microbial ecology are summarized.

I.2.2. General introduction to lipids

Membrane forming lipids are amphipathic molecules composed of a hydrophilic polar headgroup linked to a hydrophobic glycerol- or sphingoid-based backbone (Figure I.4.). This physical property is essential for the arrangement of a typically ~30 Å biological membrane which separates the cell’s interior from the surrounding environment (White et al., 2001; Dowhan and Bogdanov, 2002). While the structural composition of essential biomolecules such as DNA and proteins is determined by 4 nucleotide bases and 23 amino acids, respectively, the diversity of lipid structures may reach staggering numbers. Without taking into account all of the possible double bond positions, backbone substitutions, and stereochemistry, there are over 180,000 possible lipid species (Yetukuri et al., 2008). The total diversity in lipid structures is estimated at several million potential lipids when all these structural differences are accounted for (Koelmel et al., 2017). According to modern lipidomics analysis, thousands of different lipids are found in eukaryotic cells, which devote ca. 5% of their genes to the synthesis of these lipids (Sud et al., 2007). These data suggest that there must exist evolutionary advantages linked to this complex lipid repertoire and that the diversity of lipid structures may support diverse cellular functions. Lipids have at least three major functions (van Meer et al., 2008). The first involves energy storage primarily as triacylglycerol or steryl esters in lipid droplets. This efficient reserve of energy may also serve as important storage of fatty acids and sterols that are important components of cell membranes. Secondly, lipids are recognized as primary and secondary messengers in signal transduction and molecular recognition processes. For instance, production of eicosanoids, which are implicated in tissue homeostasis and/or inflammation, is initiated by the release of C20-polyunsaturated fatty acids, such as arachidonic acid (C20:4), from phospholipids or diacylglycerol (e.g. Boyce,

Chapter I

Figure I.4. | Diversity of polar membrane lipid structures of Archaea, Bacteria and Eukarya. A) Schematic illustration of the cytoplasmic membrane showing lipid molecules, forming either a mono- or a bilayer, and embedded proteins. Archaeal membrane forming lipids are displayed in red and bacterial/eukaryotal lipids in green. B) Detailed structures and characteristics of the archaeal and bacterial/eukaryotal glycerol backbone including stereochemistry and side chain linkage. C) Structures of archaeal lipids: I, acyclic glycerol dibiphytanyl glycerol tetraether (GDGT) with additional methyl groups (nMe) and a covalent bond (H- shape) between the isoprenoidal side chains; II, acyclic H-GDGT; III, cyclopentane ring-containing GDGT; V, glycerol trialkyl glycerol tetraether; VI, archaeol (AR) with unsaturations. Structures of bacterial lipids: VII-XII, diacylglycerols (DAG) containing different combinations of fatty acids. VII, C14:1ω7/C14:1ω7; VIII, C15:0/iC15:0; IX, C14:0/C18:3; X, C15:0/aiC15:0. XI, other core lipid structures of bacteria and eukarya. D) Polar headgroups of Archaea, Bacteria and Eukarya. E) Different sterols functioning as membrane rigidifiers in eukaryotic membranes. Figure modified and adapted from Kellermann (2012).

Introduction and Methods

2007). Finally, lipids modulate the physical properties of membranes including surface electrostatics, phase behavior, membrane curvature and hydrogen-bonding capacity (e.g. Kučerka et al., 2015). Such a role is played largely by differences in the length of hydrocarbon chains, the degrees and locations of unsaturation, density of charges, and the polarity and size of the headgroup (de Kroon et al., 2013).

I.2.3. Lipids and the domains of life

Membrane lipids are one of the most prominent features differentiating Archaea from Bacteria and Eukarya (Kates, 1978; Koga et al., 1993). Profound differences in the lipid stereochemistry of the glycerol backbone distinguish Archaea (headgroup attached to sn-1-glycerol) from Bacteria and Eukarya (headgroup attached to sn-3-glycerol)

(Kates, 1978). Furthermore, the synthesis of isoprenoidal side chains (C20, C25 or C40, respectively, phytanyl, sesterterpanyl and biphytanyl) attached via di- or tetraether linkage to the glycerol backbone distinguishes the unique lipid characteristics of the Archaea. Bacterial glycerolipids as well as those from eukarya consist of ester-linked fatty acids, with some exceptions being plasmanyl and plasmalogens that have one of their alkyl chains ether-linked to the glycerol backbone (Goldfine, 2010; Řezanka et al., 2012). Another very peculiar group of lipids found exclusively in bacteria and eukarya are sphingolipids (Olsen and Jantzen, 2001; Muralidhar et al., 2003) (Figure I.4.) while ornithine lipids so far seems to be restricted to the domain of Bacteria. Ornithine lipids exhibit an amide bound 3-hydroxy fatty acid, which is esterified via its hydroxyl group to a second fatty acid (Figure I.4.). A prominent feature of eukaryotal membranes are sterols, which are important as cell rigidifiers that reduce membrane fluidity and permeability.

I.2.4. A brief history of the microbial lipid ecology

Microbial lipid ecology is a field that involves the study of adaptations of microbial membranes through their lipid composition to environmental conditions (Zhang and Rock, 2008). By regulation of the polar headgroup and/or the hydrophobic tail composition in a biological membrane (Figure I.4.), organisms control membrane permeability and fluidity thereby adapting to changing environmental conditions (e.g. temperature, pH and pressure). The following section will introduce important contributions of researchers to the field of lipidomics and also technical developments. For more detailed information the reader is referred to comprehensive reviews available in the literature (e.g., Vestal and White, 1989; Zhang and Rock, 2008). 16

Chapter I

The laboratory of Prof. Wieslander was arguably one of the first to study lipids as adaptive strategy of microbial membranes using Acholeplasma laidlawii as model system. The scientific contributions of this group (between the 70’s and 90’s) were mainly related to the role of lipids in the regulation of cell membrane homeostasis, including changes in glycolipid composition, configuration of incorporated fatty acids and cholesterol content in response to elevated temperatures (e.g. Wieslander and Rilfors, 1977; Christiansson and Wieslander, 1980). At that time, lipid membrane homeostasis was also intensely studied by other laboratories interested in the modulation of lipid composition according to environmental conditions (e.g. Cronan and Gelmann, 1975; Quinn, 1981; McElhaney, 1974; Melchior, 1982; De Mendoza and Cronan, 1983; Hazel and Williams, 1990). The research on lipid ecology gained momentum in the late 90´s with the work conducted at the laboratory of Prof. Konings. These studies involved several cultured strains of microbes, including archaea and bacteria, and essentially highlighted membrane lipids as major factors controlling ion permeability. More specifically, several strains isolated from hydrothermal systems displayed the lowest permeability against ions at elevated temperatures (van de Vossenberg et al., 1995; Konings et al., 2002). Thomas H. Haines was also a pioneer in the study of lipids as permeability barriers, publishing an innovative paper in 2001 that summarized membrane adaptations observed in membranes of bacteria and the role of sterols in eukaryotic membranes (Haines, 2001). A few years later David L. Valentine (2007) used principles established by these early investigations to conclude that lipids are essential for archaea to conserve energy under stressful conditions compared to the other domains of life. This topic was later further explored by Valentine and co-workers (Valentine and Valentine, 2009; 2013; 2015; Yoshinaga et al., 2016), who also published an article about the importance of lipids in the bioenergetics of haloarchaea recently (Kellermann et al., 2016). Whereas the concepts developed by the above-mentioned studies evolved from investigations performed with cultured microbes, this thesis focused on understanding how environmental conditions may affect microbial membranes in nature. Another important aspect from this thesis is the detection and accurate monitoring of a large number of lipids (> 1,000 individual polar lipids/molecular species), only possible due to modern analytical techniques in the field of lipidomics. This large structural diversity of lipids is hypothesized as a chief requirement for prokaryotes, and also eukaryotes, to colonize many extreme environments, as exemplified throughout this thesis.

Introduction and Methods

I.2.5. A historical perspective of lipidomics and its application in marine microbial ecology

In recent years, advances in mass spectrometry (MS) were one of the major drivers that have pushed the field of lipidomics forward (e.g. Han and Gross, 2003; Piomelli et al., 2007; Wenk, 2010; Spickett and Pitt, 2015; Kohlwein, 2017). Notably, these advances in lipidomics are directly linked to the application of electrospray ionization (ESI) to crude lipid extracts. The field of lipidomics is concerned with the identification and quantification of lipids within a given biological system, but also seeks to elucidate how individual molecular species affect lipid metabolism and the function (or dysfunction) of the system as a whole (Naudí et al., 2015). The history of lipidomics in microbial ecology coincides with the history of lipidomics using ESI-MS. While lipidomics was initially used to investigate physiological changes in a given cell or tissue for clinical/medical purposes (e.g. Han and Gross, 2003), bacterial cultures were almost concomitantly screened for their lipid composition by liquid chromatography (LC)-MS (Fang and Barcelona, 1998). This methodology was applied in marine microbiology with the pioneering work performed at Prof. Rullkötter’s laboratory with pure cultures of bacteria only a few years later (Rütters et al., 2001, 2002a). The idea at that time was to establish a robust culture-independent protocol, with which polar lipids could potentially be used as microbial biomarkers in the marine realm (e.g. deep biosphere, anoxic water columns, cold seeps), where most microbes are still uncultivated (Rütters et al., 2002b; Zink et al., 2003; Sturt et al., 2004). Lipid biomarker studies using LC-ESI-MS, however, have flourished in the laboratory of Prof. Hinrichs. Not only it was evident that his group was in a quest to describe lipids from diverse environmental settings (e.g. Biddle et al., 2006; Schubotz et al., 2009; Rossel et al., 2008; Kellermann et al., 2012), but they also tackled fundamental issues regarding microbial ecology. The controversial paper by Lipp and coworkers (2008) was a breakthrough in the microbial ecology of the deep biosphere showing a clear dominance of archaea over bacteria in these deeply buried sediments based on polar lipids. Such an important finding triggered feedback reactions from the community leading to publications questioning their results (Schouten et al., 2010; Logemann et al., 2011). Recently, Xie et al. (2013) showed by in vitro experiments that archaeal ether- linked lipids are indeed extremely resilient to degradation and might majorly represent molecular fossils in deeply buried marine sediments. Chapter IV of this thesis focuses on

Chapter I archaeal lipids, more specifically on the potential significance of glycosidic tetraethers as one of the most abundant archaeal polar lipid detected in environmental samples. In year 2013 several analytical improvements (both in LC and in MS) were published by the Hinrichs group (Becker et al., 2013; Wörmer et al., 2013; Zhu et al., 2013), expanding the detection window from already hundreds of lipids to more than 2000 lipids (including > 1000 polar lipids, storage lipids, sterols etc.) as exemplified by Chapter V of this thesis (Figure I.5.). These analytical advances opened the opportunity to study these molecules not only for microbial ecology, but also as mentioned before as potential natural products with translational applications. For instance, the detection and characterization of several quinones (Elling et al., 2016) and carotenoids (Kellermann et al., 2016) produced by archaea may potentially serve as platform for isolation of key compounds for protecting cells against oxidative stress.

Figure I.5. | Timeline showing a selection of publications and the amount of identified molecular species of archaeal and non-archaeal polar lipids from 2008 until 2017.

Introduction and Methods

I.2.6. Lipids as molecular tools to study the microbial ecology of hydrothermal systems

Lipids were one of the pioneering molecular biomarkers applied to the microbial ecology of deep-sea vents (Hedrick et al., 1992). These authors investigated microbial lipids in sulfide deposits from the Endeavor Ridge hydrothermal vent site and found ether lipids from archaea distributed abundantly at the hotter portions of the sulfide structure. In contrast, bacterial fatty acids were distributed toward the cooler surfaces. With the advance of GC coupled to isotope ratio mass spectrometer (irMS), the field of lipids used as biomarkers moved forward towards the evaluation of microbial pathways for carbon fixation (Hayes, 1993). This analytical tool was successfully employed in studies at hot springs (Jahnke et al., 2001; Van der Meer, 2007; Havig et al., 2011), but to a great extent δ13C lipid data were generated at hydrothermal vents in the deep sea (Pond et al., 1997a, 1997c, 1998, 2002; Blumenberg et al., 2007; Naraoka et al., 2008; Biddle et al., 2012; Kellermann et al., 2012; Reeves et al., 2014). A study by Havig et al. (2011) has shown a spatial gradient in the microbial community distribution at hot springs, from a cyanobacteria-dominated community to the dominance of the phylum Aquificaea towards the source of the hot spring, based on the analysis of fatty acids and their stable carbon isotopic signatures. In deep-sea vents, δ13C composition of fatty acids was used to study the carbon fixation pathway of microbes (e.g. methanogenesis by Bradley et al., 2009b; reverse tricarboxylic acid cycle by Reeves et al., 2014) and type of symbionts from macrofauna and macrofaunal lipids (Rieley et al., 1995; Pranal et al., 1996; Limén et al., 2008, Pond et al., 1997a, 2000, 2002, 2008; Phleger et al., 2005a, 2005b; Kouris et al., 2010; Kellermann et al., 2012). One controversial issue attempted by Pond and colleagues (Pond et al., 1997a, 1997c; Allen-Copley et al., 1998; Gebruk et al., 2000) was the origin of polyunsaturated fatty acids (PUFA) observed in considerable proportions in macrofauna such as the hydrothermal vent shrimps Mirocaris fortunate, Rimicaris exoculata and Alvinocaris markensis. These authors proposed the hypothesis that juvenile forms of macrofauna could obtain their PUFA content by swimming to surface waters migrating back to the deep sea to live the adult stage (Pond et al., 1997b). This issue is revisited in Chapter V of this thesis. It was only in the last decade that LC-MS techniques and lipid biomarker studies were repeatedly applied to deep-sea vents. Studies performed at Lost City Hydrothermal Field (Bradley et al., 2009a, 2009b), symbiotic mussels (Kellermann et al., 2012) and

Chapter I sulfide structures of deep-sea vents (Gibson et al., 2013; Reeves et al., 2014) revealed a great diversity in microbial polar lipids, but also highlighted archaeal lipids into the biomarker picture – since these lipids are mostly undetectable using conventional gas chromatographic methods. In addition, numerous other investigations were performed at terrestrial hot springs using LC-MS techniques with a focus on archaeal lipids (e.g. Boyd et al., 2013; Pearson et al., 2004, 2008; Zhang et al., 2004, 2007; Schubotz et al., 2013). The great advantage of LC-MS is that lipids can be studied in their natural intact forms, giving rise to a myriad of lipid molecular species. In particular several novel molecular species were described for archaea (Yoshinaga et al., 2011, 2012; Liu et al., 2012; Zhu et al., 2014; Bauersachs and Schwark, 2016), and some of these structures are intensely discussed in chapter III of this thesis. The variety of molecular species of lipids detected by LC-MS not just established the use of lipids as biomarkers, but also opens new interpretation possibilities about their structure-function in microbial ecology, a topic which this thesis is mainly focused on.

I.3. References

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Introduction and Methods

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Chapter I

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Introduction and Methods

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Chapter II

CHAPTER II – STRUCTURE AND OBJECTIVES

Structure and main objectives of this thesis

In this thesis, three novel applications of lipids as tools to study the microbial ecology were investigated in submarine hydrothermal systems and are listed below. [Chapter III] deals with the lipid ecology of microbes inhabiting the sediments of shallow-water hydrothermal sediments. The investigated sediments off Milos Island represent ideal outdoor laboratory conditions to study microbial cell membrane adaptations due to the presence of thermal gradients, which are difficult to capture sampling deep-sea vent chimneys (Kormas et al., 2006). Sediment samples were precisely taken along a thermal gradient covering an overall temperature increase from 18 °C to a maximum of 101 °C, which is close to the so far determined temperature limit of life (Kashefi and Lovley, 2003; Takai et al., 2008). Polar lipids analyzed by this study amounted to ca. 430 molecular species, from which major structural characteristics (e.g. fatty acid chain length and unsaturations; polar headgroups) were correlated with sediment temperature.

Research questions:

- What is the significance of bacterial versus archaeal polar lipids in these heated sediments? - Is it possible to identify patterns of polar lipid distribution relative to temperature that may reflect adaptations of the microbial community at the cell membrane level? - Is there any unity of membrane lipid structure-function between archaea and bacteria?

[Chapter IV] focuses on the diversity and relative abundance of archaeal membrane lipids in hydrothermally-influenced sediments off the coast of Dominica. The approach was to test the reproducibility of previously obtained results of chapter III on membrane lipid adaptations related to sediment temperature induced by underlying hydrothermal activity. Two distinct sediment cores were taken off the coast of Dominica at Soufrière and Portsmouth. Geochemical characterization was different to sediment samples recovered from Milos Island, however, the overall temperature gradient was comparable

Structure and Objectives and ranged from 28 to approx. 104 °C. Additionally, the compilation of both data sets, Dominica and Milos, including lipid diversity and abundance as well as geochemical data, allowed for multivariate analyses and to determine the influence of temperature and other geochemical drivers, on the lipid distribution in these sediments.

Research questions:

- Is there a universal response of certain archaeal membrane lipids to temperature in sediments of hydrothermal activity? - What is the significance and influence of other environmental parameters on the distribution of archaeal polar lipids in hydrothermally-influenced sediments?

[Chapter V] combines the analysis of a broad spectrum of lipids by lipidomics (> 1500, including storage lipids, sterols etc.) and stable carbon isotopic composition of fatty acids to identify the microbial biomass associated with diffuse fluids (13 to 25 °C in temperature). The samples were taken filtering hundreds of liters of fluids collected from an opening (hollow lava tube) into the subsurface hydrothermal aquifer from the East Pacific Rise 9°50’N. Although these fluids are presumably enriched in bacterial biomass (especially abundant Epsilonproteobacteria from the subsurface; McNichol et al., 2016), the data presented in this study revealed that ca. 90% of total lipids consisted of storage lipids (such as triacylglycerol), which are not typically found in bacteria. Moreover, major classes of phospholipids were enriched in polyunsaturated fatty acids, another uncommon lipid signature of bacteria.

Research questions:

- What are the major sources of lipids associated with diffuse hydrothermal fluids and adjacent bottom waters from the East Pacific Rise? - What are the potential sources of storage lipids and polyunsaturated fatty acids? - Can trophic upgrading explain the distribution of lipids in the fluid samples? - What is the ecological significance of these findings for these diffusive systems?

Chapter II

Contributions to publication

Chapter III: Heat stress dictates microbial lipid composition along a thermal gradient in marine sediments

Miriam Sollich1, Marcos Y. Yoshinaga1,2, Stefan Häusler3, Roy E. Price1,4, Kai-Uwe Hinrichs1, Solveig I. Bühring1

1 University of Bremen, MARUM Center for Marine Environmental Sciences, Bremen, Germany 2 Institute of Chemistry, University of São Paulo, São Paulo, Brazil 3 Department of Molecular Ecology, Max Planck Institute for Marine Microbiology Bremen, Germany 4 School of Marine and Atmospheric Sciences, Stony Brook University, Stony Brook, NY, United States

Published in Frontiers in Microbiology, 2017, 8: 1550. doi: 10.3389/fmicb.2017.01550.

MS, K-UH, and SB designed the research; MS, SH, REP and SIB carried out field sampling, MS and SH performed lab work; MS analyzed data with help from MYY; MS wrote the manuscript with help from MYY. All authors contributed to manuscript preparation.

Structure and Objectives

Contributions to anticipated publications

Chapter IV: Shallow-water hydrothermal systems offer ideal conditions to study archaeal lipid membrane adaptations to environmental extremes

Miriam Sollich1; Marcos Y. Yoshinaga1,2; Raymond C. Valentine3; Matthias Y. Kellermann4; Thomas Pichler1; Solveig I. Bühring1

1 University of Bremen, MARUM Center for Marine Environmental Sciences, Bremen, Germany 2 Institute of Chemistry, University of São Paulo, São Paulo, Brazil 3 University of California, Davis, USA 4 Institute for Chemistry and Biology of the Marine Environment, Carl von Ossietzky University, Wilhelmshaven, Germany

In preparation for Organic Geochemistry

MS, MYY and SIB designed the research; MS, TP and SIB carried out field sampling; MS and TP performed lab work; MS analyzed data; MS and MYY interpreted data with help from RCV and MYK; MS wrote the current draft with help from MYY.

Chapter V: Transfer of chemosynthetic fixed carbon and its ecological significance revealed by lipid analysis of fluids at diffuse flow deep-sea vents (East Pacific Rise 9°50’N)

Miriam Sollich1, Marcos Y. Yoshinaga1,2, Solveig I. Bühring1, Stefan M. Sievert3 1 University of Bremen, MARUM Center for Marine Environmental Sciences, Bremen, Germany 2 University of São Paulo, Institute of Chemistry, Brazil 3 Biology Department, Woods Hole Oceanographic Institution, United States

In preparation for Deep-Sea Research I

MS, SMS, SIB designed the research; MS performed lab work; MS analyzed data with contributions of MYY; MS and MYY interpreted data; MS wrote the current draft with help from MYY.

Chapter II

References

Kormas, K.A., Tivey, M.K., Von Damm, K.L., and Teske, A. (2006) Bacterial and archaeal phylotypes associated with distinct mineralogical layers of a white smoker spire from a deep-sea hydrothermal vent site (9°N, East Pacific Rise). Environmental Microbiology 8: 909–920. Kashefi, K., and Lovley, D.R. (2003) Extending the upper temperature limit for life. Science 301: 934–934. McNichol, J., Sylva, S.P., Thomas, F., Taylor, C.D., Sievert, S.M., and Seewald, J.S. (2016) Assessing microbial processes in deep-sea hydrothermal systems by incubation at in situ temperature and pressure. Deep-Sea Research Part I 115: 221–232. Takai, K., Nakamura, K., Toki, T., Tsunogai, U., Miyazaki, M., Miyazaki, J., Hirayama, H., Nakagawa, S., Nunoura, T., and Horikoshi, K. (2008) Cell proliferation at 122 °C and isotopically heavy CH4 production by a hyperthermophilic methanogen under high-pressure cultivation. Proceedings of the National Academy of Sciences of the United States of America 105: 10949–10954.

Structure and Objectives

Chapter III

Chapter III – MICROBIAL LIPID COMPOSITION ALONG A THERMAL GRADIENT

Heat Stress Dictates Microbial Lipid Composition along a Thermal Gradient in Marine Sediments

Miriam Sollich1*, Marcos Y. Yoshinaga1,2, Stefan Häusler3, Roy E. Price1,4, Kai-Uwe Hinrichs1, Solveig I. Bühring1

Published in Frontiers in Microbiology

August 2017 | Volume 8 | Article 1550 doi:10.3389/fmicb.2017.01550

* Corresponding author E-mail address: [email protected]

Microbial lipid composition along a thermal gradient

III.1. Abstract

Temperature exerts a first-order control on microbial populations, which constantly adjust the fluidity and permeability of their cell membrane lipids to minimize loss of energy by ion diffusion across the membrane. Analytical advances in liquid chromatography coupled to mass spectrometry have allowed the detection of a stunning diversity of bacterial and archaeal lipids in extreme environments such as hot springs, hydrothermal vents and deep subsurface marine sediments. Here, we investigated a thermal gradient from 18 to 101 °C across a marine sediment field and tested the hypothesis that cell membrane lipids provide a major biochemical basis for the bioenergetics of archaea and bacteria under heat stress. This paper features a detailed lipidomics approach with the focus on membrane lipid structure-function. Membrane lipids analyzed here include polar lipids of bacteria and polar and core lipids of archaea. Reflecting the low permeability of their ether-linked isoprenoids, we found that archaeal polar lipids generally dominate over bacterial lipids in deep layers of the sediments influenced by hydrothermal fluids. A close examination of archaeal and bacterial lipids revealed a membrane quandary: not only low permeability, but also increased fluidity of membranes are required as a unified property of microbial membranes for energy conservation under heat stress. For instance, bacterial fatty acids were composed of longer chain lengths in concert with higher degree of unsaturation while archaea modified their tetraethers by incorporation of additional methyl groups at elevated sediment temperatures. It is possible that these configurations toward a more fluidized membrane at elevated temperatures are counterbalanced by the high abundance of archaeal glycolipids and bacterial sphingolipids, which could reduce membrane permeability through strong intermolecular hydrogen bonding. Our results provide a new angle for interpreting membrane lipid structure-function enabling archaea and bacteria to survive and grow in hydrothermal systems.

III.2. Introduction

Temperature exerts a first-order control on microbial populations, as cell physiology and biochemistry are adapted to specific temperature ranges (Rothschild and Mancinelli, 2001; Schrenk et al., 2008). For instance, archaea and bacteria that use protons and/or sodium as coupling ions for bioenergetics may be affected under elevated temperatures since the ion permeability of biological membranes increases with temperature (e.g., van de Vossenberg et al., 1995). In this respect, membrane lipid

Chapter III composition plays a major role controlling the ion permeability of cells. At least two mechanisms have been proposed to explain the permeation of ions across lipid bilayers. The first involves a solubility-diffusion model from which permeability coefficients were calculated (Finkelstein, 1987). In many cases, however, discrepancies between predicted and measured permeability have raised questions about the validity of the solubility- diffusion concept (van de Vossenberg et al., 1995; Paula et al., 1996). An alternative mechanism has been proposed and concerns the formation of transient water wires across the membrane. These water wires can be formed spontaneously in membranes creating a pathway for proton transport by a von Grotthuß-type mechanism (Nagle and Morowitz, 1978; Deamer and Nichols, 1989; Paula et al., 1996; Haines, 2001). The extraordinary proton conductance via the von Grotthuß mechanism can result in futile ion cycling, i.e., inadvertent passage of ions across the membrane (Konings et al., 1995; van de Vossenberg et al., 1995, 1999; Valentine, 2007; Valentine and Valentine, 2009; Yoshinaga et al., 2016). Thus environmental conditions, such as temperature and pH, are expected to dictate cell membrane lipid composition (e.g., fatty acid chain length, types of lipid headgroups), which in turn controls the maintenance and dissipation of ion gradients across biological membranes (i.e., cell bioenergetics). Several studies have shown microbial lipid adaptation to heat stress in cultured bacteria (e.g., Ray et al., 1971; Weerkamp and Heinen, 1972; Rilfors et al., 1978; Hazel and Williams, 1990) and archaea (e.g., De Rosa et al., 1980; Sprott et al., 1991; Uda et al., 2001; Matsuno et al., 2009). For instance, the important pathogen Listeria monocytogenes is widely known as a major foodborne disease threat. This bacterium dramatically modifies its membrane fatty acids, including longer chain fatty acids and switches from anteiso- to iso-fatty acids, when transiting from a free-living life style on refrigerated food (2–4 °C) to a human pathogenic state (37 °C) (Annous et al., 1997). By investigating the polar lipids of the cultured archaeon Thermoplasma acidophilum, Shimada et al. (2008) observed an increase in glycolipids content with increasing temperature. This result was attributed to more effective hydrogen bonds between sugar headgroups of glycolipids compared to those between glycophospho- and phospholipids (e.g., Curatolo, 1987; Baba et al., 2001). In addition to studies using cultured microorganisms, a limited number of investigations have attempted to reconcile microbial membrane adaptations and lipid distributions in relation to elevated temperatures in natural settings. For instance, variations in the degree of cyclization of glycerol-based tetraether lipids are captured by

Microbial lipid composition along a thermal gradient the ring-index, which has been applied as a proxy for archaeal membrane lipid adaptation to heat stress in terrestrial hot springs (Pearson et al., 2004, 2008; Schouten et al., 2007; Boyd et al., 2013; Paraiso et al., 2013; Wu et al., 2013; Jia et al., 2014). Supported by studies in pure cultures of thermophiles (De Rosa et al., 1980; Uda et al., 2001; Shimada et al., 2008; Boyd et al., 2011), the rationale is that by increasing the number of rings, tetraethers are packed more tightly, decreasing membrane permeability under heat stress (Gliozzi et al., 1983; Gabriel and Chong, 2000; Gliozzi et al., 2002). However, most of the studies in natural settings have demonstrated that the ring-index may not be applicable as a universal proxy of archaeal membrane adaptation to extreme temperature ranges. Since controversial results in tetraether cyclization relative to pH were obtained in cultured thermoacidophilic archaea (Shimada et al., 2008; Boyd et al., 2011), a possible explanation for the deviation in the ring-index and temperature can be attributed to the considerably large variation in pH and temperature at ecosystems such as hot springs (e.g., Pearson et al., 2004, 2008; Schouten et al., 2007; Jia et al., 2014). In fact, environmental surveys attempting to probe membrane lipid adaptation or lipid/DNA biomarker distribution in relation to temperature have encountered difficulties in sampling across a thermal gradient. This is the case for sulfide deposits or black smoker chimneys in deep-sea hydrothermal vent systems. Results from lipid biomarkers and DNA evidencing a dominance of archaea vs. bacteria in the interior of chimney structures (Hedrick et al., 1992; Kormas et al., 2006) may not exclusively reflect thermal adaptation. The rationale is that the variation in temperature from 2 °C (ambient seawater) to ~350 °C (chimney’s interior) may occur in less than 5 cm toward the inside of these structures (Tivey, 2007), so that interior samples are either located at inhabitable temperature zones or may record the result of seawater entrainment during sampling (Kormas et al., 2006). In this study, we have examined a thermal gradient across a sediment field and conducted a lipidomics approach for the analysis of microbial life. For this purpose, we used a marine sediment field off the coast of Milos (Greece) featuring a point source of extreme heat generating a thermal gradient (ranging from 18 to 101 °C; Figure III.1.). This thermal gradient provides ideal outdoor laboratory conditions to test how temperature drives changes in membrane lipid molecular architecture of archaea and bacteria for bioenergetic gains. That is, although considerable changes in microbial community composition are expected along this thermal gradient, we predict that any given living cell must adjust its cell membranes to in situ temperature conditions. We thus tested the concept that membrane lipids dictate the thermodynamic ecology of bacteria

Chapter III and archaea in stressful conditions (Valentine, 2007; Valentine and Valentine, 2009; Kellermann et al., 2016a). The general strategy of this paper features a detailed lipidomics approach with the focus on membrane lipid structure-function based on experimental data from the literature (e.g., pure culture experiments and/or molecular dynamics simulations).

III.3. Materials and Methods

III.3.1. Study area

As part of the Hellenic Arc, the Milos Island is one of the largest hydrothermal systems in the Mediterranean Sea (Figure III.1A.). Extensive submarine venting is reported to occur through the sandy sediments from the intertidal zone until water depths of more than 100 m, covering an area of more than 35 km2 (Dando et al., 1995a, 2000). Temperatures of these sediments can be as high as 119 °C (Botz et al., 1996) and the pH in pore waters slightly acidic (pH 5, Dando et al., 2000; Wenzhöfer et al., 2000; Price et al., 2013a). The gasses of venting fluids are characterized by high contribution of CO2

(55–92%), with others such as CH4, H2S, and H2 representing usually less than 10% each (Botz et al., 1996; Dando et al., 2000). In addition, venting fluids contain elevated concentrations of ammonium and sulfide (up to 1 mM), manganese (up to 0.4 mM) and arsenic in the micromolar range (Fitzsimons et al., 1997; Dando et al., 2000; Price et al., 2013a). Surface sediments of diffuse hydrothermal venting are covered by yellow to orange patches and white mats, which are derived from mineral deposits such as native sulfur, arsenic sulfide and/or microbial mats (Dando et al., 1998, 2000; Price et al., 2013a). Most studies conducted in the shallow-water hydrothermal sediments off Milos Island were performed at Palaeochori Bay (e.g., Dando et al., 1995a,b, 1998; Brinkhoff et al., 1999; Sievert et al., 1999, 2000b; Giovannelli et al., 2013). Located approximately 500 m east of Palaeochori Bay (Figure III.1A.), Spathi Bay also features hydrothermal activity, including free gas emission into the water column, abundant orange to white patches in surface sediments and similar pore water geochemistry (Price et al., 2013a).

Microbial lipid composition along a thermal gradient

Figure III.1. | The thermal gradient studied in sediments of Spathi Bay, Milos Island, Greece. (A) As part of the Hellenic back-arc and fault (semi-circles and triangles, respectively), Milos Island is one of the largest hydrothermal systems of the Mediterranean Sea (modified after Giovannelli et al. (2013)). The study site Spathi Bay is located 500 m away from Palaeochori Bay, where most geochemical and microbiological investigations from Milos Island have been performed (see Main Text). (B) Modeled temperature profiles in sediments of Spathi Bay calculated for every 100 µm from surface until 20 cm of sediment depth (see Materials and Methods section). Prior to sampling, stations S1 to S5 were selected based on temperatures recorded in situ at 5 cm sediment depth. Station S1 represents the reference sediments with temperatures below 20 °C, while hydrothermally influenced stations S2 to S5 are characterized by a steep increase in temperature with depth.

III.3.2. Sampling

Sediment samples were collected by SCUBA-divers in 10 m water depth at Spathi Bay in May 2012. A permission for sample holding and processing was granted by the General Directorate of Antiquities and Cultural Heritage in Athens (permit number: Φ8/1586). The sampling site consisted of an area covered by several white patches. Before retrieving the individual sediment cores, temperature was measured in situ in 5 cm intervals by a handheld temperature-probe in a custom-built underwater housing (Max- Planck-Institute for Marine Microbiology in Bremen, Germany). In one of these white patches (ca. 7 m × 4 m in size), four sediment cores (up to 20 cm length), representing stations S2, S3, S4, and S5, were retrieved along a transect with increasing temperatures.

Chapter III

An additional core was taken a few meters away from the white patches, in an area presumably free of any major vent influence, here named the reference site (S1). Sediment cores were sliced into 2 cm sections and kept at –20 °C (49 samples in total for lipid analysis). For in situ microsensor measurements Clark-type oxygen (Revsbech and Ward,

1983), H2S (Jeroschewski et al., 1996) and manufactured pH microelectrodes (MI-407; Microelectrodes Inc., Bedford, NH, United States) together with an external reference (MI-401; Microelectrodes Inc., Bedford, NH, United States) were used and measurements were performed in all stations except for S2 due to weather constraints. All sensors were mounted on an autonomous profiling lander (Gundersen and Jørgensen, 1990; Wenzhöfer and Glud, 2002) deployed at the sediment-water interface. Depth profiles were recorded with a spatial resolution of 100 μm, and sensors were allowed to equilibrate at each depth for 5 s before the signal was recorded. Triplicate readings were averaged from each depth. Prior to each in situ measurement all microsensors were calibrated. Oxygen microsensors were calibrated by a two-point calibration, where the signal obtained in aerated seawater represented the concentration of 100% air saturation and the signal obtained in anoxic seawater (bubbled with N2 gas) was taken as zero oxygen. For H2S measurements, a 4–5 point calibration was performed in anoxic seawater of pH lower than 2. Aliquots of Na2S (1 M) were added stepwise into the calibration solution and the sensor signal was recorded. Subsamples, taken after each aliquot’s addition, were fixed in 2% zinc-acetate and stored at 4 °C. H2S concentration of the subsamples was determined according to Cline (1969) by using a spectrophotometer (UV-160A Spectrophotometer, SHIMADZU GmbH, Düsseldorf, Germany). pH sensors were 2-point-calibrated using commercial buffer solutions (Mettler Toledo A.G. Analytical, Schwerzenbach, Switzerland).

III.3.3. Temperature modeling

In situ temperature measurements (every 5 cm) were used as input for a thermal diffusivity model run in R v. 2.9.1 (R Development Core Team, 2009)3 using vegan (Oksanen et al., 2011) and custom R scripts. For this model, we assumed a thermal diffusivity of 3 × 10−7 m2 s−1 (for sandy sediments) and steady flow velocity of 0.021 m h−1. Temperature values for sediment sections of 100 μm were determined. Temperature values for S2 were predicted based on bottom water temperature and a single in situ value ( 40 °C) obtained at 5 cm sediment depth. Fluid flow velocity and

3 http://www.R-project.org 49

Microbial lipid composition along a thermal gradient diffusivity were modeled for S2 based on the values obtained for the hydrothermal influenced sediments at stations S3 to S5 (Figure III.1B.).

III.3.4. Lipid extraction

Lipids were extracted from 30 to 60 g of freeze-dried sediments using a modified Bligh and Dyer method as in Sturt et al. (2004). In brief, an internal standard 1,2- dihenarachidoyl-sn-glycero-3-phosphocholine (C21:0/C21:0-PC, Avanti Lipids) and a mixture of dichloromethane/methanol/buffer (DCM/MeOH/buffer, 1:2:0.8; v/v/v) was added to the sediment and ultrasonicated for 10 min in four steps. For the first two extraction steps a phosphate buffer was used (pH 7.4), and, for the last two steps, a trichloroacetic acid buffer (50 g L−1, pH 2.0). After each ultrasonication, samples were centrifuged and the supernatant collected in a separatory funnel. For phase separation equal amounts of DCM and deionized Milli-Q water were added to a final volume of 1:1:0.8 (MeOH/DCM/buffer, v/v/v). The organic phase was separated and the remaining aqueous phase washed three times with DCM. Subsequently the DCM phase was washed three times with deionized Milli-Q water, evaporated close to dryness under a stream of nitrogen at 37 °C and stored at –20 °C as total lipid extract (TLE) until further analysis.

III.3.5. Analyses and quantification of polar lipids

An aliquot of each TLE was analyzed for polar lipid quantities on a Dionex Ultimate 3000 high performance liquid chromatography (HPLC) system connected to a Bruker maXis Ultra-High Resolution quadrupole time-of-flight tandem mass spectrometer (qToF-MS) equipped with an ESI ion source (Bruker Daltonik, Bremen, Germany). Polar lipid analyses by HPLC-MS were performed using three different methods: normal and reverse phase following the protocol of Wörmer et al. (2013) to quantify non-archaeal and archaeal polar lipids, respectively; and reverse phase according to Zhu et al. (2013) for ring, unsaturation, methylation indices for both core and polar archaeal tetraethers. Detection of lipids was performed in positive and/or negative ionization mode while scanning a mass-to-charge (m/z) range from 150 to 2,000. MS2 scans were obtained in data-dependent mode, for each MS full scan up to three MS2 experiments were performed, targeting the most abundant ions. Active exclusion limits the times a given ion is selected for fragmentation (three times every 0.5 min) and thus allowed to also obtain MS2 data of less abundant ions. Lipid identification was achieved by monitoring exact masses of + + + possible parent ions (present as either H , NH4 , or Na adducts) in combination with characteristic fragmentation patterns as outlined by Sturt et al. (2004) and Yoshinaga et 50

Chapter III al. (2011). Polar lipid quantification was performed by comparison of parent ion responses relative to known amounts of an internal standard (i.e., C21:0/C21:0-PC) and normalized to gram of dry sediment weight. Reported concentrations were corrected for response factors using commercially available or purified standards (see Supporting Information Table III.S1.). Additional analyses of archaeal core tetraethers, which lack a polar headgroup, were performed according to Becker et al. (2013).

III.3.6. Correlation analysis

Given that the data points were not normally distributed, we conducted a two-sided Spearman’s rank correlation test for several archaeal and non-archaeal lipid parameters against temperature. Spearman’s rank correlations were calculated using the software of Wessa (2017). Parameters included the total number of individual archaeal and non- archaeal polar lipids from each sample, which was defined as lipid diversity. For bacterial polar lipids, we calculated the averaged number of unsaturation(s) and chain length of lipids from specific compound classes (e.g., SQ, CL, and PC). That is, the number of double bonds and chain length of each individual lipid were multiplied by the relative abundance within a compound class and summed up. For details in core and polar archaeal tetraether indices calculation (number of cyclopentane rings, degree of unsaturation and additional methyl groups) please see the Supporting Information.

III.4. Results

III.4.1. The thermal gradient in sediments from Spathi Bay

Figure III.1B. displays the modeled temperature profiles from surface sediments until 20 cm sediment depth of stations S1–S5. Temperature profiles from diffuse hydrothermal venting stations showed a uniform trend of continuous and steep increasing values within the first centimeters and a flattened slope at greater depths. For instance, station S5 displayed the strongest temperature gradient (ΔT > 80 °C from top to bottom) and the highest temperature recorded in Spathi sediments (101 °C at 20 cm sediment depth). The temperature of the reference site S1 was relatively constant throughout the core, increasing only slightly from 18 °C at surface to 20 °C at 20 cm sediment depth. This thermal gradient in Spathi sediments is generated by the venting fluids as evidenced in the in situ profiles of pH and H2S recorded by microsensors (Supporting Information Figure III.S1.). As a remarkable characteristic of the influence of venting fluids in sediments with temperatures > 20 °C, we observed a downcore decrease in pH

Microbial lipid composition along a thermal gradient

(from seawater pH of 8.4 to 5.5) and an increase in H2S concentrations (up to ca. 2 mM; Supporting Information Figure III.S1.). The investigated sediments displayed an abrupt decrease in oxygen concentration to anoxic conditions within the first millimeters (Supporting Information Figure III.S1.). Oxygen depletion within the first cm of sediments was observed in all stations, including S1, indicating that sediments analyzed in this study were mostly associated with anoxic conditions.

III.4.2. Polar lipid distribution along a thermal gradient in marine sediments

With some exceptions, total polar lipid content in the sediments decreased downcore (Figure III.2.). Averaged concentrations of polar lipids were highest at S2, S4, and S1 (> 0.31 μg g−1), followed by S5 and S3. The highest total polar lipid concentration of > 1.1 μg g−1 sediment was recorded at the surface layer of S4, whereas the lowest concentration was detected at S3 (12–14 cm) with 0.025 μg g−1. While the relative abundance of non-archaeal lipids was highest at the reference site (S1) and in surface layers from hydrothermally influenced sediments (in general < 12 cm), the contribution of archaeal lipids increased with temperature of the sediments (i.e., downcore and toward S5).

III.4.3. Archaeal polar lipid distribution

Archaeal polar lipids were identified and quantified by high performance liquid chromatography coupled with mass spectrometry (HPLC-MS). Archaeal polar lipids consisted exclusively of glycosidic headgroups and were categorized in six classes according to their isoprenoidal side chains characteristics (see Supporting Information Figure III.S2.). Polar lipids included a monoglycosyl archaeol (G-AR) as the only archaeal diether and a variety of different glycerol-dibiphytanyl-glycerol-tetraethers (GDGT) with monoglycosyl (G) and minor contributions of diglycosyl (2G) headgroups. Notably, archaeal polar lipids were dominated by tetraethers over diethers (Figure III.3A.). Archaeal tetraethers included acyclic to pentacyclic GDGT (GDGT-0 to -Cren, or caldarchaeol to crenarchaeol), unsaturated tetraethers (Uns-GDGT, with up to four double bonds), GDGT with a covalent bond linking the two biphytanyl chains into an H- shaped structure (H-GDGT), and presence of one to four additional methyl groups (nMe) in the isoprenoidal side chains (both nMe-GDGT and H-nMe-GDGT). H-GDGT and H-nMe-GDGT consisted of acyclic to tetracyclic compounds.

Chapter III

Figure III.2. | Sediment profiles of concentration of total microbial polar lipids (dots) and distribution of archaeal vs. non-archaeal lipids (horizontal bars) in Spathi Bay. Samples were analyzed in 2 cm horizons from surface until 20 cm of sediment depth. Adjacent vertical bars indicate the thermal gradient of each station with upper, mid and bottom sediments temperatures in °C. n.d., no data.

Microbial lipid composition along a thermal gradient

Figure III.3. | Sediment profiles displaying the abundance of polar lipid classes relative to total archaeal (A) and total non-archaeal (B) lipids. Note that this figure should be interpreted together with the relative abundance of archaeal and bacterial polar lipids shown in Figure III.2. Samples were analyzed in 2 cm horizons from surface until 20 cm of sediment depth. Adjacent vertical bars indicate the thermal gradient of each station with upper, mid and bottom sediments temperatures in °C. n.d., no data. Abbreviations: monoglycosidic archaeol (AR); monoglycosidic unsaturated (Uns) acyclic glycerol-dibiphytanyl-glycerol-tetraethers (GDGT); mono- and diglycosidic GDGT with 0 to 5 rings (0–Cren); mono- and di-glycosidic GDGT with up to 4 rings divided into mono- and di-methylated (nMe), H-shaped (H) and H-nMe-GDGT with up to 4 methyl groups. Cren, crenarchaeol. For abbreviations of non-archaeal lipid classes please see Main Text. sP-Uk, unknown glycosylated phosphatidyl sphingolipids.

Chapter III

Monoglycosidic AR and GDGT were the most widely distributed archaeal polar lipids with highest contribution between 40 and 90 °C (Figure III.4A.). With highest relative abundance associated with temperatures above 50 °C, the monoglycosidic H- GDGT, H-1Me- and H-2Me-GDGT were also abundant archaeal lipids in the sediments of Spathi Bay. Particularly interesting are the high contributions of monoglycosidic H- 2Me-GDGT in sediments above 80 °C. In sediments with temperatures below 40 °C, other abundant archaeal lipids included monoglycosidic Me-GDGTs and diglycosidic forms of H-GDGT and H-nMe-GDGTs. In contrast with these lipids, the distribution of unsaturated tetraethers was mostly associated with sediments displaying temperatures between 30 and 50 °C.

Figure III.4. | Heat map distribution of archaeal (A) and non-archaeal (B) polar lipid classes in sediments of Spathi Bay. Green to dark purple scale (in %) refers to the contribution of lipid classes relative to the total polar lipid concentration. ND, not detectable. Adjacent vertical bars indicate the thermal gradient in sediments ranging from 18 to 101 °C. List of compounds in A (archaeal lipids): 1 (G-AR), 2 (G-GDGT), 3 (2G-GDGT), 4 (G-Uns-GDGT), 5 (G-Me-GDGT), 6 (G-2Me-GDGT), 7 (2G-Me- GDGT), 8 (2G-2Me-GDGT), 9 (G-H-GDGT), 10 (2G-H-GDGT), 11 (G-H-1Me- GDGT), 12 (G-H-2Me-GDGT), 13 (G-H-3Me-GDGT), 14 (G-H-4Me-GDGT), 15 (2G-H-1Me-GDGT), 16 (2G-H-2Me-GDGT), 17 (2G-H-3Me-GDGT). List of compounds in B (non-archaeal lipids): 1 (G), 2 (SQ), 3 (2G), 4 (BL), 5 (OL), 6 (PDME), 7 (PME), 8 (PE), 9 (PG), 10 (PC), 11 (CL), 12 (sPA), 13 (sP-Uk1), 14 (sP- Uk2), 15 (sP-Uk3), 16 (sP-Uk4), 17 (sPI), 18 (sPE), 19 (sPG). sP-Uk1–4: unknown glycosylated phosphatidyl sphingolipids (see Supporting Information Figure III.S3. for structure identification).

Microbial lipid composition along a thermal gradient

III.4.4. Non-archaeal polar lipid distribution

Non-archaeal polar lipids can be divided into four major groups: glycolipids, phospholipids, aminolipids, and sphingolipids (Supporting Information Figure III.S2.). Glycolipids and phospholipids were exclusively identified as diacylglycerol lipids (DAG), i.e., the side chains were represented by two fatty acids. A variety of phospholipids were detected in the sediments, including phosphatidyl-ethanolamine, -N- methylethanolamine, -N-dimethylethanolamine, -glycerol and -choline (PE, PME, PDME, PG, and PC, respectively) and cardiolipin (CL). The glycolipids were composed of sulfoquinovosyl (SQ), mono and diglycosyl (G and 2G) headgroups, and aminolipids consisted of ornithine and betaine lipids (OL and BL). The sphingolipids were composed exclusively of phosphatidyl-based headgroups such as ethanolamine (sPE), glycerol (sPG), phosphatidic acid (sPA), and inositol (sPI), with the latter two headgroups not observed among the DAG phospholipids (Figures III.3B. and III.4B.). Based on HPLC- MS experiments in comparison with sphingomyelin, we tentatively identified the side chains of these sphingolipids with a terminal methyl group (Supporting Information Figure III.S3.) as reported for Bacteroidetes and Sphingobacterium (Olsen and Jantzen, 2001; Naka et al., 2003; An et al., 2011; Wieland Brown et al., 2013). A general trend of decreasing concentrations with increasing temperature was observed for several DAG lipids including glycolipids, BL, PDME, PME, PE, and PG (Figure III.4B.). In contrast to this distribution, the sphingolipids were mostly associated with sediment temperatures ranging from 30 to 80 °C. Among the aminolipids, the highest contribution of OL occurred between 25 and 50 °C with peaks in concentration associated with sediments as hot as 80 °C. Although displaying highest contribution in sediments below 30 °C, PC together with CL were the major DAG lipids in sediments with temperatures between 60 and 85 °C. In these relatively “hot” sediments, CL and PC displayed contributions with percentages as high as the most abundant archaeal lipids (Figure III.4.).

III.4.5. Molecular species of polar lipids revealed trends in relation to sediment temperature

In order to access the information encoded in the assemblage of polar lipids, we examined a Spearman’s rank correlation between several lipid indices and temperature (Figure III.5., see Supporting Information for detailed calculations of indices). As already evidenced in Figure III.4A., the percentage of H-GDGT (including the H-nMe-GDGT)

Chapter III was positively correlated with temperature (p-value: 2.37e-07). We also observed a positive correlation between temperature and number of additional methyl groups in tetraethers (MIX-index for H-GDGT/GDGT) and number of cyclopentane rings in H- GDGT (ring-index for both polar and core lipids). Other parameters such as the ring- and unsaturation- (Uns) indices for core and polar GDGT as well as MIX for core H-GDGT were negatively correlated with temperature while the weak negative correlation of the ring-index for polar GDGT was not significant (p-value: 8.39e-02). Furthermore, our data revealed that the diversity of archaeal lipids (estimated as the number of individual lipids in a given sample) increased with temperature whereas non-archaeal lipids displayed an

Figure III.5. | Analysis of molecular species of archaeal and non-archaeal lipids as a function of temperature. Spearman’s rank correlation coefficient values corresponding to positive or negative correlation (see Materials and Methods and Supporting Information Table III.S2.) between lipid parameters and temperature are displayed as circles, with colors indicating the level of significance (p < 0.001, p < 0.05, p < 0.01 or not significant, n.s.). Archaeal/Bacterial Lip Div, archaeal/bacterial lipid diversity. Please see Supporting Information for details about lipid parameters used as input to the correlation analysis.

Microbial lipid composition along a thermal gradient opposing trend (Figure III.5.). Interestingly, for most non-archaeal lipids we observed a significant positive correlation between temperatures and degrees of unsaturation and chain length. Only a few DAG lipid classes such as G, PME, and PDME displayed a significant negative correlation between the Uns-index and temperature. The exact calculated two-sided p-values and the individual values of the Spearman’s rank correlation coefficient can be found in Supporting Information Table III.S2.

III.5. Discussion

III.5.1. Environmental setting and potential sources of polar lipids in sediments of Spathi Bay

The studied sediments of Spathi Bay covered a broad temperature range of 18– 101 °C, at the high end approaching the currently determined limit of life ( 120 °C; Kashefi and Lovley, 2003; Takai et al., 2008). Temperature gradients were observed vertically in downcore profiles as well as horizontally on the transect from S1 to S5 (Figure III.1.). These conditions are also observed at other sites of Milos Island, such as at Milos, Boudia, and Palaeochori bays (Dando et al., 1995a; Fitzsimons et al., 1997; Wenzhöfer et al., 2000; Aliani et al., 2004; Price et al., 2013a; Yücel et al., 2013). Hydrothermally affected sediments off Milos Island are characterized by steep geochemical gradients, and might undergo daily fluctuations by waves, tides, and seismic events that influence the intensity of discharged fluids (Aliani et al., 2004; Yücel et al., 2013). Steep geochemical gradients included not only suboxic to anoxic transition within the first millimeters, but also lower pH and higher H2S concentrations toward the source fluid, i.e., downcore (Supporting Information Figure III.S1.). In addition to reporting the influence of source fluids, we suggest that temperature is one of the key factors controlling microbial populations in sediments of Spathi Bay, as it has been reported for other hydrothermal environments (Schrenk et al., 2008; Boyd et al., 2013; Cole et al., 2013; Sharp et al., 2014). Furthermore, the highest concentrations of arsenic in the marine environment have been reported for sediments off Milos (Price et al., 2013a), indicating that source fluids are not only “hot,” but also toxic. In order to cope with frequent changes in temperature, and consequently in toxic conditions, bacteria and archaea inhabiting these sediments must continuously adapt their cell membrane lipid composition. The identified glycosidic diethers and tetraethers are widespread among archaea (e.g., Kates, 1993; Koga et al., 1993). However, several of these lipid features are characteristic of thermo- and/or hyperthermophilic archaea, which are known to inhabit

Chapter III the sediments of Milos (e.g., Thermococcales, Archaeoglobales, and Thermoprotei; Dando et al., 1998; Price et al., 2013b). The “thermophilic” lipid features appeared exclusively in tetraethers (see Supporting Information Figure III.S2.) and included additional methylation(s) in isoprenoidal chains as found in Methanothermobacter thermautotrophicus (Knappy et al., 2009; Yoshinaga et al., 2015a) and Thermococcus kodakarensis (Meador et al., 2014), H-shaped configuration as in Methanothermus fervidus (Morii et al., 1998), Aciduliprofundum boonei (Schouten et al., 2008), and Ignisphaera aggregans (Knappy et al., 2011), or a combination of both as in Methanopyrus kandleri (Liu et al., 2012). Thus far, these modified tetraethers have only been detected as major archaeal lipids at hydrothermally influenced systems such as deep- sea vents and hot springs (e.g., Jaeschke et al., 2012; Lincoln et al., 2013; Gibson et al., 2013; Schubotz et al., 2013; Reeves et al., 2014b; Jia et al., 2014). Archaeal polar lipids possess high preservation potential in marine sediments (Schouten et al., 2010; Logemann et al., 2011; Xie et al., 2013). In fact, several archaeal lipids that we attribute to thermophilic and/or hyperthermophilic archaea (e.g., H-GDGT, H-nMe-GDGT) were also found in the reference core S1 with temperatures below 20 °C (Figures III.3A., and III.4A.). Moreover, regular GDGTs (i.e., 1&2G GDGT 0–Cren) that are dominant archaeal polar lipids in non-hydrothermal marine sediments (Lipp and Hinrichs, 2009; Schouten et al., 2013) were found throughout the transect and at all depths. Although thermophilic Thaumarchaeota may represent potential sources of GDGT-Cren, these archaea have not been detected in 16S rRNA gene libraries from Milos sediments (Dando et al., 1998; Price et al., 2013b) and are not considered major players in marine hydrothermal settings (de la Torre et al., 2008; Pitcher et al., 2010; Sinninghe Damsté et al., 2012). We thus conclude that archaeal polar lipids in the hydrothermally heated sediments of Spathi Bay may partially reflect records of past communities – both from in situ production (in the case of H-GDGT or H-nMe-GDGT) and the water column (particularly the GDGT-Cren). Such a preservation scenario for archaeal lipids has been also reported for an inactive sulfide deposit in Manus Basin deep- sea hydrothermal system (Reeves et al., 2014b). There are at least two fundamental aspects that limit eukaryotic life in the thermophilic sediments of Milos: temperatures higher than 60 °C (e.g., Brock, 1967; Tansey and Brock, 1972; Rothschild and Mancinelli, 2001) and predominance of anoxic conditions after the first few millimeters in the sediments (Supporting Information Figure III.S1.). Although abundantly distributed in surface sediments of Milos, photosynthetic

Microbial lipid composition along a thermal gradient algae and cyanobacteria are only expected at the sediment-water interface and were reported from several studies as brownish or green tinge on top of the sediments or on top of white mats, which cover hydrothermally active sediments (Dando et al., 1995b, 2000; Thiermann et al., 1997; Sartoni and De Biasi, 1999; Sievert et al., 2000a). Photosynthetic membranes of algae and cyanobacteria are enriched in glycolipids such as G-, 2G-, and SQ-DAG (e.g., Guschina and Harwood, 2006; Wada and Murata, 2009). If photosynthetic organisms were major sources of polar lipids in surface sediments one could expect drastic changes in lipid composition from surface sediments to anoxic sediments at the reference site S1. Nevertheless, the polar lipids distribution at S1 was largely uniform throughout the sediment core (Figure III.3B.). Although there exists the possibility that these glycolipids represent fossil material, analysis of acyl chains of BL, another well- known component of photosynthetic membranes (Dembitsky, 1996; Kato et al., 1996), revealed the presence of odd chain fatty acids (e.g., C15:0 and C17:0) typical of bacteria (Schubotz et al., 2009). Among eukaryotes, nematodes and fungi are also potential sources of phospholipids and sphingolipids in the anoxic sediments of Milos, as they have been commonly found associated with hydrothermal ecosystems (e.g., Dando et al., 1995b; Thiermann et al., 1997; Le Calvez et al., 2009; Burgaud et al., 2014; Portnova et al., 2014). Whereas phospholipids such as PC and CL (Figure III.3.) are characteristic of mitochondria (Horvath and Daum, 2013), eukaryotic membranes are enriched in sugar-based ceramides and sphingomyelin (Hannun and Obeid, 2008; Merrill, 2011). The latter lipids were not apparent in our samples, instead sphingolipids in Milos sediments were dominated by uncommon phosphate-based headgroups such as PE, PG, and PI (Figure III.3., and Supporting Information Figure III.S3.). Our results thus suggest that bacteria rather than eukaryotes are likely the major sources of non-archaeal polar lipids throughout the sediments of Spathi Bay. Therefore the non-archaeal lipids are referred as mostly bacterial in origin throughout the following discussion. The bacterial polar lipid distribution according to headgroups in surface sediments at S2 to S5 was generally comparable to those at S1 (Figure III.3B.), indicating mesophilic bacteria as likely sources of lipids. In contrast to S1, the hydrothermally influenced sediments at S2 to S5 revealed significant downcore shifts in polar lipid distribution, which we attribute to the presence of thermophilic bacteria. Data on 16S rRNA gene libraries revealed dominance of mesophilic Epsilonproteobacteria in surface sediments of Palaeochori Bay, with other minor groups composed of Cytophaga-Flavobacteria-

Chapter III

Bacteroidetes, Gammaproteobacteria and Deltaproteobacteria (Sievert et al., 2000a; Giovannelli et al., 2013; Price et al., 2013b). The few 16S rRNA-gene based phylogenetic studies that investigated sediments deeper than 2 cm reported considerable shifts in bacterial community composition relative to the surface sediments, notably the presence of Bacilli, Planctomycetes and thermophilic bacteria such as Thermodesulfobacteria, Thermomicrobia and Thermotogae (Sievert et al., 2000a; Price et al., 2013b). Lipid biomarkers have proven to be important tools for monitoring microbial ecology of marine environments, where most species of archaea and bacteria are uncultured (e.g., Hedrick et al., 1992; Hinrichs et al., 1999; Schubotz et al., 2009; Kellermann et al., 2012; Gibson et al., 2013; Lincoln et al., 2013; Reeves et al., 2014b). In some cases, however, polar lipids lack the taxonomic specificity of DNA-based techniques. For instance, major DAG phospholipid classes identified in this study such as CL, PE, and PG are common among cultured bacteria (e.g., Goldfine, 1984; Dowhan, 1997; Sohlenkamp et al., 2003), including Epsilonproteobacteria that are widespread in surface sediments of Milos. Others, more specific polar lipids such as sphingolipids, may be assigned to Sphingobacteria or Bacteroidetes (e.g., Kato et al., 1995; Olsen and Jantzen, 2001), which are detected by 16S rRNA gene sequencing in Milos sediments (Price et al., 2013b). While abundant in sediments, archaeal H-nMe-GDGT have been exclusively described for the hyperthermophilic M. kandleri (Liu et al., 2012), which is not apparent in archaeal 16S rRNA gene surveys of Milos (Dando et al., 1998; Price et al., 2013b). Rather than applying lipids as chemotaxonomic markers, we attempted to reconcile microbial membrane adaptations based on polar lipid distribution along a thermal gradient in sediments of Spathi Bay.

III.5.2. Archaeal polar lipid quandary: does the dominance of archaeal lipids in sediments with elevated temperatures reflect the extremely low permeability of their membranes?

The thermal gradient sampled in sediments of Spathi Bay (Figure III.1.) allows inferring membrane lipid adaptation to temperature. Our results evidenced a higher contribution of archaeal lipids compared to bacterial lipids in deep layers of the sediments influenced by hydrothermal fluids (Figures III.2., and III.4.). As discussed earlier, a preservation scenario could explain the dominance of archaeal vs. bacterial lipids at elevated temperatures. However, the significant correlation of “thermophilic” H-GDGT and H-nMe-GDGT as polar lipids with increasing temperatures (% H-GDGT in Figure

Microbial lipid composition along a thermal gradient

III.5.) suggests active communities as the most important source of lipids in high temperature sediments. Furthermore, addition of cyclopentane rings and/or methyl groups to these archaeal H-shaped tetraethers, which are proposed to decrease membrane permeability (see Discussion below), appeared to be significantly correlated with temperature. Thus, in agreement with Valentine (2007), we propose that the dominance of archaeal polar lipids in sediments with elevated temperatures reflects the generally lower ion permeability relative to the bacterial analogs. It is important to note that in Milos sediments, hydrothermal fluid advection is associated with high temperatures and accompanying toxic compounds such as arsenic (Price et al., 2013a). Thus membrane lipid adaptations may likely reflect the extent of these conditions. Archaeal polar lipids detected in sediments of Spathi Bay were exclusively glycolipids. Glycolipid-rich membranes are proposed to be more resistant to temperature and low pH than phospholipids given the tight hydrogen bonding between sugar headgroups (Curatolo, 1987; Baba et al., 2001; Shimada et al., 2008; Wang et al., 2012). Another interesting feature of archaeal lipids observed in sediments of Spathi Bay was the dominance of tetraethers over diethers (Figure III.3A.). Controlled experiments using cultured thermophilic archaea such as methanogens (Sprott et al., 1991), Thermococcus (Matsuno et al., 2009) and Archaeoglobus fulgidus (Lai et al., 2008) have shown that the di- to tetraether ratio tends to decrease with increasing temperature. According to these data, a higher proportion of tetraether lipids over diether lipids in archaeal membranes represents a clear lipid adaptation to elevated temperatures. Although a tetraether structure itself confers low ion permeability to the membrane (Koyanagi et al., 2016), the modifications of archaeal tetraether structures revealed by our study (e.g., H-shape, additional methylation or cyclopentane rings) suggest further requirements for avoiding futile ion cycling or ion leakage in high-temperature environments. Increasing sediment temperatures correlated with increased proportions of H- shaped GDGT and tetraethers with additional methyl groups in biphytane chains, i.e., nMe-GDGT and H-nMe-GDGT (Figures III.4., and III.5.). Although these features of archaeal tetraethers have been exclusively found in cultured thermophilic archaea (Morii et al., 1998; Sugai et al., 2004; Schouten et al., 2008; Knappy et al., 2009, 2011; Liu et al., 2012; Yoshinaga et al., 2015a), little is known about their functions in the membranes. For instance, it is generally accepted that H-shaped GDGT represent an archaeal membrane adaptation to heat stress (e.g., Morii et al., 1998). We borrow from Thomas Haines (Haines, 2001) the concept of membrane bulking, which predicts that lipid

Chapter III structures such as fatty acids with additional methyl groups, sterols or squalane can crowd the hydrophobic region of bilayers making them bulkier. The net effect is that these lipid structures may effectively prevent formation of water clusters in the lipid bilayer (see Haines, 2001), thus avoiding ion leakage across the membrane. In this sense, H-shaped GDGT may reduce the fluidity of biphytanyl chains, thereby stabilizing van der Waals forces among isoprenoidal chains from adjacent lipids. In analogy to the functions of methyl-branched fatty acids in bacteria (e.g., Suutari and Laakso, 1992; Haines, 2001), additional methyl groups in biphytane(s) of tetraethers may provide extra bulking between neighbor membrane lipids, thus reducing ion permeability of archaeal membranes under heat stress. In addition to membrane bulking, recent experiments with molecular dynamics simulations revealed that methyl branching enhances membrane fluidity of membranes composed of fatty acids (Poger et al., 2014). This effect may thus also hold true for methylated tetraethers of archaea in Spathi sediments. The presence of cyclic biphytanes is proposed to reduce membrane thickness while leading to stronger interaction between neighbor isoprenoidal chains (Gabriel and Chong, 2000; Gliozzi et al., 2002). The absence of significant correlation between increasing number of rings in regular GDGT (0–Cren) with temperature (Figure III.5.) contrasts with laboratory experiments using cultured thermoacidophilic archaea (Shimada et al., 2008; Boyd et al., 2011; Jensen et al., 2015). Interestingly, the later studies reported diverging responses of tetraether cyclization relative to pH conditions. This observation in combination with our results suggest that cyclization of archaeal tetraether might reflect a net effect of several parameters important for bioenergetics rather than only membrane bulking for thermal adaptation. We consider three possibilities to explain our findings. First, that archaea producing regular GDGT (0–Cren) do not respond to increasing temperature by increasing the number of rings in their biphytanyl chains. Second, that active thermophilic archaea producing H-GDGT and H-nMe-GDGT increase the number of rings with temperature (Figure III.5.), whereas regular GDGT are mainly sourced by less active or dead archaea (see preservation scenario above). Third and the one that we favor: cyclopentane rings may not be an exclusive membrane permeability response to temperature. One fundamental aspect to consider is that the presence of methyl groups protruding from isoprenoidal side chains is the main reason explaining the higher stability of archaeal compared to bacterial lipids (e.g., Degani et al., 1980; Yamauchi and Kinoshita, 1993; Yamauchi et al., 1993; Elferink et al., 1994; Baba et al., 2001; Mathai et al., 2001).

Microbial lipid composition along a thermal gradient

Although the addition of cyclopentane rings has been proposed to decrease permeability in molecular dynamics simulations (MDS; Gabriel and Chong, 2000; Gliozzi et al., 2002), the presence of rings corresponds to a one-to-one loss of methyl groups in isoprenoidal tetraethers. Accordingly, Sinninghe Damsté et al. (2002) comparing GDGT-4 and -Cren in MDS, concluded that the latter provided less dense packing of biphytanyl chains likely resulting in a lower thermal transition point, i.e., increased fluidity. Therefore, we alternatively suggest that the presence of cyclopentane rings in archaeal tetraethers could dramatically increase membrane fluidity/motion while keeping isoprenoidal chains neighbors compacted in the hydrophobic environment. In support of our argumentation, recent MDS experiments have evidenced a dual role of cyclopropane fatty acids in stabilizing membranes and promoting their fluidity, which in general terms is distinct from the analogous unsaturated chains (Poger and Mark, 2015). Note that this “double- function” of cyclopentane rings in tetraethers is slightly different than that of mobile, but less compacted unsaturated biphytanes (Figure III.6.). In summary, the dominance of archaeal over bacterial polar lipids in sediments with high temperatures indicates that membrane lipids may be directly linked to the higher potential for energy conservation of archaea compared to bacteria under stress conditions (Valentine, 2007; Valentine and Valentine, 2009). Alternatively, this finding may reflect the high preservation potential of archaeal lipids in marine sediments (Schouten et al., 2010; Xie et al., 2013), i.e., lower degradation rates of lipids derived from archaea compared to bacteria (Logemann et al., 2011). Our data, however, support the idea that the high abundance of H-GDGT and H-nMe-GDGT in sediments with elevated temperatures is related to the low permeability properties induced by H-shape and additional methyl groups of archaeal tetraethers. Moreover, cyclic tetraethers and/or extra methyl group(s) may provide both increased fluidity/motion and reduced permeability for archaeal membranes. Finally, we cannot exclude that these adaptations at the archaeal cell membrane level may also be used as mechanisms to cope with other stresses, such as low pH, high sulfide and arsenic concentrations that are commonly encountered in the hydrothermally heated sediments of Spathi Bay (Supporting Information Figure III.S1.; Price et al., 2013a; Gomez-Saez et al., 2016).

Chapter III

III.5.3. Bacterial lipids quandary: benefit and risk of membrane fluidity and futile ion cycling

Bacterial polar lipids concentrations decreased abruptly with increasing temperature (Figures III.2., and III.4B.). A dominance of bacterial phospholipids even under the highest measured temperatures introduces a controversial picture to the energy conservation properties of archaeal glycolipids (see above). We suggest two explanations that may account for the dominance of phospholipids among bacteria. The first involves energy invested in the synthesis of complex envelope structures (e.g., outer membrane, peptidoglycan layers) that would provide a robust permeability barrier at high temperatures. The rationale is that H-bonded lipopolysaccharides of Gram-negative bacteria, e.g., lipid A (Nikaido, 2003), or several wraps of peptidoglycan layers and associated structures in cyanobacteria or Gram-positive bacteria (Ward et al., 1998; Bansal-Mutalik and Nikaido, 2014), would provide an effective protection for cells under heat stress. Recall that archaeal cell envelopes are less sophisticated than bacterial cell envelopes (Albers and Meyer, 2011), and likely explain the dominance of sugar headgroups as a hallmark of archaeal lipids in hydrothermally heated sediments of Spathi Bay. The second explanation concerns lipid adaptations at the side chain level and/or by increasing amounts of membrane-stabilizing lipids such as sphingolipids. For most of the polar bacterial DAG lipids, we observed a trend of increased average chain length with increasing temperature (Figure III.5.). Data on lipid vesicles have shown that polar lipids composed of longer fatty acids in their side chains are less permeable to ions, including H+, K+, and Cl−, than the ones linked to shorter fatty acids (Paula et al., 1996). In addition, there is robust experimental evidence suggesting that polar lipid bilayers become thinner with increasing temperature and consequently more prone to futile ion cycling (Pan et al., 2008). Based on these assumptions, we suggest that bacterial cells may purposely increase their bilayer thickness in response to elevated temperatures in sediments of Spathi Bay. The rationale is that the elongation of fatty acid side chains may strengthen the relatively weak van der Waals forces in the hydrophobic region of the bilayer, thereby decreasing membrane permeability. It is well known that microbes decrease the unsaturation levels of fatty acids with increasing temperature (Reizer et al., 1985; Suutari and Laakso, 1992; Hazel, 1995; Beranová et al., 2008). By decreasing the amount of double bonds, lipid membranes are less mobile and less fluidized, thus reducing futile ion cycling (e.g., Valentine and Valentine, 2004). This trend was indeed observed for a few lipids such as G-, PME-, and

Microbial lipid composition along a thermal gradient

PDME-DAG (Figure III.5.). However, a number of bacterial polar lipids were observed to increase their degree of unsaturation concomitantly with an increase in fatty acid chain length. Some abundant polar lipids displaying this trend included OL and CL which represented, respectively, up to 40% and ca. 80% of total bacterial lipids in sediments of temperatures of > 60 °C (Figure III.3B.). This increase in unsaturation of fatty acids with increasing temperatures introduces a dilemma concerning lipid adaptation to temperature in sediments of Spathi Bay. On the one hand, an elongated chain would provide less permeability under elevated temperatures but also low membrane fluidity. On the other hand, more unsaturation implies more fluidity, but increased futile ion cycling (Figure III.6.). Alternatively, increased membrane fluidity might represent a response to increased cell curvature stress, as it is generally accepted that higher ambient temperature results in smaller individuals (e.g., Atkinson, 1994; Morán et al., 2015). Highly unsaturated fatty acids in bacterial membranes are known to localize to the curves and control its curvature stress during cell division (Kawamoto et al., 2009; Sato et al., 2012), similarly to CL (e.g., Kawai et al., 2004). Thus, curvature stress control may also explain the relatively high abundance of CL in hotter portions of the sediments (Figure III.4.), together with a general trend of increased membrane fluidity (Figure III.5.). This membrane quandary resembles the requirement for low permeability and high fluidity of archaeal membranes under heat stress discussed above. We thus suggest that archaea and bacteria may adjust both permeability and fluidity properties of their cell membranes to cope with elevated temperatures in sediments of Spathi Bay. While monoglycosidic ceramides are minor polar lipids in the anoxic water column of the Black Sea (Schubotz et al., 2009) and in hot springs of Yellowstone National Park (Schubotz et al., 2013), phosphatidyl sphingolipids have not been reported thus far in marine sediments. We attribute the relatively high abundance of phosphate-based sphingolipids in sediments of Spathi Bay (up to 40% of total bacterial lipids; Figures III.3B., and III.4B.) to their strong potential for hydrogen bonding. A pioneer study by Pascher (1976) revealed that both the amino and the hydroxyl groups of sphingolipids side chains form intermolecular hydrogen bonding with neighbor lipid molecules. More recently, nuclear magnetic resonance and MDS experiments demonstrated the additional possibility of intramolecular hydrogen bonding of amino and hydroxyl groups with the phosphatidyl headgroup of sphingomyelin (e.g., Talbott et al., 2000; Mombelli et al., 2003; Venable et al., 2014). The net effect is that sphingolipids may lend to both high

Chapter III

Figure III.6. | Lipid chemical structures determining ion permeability (e.g., proton and sodium) and fluidity of archaeal and bacterial membranes in sediments of Milos. The arrows indicate a continuum trend from high (+) to low (–) membrane permeability and fluidity. The essential structural distinction between archaeal and bacterial lipid membranes are the ether-linked isoprenoids in the former and typical ester-linked fatty acids in the latter. The permeability and fluidity properties of bacterial membranes were mainly regulated by the degree of unsaturation (i.e., number of double bonds) and chain length of fatty acids (highlighted in red), as shown in Figure III.5. Apart from the transition diether to tetraether, i.e., bilayer to monolayer membranes, archaea featured several modifications in tetraethers that are suggested to influence the permeability and fluidity properties of their membranes (Figure III.5.). These features (in red) include unsaturation, cyclization and methylation of biphytane(s) and covalent bonds between isoprenoidal chains (H- shaped).

Microbial lipid composition along a thermal gradient internal rigidity and intermolecular order compared to DAG lipids (e.g., Pascher, 1976; Venable et al., 2014), particularly at elevated temperatures. According to our data, the distribution of sphingolipids was markedly associated with relatively “hot” sediments (40 to 80 °C, Figures III.3B., and III.4B.). Indeed, sphingolipids are characteristic lipids of some thermophilic bacteria (e.g., Tenreiro et al., 1997; Yabe et al., 2013; Anders et al., 2014). Moreover, the role of sphingolipids in thermal adaptation of yeast has been demonstrated by both suppressor mutations (Wells and Lester, 1983) and their up-regulation in response to increased temperatures (Jenkins et al., 1997). Under elevated temperatures in sediments of Spathi Bay, the high melting point and hydrogen bonding potential of sphingolipids may provide a more rigid lateral organization of biological membranes when mixed with phospholipids via formation of lipid microdomains (Goñi and Alonso, 2009). Conversely, below and above the temperature threshold of 40–80 °C, sphingolipids may not behave as bilayer structures when mixed with other bilayer-forming lipids (reviewed in Goñi and Alonso, 2006, 2009). Sphingolipid-mediated microdomains in bacteria (e.g., An et al., 2011; Wieland Brown et al., 2013) may thus stabilize DAG phospholipids such as CL and PC that displayed relatively high abundances at elevated temperatures (Figures III.3B., and III.4B.).

III.5.4. Environmental conditions dictate microbial membrane lipid composition: a working hypothesis toward a unified concept

Our study provides evidence for a diverse array of molecular architecture of archaeal lipids to cope with heat stress (see Polar Lipid Distribution along a Thermal Gradient in Marine Sediments). It is interesting to notice that archaea inhabiting cold to moderate temperature environments generally lack the membrane bulking features identified by our study (e.g., H-GDGT, H-nMe-GDGT; Figure III.6.). Recent advances in polar lipid analysis by HPLC-MS have allowed the description of several novel archaeal lipids (e.g., Knappy et al., 2009; Yoshinaga et al., 2011; Liu et al., 2012; Zhu et al., 2014). Those include unsaturated tetraethers, which thus far were only reported to occur in marine sediments (Zhu et al., 2014) and in a few thermophilic archaea such as Thermoplasmatales (Bauersachs et al., 2015; Yoshinaga et al., 2015b). Although the distribution of unsaturated tetraethers in the marine environment is not yet fully understood, in the studied sediments they were found more abundantly in temperatures between 30 and 55 °C (Figures III.3A., and III.4A.). Thus, we propose that unsaturated

Chapter III tetraethers do not likely reflect an archaeal membrane adaptation to extreme high temperatures, as suggested recently (Bauersachs et al., 2015). Conversely, unsaturation might be critical for motion of the rigid isoprenoidal membranes (Kellermann et al., 2016a). Supporting the idea that unsaturation of isoprenoids may not directly represent a thermal adaptation, unsaturated diethers are observed in several archaea ranging from psychrophilic to hyperthermophilic (Gonthier et al., 2001; Nishihara et al., 2002; Nichols et al., 2004; Gibson et al., 2005; Kellermann et al., 2016a). The low permeability of archaeal membranes may not only explain the predominance of archaea in hotter sediments of Spathi Bay, but also in hotter portions of chimneys from deep-sea hydrothermal vents (Gibson et al., 2013; Reeves et al., 2014b). In those environments, a higher proportion of archaeal over bacterial lipids could be used to predict the hotter portions of chimney structures, represented in Gibson et al. (2013) by the Rainbow hydrothermal field and in Reeves et al. (2014b) by the RMR5-dark. Coincidently, both of these structures (characterized by the highest contribution of archaeal polar lipids among all samples) displayed PC as the major bacterial polar lipids, which otherwise were only found as minor components. This trend is observed in our study (Figure III.4B.), suggesting that PC may represent a common membrane lipid adaptation of bacteria to heat stress. The rationale is that temperature influences the phase behaviors of polar lipids (Quinn, 1985; Hazel and Williams, 1990): bilayer-stabilizing lamellar phase (e.g., PC, sphingolipids and 2G) or bilayer-destabilizing non-lamellar phase (e.g., PE and G). Although the acyl chain composition may also influence lipid phase behavior, increased temperatures generally induce the formation of non-lamellar phase membranes (Quinn, 1985; Hazel and Williams, 1990). Thus the ratio of membrane- stabilizing PC is predicted to increase relative to PE, and perhaps PME and PDME, with increasing growth temperatures (Hazel, 1995), as suggested by our data (Figure III.4B.). Polar lipids of archaea in sediments of Spathi Bay were largely composed of glycolipids, likely as a thermal adaptation strategy. This glycolipid strategy for heat stress is not only consistent with cultured thermophilic archaea (Shimada et al., 2008), but also with the high abundance of archaeal glycolipids in hydrothermal systems (Gibson et al., 2013; Schubotz et al., 2013; Reeves et al., 2014b; Kellermann et al., 2016b). While the glycolipid strategy was not evidenced for bacteria in sediments of Spathi Bay, a study in Lost City Hydrothermal Field attributed the dominance of bacterial glyco- over phospholipids to a possible phosphate limitation (Bradley et al., 2009). Given that these bacteria might inhabit the mixing zone between the source fluid (pH 10, low phosphate

Microbial lipid composition along a thermal gradient and 90–100 °C; Reeves et al., 2014a) and seawater replete with phosphate, we suggest the glycolipid strategy for thermal adaptation as an additional explanation for the dominance of bacterial glycolipids at Lost City. Given the strong hydrogen bonding network between one another, glycolipids appear as a signature lipid of many thermophilic and thermoacidophilic archaea and bacteria (e.g., Sprott et al., 1991; Yang et al., 2006; Shimada et al., 2008; Kellermann et al., 2016b). Under phosphate limitation, however, glycolipids can indeed substitute for phospholipids in photosynthetic, bacterial, and archaeal membranes (Van Mooy et al., 2006; Carini et al., 2015; Yoshinaga et al., 2015b). In addition to phosphate limitation, this glycolipid substitution has been also observed in cultured methanogens grown under hydrogen limitation (Yoshinaga et al., 2015b). We suggest that sugar headgroups may represent a common microbial strategy for energy conservation at the cell membrane level, including high temperature, low pH, phosphate limitation and perhaps substrate limitation. Our study revealed that non-DAG lipids such as sphingolipids and OL are relatively enriched in sediments above 60 °C (Figures III.3B., and III.4B.). A distinctive characteristic of sphingolipids and OL compared to DAG lipids is the availability of an amino group close to the lipid headgroup in position to hydrogen bonding, which may confer an effective protection for cells under elevated temperatures (see above Discussion). Indeed, an upregulation of OL has been reported as the main membrane adaptation of cultured sulfate-reducing bacteria to increased temperatures (Seidel et al., 2013). In analogy to sphingolipids and OL, we suggest that lipid headgroups such as N- acetylated hexosamines and aminopentanetetrol characteristic of several deep-branching thermophilic bacteria such as Thermus, Thermodesulfobacterium, and Aquificales (e.g., Ferreira et al., 1999; Sturt et al., 2004; Yang et al., 2006) may also feature potential for strong intermolecular hydrogen bonding. This aspect could explain the widespread distribution of OL, N-acetylated hexosamines and aminopentanetetrol in hydrothermal settings (Gibson et al., 2013; Schubotz et al., 2013; Reeves et al., 2014b). While ubiquitous in eukaryotic cells, sphingolipids are generally absent in most bacteria. Sphingolipid-containing bacteria, however, are highly represented in Bacteroidetes (Kato et al., 1995; Olsen and Jantzen, 2001), and sphingolipid-mediated microdomains have been linked to the capacity of Bacteroides fragilis to overcome stressful conditions in mammalian intestine (An et al., 2011). It is fascinating that similar properties of sphingolipids as permeability barrier in bacterial outer membranes (e.g., against antibiotics and salt; Nikaido, 2003) or as key components of “lipid rafts” in

Chapter III mammalian cells (e.g., Simons and Ikonen, 1997; Hannun and Obeid, 2008; Lingwood and Simons, 2010) may apply to lipid microdomains in bacterial membranes under heat stress. We suggest that sphingolipid-mediated microdomains may stabilize DAG phospholipids (e.g., CL and PC), enabling bacterial membranes to achieve both low permeability and a more fluidized configuration that are apparently required under elevated temperatures in sediments of Spathi Bay (Figure III.5.). As described in our study, archaea and bacteria modulate their membrane architecture in response to temperature. Surprisingly, a balance between low permeability and increased fluidity (i.e., motion) appears as a unified property of microbial membranes to cope with heat stress. For instance, a higher degree of bulking (e.g., H-shaped) and fluidity (i.e., cyclization) of archaeal tetraethers were observed in concert with elevated temperatures. We propose that a more fluidized configuration of cell membranes may be beneficial for both cell bioenergetics (e.g., Valentine and Valentine, 2004, 2009; Kellermann et al., 2016a) and perhaps membrane curvature stress control (Kawamoto et al., 2009; Mouritsen, 2011; Sato et al., 2012). At the headgroup level, heat stress adaptations included membrane-stabilizing glycolipids in archaea. In bacteria, abundant CL in high temperature sediments may reflect curvature stress control and PC, OL and sphingolipids may potentially form rigid microdomains. The possibility for membrane domain formation under energy stress may provide a new dimension for interpreting lipid distribution in response to environmental conditions. That is, we are considering the possibility that lipid responses may in fact reflect a general energy conservation strategy rather than a limited effect of single stressors (e.g., temperature, pH or nutrients; see Valentine, 2007 and Kellermann et al., 2016a). It will be very exciting in future studies to interpret lipids with the idea that they are crucial for the bioenergetics of bacteria and archaea in natural systems.

III.6. Funding

This study was supported by the Deutsche Forschungsgemeinschaft (DFG, Germany) through the Emmy Noether-Program (grant BU 2606/1-1 to SB) and the Gottfried Wilhelm Leibniz Prize (grant HI 616/16 to K-UH).

Microbial lipid composition along a thermal gradient

III.7. Acknowledgments

The authors would like to thank all members of the scientific expedition to Milos. Thomas Pichler is acknowledged for assistance during fieldwork. Tomas Feseker, Ioulia Santi, Kevin W. Becker, Felix J. Elling, and Julius S. Lipp are thanked for their help during data analysis. We are in debt to Lars P. Wörmer, Florence Schubotz, and Dirk de Beer for support and discussions. Special thanks go to Raymond C. Valentine and Matthias Y. Kellermann from the Membrane Lipid Code Crackers Society (http://mlccs.jimdo.com/) who provided ideas about lipid structure-function throughout the preparation of the manuscript.

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III.10. Supporting Information

III.10.1. Supporting Methods

III.10.1.1. Quantification of polar lipids based on response factors of standards

Microbial polar lipids were quantified by HPLC-MS and the reported concentrations corrected for response factor of commercially available or purified standards as described in Table III.S1. Concentrations of mono- and diglycosidic archaeal tetraethers (including all saturated and unsaturated GDGT as well as the nMe- and/or H- (nMe)-GDGT) were corrected, respectively, for the response factor of G-GDGT 0 and 2G-GDGT 0 purified from Archaeoglobus fulgidus as described in Zhu et al. (2013). The monoglycosidic archaeal diether G-AR was corrected for PC-AR. Most of the bacterial polar lipids were corrected for response factors of commercial standards according to headgroup. Due to the lack of authentic standards phosphatidyl sphingolipids and ornithine lipids were corrected for the response factors of glucosylceramide and

C21:0/C21:0-PC, respectively.

Table III.S1. | Polar lipids were quantified by HPLC-MS and concentrations were corrected for response factors based on commercial or purified standards as listed below.

# Standard Source Polar Lipids* 1 G-GDGT 0 Zhu et al. (2013) G-(H)-n(Me)-GDGT(0-5) 2 2G-GDGT 0 Zhu et al. (2013) 2G-(H)-n(Me)-GDGT(0-5) 3 PC-AR Avanti Polar Lipids Inc., USA G-AR 4 Glucosylceramide Avanti Polar Lipids Inc., USA Sphingolipids 5 DGTS Avanti Polar Lipids Inc., USA BL

6 C16:0/C16:0-PE Avanti Polar Lipids Inc., USA PE

7 C16:0/C16:0 dimethyl-PE Avanti Polar Lipids Inc., USA PME, PDME 8 MGlc-DAG Avanti Polar Lipids Inc., USA G-DAG

9 C18:0/C18:0-DGal Biotrend Chemikalien GmbH DGDG, SQDG

10 C18:1/C18:1-CL Avanti Polar Lipids Inc., USA CL

11 C21:0/C21:0-PC Avanti Polar Lipids Inc., USA PC, OL

12 C16:0/C16:0-PG Avanti Polar Lipids Inc., USA PG #1. monoglycosyl glycerol dibiphytanyl glycerol tetraether; 2. diglycosyl glycerol dibiphytanyl glycerol tetraether; 3. 1,2-di-O-phytanyl-sn-glycero-3-phosphocholine; 4. D-glucosyl-1,1'-N- stearoyl-D-erythro-sphingosine; 5. 1,2-dipalmitoyl-sn-glycero-3-O-4’-(N,N,N-trimethyl)- homoserine; 6. 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine; 7. 1,2-dipalmitoyl-sn- glycero-3-phosphoethanolamine-N,N-dimethyl; 8. 1,2-diacyl-3-O-(α-D-glucopyranosyl)-sn- glycerol; 9. digalactosyldiacylglycerol; 10. 1’,3’-bis[1,2-dioleoyl-sn-glycero-3-phospho]-sn- glycerol; 11. 1,2-dihenarachidoyl-sn-glycero-3-phosphocholine; 12. 1,2-dipalmitoyl-sn-glycero- 3-phospho-(1’-rac-glycerol). *please see Main Text for abbreviations.

Chapter III

III.10.1.2. Calculation of lipid indices used as input for Figure III.5. (Main Text)

The percent of polar H-GDGT displayed in Figure III.5. was calculated as the sum of all H-containing tetraethers (i.e. H-GDGT and H-nMe-GDGT) divided by the sum of all archaeal polar lipids. The ring-index for monoglycosidic archaeal tetraethers (G- GDGT and G-H-GDGT), and respective core lipids ((H)-cGDGT) was calculated using a modified equation after Pearson et al. (2004), as follows:

It is worth noting that crenarchaeol (Cren) was not detected among H-GDGT and thus were not used in ring-index calculation for H-shaped tetraethers. A similar rationale was used to calculate the unsaturation index of monoglycosidic unsaturated GDGT:

The methylation index (MIX) was determined for G-nMe-GDGT, G-H-nMe- GDGT and H-nMe-cGDGT as follows:

For the MIX, only the number of methylation(s) was taken into account, so that methylated tetraethers containing different number of rings were summed up. For bacteria polar lipids, we calculated the averaged number of unsaturation(s) and chain length of lipids from specific compound classes (e.g. SQ, CL, PC). That is, the number of double bonds and chain length of each individual lipid were multiplied by the relative abundance within a compound class and summed up. Finally the diversity of polar lipids was simply estimated by the number of individual archaeal and bacterial lipids in a sample.

Microbial lipid composition along a thermal gradient

III.10.2. Supporting Figures

Figure III.S1. | In situ microsensor profiles of pH, oxygen and hydrogen sulfide concentrations in the upper 3 to 4 cm in sediments of Spathi Bay. For detailed information about microsensor calibration and data analysis please see Methods section. Measurements were not performed at station S2.

Chapter III

Figure III.S2. | Non-archaeal polar lipid classes and core lipid composition of archaeal glycolipids detected in sediments of Spathi Bay. Highlighted in red are chemical structures including variable numbers of glycosidic headgroups (n), presence of unsaturation and terminal methyl branched groups in phosphatidyl sphingolipids, and tetraether modifications (unsaturation, cyclization and methylation of biphytane(s) and covalent bonds between isoprenoidal chains). List of diacylglycerolipids (DAG): (a) mono- (G) and diglycosyl (2G), n = 1 and 2, respectively; (b) sulfonoquinovosyl (SQ); (c) phosphatidylcholine (PC); (e) betaine lipids (BL); (f) phosphatidyldimethylethanolamine (PDME); (g) phosphatidylethanolamine (PE); (h) phosphatidylmethylethanolamine (PME); (i) phosphatidylglycerol (PG); (j) cardiolipin (CL). List of non-DAG lipids: (d) ornithine lipids (OL); (k) exemplary phosphatidylinositol sphingolipid (sPI). Phosphatidyl sphingolipids included PE, PG, phosphatidic acid (PA) and unknown glycosylated phosphatidyl sphingolipids (sP-Uk) as polar headgroups. List of archaeal core lipids: (l) diether or archaeaol (AR); (m) glycerol-dibiphytanyl- glycerol-tetraethers (GDGT).

Microbial lipid composition along a thermal gradient

Figure III.S3. | Fragmentation pattern of sphingomyelin from mammalian cell and phosphatidylinositol sphingolipid during MS/MS experiments in HPLC-ESI-MS. (A) MS/MS experiments of sphingomyelin typically yield fragments such as m/z 586 and 264 related respectively to ceramide (–H2O) and sphingosine (–H2O). (B) Tentatively identified phosphatidyl sphingolipid (sPI) structure, which was drawn according to sphingolipids of bacteria (e.g. Olsen and Jantzen, 2001; Naka et al., 2003). Note that in contrast to sphingomyelin, the MS/MS experiment of sPI yields a major ceramide fragment (m/z 536) without a water loss and no indication for a fragment related to the sphingosine as in (A) is observed. Unknown phosphatidyl sphingolipids (sP-Uk) reported in Figures III.3. and III.4. (Main Text) yielded MS/MS spectra with a major hexose loss from MS1, similar to those of sPI. We suggest that sP-Uk are likely similar to glycosylated phosphatidyl sphingolipids detected in Sphingobacterium spiritivorum (cf. Naka et al., 2003).

Table III.S2. | Spearman’s rank correlation coefficient (ρ), n = number of pairs (samples) and corresponding p-value of the two-tailed tests represented in Figure III.5.

n parameter ρ two-sided p-value 49 % H-GDGT 0.68 2.37e-07 49 RI H-cGDGT 0.52 1.77e-04 49 MIX H-GDGT 0.40 4.73e-03 35 RI H-GDGT 0.35 4.27e-02 45 MIX GDGT 0.30 4.76e-02 49 RI cGDGT -0.68 1.96e-07 48 Uns GDGT -0.60 6.35e-06 49 MIX H-cGDGT -0.53 1.06e-04 48 RI GDGT -0.25 8.39e-02

Chapter III

49 Bacterial Lip Div* -0.77 7.91e-11 49 Archaeal Lip Div* 0.60 5.86e-06

Unsaturation: 42 1G -0.45 2.99e-03 36 SQ 0.34 4.32e-02 35 2G 0.76 1.17e-07 46 BL 0.25 8.93e-02 41 OL 0.70 1.15e-06 44 PE 0.66 8.92e-07 33 PME -0.52 2.27e-03 26 PDME -0.63 5.22e-04 47 PC -0.03 8.24e-01 38 PG 0.33 4.28e-02 37 CL 0.49 2.03e-03 30 sPG 0.26 1.71e-01 30 sPE 0.76 2.68e-06 12 sPA 0.50 9.88e-02 24 sPI 0.42 4.23e-02

Chain length: 42 1G 0.03 8.30e-01 36 SQ 0.54 5.91e-04 35 2G 0.74 4.32e-07 46 BL 0.11 4.61e-01 41 OL 0.72 4.95e-07 44 PE 0.53 2.16e-04 33 PME 0.05 8.03e-01 26 PDME 0.66 3.32e-04 47 PC 0.43 2.51e-03 38 PG 0.42 9.42e-03 37 CL 0.56 3.08e-04 30 sPG 0.42 1.98e-02 30 sPE 0.96 2.20e-16 12 sPA 0.49 1.10e-01 24 sPI 0.91 2.61e-06 * Lip Div = Lipid Diversity

Microbial lipid composition along a thermal gradient

III.10.3. References

Naka, T., Fujiwara, N., Yano, I., Maeda, S., Doe, M., Minamino, M., Ikeda, N., Kato, Y., Watabe, K., Kumazawa, Y., Tomiyasu, I., and Kobayashi, K. (2003) Structural analysis of sphingophospholipids derived from Sphingobacterium spiritivorum, the type species of genus Sphingobacterium. Biochimica et Biophysica Acta 1635: 83–92. Olsen, I., and Jantzen, E. (2001) Sphingolipids in bacteria and fungi. Anaerobe 7, 103– 112. Pearson, A., Huang, Z., Ingalls, A.E., Romanek, C.S., Wiegel, J., Freeman, K.H., Smittenberg, R.H., and Zhang, C.L. (2004) Non-marine crenarchaeol in Nevada hot springs. Applied Environmental Microbiology 70: 5229–5237. Zhu, C., Lipp, J.S., Wörmer, L., Becker, K.W., Schröder, J., and Hinrichs, K.-U. (2013) Comprehensive glycerol ether lipid fingerprints through a novel reversed phase liquid chromatography-mass spectrometry protocol. Organic Geochemistry 65: 53–62.

Chapter IV

CHAPTER IV – GEOCHEMICAL INFLUENCE ON THE ARCHAEAL MEMBRANE LIPID DIVERSITY

Shallow-water hydrothermal systems offer ideal conditions to study archaeal lipid membrane adaptations to environmental extremes

IV.1. Abstract

Hydrothermal systems are considered as one of the most extreme environments on this planet, however they harbor rich microbial communities. A previous study explored hydrothermally-influenced sediments off the shallow coast of Milos Island and showed that archaea adapted their lipid membrane inventory along the investigated sediment temperature gradient from 18 to 101 °C. Archaea tended to produce higher amounts of specialized glycosidic tetaether lipids like H-GDGT and H-(n)Me-GDGT with increasing sediment temperature. Moreover, archaea modified their tetraethers by incorporation of additional methyl groups and extra cyclopentane rings along the investigated gradient, which was hypothesized to balance the relatively low permeability but increased fluidity of the archaeal cell membrane to sustain its functionality at higher temperatures. In this study, we tested if these previous findings are also applicable to other shallow-water hydrothermal sediments. Therefore we analyzed the archaeal lipid diversity of two, geochemically heterogeneous, sediment cores off the hydrothermally-active coast of Dominica and compared lipid findings with previously obtained results from Milos. In fact, the archaeal lipid diversity in sediments from Dominica was comparable with the lipid diversity found at Milos. Specialized lipids e.g. monoglycosidic H-GDGT and H-(n)Me-GDGT dominated in higher-temperature sediments, showing a positive correlation with increasing sediment temperature. Due to a smaller sample size taken at Dominica, ring- and methylation-indices were not completely consistent with previous findings. Nevertheless, this study supports the former results, identifiying temperature as an important parameter influencing the lipid distribution, especially of H-GDGT in hydrothermally-influenced sediments. Furthermore, this study revealed that besides temperature, salinity and hydrogen sulfide concentrations also have a significant influence on the polar lipid distribution, especially on the relative abundance of H-(n)Me-GDGT in hydrothermally-active sediments.

Geochemical influence on the archaeal membrane lipid diversity

IV.2. Introduction

Areas of submarine hydrothermal activity are characterized by extreme and ever- changing physico-chemical conditions (Hessler and Kaharl, 1995). Although hydrothermal systems are defined as one of the most extreme environments on Earth they usually support dense microbial communities and often crowded megafaunal populations (Hessler and Kaharl, 1995). Since their famous discovery in 1977, great scientific attention has been drawn to deep-sea hydrothermal vent systems (Ballard, 1977; Lonsdale, 1977; Corliss et al., 1979), although there are relatively few scientific investigations of their shallow-water counterparts (Price and Giovannelli, 2017). Thus far, fine-scaled sampling of deep-sea hydrothermal chimneys or black smokers to investigate microbial adaptations along a thermal and/or geochemical gradient remains difficult (Kormas et al., 2006). The rationale is that the temperature gradient from 2 °C (ambient seawater) to ~350 °C (chimney’s interior) may occur within less than 5 cm, so that the suitable habitat for living microorganisms is confined to only a few mm or maybe even less (Tivey, 2007). Shallow-water and deep-sey hydrothermal systems are both extreme environments with great geochemical similarities. However, due to their proximity to the coastline shallow-water hydrothermal systems offer the great advantage of a relative easy accessibility with the possibility to study microbial cell membrane adaptations in detail along physico-chemical gradients in the sediments. In a recent publication, we have performed a structure-function analysis of bacterial lipids and archaeal glycosidic tetraethers along a thermal gradient in shallow-water hydrothermal sediments off Milos Island, covering a temperature gradient from 18 to 101 °C (Sollich et al., 2017). One major finding was that the structural diversity of archaeal tetraethers (e.g. increased cyclization and methylation; Figure IV.1.) is influenced by temperature profiles in these heated sediments. We thus concluded that archaeal glycosidic tetraethers were largely sourced from living/active biomass, despite the fact that glycosidic tetraether lipids have also been shown to exhibit an extraordinary preservation potential (Schouten et al., 2010; Xie et al., 2013). The goal here is to test the range of applicability of our previous obtained lipid findings of archaeal polar and core lipid patterns from hydrothermally-influenced sediments of Milos Island (Sollich et al., 2017). Therefore, two geochemically distinct sediment cores were sampled offshore the coast of Dominica (Lesser Antilles), covering an overall temperature gradient from 28 °C to approx. 104 °C. Archaeal polar lipid

Chapter IV diversity and relative abundance were analyzed in conjunction with the ring- and methylation-indices of tetraether lipids and tested for temperature dependencies. Additionally, archaeal lipid abundance data from Milos and Dominica were combined with physico-chemical data and further investigated by multivariate analyses. The latter approach was to test whether geochemical parameters, besides temperature, may drive modifications in archaeal glycosidic tetraethers in hydrothermally-influenced sediments.

IV.3. Material and Methods

IV.3.1. Site characteristics and description

Dominica is located at the center of the approx. 850 km long active volcanic arc of the Lesser Antilles which is an expression of the westward subduction of the Atlantic plate beneath that of the Caribbean. It is recorded as the most volcanically productive island of the Lesser Antilles (Wadge, 1984) with a convergence rate of 2 cm yr-1 for the last 30 Ma (Rosencrantz and Sclater, 1986). Several hydrothermal active regions along the coastline off Dominica and on land have been documented (Johnson and Cronan, 2001; McCarthy et al., 2005; Joseph et al., 2011; Gomez-Saez et al., 2015). Especially the site “Champagne Springs” located at the south-west off Dominica has been intensively investigated for its geochemical characterization of the hydrothermally-active sediments and fluids. Typically for most shallow-water marine hydrothermal areas, hydrothermal fluids off Dominica consist of a mixture of entrained meteoric water with seawater (McCarthy et al., 2005). Fluids were reported as slightly acidic with elevated temperatures up to 74 °C, enriched in ions like - 2- - + + B, Fe, As, Sb, Mn, Si and Li and depleted in others like Br , SO4 , Cl , Na , K , Mg, and Sr2+ relative to ambient seawater (McCarthy et al., 2005). Because we detected only very low hydrothermal activity with a maximum temperature of 48 °C in 20 cm sediment depth at Champagne Springs during our sampling expedition it seems that the intensity of hydrothermal activity might vary strongly over time. This finding is already reported for other marine hydrothermal systems and was attributed to smaller seismic events and periodicity of tidal and wave action (Aliani et al., 1998, 2004; Wenzhöfer et al., 2000; Chen et al., 2005; Italiano et al., 2011; Yücel et al., 2013). Gas samples from Champagne Springs showed similarities to other typical arc-type gases and have both meteoric and magmatic signatures (CO2 >> N2 > H2S > CH4) (McCarthy et al., 2005). While McCarthy and coworkers reported hydrous ferric oxides precipitation when iron-enriched hydrothermal fluids mix with the oxygenated seawater, a recent publication documented

Geochemical influence on the archaeal membrane lipid diversity a strong complexation of Fe(II) preventing the rapid oxygenation of this molecule which might ascertain bioavailability and high productivity in the photic zone (Kleint et al., 2017).

IV.3.2. Dominica sampling

All samples were retrieved by SCUBA-divers during the rainy season in April 2013. The sampling site Soufrière, located at the south west of the island (15°13'57.1"N 61°21'43.1"W) showed reddish iron precipitates on top layers of the sandy sediment and anthropogenic input flushed in by periodic heavy rain falls. Hydrothermal activity was relatively low at this site recognized either visually by bubbles of released gas emissions or detected by elevated temperatures which were taken randomly in the bay with an handheld temperature-probe in a custom-built underwater housing (Max-Planck-Institute for Marine Microbiology in Bremen, Germany). A spot of the size of several square meters with the highest recorded temperature (55 °C in 20 cm sediment depth) in the bay was chosen for sampling. Before retrieving a sediment core until a length of 20 cm for lipid analysis, temperature was measured in situ in 5 cm intervals. On land, the sediment core was subsampled in 2 cm sections and subsequently kept at –20 °C until lipid extraction. Pore fluids at Soufrière were collected with a funnel placed on the vent orifice which opened into food-grade large volume nylon bags as described in Gomez-Saez et al. (2015). pH and salinity of hydrothermal fluids were analyzed directly in the field using a WTW pH meter 3210 with Mic-D electrode. Fluid samples were immediately separated in aliquots for total sulfide, anion and cation concentrations (for the procedure please see below). In contrast the second sampling site called “Portsmouth” is located at the north west of the island ~700 m offshore (15°33'18.4"N 61°28'09.3”W) in 27 m water depth. Hydrothermal activity was much greater and visible by vigorous gas emissions. Due to a very short bottom time for the divers, temperature was only measured randomly between 10 and 20 cm sediment depth and temperatures were modeled and interpolated afterwards (see section IV.3.5.). Pore fluids were collected in situ into BDTM (Becton, Dickinson and Company) 60 mL syringes from a tube placed at 10 cm sediment depth in 4 replicates. Fluids were filtered through 0.2 µm and three 2 mL aliquots were stored in 2 mL glass vials without head space. Total sulfide was determined spectrophotometrically (Pharo 100 Spectroquant, Merck KGaA, Darmstadt, Germany) on subsamples that were fixed with 5% zinc acetate (Cline, 1969). Subsamples for the cations of iron (Fe) and arsenic

Chapter IV

(As) were acidified to 1% with ultrapure HNO3 in the field, and stored at 4 °C until measured by inductively coupled-optical emission spectrometry (Optima 7300 ICP-OES; Perkin-Elmer, Rodgau, Germany) following established procedures (McCarthy et al., 2‒ 2005; Reeves et al., 2011). Measurements of sulfate (SO4 ) was performed by ion chromatography (Metrohm 883 IC Basic Plus; Metrohm AG, Herisau, Switzerland) on previously frozen (–20 °C) subsamples. Pore fluids at Milos were always extracted in 2 cm steps through rhizons with a pore size of 0.1 µm (Rhizosphere Research Products, Wageningen, Netherlands) of sediment cores adjacent to the sediment cores which were taken for lipid analyses. Because i) geochemistry of Portsmouth and Soufrière were taken only at single measurements and ii) geochemical results of Milos revealed great seawater interference during sampling, the most conservative layer with the lowest magnesium concentration of each core (between 6 and 12 cm sediment depth) at Milos was taken and values assumed for the full sediment core for redundancy analysis (RDA).

IV.3.3. Lipid extraction

Lipids were extracted from either 30 g (0–10 cm sediment depth) or 60 g (10–20 cm sediment depth) of freeze-dried sediments using a modified Bligh and Dyer method as described in Sturt et al. (2004). In brief, an internal standard 1,2-dihenarachidoyl-sn- glycero-3-phosphocholine (C21:0/C21:0-PC, Avanti Lipids) and a mixture of dichloromethane/methanol/buffer (DCM/MeOH/buffer, 1:2:0.8; v/v/v) was added to the sediment and subsequently ultrasonicated for 10 min in four steps. A phosphate buffer was used (pH 7.4), for the first two extraction steps and, for the last two steps, a trichloroacetic acid buffer (50 g L-1, pH 2.0). After each ultrasonication step, samples were centrifuged and the supernatant collected in a separatory funnel. For phase separation equal amounts of DCM and deionized Milli-Q water were added to a final volume of 1:1:0.8 (DCM/MeOH/buffer, v/v/v). The organic phase was separated and the remaining aqueous phase washed three times with DCM. Subsequently the DCM phase was washed three times with deionized Milli-Q water, evaporated close to dryness under a stream of nitrogen at 37 °C and stored at –20 °C as total lipid extract (TLE) until further analysis.

IV.3.4. Analyses and quantification of polar lipids

An aliquot of each TLE was analyzed for lipid quantities on a Dionex Ultimate 3000 high performance liquid chromatography (HPLC) system connected to a Bruker 95

Geochemical influence on the archaeal membrane lipid diversity maXis Ultra-High Resolution quadrupole time-of-flight tandem mass spectrometer (qToF-MS) equipped with an ESI ion source (Bruker Daltonik, Bremen, Germany). Quantification of archaeal polar and core lipids by HPLC-MS were performed following the reverse phase protocol of Wörmer et al. (2013). Detection of lipids was performed in positive ionization mode while scanning a mass-to-charge (m/z) range from 150 to 2,000. MS2 scans were obtained in data-dependent mode, for each MS full scan up to three MS2 experiments were performed, targeting the most abundant ions. Active exclusion limits the times a given ion is selected for fragmentation (three times every 0.5 min) and thus allowed to also obtain MS2 data of less abundant ions. Lipid identification was achieved + + + by monitoring exact masses of possible parent ions (present as either H , NH4 , or Na adducts) in combination with characteristic fragmentation patterns as outlined by Sturt et al. (2004) and Yoshinaga et al. (2011). Polar lipid quantification was performed by comparison of parent ion responses relative to known amounts of an internal standard

(i.e. C21:0/C21:0-PC) and normalized to gram of dry sediment weight. Reported concentrations were corrected for response factors of commercially available or purified standards. Archaeol was purchased from Avanti Polar Lipids Inc. (Alabaster, AL, USA) and GDGT-0, 1G-GDGT-0, 2G-GDGT-0, monoglycosidic archaeol (G-AR), and diglycosidic archaeol (2G-AR) were purified from extracts of Archaeoglobus fulgidus by orthogonal preparative liquid chromatography as described by Zhu et al. (2013). Due to a lack of appropriate standards the abundances of core and polar H-GDGTs and H-nMe- GDGTs were corrected for the response factor of GDGT-0, G-GDGT-0 and 2G-GDGT- 0 according to the headgroup.

IV.3.5. Temperature modeling of Soufrière and Portsmouth

In situ temperature measurements at Soufrière were taken in five 5 cm intervals and used as input for a thermal diffusivity model ran in R v. 2.9.1 (R Development Core Team, 2009).

Eq. 2

T: temperature (°C) z: depth (m) Ts: source temperature (°C) (150 °C) Tbw: bottom water temperature in °C (28 °C)

Chapter IV

v: Darcy velocity, upward is negative because z is positive downward (m/s) (–0.5 d-1) r: ratio of the volumetric heat capacity of the fluid and the bulk sediment DT: thermal diffusivity (around 3x10-7 m2 s-1 for sand)

Details of the equation can be found in Feseker et al. (2008). For this model, we assumed a thermal diffusivity of 3x10-7 m2 s-1 (for sandy sediments) and steady flow velocity of 0.021 m h-1. Temperature values for sediment sections of 100 µm were determined. Temperature values for Portsmouth have been modeled based on random one-point measurements between 10 and 20 cm sediment depth which recorded a temperature range between 90 and 111 °C.

IV.3.6. Statistical analyses/RDA model

Individual archaeal polar lipids were grouped according to their headgroups and core structure (diether, isoprenoidal tetraether, unsaturation, H-shape, H-shape with additional methyl groups) resulting in 14 different groups (G-AR, 2G-AR, G-GDGT, G- uns-GDGT, G-H-GDGT, G-H-Me-GDGT, G-H-2Me-GDGT, G-H-3Me-GDGT, G-H- 4Me-GDGT, 2G-GDGT, 2G-H-GDGT, 2G-H-Me-GDGT, 2G-H-2Me-GDGT and 2G- H-3Me-GDGT). A priori IPL patterns were subjected to a Hellinger transformation to applying linear multivariate methods, as recommended for this type of data (Legendre and Gallagher, 2001; Ramette, 2007). Principal component analysis (PCA) was performed with a focus on inter-species distances and principal axes were calculated for 69 samples in total (49 samples of Milos and 20 samples of Dominica; see Table IV.S1.). Geochemical parameters were standardized to zero mean and unit variance. A forward selection procedure was performed to retain only the spatial terms that significantly explained variation in the lipid data. The selected terms were then analyzed in concert with the other contextual parameters. The significance level in the RDA models was assessed by 1000 data permutations. All statistical analyses were performed in Rstudio (RStudio Team, 2016) version 1.0.143, using the vegan package version 2.0-10 (Oksanen et al., 2013) and custom R scripts. Polar lipid data of Milos are available and published in Sollich et al. (2017).

IV.3.7. Correlation analysis

Given that the data points were not normally distributed, we conducted a two-sided Spearman’s rank correlation test for several archaeal lipid parameters against 97

Geochemical influence on the archaeal membrane lipid diversity temperature. Spearman’s rank correlations were calculated using the software of Wessa (2017). Parameters included the relative abundance of G-H-GDT and G-H-nMe-GDGTs compared to total archaeal PL as well as ring- (RI) and methylation- (MIX) indices for G-H-GDT and G-H-nMe-GDGTs when applicable for the data set. For details in core and polar archaeal tetraether indices calculation (number of cyclopentane rings, degree of unsaturation and additional methyl groups) please see Sollich et al. (2017) and the Supporting Information.

IV.4. Results and Discussion

IV.4.1. Distribution of archaeal polar lipids in hydrothermally-influenced sediments of Dominica

Archaeal polar lipids in the hydrothermal sediments of Dominica were composed of glycosidic diethers (archaeol, AR) and tetraethers (glycerol-dibiphytanyl-glycerol- tetraether, GDGT), most of them linked to monoglycosidic headgroups (0–7% of polar GDGT possessed a 2G headgroup; 0–13% of the total polar lipid pool possessed a 2G headgroup) (Figure IV.1., and IV.2.). Like in the majority of marine sediments (Biddle et al., 2006; Lipp and Hinrichs, 2009; Schubotz et al., 2009), GDGT dominated over AR, representing 72–100% of total polar archaeal lipids. A striking finding was the exclusive detection of glycosidic membrane lipids in all investigated sediment samples of Dominica and Milos, which is consistent with other environmental ecosystems like the marine deep biosphere. Table IV.1. shows a compilation of archaeal polar lipid diversity of various ecosystems available from the literature. The table includes also the ranges of the relative abundances of archaeal phospholipids in these samples. The general predominance of archaeal glycolipids in most environmental samples could be a result of the exceptional preservation potential, which has been demonstrated previously for these kinds of lipids (Schouten et al., 2010; Logemann et al., 2011; Xie et al., 2013). On the other hand, it has been shown that glycosidic moieties are important for the protection of archaeal membranes against harmful environmental factors such as extremely low pH (Wang et al., 2012) and elevated temperatures of hydrothermal sediments (Sollich et al., 2017 and references therein). Overall, although we consider one or both of these scenarios to be likely other scenarios can not be ruled out at this point.

Chapter IV

Figure IV.1. | Core lipid structures of archaeal glycolipids detected in sediments of Spathi Bay (Milos), Soufrière and Portsmouth (Dominica). Highlighted in red are chemical structures including variable numbers of glycosidic headgroups (n = 1 or 2), and tetraether modifications (unsaturation, cyclization and methylation of biphytanes(s) and covalent bonds between isoprenoidal chains). (a) mono- (G) and diglycosyl (2G), n = 1 and 2, respectively; List of exemplary archaeal core lipids: (b) diether or archaeaol (AR); (c–g) glycerol-dibiphytanyl-glycerol-tetraethers (GDGT); (c) unsaturated GDGT; (d) GDGT-0; (e) GDGT-4; (f) H-GDGT-2; (g) H- nMe-GDGT-2.

Geochemical influence on the archaeal membrane lipid diversity

Figure IV.2. | Sediment profiles displaying the relative abundance of lipid classes and total archaeal polar lipids in sediments of Soufrière (A) and Portsmouth (B) off the coast of Dominica. Sediment samples were analyzed in 2 cm sections from surface until 20 cm sediment depth. Please note the different scaling of the x-axes of polar lipid concentrations of Soufrière and Portsmouth. Abbreviations: monoglycosidic archaeol (G-AR); monoglycosidic unsaturated (uns) acyclic glycerol-dibiphytanyl-glycerol-tetraethers (GDGT); mono- and diglycosidic GDGT with 0 to 5 rings (0–Cren); mono- and di-glycosidic H-shaped (H) and H-nMe-GDGT with up to 4 methyl groups and max 4 rings. Cren, crenarchaeol.

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Table IV.1. | Diversity of tetraether headgroups and core structures in different environmental ecosystems.

Location Polar GDGTs GDGT types References Water column Gly, Phos (HPH)* i, OH Schubotz et al., 2009; Zhu et al., 2016*; Xie et al., 2014 Marine surface sediments Gly, Phos (HPH)* i, OH, GDNT Schubotz et al., 2009; Liu et al., 2011; Lengger et al., 2012*; Evans et al., 2017* Deep marine biosphere Gly i, OH, GDNT Lipp et al., 2008; Lipp and Hinrichs, 2009; Liu et al., 2011; Lengger et al., 2014 Methane seeps Gly, Phos (PG**) i, OH, uns Schubotz et al., 2011a; Rossel et al., 2008; 2011b; Yoshinaga et al., 2015bc Hydrothermal vents Gly, Phos (PG**) i, H Reeves et al., 2014d**; Gibson et al., 2013 Shallow marine Gly i, uns, H, Sollich et al., 2017 hydrothermal systems nMe, H-nMe Hot springs Gly, Phos (HPH) i Schubotz et al., 2013e Peat bog soil Gly i, GDNT Liu et al., 2010 *hexose-phosphate-hexose (HPH) were detected using quadrupole MS in MRM mode, and represented therefore just a minor (likely < 1% of total polar tetraethers). **PG = phosphatidylglycerol; Gly = glycosidic; Phos = phosphatidic; HPH = hexose- phosphate-hexose; i = isoprenoidal; OH = hydroxylated; uns = unsaturated; H = H- shaped; nMe = number of additional methylations; a-erelative abundance of phosphatidic tetraethers from total archaeal polar lipids; a0 to ~65%; b0 to ~90%; c5 to ~42%; dnot determined; e0 to ~55%

IV.4.2. Influence of sediment temperature on archaeal polar lipid diversity

The distribution of polar lipids in the sediments of Dominica was marked by a decrease in AR and regular GDGT with depth, which corresponded to an increase with temperature especially for Portsmouth (Figure IV.2.) while Soufrière showed an increase of regular GDGT on the expense of AR. Furthermore, we observed an increase in the relative abundance of H-shaped GDGT with depth at Portsmouth. The latter molecules consisted of H- and H-nMe structures (Figure IV.1.), both exclusively reported in hydrothermal systems (Jaeschke et al., 2012; Gibson et al., 2013; Lincoln et al., 2013; Schubotz et al., 2013; Jia et al., 2014; Reeves et al., 2014) and in thermophilic strains of cultured archaea (Morii et al., 1998; Schouten et al., 2008; Knappy et al., 2011; Liu et al., 2012; Yoshinaga et al., 2015a). The fact that the contribution of these specialized H- shaped tetraethers increase with temperature seems in line with our previous investigation in the hydrothermal sediments off Milos Island (Sollich et al., 2017). That is, the relative abundance of glycosidic H- and H-(n)Me-GDGT is positively correlated with temperature 101

Geochemical influence on the archaeal membrane lipid diversity

(Table IV.2.) whereas relative contributions of regular GDGT are negatively correlated with increasing temperature (Table IV.2.)

Table IV.2. | Analysis of molecular species of archaeal lipids as a function of temperature. Spearmans’s rank correlation coefficient values (Spearman Rho) correspond to positive or negative correlation and the 2-sided p-value indicates the level of significance. Numbers in red indicate values of no significance. Please see Supporting Information for details about lipid parameters used as input to the correlation analysis.

Milos Dominica no. of Spearman 2-sided no. of Spearman 2-sided samples Rho p-value samples Rho p-value % G-GDGT 49 -0.73 1.22E-08 20 -0.62 4.22E-03 % G-H-GDGT 49 0.84 8.74E-14 20 0.72 5.00E-04 % G-H-nMe-GDGT 49 0.56 4.65E-05 20 0.60 4.85E-03 RI GDGT 49 -0.25 8.39E-02 19 -0.56 1.38E-02 RI H-GDGT 35 0.34 4.27E-02 19 -0.33 1.73E-01 RI c-GDGT 49 -0.68 1.96E-07 20 0.39 9.06E-02 RI c-H-GDGT 49 0.52 1.77E-04 20 0.48 3.27E-02 MIX G-H-nMe-GDGT 49 0.40 4.73E-03 8 -0.10 8.40E-01 MIX c-H-nMe-GDGT 49 -0.53 1.06E-04 20 -0.45 4.82E-02

These results prompted the query as to whether the structural variations of these specialized tetraethers (e.g. number of rings or methyl groups, Figure IV.1.) would be influenced by temperature as reported in Sollich et al. (2017). The data presented in Table IV.2. was an attempt to identify a relationship of the RI- and the MIX-index from major GDGT and temperature comparing two geochemically different shallow-water hydrothermal sediments off Dominica and Milos. The results revealed contrasting trends of RI and MIX for the two sites (Table IV.2.). In the one hand, it is surprising that the increase in both RI and MIX with temperature attributed to living/active archaeal biomass in Milos sediments (Sollich et al., 2017) was not apparent in Dominica sediments. On the other hand, the limited number of samples for Dominica (n = 8 to 20) or other environmental parameters rather than temperature (Table IV.3.) might have influenced the composition and relative abundance of glycosidic tetraethers.

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Table IV.3. | Selected geochemical parameters, which were tested for statistical significance of the two hydrothermal active sediments at Dominica (Soufrière and Portsmouth) and of the five sediment cores taken off the coast of Milos Island at Spathi Bay. If not reported, geochemical data are represented in mM. *indicates geochemical parameters which were used as input for the RDA model after reducing the model for not significant and co-correlating environmental parameters.

Salinity Temperature 2- sample name pH ‰* SO4 As Fe* H2S* (°C)* Soufrière 6.33 10.90 9.53 0.0133 0.2147 0.0013 31-54 Portsmouth 5.87 18.90 9.30 0.0133 0.0480 0.0518 46-104 Milos_1 7.68 37.50 31.75 0.0014 0.0120 0.0009 18-20 Milos_2 6.10 38.00 31.95 0.0202 0.0280 0.0473 24-62 Milos_3 6.01 35.90 29.54 0.0392 0.0091 1.4554 26-71 Milos_4 5.88 32.50 24.26 0.0002 0.0018 1.2549 30-81 Milos_5 6.12 36.20 29.77 0.0210 0.1421 0.8581 41-101

IV.4.3. Major environmental parameters affecting the distribution of archaeal polar lipids in shallow-water hydrothermal sediments

The redundancy analysis (RDA) was performed to determine the influence of each environmental variable (displayed in Table IV.3.) on the distribution of archaeal polar lipids from the hydrothermal sediments of Dominica and Milos. In this analysis, we used the relative abundances of 14 archaeal polar lipid classes rather than RI or MIX indices to fulfil statistical requirements of this test. The results of the RDA revealed that axes 1 and 2 explain 61% of the relationship between archaeal polar lipids and the four significant environmental variables: temperature, hydrogen sulfide, salinity and iron (Figure IV.3.). RDA axis 1 captured the major trend of high sediment temperatures at Portsmouth and station 5 at Milos, contrasting with the relatively lower temperatures of other samples (Table IV.S1.). A higher proportion of glycosidic AR and regular GDGT was the hallmark of the latter stations. While the distribution of monoglycosidic H-GDGT was clearly driven by elevated temperatures, high concentrations of sulfide and high salinity (characteristics of hot sediments at Milos) likely played an additional role influencing the distribution of monoglycosidic H-nMe-GDGT. RDA axis 2 captured the major geochemical differences between the two shallow hydrothermal fields. Apart from the relatively high iron concentrations, Dominica sediments are characterized by lower salinity and concentrations of sulfide than Milos (Table IV.3.). These characteristics had apparently a significant but minor impact on the variance in archaeal polar lipids distribution in the sediments.

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Figure IV.3. | RDA plot showing the distribution of samples and archaeal polar lipids (grouped according to headgroup and core lipid structure) in relation to environmental variables that explain most of the variability. Environmental variables are shown in blue arrows. Temp, temperature; Fe, iron concentration; Sal, salinity; H2S, hydrogen sulfide concentration. Symbol code is according to the investigated sites (triangle, Portsmouth; square, Soufrière; circle, Milos), color code of Milos samples represent samples of the same core. As an example, sediments dominated by G-H-GDGT (black triangles) were characterized by high sediment temperatures.

Sampling along a thermal gradient in a natural setting is extremely difficult. Shallow hydrothermal sediments thus offer a unique opportunity to probe the membrane ecology of microbial communities (Sollich et al., 2017). Our results from the RDA analysis suggest temperature as the most important environmental variable influencing the distribution of monoglycosidic H-GDGT in shallow hydrothermal sediments greatly. Glycosidic AR and regular GDGT, both major archaeal polar lipids reported in typical marine sediments (Schouten et al., 2013; Lipp and Hinrichs, 2009), seem to display an inverse relationship with sediment temperatures (Figure IV.3.). In addition, our RDA analysis shows that other variables rather than temperature (e.g. hydrogen sulfide and salinity) can significantly impact the distribution of monoglycosidic H-nMe-GDGT.

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IV.5. Conclusions

Archaeal polar lipid distribution in hydrothermally-influenced sediments from Dominica showed strong temperature dependencies as reported previously for hydrothermally-affected sediments from Milos Island. The relative abundance of modified tetraethers, e.g. H-GDGT and H-nMe-GDGT, correlated positively with increasing sediment temperature. Surprisingly, calculated ring- and methylation indices of polar and core GDGT were partly inconsistent with findings of Milos sediments when tested for temperature correlation. However, this discrepancy can probably be related to the much smaller sample size of Dominica compared with the sample size investigated from Milos’ sediments. Nevertheless, we were able to demonstrate statistically that temperature is not the only factor influencing the lipid distribution in hydrothermally-influenced sediments. Geochemical parameters like hydrogen sulfide, salinity and iron are additional major drivers.

IV.6. Acknowledgements

The authors would like to thank all members of the scientific expedition to Dominica. We thank Prof. Kai-Uwe Hinrichs (KUH) for providing lab facilities and technical support. Tomas Feseker is acknowledged for modeling sediment temperature profiles. This study was supported by the Deutsche Forschungsgemeinschaft (DFG, Germany) through the Emmy Noether-Program (grant BU 2606/1-1 to SIB) and the Gottfried Wilhelm Leibniz Prize (grant HI 616/16 to KUH).

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IV.8. Supporting Information

IV.8.1. Supporting Methods

IV.8.1.1. Calculation of lipid indices used as input for Table IV.2. (Main Text)

The percent of G-H-GDGT used as input for Table IV.2. was calculated as the sum of G-H-GDGT (sum of G-H-GDGT with 0 to 4 rings) divided by the sum of all archaeal polar lipids. The same procedure was applied for the percent of G-H-nMe-GDGT. The ring-index for monoglycosidic archaeal tetraethers (G-GDGT and G-H-GDGT), and respective core lipids ((H)-cGDGT) was calculated using a modified equation after Pearson et al. (2004), as follows:

The methylation index (MIX) was determined for G-nMe-H-GDGT and nMe-H- cGDGT as follows:

For the MIX, only the number of methylation(s) was taken into account, so that methylated tetraethers containing different number of rings were summed up.

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IV.8.2. Supporting Tables

Table IV.S1. | Geochemical parameters which has been tested for statistical significance of the two hydrothermal active sediments at Dominica (Soufrière and Portsmouth) and of the five sediment cores taken off the coast of Milos at Spathi Bay. If not recorded, geochemical data are represented in mM.

Salinity Temperature 2- sample name pH ‰ SO4 As Fe H2S (°C) S_1 6.33 10.90 9.53 0.0133 0.2147 0.0013 30.84 S_3 6.33 10.90 9.53 0.0133 0.2147 0.0013 36.52 S_5 6.33 10.90 9.53 0.0133 0.2147 0.0013 42.20 S_7 6.33 10.90 9.53 0.0133 0.2147 0.0013 44.80 S_9 6.33 10.90 9.53 0.0133 0.2147 0.0013 47.40 S_11 6.33 10.90 9.53 0.0133 0.2147 0.0013 49.54 S_13 6.33 10.90 9.53 0.0133 0.2147 0.0013 51.22 S_15 6.33 10.90 9.53 0.0133 0.2147 0.0013 52.90 S_17 6.33 10.90 9.53 0.0133 0.2147 0.0013 53.58 S_19 6.33 10.90 9.53 0.0133 0.2147 0.0013 54.26 P_1 5.87 18.90 9.30 0.0133 0.0480 0.0518 45.99 P_3 5.87 18.90 9.30 0.0133 0.0480 0.0518 70.20 P_5 5.87 18.90 9.30 0.0133 0.0480 0.0518 84.30 P_7 5.87 18.90 9.30 0.0133 0.0480 0.0518 92.52 P_9 5.87 18.90 9.30 0.0133 0.0480 0.0518 97.31 P_11 5.87 18.90 9.30 0.0133 0.0480 0.0518 100.10 P_13 5.87 18.90 9.30 0.0133 0.0480 0.0518 101.73 P_15 5.87 18.90 9.30 0.0133 0.0480 0.0518 102.68 P_17 5.87 18.90 9.30 0.0133 0.0480 0.0518 103.23 P_19 5.87 18.90 9.30 0.0133 0.0480 0.0518 103.55 M1_1 7.68 37.50 31.75 0.0014 0.0120 0.0009 18.12 M1_3 7.68 37.50 31.75 0.0014 0.0120 0.0009 18.36 M1_5 7.68 37.50 31.75 0.0014 0.0120 0.0009 18.59 M1_7 7.68 37.50 31.75 0.0014 0.0120 0.0009 18.83 M1_9 7.68 37.50 31.75 0.0014 0.0120 0.0009 19.06 M1_11 7.68 37.50 31.75 0.0014 0.0120 0.0009 19.29 M1_13 7.68 37.50 31.75 0.0014 0.0120 0.0009 19.53 M1_15 7.68 37.50 31.75 0.0014 0.0120 0.0009 19.76 M1_17 7.68 37.50 31.75 0.0014 0.0120 0.0009 19.99 M1_19 7.68 37.50 31.75 0.0014 0.0120 0.0009 20.23 M2_1 6.10 38.00 31.95 0.0202 0.0280 0.0473 23.55 M2_3 6.10 38.00 31.95 0.0202 0.0280 0.0473 32.73 M2_5 6.10 38.00 31.95 0.0202 0.0280 0.0473 39.82 M2_7 6.10 38.00 31.95 0.0202 0.0280 0.0473 45.43 M2_9 6.10 38.00 31.95 0.0202 0.0280 0.0473 49.89 M2_11 6.10 38.00 31.95 0.0202 0.0280 0.0473 53.43 M2_13 6.10 38.00 31.95 0.0202 0.0280 0.0473 56.23 M2_15 6.10 38.00 31.95 0.0202 0.0280 0.0473 58.46 M2_17 6.10 38.00 31.95 0.0202 0.0280 0.0473 60.22 M2_19 6.10 38.00 31.95 0.0202 0.0280 0.0473 61.62 M3_1 6.01 35.90 29.54 0.0392 0.0091 1.4554 26.17

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M3_3 6.01 35.90 29.54 0.0392 0.0091 1.4554 39.30 M3_5 6.01 35.90 29.54 0.0392 0.0091 1.4554 48.63 M3_7 6.01 35.90 29.54 0.0392 0.0091 1.4554 55.46 M3_9 6.01 35.90 29.54 0.0392 0.0091 1.4554 60.46 M3_11 6.01 35.90 29.54 0.0392 0.0091 1.4554 64.13 M3_13 6.01 35.90 29.54 0.0392 0.0091 1.4554 66.81 M3_15 6.01 35.90 29.54 0.0392 0.0091 1.4554 68.78 M3_17 6.01 35.90 29.54 0.0392 0.0091 1.4554 70.22 M3_19 6.01 35.90 29.54 0.0392 0.0091 1.4554 71.27 M4_1 5.88 32.50 24.26 0.0002 0.0018 1.2549 30.36 M4_3 5.88 32.50 24.26 0.0002 0.0018 1.2549 49.05 M4_5 5.88 32.50 24.26 0.0002 0.0018 1.2549 60.78 M4_7 5.88 32.50 24.26 0.0002 0.0018 1.2549 68.39 M4_9 5.88 32.50 24.26 0.0002 0.0018 1.2549 73.32 M4_11 5.88 32.50 24.26 0.0002 0.0018 1.2549 76.52 M4_13 5.88 32.50 24.26 0.0002 0.0018 1.2549 78.60 M4_15 5.88 32.50 24.26 0.0002 0.0018 1.2549 79.95 M4_17 5.88 32.50 24.26 0.0002 0.0018 1.2549 80.82 M5_1 6.12 36.20 29.77 0.0210 0.1421 0.8581 41.06 M5_3 6.12 36.20 29.77 0.0210 0.1421 0.8581 71.49 M5_5 6.12 36.20 29.77 0.0210 0.1421 0.8581 86.34 M5_7 6.12 36.20 29.77 0.0210 0.1421 0.8581 93.86 M5_9 6.12 36.20 29.77 0.0210 0.1421 0.8581 97.67 M5_11 6.12 36.20 29.77 0.0210 0.1421 0.8581 99.60 M5_13 6.12 36.20 29.77 0.0210 0.1421 0.8581 100.57 M5_15 6.12 36.20 29.77 0.0210 0.1421 0.8581 101.07 M5_17 6.12 36.20 29.77 0.0210 0.1421 0.8581 101.32 M5_19 6.12 36.20 29.77 0.0210 0.1421 0.8581 101.45 S = Soufrière; P = Portsmouth; M1 to M5 = Milos; following numbers (1 to 19) indicate the corresponding depth layer, e.g. 1 = 0–2 cm, 3 = 2–4 cm etc.

IV.9. References

Pearson, A., Huang, Z., Ingalls, A.E., Romanek, C.S., Wiegel, J., Freeman, K.H., Smittenberg, R.H., and Zhang, C.L. (2004) Nonmarine crenarchaeol in Nevada hot springs. Applied Environmental Microbiology 70: 5229–5237.

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CHAPTER V – LIPID ANALYSES IN DEEP-SEA HYDROTHERMAL FLUIDS

Transfer of chemosynthetic fixed carbon and its ecological significance revealed by lipid analysis of fluids at diffuse flow deep-sea vents (East Pacific Rise 9°50’N)

V.1. Abstract

In deep-sea vent research, much attention has been given to black smokers, which results from deeply circulating seawater heated to extreme high temperatures (often > 300 °C) and discharged at the seafloor. Nevertheless, far more seawater circulates in the shallow subsurface and discharges mildly heated fluids (up to 100 °C) in diffuse flow hydrothermal systems. From a deep-sea research perspective, the extent of the subsurface biosphere at diffuse flow systems is one of the major scientific challenges. In this context, our study was aimed at quantifying the prokaryotic communities associated with diffuse flow fluids by the analyses of microbial lipids. However, our results provide evidence for small eukaryotes rather than bacteria as the main sources of major lipid classes of our samples. Those diagnostic lipids included to a great extent storage lipids such as triacylglyceroles and wax esters (ca. 90% of total lipids), phosphatidylcholine as the major polar lipid and sterols. Though, the obtained quantitative lipid results can probably not be extrapolated to the overall system. Further analysis of stable carbon isotopes of fatty acids and cholesterol revealed trophic transfer of chemosynthetic Epsilonproteobacteria to eukaryotes in fluid discharge sites. The accumulation of storage lipids in small eukaryotes suggests an intimate energy flux from chemosynthetic bacteria to higher trophic levels at diffuse flow systems. Lower temperatures and less toxic sulfide concentrations relative to black smoker areas may provide ideal conditions for development and/or dispersal of small eukaryotes such as microzooplankton, meiofauna and/or larval stages of macrofauna from deep-sea hydrothermal vents.

V.2. Introduction

Hydrothermal vent areas create oases for life in the otherwise hostile and desolated deep sea. Much attention has been given to the study of chimneys or black smokers of

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Lipid analyses in deep-sea hydrothermal fluids vigorous venting and focused discharge of deeply circulating hydrothermal fluids general exceeding 410 °C and 300 bars (Von Damm, 2004; Foustoukos and Seyfried, 2007; Fontaine et al., 2009; Fornari et al., 2012). However, adjacent to these systems there exist extensive areas of diffuse flow venting (Orcutt et al., 2011; Mittelstaedt et al., 2012), which may account for 50–90% of the total hydrothermal heat loss (Bemis et al., 2012). These diffuse flow systems result from seawater circulating in the shallow subsurface and discharge mildly heated fluids usually between < 0.2 °C and ~100 °C (Bemis et al., 2012). The upper temperatures of diffuse flow systems are below the currently determined limit of life (~120 °C; Kashefi and Lovley, 2003; Takai et al., 2008) and therefore may support great species diversity, richness and biomass (Butterfield et al., 2004). In fact, determining the extent of the subsurface biosphere in these ecosystems is one of the major scientific challenges in deep-sea research (e.g. Huber et al., 2007; Wankel et al., 2011; Bemis et al., 2012). The East Pacific Rise (EPR) is a fast-spreading mid-ocean ridge system and one of the best studied areas of hydrothermal activity since the famous discovery of the Galápagos vents in 1977 (Ballard, 1977; Lonsdale, 1977; Corliss et al., 1979). Especially the segment around 9°50’N has been of interest in many multidisciplinary investigations for the last 20 years (Fornari et al., 2012). One particularly suitable location in this area to study low temperature diffuse flow fluids is called “Crab Spa”, an opening (lava tube feature) into the subsurface hydrothermal aquifer. Crab Spa was formed after the volcanic eruption in the year 2006 at EPR 9°50’N (Tolstoy et al., 2006; Fornari et al., 2012), and underwent a successive biofaunal recolonization over the following years (Mullineaux et al., 2010). Interestingly, temperature around 23 °C and other parameters such as pH, sulfide concentrations and oxygen levels in fluids from Crab Spa have been seemly constant since the first investigation in 2007 (see Materials and Methods). Similarly to other low temperature fluids (e.g. Huber and Holden, 2008; Akermann et al., 2013; Meier et al., 2016), chemosynthetic processes appear to be dominated by the metabolic activity of Epsilonproteobacteria in Crab Spa fluids (McNichol et al., 2016). Early work by e.g. Hessler and Kaharl (1995) and Fisher (1995) pushed the field of hydrothermal research toward a more comprehensive understanding of community structures, species inventories and food web complexities at hydrothermal vents. Excellent reviews, available in the literature, can be find e.g. by Lutz and Kennish (1993) and by Van Dover (2000). However, until today there are still many hidden complexities of the food chain at hydrothermal vents (Grassle, 1986; Tunnicliffe, 1991; Van Dover, 1995; Hessler and

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Kaharl, 1995) and it is surprisingly little known about the energy transfer from the basis of the food web to higher trophic levels (Govenar, 2012). To gain further insights into the prokaryotic communities associated with low temperature diffuse flow fluids, we aimed at quantifying the microbial biomass at Crab Spa and the newly discovered area of Teddy Bear at the EPR 9°50’N (see Materials and Methods). For this purpose, we have used large volume pumps to filter > 200 L of fluids and employed modern lipidomic analysis. We found that lipid inventories were mainly composed of eukaryotic-derived storage lipids, leading to the opportunity to access how effectively energy transfer occurs from the subsurface geothermal source to higher trophic levels.

V.3. Materials and Methods

V.3.1. Field site and sampling of diffuse hydrothermal fluids

Diffuse flow hydrothermal fluids were collected from two locations at the East Pacific Rise during the research cruise AT26-10 in January 2014. The region called Crab Spa is a lava tube feature in the basalt of diffuse flow venting, located at 9°50.40’N and 104°17.49’W in 2506 m water depth (see Supporting Information Figure V.S1A.). This site has been studied since 2007, and recorded a relatively constant temperature of ~23 °C as well as major physico-chemical characteristics (pH ~6, sulfide ~180 µM, oxygen 4 µM, chloride ~560 mM kg-1, nitrate ~5 µM; from Sievert, Seewald, Le Bris and Luther, unpublished data) throughout the years (Reeves et al., 2014a; McNichol et al., 2016; Le Bris et al., unpublished). A newly discovered diffuse-flow hydrothermal vent was named Teddy Bear during the AT26-10 cruise in 2014 (9°50.50’N, 104°17.51’W; 2514 m water depth; ~13 °C; Supporting Information Figure V.S1B.). This site is characterized by colonization of filamentous sulfide-oxidizing bacteria, likely Beggiatoa, on the seafloor (pers. oberv.). Collectively, these areas provide an exceptional opportunity to comparatively study the microbial community composition of low-temperature hydrothermal vents. Overgrowing megafauna of Riftia pachyptila and Bathymodiolus sp. at Crab Spa were cleared out and fluid venting to the basalt opening was allowed to stabilize before positioning the first large volume pump (LVP) to minimize sea water contamination. The LVPs were prepared with a GF-filter of 0.3 µM pore size (Sterlitech, Kent, USA). Start point and duration of the LVP was set manually before deployment and lasted between 6 and 12 hours. Hydrothermal fluids of Crab Spa and Teddy Bear were taken in

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Lipid analyses in deep-sea hydrothermal fluids duplicates. Two additional filters, here referred as reference samples, were obtained from the base of the Crab Spa mount including a small amount of Crab Spa fluids, as the snorkel was positioned in hydrothermal fluids first, but fell down shortly thereafter. Unfortunately, a real control seawater sample, ideally taken hundreds of meters away from any hydrothermal activity, could not be obtained due to an emergency on board of the research vessel and dive limitation. Total filtered volumes varied between 236 and 405 L bottom seawater/fluids (see Supporting Information Table V.S1.). After LVP retrieval, filters were inspected for visible macrofauna (> 1 mm) and divided into quarters for different scientific purposes in the cold room at +4 °C. GF-filter pieces for lipid analyses were stored in combusted aluminum foil and kept frozen at –80 °C until further analysis.

V.3.2. Lipid extraction

Lipids were extracted from one quarter of each GF-filter using a modified Bligh and Dyer method as described in Sturt et al. (2004). In brief, an internal standard 1,2- dihenarachidoyl-sn-glycero-3-phosphocholine (C21:0/C21:0-PC, Avanti Lipids) was added to the sediment together with dichloromethane/methanol/buffer (DCM/MeOH/buffer, 1:2:0.8; v/v/v). This mixture was ultrasonicated for 10 min in four steps (ultrasonic bath and ultrasonic sticks alternating). For the first two extraction steps a phosphate buffer was used (pH 7.4) and, for the last two steps, a trichloroacetic acid buffer (50 g L-1, pH 2.0). After each ultrasonication, samples were centrifuged and the supernatant collected in a separatory funnel. For phase separation, equal amounts of DCM and deionized Milli-Q water were added to a final volume of 1:1:0.8 (DCM/MeOH/buffer, v/v/v). The organic phase was separated and the remaining aqueous phase washed three times with DCM. Subsequently the DCM phase was washed three times with deionized Milli-Q water, evaporated close to dryness under a stream of nitrogen at 37 °C and stored at –20 °C as the total lipid extract (TLE) until further analysis.

V.3.3. Lipid analyses by liquid chromatography mass spectrometry

Chapter V performed in positive and/or negative ionization mode while scanning a mass-to-charge (m/z) range from 150 to 2,000 Da. MS2 scans were obtained in data-dependent mode, and for each MS full scan up to three MS2 experiments were performed targeting the most abundant ions. Active exclusion was used to limit the time a given ion is selected for fragmentation (three times every 0.5 min), thus allowing to obtain MS2 data of less abundant ions. Lipid identification was achieved by monitoring exact masses of possible + - + - + parent ions (present as either H / , NH4 /HCOO , or Na adducts) in combination with characteristic fragmentation patterns as outlined by Sturt et al. (2004) and Yoshinaga et al. (2011). Polar lipid quantification was performed in positive ionization mode by comparison of parent ion responses relative to known amounts of an internal standard

(i.e. C21:0/C21:0-PC) and normalized to filtered volume (in L). Reported concentrations were corrected for response factors using commercially available or purified standards (see Supporting Information Table V.S2.). In addition to polar lipids, triacyglycerols (TAG), wax esters (WE) and alkyldiacylglycerols (ADG) could be detected and were identified in all samples. The amount of TAG was corrected for both solvent contamination and response factor (Supporting Information Table V.S2.). Molecular species of lipids (i.e. side chain fatty acids combination) were determined by analysis of MS2 fragmentation patterns in negative ionization mode for the most abundant phospholipids and in positive ionization mode for TAG.

V.3.4. Hydrolysis of TLE for fatty acid methyl esters (FAMES) and sterol analyses by gas chromatography

In order to obtain details of the molecular structure and stable carbon isotopic values of free and bound fatty acids, we have performed a base hydrolysis of the TLE for analysis by gas chromatography (GC). An aliquot of the TLE (20%) was saponified according to the method described by Elvert et al. (2003). Briefly, an aliquot of the TLE was dissolved in 2 mL of methanolic KOH (6% KOH in methanol, w/v). Reaction took place for 3 h at 80 °C. The mixture was shaken with a vortex several times during incubation. After cooling down to room temperature, 2 mL of a 0.05 M KCl solution were added and a neutral fraction was extracted by four times shaking with 2 mL n-hexane. After collecting the neutral fraction, the pH was adjusted to pH 1 with 25% HCl and fatty acids (FA) were extracted by shaking with 2 mL n-hexane. The neutral fraction was dried close to dryness and derivatized with 50 µL of bis-(trimethylsilyl)trifluoroacetamide (BSTFA, Merck, Germany) and 100 µL pyridine for 1 hour at 70 °C, yielding trimethylsilyl(TMS)-

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Lipid analyses in deep-sea hydrothermal fluids derivatives. The FA fraction was dried close to dryness and treated with 100 µL of BF3 in methanol for 1 hour at 70 °C yielding FAMES. The TMS-derivatives and FAMES were dried under nitrogen, redissolved in 100 µL n-hexane and stored at –20 °C until further GC analyses.

V.3.5. Gas chromatography analyses

TMS-derivatives and FAMES were analyzed by gas chromatography (GC) using three different detectors: (i) GC-flame ionization detection (FID) for quantification of compounds, (ii) GC-mass spectrometry (MS) for identification of compounds, and (iii) GC-isotope ratio (ir) MS for determination of the stable carbon isotopic compositions. Given their high abundance, only data on sterols are reported for the neutral fraction in this study. TMS-sterols and FAMES were dissolved in n-hexane and the GC conditions were the same for FID, MS and irMS. The initial oven temperature was held at 60 °C for 1 min, increased to 150 °C with a rate of 10 °C min-1, then raised to a temperature of 310 °C with a rate of 4 °C min-1 and held at 310 °C for 27.5 min. The carrier gas was helium with a constant flow of 1.0 mL min-1. The ThermoFinnigan Trace GC was coupled to a ThermoFinnigan Trace MS and used for structural identification through mass spectral information. The MS was operated in electron impact mode at 70 eV with a full scan mass range of m/z range from 40 to 800. Compound-specific stable carbon isotopic compositions were determined on a Thermo Scientific Trace GC Ultra with a GC-Isolink coupled to a Delta V plus isotope ratio MS via conflo IV interface. The isotopic compositions of FAMES and TMS-sterols were not corrected for the additional methyl groups induced through the derivatization process. All isotopic values are reported in the delta notation as δ13C relative to the Vienna PeeDee Belemnite (VPDB) Standard.

V.4. Results

V.4.1. Lipid diversity, polar lipids and fatty acid composition

Four major lipid classes were detected in both reference samples and hydrothermal fluid samples by HPLC-MS: triacylglyceroles (TAG), wax esters (WE), acyldialkylglyceroles (ADG) and polar lipids. The most abundant lipid group was represented by TAG, with concentrations at least 10 times higher than the other lipid classes in all samples (Figure V.1A.). TAG concentrations were highest in the reference samples (with an average value of 5329 ng L-1), followed by Crab Spa and Teddy Bear

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Figure V.1. | (A) Concentrations of total polar lipids (archaeal and non-archaeal polar lipids) and identified lipid classes triacylglycerols (TAG), wax esters (WE) and alkyldiacylglycerols (ADG) from reference samples and 2 different diffuse hydrothermal fluids (Crab Spa and Teddy Bear) from the East Pacific Rise. Values shown are averages from 2 filter replicates with the standard range as error. (B) Relative abundance of dominant non-archaeal polar lipids from reference samples, Crab Spa and Teddy Bear. Values are displayed as mean values of the 2 filter replicates. Abbreviations: phosphatidyl-choline (PC); -ethanolamine (PE); -glycerol (PG); cardiolipin (CL); diacylgylcerols (DAG); acyletherglycerols (AEG). Amino lipids and others are described in the main text. fluids displaying the lowest concentrations (4158 and 3445 ng L-1, respectively). In contrast, WE, ADG and polar lipids were most abundant in Crab Spa fluids (465, 296 and 382 ng L-1, respectively) followed by reference samples (249, 265 and 187 ng L-1), and the lowest abundance recorded in Teddy Bear fluids (66, 76 and 106 ng L-1). Phospholipids represented 97 ± 0.5% of total polar lipids (Figure V.1B.). The major phospholipid classes were represented by phosphatidyl-choline (PC), -ethanolamine (PE) and -glycerol (PG), followed by cardiolipin (CL). Among the phospholipids, diacylgylcerols (DAG) were the most abundant, accounting for 68 to 78% of total polar lipids. A relatively high amount of acyletherglycerols (AEG), mainly linked to PC as headgroup, was found in all samples with relative abundances ranging from 14 to 26% of total polar lipids. Other polar lipids found in these samples included dietherglycerols of PC, PE, PG, phosphatidyl-(N)-methylethanolamine (PME) and phosphytidyl-(N,N)- dimethylethanolamine (PDME), PDME/PME/PG-AEG, phosphoinositol (PI)/PDME/PME-DAG, mono- and dihydroxylated PE/PC-DAG (PE-(2)-OH), monoglycosidic DAG, aminolipids (ornithine (OL) and betaine lipids (BL)), sphingolipids and unknown lipids (UKs) that together accounted for 6 to 8% of total polar lipids. Archaeal polar lipids were also detected but accounted for less than 0.05% of total polar lipids (30–150 pg L-1). While abundant archaeal lipids are frequently reported in high temperature deep-sea hydrothermal systems (Jaeschke et al., 2012; Lincoln et al., 2013; Gibson et al., 2013; Reeves et al., 2014b), archaea seem to play only a minor role

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Lipid analyses in deep-sea hydrothermal fluids in low temperature diffuse flow hydrothermal fluids (Huber et al., 2007; Fortunato and Huber, 2016). Given their low abundance, the sources of archaeal lipids will not be further discussed. The molecular composition of TAG and polar lipids was determined by the averaged chain length and unsaturation of fatty acids linked to these abundant lipid classes (Figure V.2.). For instance, a PC-DAG with a fatty acid combination of 36 carbons and 4 unsaturations (PC36:4) would result in averaged fatty acid chain length and unsaturation of 18 and 2, respectively. These values were calculated from individual lipids taking into account their abundance within a lipid class and a mass-weighted average for both chain length and degree of unsaturation was created for TAG and major phospholipids. The data shown in Figure V.2. reveal a relatively variable fatty acid composition among selected lipid classes and sampling sites. In general, PC (both DAG and AEG) displayed the longest chain length and the highest degree of unsaturation (18.1 ± 0.4 and 1.8 ± 0.3 in average, respectively), contrasting with PE- and CL-DAG (16.7 ± 0.5 and 1.1 ± 0.2). This difference can be attributed to a higher contribution of polyunsaturated fatty acids (PUFA) linked to PC-DAG/AEG such as linoleic (18:2), linolenic (18:3), arachidonic (ARA, 20:4) and docosohexaenoic (DHA, 22:6) acids than to PE and CL (data not shown). Comparatively, the mass-weighted average of chain length and degree of unsaturation for TAG and PG-DAG revealed intermediate values and less variability among sampling sites. For comparison, we have compiled data available in the literature to assign a mass-weighted average chain length and degree of unsaturation for cultured Epsilonproteobacteria (see Supporting Information Table V.S3.), which are potentially the most prominent source of microbial lipids in these hydrothermal systems (Gulmann et al., 2015; McNichol et al., 2016). Surprisingly, mass-weighted average values for fatty acids of cultured Epsilonproteobacteria (16.5 ± 0.5 and 0.5 ± 0.2) fall short both in chain length and unsaturation compared to TAG and major phospholipids (Figure V.2.).

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Figure V.2. | Mass-weighted averaged chain length and unsaturation of fatty acid of major polar lipids and TAG from reference samples in black, Crab Spa (CS) in grey and Teddy Bear (TB) in white. The molecular composition of fatty acids of Epsilonproteobacteria (Epsilon) is shown in blue. The latter data were retrieved from the literature depicted in Supporting Information Table V.S3.

V.4.2. Molecular characterization and stable carbon isotopes (δ13C) of total fatty acids and sterols

The hydrolyzed TLE yielded total fatty acids (FA), which represent the sum of free and bound FA in each sample. In the investigated filters, the majority of bound FA can be assigned to TAG, and to a lesser extent to polar lipids, WE and ADG (Figure V.1.).

The total FA were dominated by saturated and monounsaturated C16 and C18 FA (Figure V.3.), and the mass-weighted average value for total FA was 16.8 ± 0.04 and 0.8 ± 0.04 for chain length and degree of unsaturation, respectively. This fatty acid composition appears to fall within the values observed for major lipid classes (Figure V.2.). In line with the analysis of molecular species of phospholipids such as PC-DAG and PC-AEG

(data not shown), PUFA accounted for 3 to 9% of total FA. Minor amounts of C14, C15,

C20 and C22 saturated and monounsaturated FA were also detected in the samples. Although the mass-weighted chain length and degree of unsaturation of total FA were very similar among samples, marked differences in δ13C were observed

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Figure V.3. | A) Molecular and stable carbon isotopic composition of total fatty acids. Grey bars show the relative abundance of fatty acids (averaged from two replicates) and dots represent their respective stable carbon isotopic composition (δ13C). Note that for the reference sample the reported results are from a single sample. Dashed lines represent the stable carbon isotopic composition of dissolved 5 inorganic carbon (DIC, black), cholesterol (C27Δ , red) and mass weighted average of fatty acids (MWA FA, white). Values for δ13C of DIC were taken from Proskurowski et al. (2008). Abbreviations: ai = anteiso, i = iso, a/b = FA with same carbon number and degree of unsaturation. B) For comparison, the maximum known ranges of bacterial δ13C lipids for three pathways are shown in bars (values from van der Meer et al. (1998) and Sakata et al. (1997)).

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(Figure V.3.). Hydrothermal fluids sampled at Crab Spa and Teddy Bear showed distinctively less negative mass-weighted averaged δ13C values (–8.8 and –12.8‰, respectively) than the reference samples (–34.5‰). Abundant saturated and 13 monounsaturated FA (C16, C18 and C20) displayed striking differences in δ C values between hydrothermal fluids and reference samples. It is worth noting that relatively 13C- enriched FA (ca. –10‰) in the fluids of Crab Spa and Teddy Bear included PUFA such as ARA and DHA (Figure V.3.). All investigated samples contained similar sterol diversity with cholesterol being the most abundant sterol accounting for ~58 to 71%, except for one reference sample that contained exclusively this sterol (Table V.1). Other sterols present in all samples are desmosterol, 22-dehydrocholesterol, 24-methylcholesta-5,24(28)-dien-3β-ol and cholestanol followed by smaller amounts of sitosterol. In addition, fucosterol was only present in trace amounts in Crab Spa samples (Table V.1). Stable carbon isotopes were determined only for cholesterol and revealed a similar pattern as for FA, with less negative δ13C values in hydrothermal fluids of Crab Spa and Teddy Bear (–16.5 and -18.7‰, respectively) than the reference (–20.3‰) (Figure V.3.).

Table V.1. | Relative abundance of detected sterols in filters from the EPR 9°50’N, detected by gas chromatography. RS = reference sample; CS = Crab Spa; TB = Teddy Bear

5,22a 5b 0c 5,24d 5,24(28)e 5f 5,24(28)g sample C27∆ C27∆ C27∆ C27∆ C28∆ C29∆ C29∆ RS 1 0.0 100.0 0.0 0.0 0.0 0.0 0.0 RS 2 3.8 68.9 5.6 10.0 8.8 2.9 0.0 CS 1 1.4 62.7 4.0 15.6 12.6 2.8 0.9 CS 2 1.3 58.2 5.2 16.8 14.2 2.6 1.7 TB 1 6.5 67.6 9.3 4.6 5.6 6.5 0.0 TB 2 4.5 71.2 9.0 5.4 5.4 4.5 0.0

a) 22-dehydrocholesterol/cholesta-5,22E-dien-3β-ol; b) Cholesterol/cholest-5-en- 3β-ol; c) Cholestanol/5α(H)-cholestan-3β-ol; d) Desmosterol/cholesta-5,24-dien-3β- ol; e) 24-methylcholesta-5,24(28)-dien-3β-ol; f) Sitosterol/24-ethylcholest-5-en-3β- ol; g) Fucosterol/24-ethylcholesta-5,24(28)E-dien-3β-ol

V.5. Discussion

While widespread seafloor features on and around hydrothermal vents at mid-ocean ridges, diffuse flow systems have received much less scientific attention than highly heated fluids also known as black smokers (Bemis et al., 2012). Quantifying the extent of the subsurface biosphere in diffuse flow systems is one critical challenge in deep-sea

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Lipid analyses in deep-sea hydrothermal fluids ecology research. Our study was designed to probe the prokaryotic communities associated with diffuse flow fluids of EPR 9°50’N using a comprehensive analysis of lipids (both molecular and stable carbon isotope compositions). The results presented here, however, do not support a major bacterial lipid source, e.g. Epsilonproteobacteria, as expected from these fluids (Gulmann et al., 2015; McNichol et al., 2016). In the following sections, we present evidence for eukaryotic sources of major lipid classes and lipid’s stable carbon isotope data indicating that Epsilonproteobacteria are likely the basis of the food web in diffuse hydrothermal systems of EPR 9°50’N.

V.5.1. Evidences for eukaryotic sources of major lipids

Storage lipids such as TAG, ADG and WE represented 96 ± 2% of total lipids in all samples (Figure V.1.). In the marine environment, these lipids are characteristic of photosynthetic algae and zooplankton (Lee et al., 2006; Kattner et al., 2007). For instance, zooplanktonic crustaceans from surface waters are known to produce higher amounts of TAG relative to phospholipids (Lee et al., 2006). The few studies conducted in hydrothermal vents have also revealed that storage lipids can be more abundant than phospholipids in macrofauna (Pond et al., 1997a, 2000; Phleger et al., 2005). In contrast to eukaryotes, bacteria capable of producing TAG and WE are limited to Acinetobacter species and the actinomycetes group (Alvarez and Steinbüchel, 2002; Ishige et al., 2003; Kalscheuer, 2010), which do not appear as major bacterial groups in hydrothermal systems of the EPR or elsewhere (Huber and Holden, 2008; Akerman et al., 2013; Gulmann et al., 2015; McNichol et al., 2016; Meier et al., 2016). Based on the prevalence of TAG, ADG and WE among all lipid classes, our data indicate eukaryotes rather than bacteria as major sources of storage lipids in filters collected at EPR 9°50’N. Nevertheless, obtained quantitative lipid results should be taken with caution and extrapolation to the overall abundances of the entire system might not be appropriate. PC is a signature polar lipid of eukaryotes accounting for more than 50% of total phospholipids (e.g. van Meer et al., 2008) and represented the major class of phospholipids found in our samples (Figure V.1B.). Although a few bacterial taxa are capable of synthesizing these phospholipids, major polar lipids in bacteria are PE, PG and CL (Sohlenkamp et al., 2003; Geiger et al., 2013; López-Lara and Geiger, 2016), such as in Epsilonproteobacteria (Mino et al., 2014; Giovannelli et al., 2016). Acyletherglycerols (AEG), the majority of which occurring as plasmalogens, accounted for 18 to 33% of total PC. Plasmalogens are known to occur in specialized bacteria, notably Clostridium

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Chapter V species (Goldfine, 2010; Oberg et al., 2012), but they have usually PE, PG and CL as headgroups and are associated with saturated and mono-unsaturated FA (Řezanka et al., 2012). This is in contrast to PUFA such as DHA as major FA linked to plasmalogens in eukaryotes (e.g. Braverman and Moser, 2012), as observed for several plasmalogens PC from this study (data not shown). Although PUFA might be synthesized by psychrophilic and barophilic marine bacteria such as Shewanella and Moritella (DeLong and Yayanos, 1986; Kato et al., 1998), these bacteria are not commonly reported to be dominant at hydrothermal systems (e.g. Takai et al., 2006; Orcutt et al., 2011). We thus attribute PC- DAG and PC-AEG to eukaryotes, which would represent a more plausible source for PUFA associated with these phospholipids. Similarly to storage lipids, PC and PUFA, bacteria are also unlikely producers of extensive amounts of cholesterol or any of the major sterols found in our study (Table V.1., Volkman, 2003; Pearson et al., 2003; Wei et al., 2016). Although the lipidomic analysis of fluids at EPR 9°50’N indicates that major sources of lipids are not bacterial in origin, we compiled the mass-weighted average chain length and degree of unsaturation of FA from potential biological sources and compared to our results from Figure V.2. Data shown in Figure V.4. display the mass-weighted average chain length and unsaturation of FA from typical bacteria and macrofauna of hydrothermal systems. While Epsilonproteobacteria and Beggiatoa display the shortest chain length and the least unsaturated FA, the mass-weighted average fatty acid composition of macrofauna are clearly more variable. However, macrofauna FA show consistently longer chain length and higher degree of unsaturation than the bacterial end-members. As expected, mass- weighted average chain length and unsaturation of fatty acids linked to typical bacterial phospholipids such as PE (see also PG and CL in Figure V.2.) display a trend towards shorter chain length and lower unsaturation relative to PC, more similar to the bacterial end-members. In contrast, FA linked to PC (both DAG and AEG) tend to display longer chain length and higher degree of unsaturation than other phospholipids, and thus more similar to macrofauna. The mass-weighted average chain length and unsaturation of fatty acids linked to TAG is positioned between macrofaunal and bacterial end-members. The comparative analysis of FA compositions displayed in Figure V.4. strongly supports the evidence for small eukaryotes, rather than bacteria, as major sources of lipids in filters collected at EPR 9°50’N. Because these filters were previously inspected with the naked eye for potential filtered fauna (compare section V.3.1.), it is likely that the source of eukaryotic lipids could be not visible species, like meiofauna and/or microzooplankton.

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However, it is important to keep in mind, that the group of microzooplankton is rather diverse and only sorted according to size. Therefore this group usually encompasses both protists and metazoa and might contain copepod nauplii and various larvae of macrofaunal species (Elangovan et al., 2012). To our knowledge, there are only very few studies of lipid species of vent-related microzooplankton available in the literature, thus we used vent-related macrofaunal lipid species as a proxy, especially because most of the microzooplankton at hydrothermal systems probably derives from reproduction of the surrounding macrofauna.

Figure V.4. | Mass-weighted average chain length and unsaturation of fatty acids from typical bacteria and macrofauna of hydrothermal vent systems (colored symbols) and data from this study (major phospholipids and TAG, empty squares and triangle respectively). Dashed lines mark the sector of fatty acid chain length and unsaturation of typical hydrothermal vent fauna. For error bars please see Supporting Information Figure V.S2. The references used as data input for bacterial and macrofauna fatty acids are detailed in Supporting Information Table V.S3., and Table V.S4. Abbreviations: Tw = Tubeworm; Pq = Polychaete; Sh = Shrimp; Cb = Crab; Gt = Gastropod; Ms = Mussel; Lp = Limpet; Amp = Amphipod; Cil = Ciliate; Beg = Beggiatoa; Cop = Copepod; Rimicaris_TAG = Triacylglycerols of Rimicaris exoculata; Epsilon = Epsilonproteobacteria. EPR = major phospholipid classes of different samples from the EPR and EPR_TAG = triacylglycerols from the EPR (cf. Figure V.2.).

V.5.2. Trophic transfer of chemosynthetically fixed carbon revealed by stable carbon isotopes

Before discussing our results, it is important to mention that data presented in Figure V.3. are within the range of stable carbon isotopes values reported for lipids of vent

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Chapter V macrofauna (Limén et al., 2008; Pond et al., 1997b, 2002; Hu et al., 2012). Based on the δ13C composition of DIC (Proskurowski et al., 2008), the less negative δ13C of fatty acids in Crab Spa (CS) and Teddy Bear (TB) fluids (Figure V.3.) can be attributed to carbon metabolism involving the reverse tricarboxylic acid cycle (rTCA), likely carbon fixation by abundant Epsilonproteobacteria in hydrothermal systems (Hügler and Sievert, 2011). However, as already discussed above, the majority of fatty acids analyzed for δ13C are derived from major lipids such as TAG and PC which in turn are sourced by eukaryotic biomass. Thus, our results from fatty acids δ13C composition likely reflect an effective trophic transfer from Epsilonproteobacteria to small eukaryotes in these hydrothermal systems. This is reinforced by δ13C values of typical eukaryotic PUFA (DHA, EPA and ARA) and cholesterol at CS and TB that are in the range of less negative fatty acids such as saturated and monounsaturated FA typically attributed to bacteria (Figure V.3.). More interesting however, is the striking difference in fatty acids δ13C composition observed between reference samples and the hydrothermal fluids (CS and TB). That is, reference samples displayed consistently more negative δ13C values of FA than the hydrothermal fluids, highlighting the importance of Epsilonproteobacteria for the trophic ecology at CS and TB. These findings indicate that subsurface Epsilonproteobacteria likely provide the basis of food web transfer to small eukaryotic biomass in areas of fluid discharge, whereas small eukarotes (possibly microzooplankton) in adjacent reference samples might also rely on other bacteria rather than Epsilonproteobacteria.

V.6. Conclusions

Lipidomic analysis of filters sampled at diffuse flow hydrothermal systems of EPR 9°50’N provides evidence for a major contribution of small eukaryotes as main lipid sources. Storage lipids, primarily as TAG, represented by far the most abundant lipid class. Among zooplankton researchers, there is a lack of consensus concerning the relative importance of lipids in buoyancy and energy storage (e.g. Lee et al., 2006; Pond, 2012). However, for instance, large amounts of lipids are stored as WE by herbivorous zooplankton (90% of total lipids in calanoid copepods), especially at higher latitudes to survive long periods of food scarcity in resting stages (Kattner et al., 2007). By contrast, TAG are widely used lipid storage in zooplankton species of surface waters that remain active throughout the year (Kattner et al., 2007). The accumulation of lipids in small eukaryotes as TAG (> 75% of total lipids), exceeding the amounts observed in vent macrofauna (e.g. Pond et al., 1997a, 2000; Phleger et al., 2005), suggests an intimate

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Lipid analyses in deep-sea hydrothermal fluids energy flux from chemosynthetic fixed carbon to higher trophic levels in diffuse flow systems. This aspect may be critical in the ecology of deep-sea hydrothermal vents, particularly CS that has been relatively stable for the last 7–8 years in terms of fluid temperature and associated parameters (e.g. sulfide and oxygen concentrations and pH) (Reeves et al., 2014a; McNichol et al., 2016; from Sievert, Seewald, Le Bris and Luther, unpublished data). Phosphatidylcholine was the most abundant class of polar lipids and given the fairly high contribution of PUFA linked to PC-DAG and PC-AEG, they consistently displayed the longest chain length and highest degree of unsaturation (Figure V.2.). While the relatively high proportion of PUFA is commonly reported in tissues of hydrothermal vent macrofauna (Figure V.4.), the origin of PUFA in deep-sea vents has been linked to phytoplankton via trophic upgrading (Pond et al., 1997b, 2002; Rieley et al., 1995). However, our analysis of PUFA δ13C composition in CS and TB revealed a trophic level transfer from Epsilonproteobacteria to small eukaryotes (see discussion above). This finding opens the possibility for de novo synthesis of PUFA in hydrothermal vents. Since Epsilonproteobacteria do not figure among the bacteria capable of producing PUFA (e.g. Shulse and Allen, 2011), these fatty acids are likely produced by small eukaryotes. Possible candidates in the filters collected at CS and TB are heterotrophic protists and nematodes that may produce PUFA de novo (Metz et al., 2001; Wallis et al., 2002) and are found in relatively high abundance inhabiting hydrothermal vent systems (Atkins et al., 2000; Moreira and López-García, 2003; Coyne et al., 2013; Stokke et al., 2015). These small eukaryotes could be the source of essential PUFA in these ecosystems, as reported for trophic level upgrading of PUFA in other aquatic systems (Klein Breteler et al., 1999; Bec et al., 2006, 2010). It is known that marine invertebrates require essential fatty acids (18:2ω6 and 18:3ω3), but possess the enzymatic machinery (e.g. elongases and desaturases) for producing their highly unsaturated fatty acids such as ARA and DHA (Monroig et al., 2013). Therefore HUFAs such as EPA and DHA characteristic from higher organisms of vents (e.g. shrimps (Pond et al., 1997b, 2000) and tube worms (Pond et al., 2002; Phleger et al., 2005)) may derive from in situ production of PUFA by small/invisible eukaryotes. The variability in δ13C values of FA between reference samples and hydrothermal fluids (Figure V.3.) is consistent with distinct dietary sources for microzooplankton, possibly different bacterial groups, occurring on relatively small spatial scales (i.e. within 2–5 meters from the fluid discharge). Small eukaryotes thriving close to the fluid discharges definitely benefit from high biomass of

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Epsilonproteobacteria, likely sourced by the subsurface biosphere. However, adjacent areas to fluid discharge may support more diverse bacterial groups on small (few cm to 1 m) spatial scales (Meier et al., 2016), explaining the remarkable differences in δ13C of fatty acids revealed by our study. Low temperatures and low sulfide concentrations in concert with food availability in diffuse flow systems might represent ideal conditions for the proliferation of small eukaryotes such as meiofauna, which in areas of highly heated fluids close to tubeworm fields and mussel beds are low in abundance and diversity relative to other deep-sea environments (Gollner et al. 2010). Furthermore, these environmental conditions coupled to the stability of CS (and perhaps TB) on time scales of nearly a decade may play a major role as feeding grounds for larval development and dispersal, with broad implications for the biogeography of deep-sea vent fauna.

V.7. Acknowledgements

We thank the officers, crew and pilots of the R/V Atlantis and ROV Jason for their help at sea and efforts obtaining the samples used in this study. Special thanks go to Craig D. Taylor for preparation and set-up of the large volume pumps. We are in debt to Eoghan P. Reeves and Raymond C. Valentine for fruitful discussions during manuscript preparation. We are also thankful to Ann Vanreusel for sharing fatty acids data used in this study. This study was supported by the Deutsche Forschungsgemeinschaft (DFG, Germany) through the Emmy Noether-Program (grant BU 2606/1-1 to SIB), the Gottfried Wilhelm Leibniz Prize (grant HI 616/16 to KUH) and the Dimensions of Biodiversity program of the National Science Foundation (NSF grant OCE-1136727 to SMS).

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V.9. Supporting Information

V.9.1. Sampling

V.9.1.1. Sample collection

Hydrothermal fluid samples were collected by large volume pumps (LVP) from two low-temperature diffuse flow hydrothermal systems named Crab Spa and Teddy Bear (Figure V.S1.) at the EPR 9°50’N. Reference samples were obtained within ~5 m of Crab Spa. The individual sample names, locations and filtered volumes are shown in Table V.S1.

Figure V.S1. | Photographs from the two low-temperature diffuse flow hydrothermal sites: A) Crab Spa and B) Teddy Bear (images: courtesy of Woods Hole Oceanographic Institution)

Table V.S1. | Description of samples, locations and filtered volumes (L).

Samples Filtered volume (L) Reference 1 271 Reference 2 236 Crab Spa 1 406 Crab Spa 2 345 Teddy Bear 1 326 Teddy Bear 2 257

V.9.2. Supporting Methods

V.9.2.1. Quantification of polar lipids based on response factors of standards

Polar lipids, triacylglycerols (TAG), wax esters (WE) and alkyldiacylglycerols (ADG) were quantified by HPLC-MS and the reported concentrations corrected for response factor of commercially available standards as described in Table V.S2. Due to the lack of authentic standards of ornithine lipids, values were corrected for the response

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Chapter V factor of structural similar betaine lipids. In addition, ADG and WE were corrected for TAG given the lack for authentic standard.

Table V.S2. | Commercially standards used to quantify lipid classes by HPLC-MS.

# Standard Source Polar Lipids* 1 Glucosylceramide Avanti Polar Lipids Inc., USA Sphingolipids 2 DGTS Avanti Polar Lipids Inc., USA BL, OL, UKs

3 C16:0/C16:0-PE Avanti Polar Lipids Inc., USA PE, PE-(2)-OH

4 C16:0/C16:0 dimethyl-PE Avanti Polar Lipids Inc., USA PDME 5 MGlc-DAG Avanti Polar Lipids Inc., USA G-DAG

6 C16:0/C16:0 methyl-PE Avanti Polar Lipids Inc., USA PME

7 C18:1/C18:1-CL Avanti Polar Lipids Inc., USA CL 8 o-PC Avanti Polar Lipids Inc., USA PC-DEG

9 C21:0/C21:0-PC Avanti Polar Lipids Inc., USA PC, PI-DAG

10 C16:0/C16:0-PG Avanti Polar Lipids Inc., USA PG

11 C16:0/C16:0/C16:0-TAG Sigma Aldrich, St. Louis, USA TAG, ADG, WE #1. 1. D-glucosyl-1,1'-N-stearoyl-D-erythro-sphingosine; 2. 1,2-dipalmitoyl-sn- glycero-3-O-4’-(N,N,N-trimethyl)-homoserine; 3. 1,2-dipalmitoyl-sn-glycero-3- phosphoethanolamine; 4. 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N,N- dimethyl; 5. 1,2-diacyl-3-O-(α-D-glucopyranosyl)-sn-glycerol; 6. 1,2-dipalmitoyl- sn-glycero-3-phosphoethanolamine-N-methyl; 7. 1’,3’-bis[1,2-dioleoyl-sn-glycero- 3-phospho]-sn-glycerol; 8. 1,2-di-O-hexadecyl-sn-glycero-3-phosphocholine; 9. 1,2-dihenarachidoyl-sn-glycero-3-phosphocholine; 10. 1,2-dipalmitoyl-sn-glycero- 3-phospho-(1’-rac-glycerol); 11. glyceryl tripalmitate. *please see Main Text for abbreviations.

V.9.2.2. Supporting references used as input for Figure V.2., and Figure V.4.

Table V.S3. | List of references from which fatty acid data were compiled from different species of Epsilonproteobacteria. This information was used as input for Figure V.2., and Figure V.4.

Species Reference Sulfurovum aggregans Mino et al., 2014 Sulfurovum lithotrophicum Inagaki et al., 2004 Nitratifractor salsuginis Nakagawa et al.,2005a Nitratiruptor tergarcus Nakagawa et al., 2005a Sulfurimonas paralvinellae Takai et al., 2006 Sulfurimonas autotrophica Inagaki et al., 2003 Thioreductor micantisoli Nakagawa et al., 2005b Thiofractor thiocaminus Makita et al., 2012 Nautilia profundicola Smith et al., 2008 Lebetimonas acidiphila Takai et al., 2005 Hydrogenimonas thermophila Takai et al., 2004

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Table V.S4. | List of organisms from which fatty acid data were used as input for Figure V.4., and the respective references.

Organism Species Abbreviation Type of FA analysis Reference Tubeworm Ridgea piscesae Tw Free FA Pond et al., 2002 Tubeworm Riftia pachyptila Tw Base hydrolysis Phleger et al., 2005a Polychaete Protis hydrothermica Pq Free FA Pond et al., 2002 Polychaete Alvinella pompejana Pq Base hydrolysis Phleger et al., 2005b Polychaete Alvinella caudata Pq Base hydrolysis Phleger et al., 2005b Polychaete Paralvinella grasslei Pq Base hydrolysis Phleger et al., 2005b Polychaete Hesiolyra bergii Pq Base hydrolysis Phleger et al., 2005b Polychaete Paralvinella palmiforis Pq Base hydrolysis Limén et al., 2008 Polychaete Paralvinella sulfincola Pq Base hydrolysis Limén et al., 2008 Copepod Benthoxinus spiculifer Cop Base hydrolysis Limén et al., 2008 Copepod Stygiopontius Cop Base hydrolysis Limén et al., 2008 quadrispinosus Limpet Lepetodrillus elevatus Lp Free FA Pond et al., 2008 Limpet Lepetodrilus spp. Lp Base hydrolysis Phleger et al., 2005a Amphipod Halice hesmonectes Amp Free FA Pond et al., 2008 Amphipod Ventriella sulfuris Amp Free FA Pond et al., 2008 Shrimp Alvinocaris markensis Sh Acid hydrolysis Pond et al., 1997 Shrimp Rimicaris exoculata Sh Acid hydrolysis Pond et al., 1997, 2000 Ciliate Folliculinopsis sp. Cil Free FA Kouris et al., 2010 Gastropod Ifremena nautilei Gt Acid hydrolysis Pranal et al., 1996 Gastropod Alviniconcha hessleri Gt Acid hydrolysis Pranal et al., 1996 Crab Munidopsis Cb Base hydrolysis Phleger et al., 2005a subsquamosa Crab Bythograea thermydron Cb Base hydrolysis Phleger et al., 2005a Mussel Bathymodiolus sp. Ms Base hydrolysis Phleger et al., 2005a Bathymodiolus Kellermann et al., Mussel thermophila Ms Base hydrolysis 2012 Van Gaever et al., Bacteria Beggiatoa Beg Base hydrolysis 2009

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V.9.3. Supporting Figures

Figure V.S2. | Mass-weighted average chain length and unsaturation of fatty acids from typical bacteria and FA and TAG of macrofauna of hydrothermal systems, including error bars of standard deviation (n ≥ 3) or range (n = 2). Mass-weighted average chain length and unsaturation of major phospholipids from EPR samples are displayed in empty squares (for details see Figure V.2.), and for TAG in empty triangles. Abbreviations: Tw = Tubeworm; Pq = Polychaete; Sh = Shrimp; Cb = Crab; Gt = Gastropod; Ms = Mussel; Lp = Limpet; Amp = Amphipod; Cil = Ciliate; Beg = Beggiatoa; Cop = Copepod; Rimicaris_TAG = Triacylglycerols of Rimicaris exocultata; Epsilon = Epsilonproteobacteria; EPR = major phospholipid classes (cf. Figure V.2.) from different samples from the East Pacific Rise; EPR_TAG = triacylglycerols from the EPR.

V.9.4. References

Inagaki, F., Takai, K., Kobayashi, H., Nealson, K.H., and Horikoshi, K. (2003) Sulfurimonas autotrophica gen. nov., sp. nov., a novel sulfur-oxidizing ε- proteobacterium isolated from hydrothermal sediments in the Mid-Okinawa Trough. International Journal of Systematic and Evolutionary Microbiology 53: 1801–1805. Inagaki, F., Takai, K., Nealson, K.H., and Horikoshi, K. (2004) Sulfurovum lithotrophicum gen. nov., sp. nov., a novel sulfur-oxidizing chemolithoautotroph within the ε-Proteobacteria isolated from Okinawa Trough hydrothermal sediments. International Journal of Systematic and Evolutionary Microbiology 54:1477–1482. Kellermann, M.Y., Schubotz, F., Elvert, M., Lipp, J.S., Birgel, D., Prieto-Mollar, X., Dubilier, N., and Hinrichs, K.-U. (2012) Symbiont–host relationships in chemosynthetic mussels: a comprehensive lipid biomarker study. Organic Geochemistry 43: 112–124. Kouris, A., Limén, H., Stevens, C.J., and Juniper, S.K. (2010) Blue mats: faunal composition and food web structure in colonial ciliate (Folliculinopsis sp.) mats at Northeast Pacific hydrothermal vents. Marine Ecology Progress Series 412: 93–101.

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Limén, H., Stevens, C.J., Bourass, Z., and Juniper, S.K. (2008) Trophic ecology of siphonostomatoid copepods at deep-sea hydrothermal vents in the northeast Pacific. Marine Ecology Progress Series 359: 161–170. Makita, H., Nakagawa, S., Miyazaki, M., Nakamura, K., Inagaki, F., and Takai, K. (2012) Thiofractor thiocaminus gen. nov., sp. nov., a novel hydrogen-oxidizing, sulfur- reducing epsilonproteobacterium isolated from a deep-sea hydrothermal vent chimney in the Nikko Seamount field of the northern Mariana Arc. Archieves of Microbiology 194: 785–794. Mino, S., Kudo, H., Arai, T., Sawabe, T., Takai, K., and Nakagawa, S. (2014) Sulfurovum aggregans sp. nov., a hydrogen-oxidizing, thiosulfate-reducing chemolithoautotroph within the Epsilonproteobacteria isolated from a deep-sea hydrothermal vent chimney, and an emended description of the genus Sulfurovum. International Journal of Systematic and Evolutionary Microbiology 64: 3195–3201. Nakagawa, S., Takai, K., Inagaki, F., Horikoshi, K., and Sako, Y. (2005a) Nitratiruptor tergarcus gen. nov., sp. nov. and Nitratifractor salsuginis gen. nov., sp. nov., nitrate- reducing chemolithoautotrophs of the ε-proteobacteria isolated from a deep-sea hydrothermal system in the Mid-Okinawa Trough. International Journal of Systematic and Evolutionary Microbiology 55: 925–933. Nakagawa, S., Inagaki, F., Takai, K., Horikoshi, K., and Sako, Y. (2005b) Thioreductor micantisoli gen. nov., sp. nov., a novel mesophilic, sulfur-reducing chemolithoautotroph within the ε-proteobacteria isolated from hydrothermal sediments in the Mid-Okinawa Trough. International Journal of Systematic and Evolutionary Microbiology 55: 599–605. Phleger, C.F., Nelson, M.M., Groce, A.K., Cary, S.C., Coyne, K.J., and Nichols, P.D. (2005a) Lipid composition of deep-sea hydrothermal vent tubeworm Riftia pachyptila, crabs Munidopsis subsquamosa and Bythograea thermydron, mussels Bathymodiolus sp. and limpets Lepetodrilus spp. Comparative Biochemistry and Physiology Part B 141: 196–210. Phleger, C.F., Nelson, M.M., Groce, A.K., Cary, S.C., Coyne, K., Gibson, J.A., and Nichols, P.D. (2005b) Lipid biomarkers of deep-sea hydrothermal vent polychaetes– Alvinella pompejana, A. caudata, Paralvinella grasslei and Hesiolyra bergii. Deep- Sea Research I 52: 2333–2352. Pond, D.W., Allen, C.E., Bell, M.V., Van Dover, C.L., Fallick, A.E., Dixon, D.R., and Sargent, J.R. (2002) Origins of long-chain polyunsaturated fatty acids in the hydrothermal vent worms Ridgea piscesae and Protis hydrothermica. Marine Ecology Progress Series 225: 219–226. Pond, D.W., Dixon, D.R., Bell, M.V., Fallick, A.E., and Sargent, J.R. (1997) Occurrence of 16:2 (n-4) and 18:2 (n-4) fatty acids in the lipids of the hydrothermal vent shrimps Rimicaris exoculata and Alvinocaris markensis: nutritional and trophic implications. Marine Ecology Progress Series 156: 167–174. Pond, D.W., Fallick, A.E., Stevens, C.J., Morrison, D.J., and Dixon, D.R. (2008) Vertebrate nutrition in a deep-sea hydrothermal vent ecosystem: fatty acid and stable isotope evidence. Deep-Sea Research I 55: 1718–1726. Pond, D.W., Gebruk, A., Southward, E.C., Southward, A.J., Fallick, A.E., Bell, M.V., and Sargent, J.R. (2000) Unusual fatty acid composition of storage lipids in the bresilioid shrimp Rimicaris exoculata couples the photic zone with MAR hydrothermal vent sites. Marine Ecology Progress Series 198: 171–179. Pranal, V., Fiala Medioni, A., and Guezennec, J. (1996) Fatty acid characteristics in two symbiotic gastropods from a deep hydrothermal vent of the West Pacific. Marine Ecology Progress Series 142: 175–184.

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Smith, J.L., Campbell, B.J., Hanson, T.E., Zhang, C.L., and Cary, S.C. (2008) Nautilia profundicola sp. nov., a thermophilic, sulfur-reducing epsilonproteobacterium from deep-sea hydrothermal vents. International Journal of Systematic and Evolutionary Microbiology 58: 1598–1602. Takai, K., Hirayama, H., Nakagawa, T., Suzuki, Y., Nealson, K.H., and Horikoshi, K. (2005) Lebetimonas acidiphila gen. nov., sp. nov., a novel thermophilic, acidophilic, hydrogen-oxidizing chemolithoautotroph within the ‘Epsilonproteobacteria’, isolated from a deep-sea hydrothermal fumarole in the Mariana Arc. International Journal of Systematic and Evolutionary Microbiology 55: 183–189. Takai, K., Nealson, K.H., and Horikoshi, K. (2004) Hydrogenimonas thermophila gen. nov., sp. nov., a novel thermophilic, hydrogen-oxidizing chemolithoautotroph within the ε-Proteobacteria, isolated from a black smoker in a Central Indian Ridge hydrothermal field. International Journal of Systematic and Evolutionary Microbiology 54: 25–32. Takai, K., Suzuki, M., Nakagawa, S., Miyazaki, M., Suzuki, Y., Inagaki, F., and Horikoshi, K. (2006) Sulfurimonas paravinellae sp. nov., a novel mesophilic, hydrogen- and sulfur-oxidizing chemolithoautotroph within the Epsilonproteo- bacteria isolated from a deep-sea hydrothermal vent polychaete nest, reclassification of Thiomicrospira denitrificans as Sulfurimonas denitrificans comb. nov. and emended description of the genus Sulfurimonas. International Journal of Systematic and Evolutionary Microbiology 56: 1725–1733. Van Gaever, S., Moodley, L., Pasotti, F., Houtekamer, M., Middelburg, J.J., Danovaro, R., and Vanreusel, A. (2009) Trophic specialisation of metazoan meiofauna at the Håkon Mosby Mud Volcano: fatty acid biomarker isotope evidence. Marine Biology 156: 1289–1296.

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CHAPTER VI – CONCLUDING REMARKS AND FUTURE PERSPECTIVES

The investigation of microbial lipids in extreme environments rather than in pure cultures represents an ideal scientific platform to explore the ecology of membranes exposed to various stressors. Bacteria, archaea and eukaryotes inhabiting these ecosystems have to adjust their membrane permeability and fluidity properties in order to generate ion gradients essential for energy homeostasis (i.e. ATP synthesis). The advance of ESI-MS based techniques for lipidomics allowed the detection of more than one thousand individual molecular species of lipids in environmental samples. Understanding the structure-function of these molecules in nature may provide a meaningful pathway to interpret the lipid ecology of cells in general. For example, in chapter III bacterial sphingolipids were abundant lipids at intermediate to high temperatures (30 to 80 °C) (Figure III.4.). This finding prompted the hypothesis that the high melting point and hydrogen bonding potential of sphingolipids may provide a more rigid lateral organization of membranes via the formation of lipid microdomains. Modulation of sphingolipids in mammalian cells have been implicated in a number of biological processes such as signaling in autophagy and apoptosis (e.g. Grösch et al., 2012), and in several human ailments including lipid viruses (Gerl et al., 2012), lysossomal diseases (e.g. Gaucher disease), diabetes and neurodegenerative diseases (Kolter and Sandhoff, 1999; Siddique et al., 2015). For future studies it would be very interesting to survey the distribution of bacterial sphingolipids in several natural systems in order to understand the environmental conditions governing their occurrence. For instance, bacterial sphingolipids were recently detected in authigenic carbonate samples from cold-seeps (Case et al., submitted; V. Orphan’s lab, Caltech, US). Furthermore, Bacteroidetes that are known to produce sphingolipids in the human gut are the most likely sources of these lipids in cold seeps and shallow-water hydrothermal systems. The major membrane quandary raised in chapter III concerned the increase in membrane fluidity in concert with reduced permeability as adaptive response of both archaea and bacteria to elevated sediment temperatures. In bacteria, the data point out to an increase in fatty acid chain length concomitantly with an increased degree of unsaturation. However, the abundance of several lipid classes was altered in response to elevated temperatures. Apart from sphingolipids, cardiolipin (CL) was one of the

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Conlusions and future perspectives bacterial polar lipids enriched in high temperature sediments. Because of the role of CL in controlling membrane curvature stress, it was hypothesized that bacterial cells might control homeostasis by reducing their size in response to heat stress. This hypothesis is in line with the enrichment in CL within membranes of two major human pathogens categorized as nano-bacteria: Mycoplasma and mini-cells of E. coli (Kornspan and Rottem, 2012; Koppelman et al., 2001). In archaea, the tetraether core lipids were intensely modulated with elevated temperatures. Here, it was hypothesized that cyclopentane rings and additional methyl groups in the C40 isoprenoidal chains lead to more fluid membranes, whereas the covalent bond between two C40 isoprenoids lead to an increase in membrane robustness (i.e. lowering permeability). The addition of rings and methyl groups in tetraethers may thus alter substantially the membrane fluidity, which might in turn influence the size of archaeal cells under stress. This could be evaluated in future studies investigating archaeal pure cultures. Chapter IV contains data which confirms major lipid findings of chapter III in a different shallow-water hydrothermal system off the coast of Dominica. Diversity of archaeal polar lipids were comparable with the lipid diversity in Milos’ sediments. Furthermore, temperature-related lipid modifications, e.g. H-GDGT and H-(n)Me- GDGT, could be identified and were more abundant at elevated temperatures. Additionally, multivariate analyses revealed that H-(n)Me-GDGT also correlated strongly with geochemical parameters like elevated hydrogen sulfide concentrations and salinity. However, glycolipids, especially the neutral mono- and di-glycosidic tetraethers, were the dominant archaeal lipids and a hallmark in all investigated samples in shallow- water hydrothermal sediments off the coast of Milos and Dominica. While this trend is consistent with findings from various other marine settings like the deep marine biosphere, hot springs and the water column it shows striking descrepancies compared to GDGT-producing archaeal cultures, such as methanogens (Koga et al., 1998; Yoshinaga et al., 2015; Becker et al., 2016), thermophilic archaea (Nemoto et al., 2003; Jahn et al., 2004; Lai et al., 2008; Shimada et al., 2008; Munk Jensen et al., 2015; Gagen et al., 2016; Golyshina et al., 2016) and Nitrosopumilus maritimus (Elling et al., 2014; 2017), which comprise between ~7.5 and 100% of anionic phospholipids of the total polar lipid content. Whether the predominance of archaeal glycosidic lipids in shallow-water hydrothermal systems reflects the adaptation and protection of archaeal cell membranes against environmental insults such as extremely low pH (Wang et al., 2012) and elevated temperatures of hydrothermal sediments (Sollich et al., 2017 and references therein) or is

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Chapter VI the results of an exceptional high preservation potential of glycolipids, remains an open question with potentially profound implications for microbial ecology. The final chapter of this thesis revealed the carbon transfer from chemosynthetic bacteria to higher trophic levels in the fluids from diffuse-flow vents of the East Pacific Rise 9°50’N (Figure VI.1.). The combined lipidomic approach with stable carbon isotopic analysis of lipids allowed predicting that eukaryotes rather than bacteria as the dominant source of lipids in the samples (mostly composed of triacylglycerols and wax esters). Interestingly, however, polyunsaturated fatty acids (PUFA) such as the docosahaexanoic and arachidonic acids (DHA and ARA; to a great extent esterified in phospholipids) showed δ13C signatures typical of bacterial lipids fixing carbon via the reverse tricarboxylic acid cycle. The possibility for PUFA synthesis at these hydrothermal systems raises the potential for small eukaryotes such as protists or ciliates/flagellates being the major source of abundant PUFA present in vent macrofauna. Metagenomic studies in deep-sea vents have increased awareness for an abundant and diverse component of the food-web composed of small eukaryotes (Moreira and López-García, 2003; Anantharaman et al., 2016). Because most of the benthic macrofauna associated with these ecosystems are described to possess variable levels of PUFA in their membranes, it can be concluded that they are essential for these organisms to survive the extreme environmental conditions faced at deep-sea vents. It will be thus interesting to examine genome sequences related to the synthesis of these PUFA in future genomics studies of small eukaryotes in these ecosystems. Furthermore, it would be interesting to investigate the archaeal lipid diversity and abundance to the total microbial community in future surveys. Exluding eukaryotic lipids may reveal a significant archaeal contribution of the microbial community in hydrothermal diffuse flow systems.

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Figure VI.1. | Sampling locations investigated in this thesis.Crab Spa and Teddy Bear images: courtesy of Woods Hole Oceanographic Institution. This figure was made with Generic Mapping Tools (GMT) (Wessel et al., 2013) and included the bathymetric map of Amante and Eakins (2009).

VI.1. References

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