The Dead Sea

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Nov 23, 2016 - The DNA recovered in the ICDP cores suggests that different microbial ... evaporation-precipitation ratio in the region, our data suggest that ...... The activity of the microbial community in the sediments is however still hard ..... methanogenesis primarily drive microbial colonization of the highly sulfidic Urania.
Thesis

Investigating the subsurface biosphere of a hypersaline environment the Dead Sea (Levant)

THOMAS, Camille

Abstract In the framework of the Dead Sea Deep Drilling Project, a geomicrobiological investigation has taken place to understand the extent and characteristics of life in the hypersaline sediment of the Dead Sea. The DNA recovered in the ICDP cores suggests that different microbial assemblages are associated with particular sedimentary facies, regardless of their depth and in situ salinity. Since this facies are controlled by changes in the evaporation-precipitation ratio in the region, our data suggest that subsurface microbial assemblages are themselves highly influenced by climatic variations at the time of sedimentation. In particular, humid periods allow the development of varied metabolisms such as sulfate reduction, methanogenesis and potentially anaerobic methane oxidation, deeply influencing the carbon and sulfur cycles of the lake, and subsequently allowing the formation of diagenetic Fe-S minerals. These results reveal the importance of considering microbial impact on archives retrieved from lacustrine drilling project, even in extreme environments.

Reference THOMAS, Camille. Investigating the subsurface biosphere of a hypersaline environment - the Dead Sea (Levant). Thèse de doctorat : Univ. Genève, 2015, no. Sc. 4769

URN : urn:nbn:ch:unige-737244

Available at: http://archive-ouverte.unige.ch/unige:73724 Disclaimer: layout of this document may differ from the published version.

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UNIVERSITE DE GENEVE

FACULTE DES SCIENCES

Département des sciences de la Terre

Professeur D. Ariztegui

Investigating the Subsurface Biosphere of a Hypersaline Environment – The Dead Sea (Levant)

THESE présentée à la Faculté des sciences de l’Université de Genève pour obtenir le grade de Docteur ès sciences, mention sciences de la Terre

par

Camille Thomas de Dijon (France)

Thèse N° 4769

GENEVE Atelier de reprographie ReproMail 2015

This thesis was accomplished with the financial support of the Swiss National Science Foundation through grants 200021-132529 and 200020-149221/1 at the Earth Science Department of the University of Geneva, Geneva, Switzerland

Remerciements

S’il est vrai que la thèse peut parfois décourager un doctorant du monde de la recherche, j’ai peur que pour moi elle ne l’ait rendu difficilement remplaçable. Les opportunités qui m’ont été offertes, les rencontres que j’y ai faites et la confiance qui m’a été accordée ont certainement contribué à faire de moi ce que je suis aujourd’hui et je serai toujours reconnaissant envers ces personnes qui m’ont accompagné pendant ces quatre années. En particulier, je remercie mon directeur de thèse, qui m’a fait confiance, qui m’a toujours incité à étendre mes connaissances, a soutenu chacune de mes initiatives et a su me tirer vers le haut et me redonner confiance lors des moments de doute et de remise en question. Daniel est un scientifique formidable, un superviseur disponible et attentif, un collègue altruiste et juste, bref un directeur de thèse exceptionnel. Sans lui, cette thèse serait bien évidemment différente, et moi-même je le serais. Merci Daniel. Je tiens aussi à remercier mon collègue et collaborateur Danny Ionescu. Danny taught me a lot, chaperoned me in the field of microbial ecology and shared a lot with me. He also involved much of his time in the writing of the articles, and I am grateful of all he did for me. I hope to continue working with him. And of course, I thank Mina, whom I know was always present in the background when Danny was reviewing my work on Sunday nights… I learnt a lot during this PhD, I thus want to thank all the people who shared their knowledge in the lab with me, from Emanuela Reo, Franck Lejzerowicz, Laure Apothéloz-Perret-Gentil, Jan Pawlowski, to Stefan “Zwiebel” Thiele, Muriel Pacton, Arnauld Vinçon-Laugier et bien sûr Vincent Grossi, qui m’a accompagné lors de mon master, et avec qui j’ai toujours plaisir à travailler et (essayer d’) apprendre. Je remercie aussi ceux qui m’ont aidé un jour aiguillé ou conseillé, je pense notamment à Christophe Dupraz, à Robert Moritz, à Pilar Junier, à Kurt Hanselmann, ainsi qu’à Véronique Gardien, dont les recommandations ont certainement contribuées à mon engagement dans cette thèse. Merci également à Jean-Michel Jaquet et Christos Kanellopoulos pour leur confiance. Je remercie aussi tous les gens dont j’ai croisé la route au cours de ces années et avec qui j’ai partagé sciences, rires et bières. Notamment les étudiants de l’université d’Osnabrück, les compagnons microbiologistes et pédologues de Neuchâtel et Lausanne, les vacanciers géologues abonnés aux cours CUSO… I also wish to thank my colleagues remotely or closely linked to the Dead Sea project. Those that helped logistically and humanly to the core opening parties, namely Stefi, Florian, Romain… Also those that contributed to great scientific and social exchanges, Elan, Gilad, Orit, Nicolas, Elisa, Yossi, Ittai, Moti, Adi, Yael E and Yael K, Ron, Daniel, Norbert, Achim, Markus, and of course Ina. Je remercie également les membres de l’unige: Fred, François, Jacqueline, Elisabeth, Christine, Elias, Rossana, Guy, Sébastien, Georges et Eric, pour leur gentillesse et leur disponibilité. Merci à la team Corse, ceux qui ont partagés la dureté du sol sous la tente, le froid des nuits Cortenaise, les griffures du maquis, la brulure du soleil printanier, la houle méditerranéenne, les vents hurlants de Nonza, la faune agressive, la flore i

cracheuse. Antonio, Elme, et bien sûr Chloé vous étiez là et vous avez survécu. Merci d’avoir tenu le coup et d’avoir fait de ces moments d’adversité des souvenirs impérissables. Merci aussi à Monique maman locale et réconfort incarné. Enfin merci à Mario également pour tous ces moments, pour sa bonté, son sourire, tout ce que tu transmets aux étudiants et personnes qui t’entourent. J’en viens à la longue liste des collègues et amis de l’université de Genève, qui ont fait de ces années de thèse des moments inoubliables, autant dans l’adversité que dans la fête. Mes collègues du hip-hop bureau Jay Rag et SmArTy MaRtY qui ont amené et maintenu le groove, la vibe et le swag au bureau. Je remercie aussi Aurèle, pour le temps qu’il ma consacré, les connaissances (innombrables) qu’il a voulu partager avec moi, et les grands moments passés ensemble, au labo, dans le bureau, sur le terrain et en vacances ! Merci à mes amies et (gossip) girls préférées, Lina, Mélanie, Cristina, Katrina. Vous me manquez. Merci à leur pendant masculin Camille, toujours prêt pour partager les meilleures nouvelles scientifiques et people du moment. A celui qui a partagé ma vie, qui m’a nourri et blanchi pendant deux années. Merci Valentin pour tout ! Merci à Stéphanie Girardclos pour ses conseils, ses grandes discussions (son whisky). Merci à mon équipier gagnant Cyril Chelle-Michou. Merci à notre adversaire Nicolas Saintilan. La victoire est belle car l’adversaire est beau. Aux surhommes géologiques, Arnoud, Gabriel, Aymeric. A la squadra azzurra Federico, Eduardo, et à celle qui gagne, Haseeb, Mortaza. Merci Agathe pour les tête-à-tête à balayage électronique. Merci Noel, Hosseini, Chen Chen, merci les frenchies Vincent, Bertrand, merci les bahamiennes Erika, Jennifer, merci Tiago, merci Neda, merci Paula, merci les petits nouveaux Luis et Ines. Merci les maraichers, Alexandra, Roelant, Sylvain, Jerôme, Matar, Chadia et tous ceux que j’oublie. J’en rajoute encore, Laure, Stéphane, Julien, Marie, Camille, Basti, Chia-i, Hervé, Alexia, Maria, Christian. Merci aussi aux étudiants, qui ont contribué à mon bien être ici. Si j’ai envie de continuer aujourd’hui, c’est aussi parce qu’ils sont géniaux. Merci aux volées corses, aux membres de l’AEST (Antoine en particulier), et à tous ceux qui ont participé de près ou de loin à la vie maraichère : la Yourghurteria, le Volt, le Lys, le Trulli, le MacSorleys, la Ferblanterie, Kendrick, Frank, Jonsi, Thom, Beth and Geoff, tous. Il y a quelques personnes clés qui ont contribué à ce que j’ai pu entreprendre dans la géologie. Merci Lydie Prieur, pour m’avoir conseillé puis soutenu. Je suis fier aujourd’hui d’avoir été votre étudiant, et de pouvoir vous montrer quelques résultats géologiques ET biologiques. I also wish to thank Salik and Minik Rosing, who have somehow, changed the course of my short geology career. Enfin merci à Fabrice Cordey et Nicolas Olivier, qui m’ont fait confiance pendant mon master et qui ont contribué à mon entrée dans la recherche. Leur patience, leurs conseils et leur soutien m’ont permis d’entreprendre cette thèse avec confiance et optimisme. J’en viens au privé. Merci à tous mes amis, ceux de Lyon, d’Islande, du Danemark et surtout ceux de Dijon. Mes meilleurs amis à qui je n’ai pas pu consacrer beaucoup de temps au milieu de tout cela. J’ai aussi pu faire cette thèse car je sais que vous êtes à mes côtés. En particulier, merci Antoine, merci Caroline, merci Fabien, merci Mathilde C., merci Mathilde S., merci Maxime, merci Olivier. ii

Je remercie aussi toute ma famille, qui m’a toujours soutenu. En particulier, merci à ma mère et mon père de croire en moi, et d’être fantastiques. Merci également à mes sœurs Juliette et Léa pour qui je n’ai pas beaucoup été là ces derniers temps, mais qui m’aident et m’équilibrent. J’espère leur apporter autant que ce qu’elles me donnent. Enfin, je terminerai par celle à qui j’ai certainement volé le plus de temps, et celle qui a dû faire plus de sacrifices que tous les autres. Merci Marie de m’avoir soutenu, merci de m’avoir accompagné, et merci pour ce que tu amènes dans ma vie. Sans toi je n’aurais pu accomplir tout ça. Je t’en remercie profondément, et suis impatient d’aborder la suite avec toi.

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Abstract

The Dead Sea Deep Drilling Project is an ICDP sponsored project that aims to reconstruct the Quaternary paleoenvironments of the Dead Sea Basin. Within this framework, a geomicrobiological investigation has been realized in order to assess the potential effect of microbial communities on the sediment, as well as their extent. Similarly to wellestablished studies in the oceanic subsurface, geomicrobiological analysis are now employed in lacustrine subsurface, an environment that is allegedly more susceptible to environmental and climatic disturbance than its marine counterpart. In the Dead Sea, the study also aims at qualifying the development of microbial life in extreme conditions of salinity.

A 457 m-long sedimentary core has been retrieved from the middle of the Dead Sea, in winter 2010-2011. Contrasting climatic intervals were highlighted by distinctive lithological facies such as laminated aragonitic mud for humid periods and glacial stages and halite-gypsum deposits for interglacial intervals dominated by high aridity. For the first time a deep subsurface life could be evidenced in the Dead Sea sediments through its DNA. Although living conditions are extremely harsh in this environment, a specific population has adapted and survives at depths down to at least 90 m below the present water-sediment interface, and probably deeper (200 m). Different lithological types hold specific microbial populations representative of changing environments. Members of bacterial KB1 and MSBL1 Candidate Divisions could take advantage of the osmotic solutes such as glycine betaine, available in the aragonitic sediments. Halite and gypsum sediments are dominated by extreme halophilic Archaea of the Halobacteriaceae family, which are today the only inhabitants of the lake water column. The similarity shared by gypsum-halite samples communities, and the importance of freshwater inputs for assemblages of the aragonitic sediment imply that salinity is not the only parameter influencing microbial development, but that the whole lake water balance at time of sedimentation is in part responsible for the current distribution of microbes in the sedimentary column.

Within the two main sedimentary facies previously established, archaeal metabolisms were deeply investigated using high-throughput DNA sequencing. We show that the communities are well adapted to the peculiar environment of the Dead Sea subsurface. They are able to deal with osmotic pressure using high- and low-salt-in strategies, and can also cope with high concentrations of heavy metals. Methanogenesis (from methylated compounds), for the first time identified in the Dead Sea, is an important metabolism in the aragonite sediment. Fermentation of organic matter, probably performed by some members of the Halobacteria class is common to both aragonite and gypsum-rich sediments. Genes associated with sulfur reduction have also been revealed and are associated in the sediment with microbial EPS degradation and Fe-S mineralization as revealed by SEM imaging. v

Through comparison with an active microbial mat of the Dead Sea shore, keys towards the recognition of Fe-S mineralizations of biological origin have been acquired, emphasizing the influence of a microbial sulfur cycle on mineral and organic matter. The occurrence of EPS is interpreted as an indicator of the activity of microbes in the sediment. Degradation of EPS and that of allochthonous organic matter by putative dissimilatory sulfate reduction releases H2S, enabling the formation of Fe-S precursors, which can form pyrite spherulites. Mineralizations are however small and rarely complete, supporting an incomplete sulfate reduction pathway for microbes in the Dead Sea sediment. Analogously, nanoglobules occurrence and their tight association with aragonite needles also support the use of EPS as a matrix for biologically-influenced mineralization of calcium carbonates. These results have strong implications on the fate of organic matter and on processes involved in mineralization and porosity development in the perspective of diagenetic evolution of hypersaline subsurface systems.

Higher concentration of organomineralization under the form of euhedral pyrite suggests an increase sulfate reduction activity in particular in one precise interval of the Dead Sea core. In this specific early Holocene interval, combined use of elemental and isotopic profiles of the pore water chemistry, high-resolution lithological description and lipid analysis indicate a biological signature during periods when rain dominated over evaporation. This interval also comprises a set of lipidic biomarkers indicative of anaerobic oxidation of methane. Among others, the presence of hydroxyarchaeol, pentamethyleicosene and extended archaeol highlights the reworking of biogenic methane by potential ANME Archaea. Anaerobic oxidation of methane associated with bacterial sulfate reduction is thus suggested to have disturbed organic proxies in this early Holocene interval. In conclusion, as life extent is now better constrained in the Dead Sea subsurface, influence of the environment over its development is clear. Hypersalinity selects for hyper-halophilic groups. Additionally, humid episodes allow for development of diverse metabolisms in the water column, the latter being reflected in today’s subsurface communities. Such impact shows the need for routinely applying geomicrobiological investigations in the lacustrine realm, and of unifying methods for such research in the context of International Continental Drilling Projects. Microbial effects on mineral precipitation, isotope fractionation and organic matter preservation clearly exist in extreme environments like the Dead Sea and should always be taken into account for further paleoenvironmental reconstruction and future ecological and climatic models.

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Ré sumé

Le Projet DSDDP (projet de forage profond de la Mer Morte), cofinancé par le consortium international pour les forages continentaux (ICDP), a pour objectif de reconstruire les paléo-environnements quaternaires du bassin de la Mer Morte, au Proche-Orient. Au sein de ce projet, une étude des interactions entre minéral et vivant a été entreprise afin de contraindre la potentielle influence des microbes sur leur environnement. De façon similaire à ce qui a été entrepris depuis plusieurs décennies maintenant au sein du consortium international pour les forages océaniques (IODP), des études géomicrobiogiques sont maintenant menées dans les environnements lacustres. Ces derniers sont d’ailleurs plus susceptibles aux variations environnementales et climatiques que le fond des océans. Dans le cas de la Mer Morte, l’extrême salinité de cet environnement présente également un grand intérêt quant à la compréhension des limites du vivant. Une carotte sédimentaire de plus de 450 m de long a été forée au milieu de la Mer Morte en hiver 2010-2011. Son étude montre que les variations sédimentologiques reflètent les contrastes climatiques des 230 000 dernières années. Un facies de lamines aragonitiques marque les périodes relativement humides, caractéristiques des stades glaciaires, tandis que des unités de gypse et halite caractérisent l’augmentation de l’aridité pendant les périodes interglaciaires. Pour la première fois, la présence de vie profonde au sein du sédiment de la Mer Morte a été mise en évidence à travers son ADN. Bien que les conditions de vie soient extrêmes, des communautés microbiennes spécifiquement adaptées survivent jusqu’à des profondeurs de 90 m dans le sédiment, et s’étendent possiblement jusqu’à 200 m de profondeur. Ces communautés diffèrent en fonction de la lithologie du sédiment. Des membres des Divisions Candidates bactériennes KB1 et MSBL1 profitent des solutés osmotiques potentiellement disponibles dans le sédiment aragonitique. La classe d’Archaea hyper-halophile Halobacteriaceae, aujourd’hui principale représentante dans l’eau du lac, domine quant à elle les niveaux de gypse et halite. Les similarités d’assemblages partagées par les niveaux de gypse et halite, et l’importance des apports d’eau douce pour les communautés présentes dans les niveaux aragonitiques suggèrent que les conditions hydrologiques et limnologiques dominantes lors de la mise en place des dépôts ont une grande influence sur les communautés que l’on retrouve aujourd’hui dans le sédiment. Au sein des deux principaux facies décrits ci-dessus, les métabolismes archées ont été analysés par le biais de séquençage d’ADN à haut rendement. Ces communautés semblent capables de gérer l’hypersalinité du milieu en utilisant les deux principales méthodes d’équilibrage osmotique, et peuvent aussi s’accommoder d’importantes concentrations en métaux lourds. Pour la première fois identifiée en Mer Morte, la méthanogénèse (à partir de composés méthylés) est un métabolisme clé du sédiment aragonitique. La fermentation de la matière organique résiduelle par des Halobacteria a lieu dans les deux types de sédiment. Des gènes de sulfato-réduction ont aussi été vii

détectés aux seins des métagénomes, en association avec la dégradation de biofilms microbiens et la présence de minéralisations de Fe et S.

A travers une comparaison avec un tapis microbien actif des bords de la Mer Morte, des clés de reconnaissances de minéralisations Fe-S d’origines microbiennes ont été acquises, et permettent de confirmer l’existence d’un cycle microbien du S en Mer Morte. La présence d’EPS témoigne de l’activité microbienne au sein du sédiment, et leur dégradation par sulfato-réduction produit des sulfures permettant la formation de minéraux précurseurs de la pyrite. Les minéralisations sont toutefois incomplètes, témoignant d’un processus incomplet de sulfato-reduction au sein du sédiment. De manière analogue, une relation intriquée entre EPS, nanoglobules et aiguilles d’aragonite suggère une influence microbienne sur la précipitation des carbonates de calcium en Mer Morte. Ces résultats ont de grandes implications en ce qui concerne la préservation de la matière organique et le développement des minéraux et de la porosité pendant les processus précoce de diagénèse en milieux hypersalins.

La mise en exergue d’une plus grande concentration d’organominéralisation de sulfure de fer dans un intervalle du début de l’Holocène, suggère une augmentation des taux de sulfato-réduction, pour cet intervalle précis. L’utilisation combinée de profiles élémentaires (sulfate) et isotopiques (C inorganique, S et O des sulfates) de l’eau interstitielle de la carotte, d’une description lithologique à haute résolution et des variations de signature lipidiques indiquent une période d’intense activité microbienne, en contexte relativement humide. Cet intervalle présente notamment un éventail de biomarqueurs indicatif d’une activité d’oxydation anaérobique du méthane. Entre autres, l’association de pentamethilycosene, archaeol et hydroxyarchaeol suggère la présence d’Archaea méthanotrophes du groupe ANME. L’oxydation anaérobique du méthane par ce groupe, associé avec des bactéries sulfato-reductrices semble avoir influencé certains proxies utiles aux reconstructions climatiques du début de l’Holocène. En conclusion, la présence de vie et son extension sont aujourd’hui mieux contraintes dans les sédiments de la Mer Morte, et l’influence de l’environnement sur son développement est claire. La salinité est le principal vecteur de sélection des assemblages microbiens. Toutefois, l’apport d’eau douce en période humide permet le développement d’espèces aux métabolismes plus variés. Leur activité passée est aujourd’hui reflétée par les assemblages aujourd’hui présents dans le sédiment. En contexte lacustre, l’impact climatique sur les communautés microbiennes témoigne du besoin de régulièrement prendre en compte la microbiologie du sédiment, et également d’unifier les méthodes d’investigation au sein des projets ICDP. L’activité microbienne a un effet sur les signatures élémentaires et isotopiques de nombreux éléments, ainsi que sur la préservation de la matière organique, et ce même en conditions d’extrême salinité. Ce travail démontre donc la nécessité de systématiquement prendre en compte le rôle des microbes lors d’études paléoenvironnementales et lors de la construction de futurs modèles écologiques et climatiques. viii

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TABLE OF CONTENTS

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Remerciements ………………………………………………………………………………………….. i Abstract …………………………………………………………………………………………………….. v Résumé ……………………………………………………………………………………………………. vii Table of contents …………………………………………………………………………………….... xi List of tables ……………………………………………………………………………………..……… xv List of figures …………………………………………………………………………………….……. xvi I. Introduction: geomicrobiology and the Dead Sea Deep Drilling Project….. 1 1.1 Geomicrobiology and the importance of the microbial world to geology………………………….. 3

1.2 The Dead Sea Deep Drilling Project ……………………………………………………………………………….. 7

II. Current microbial life in the Dead Sea sediment …………………………………. 17 2.1 Introduction ………………………………………………………………………………………………………………. 19

2.2 Impact of paleoclimate on the distribution of microbial communities in the subsurface

sediment of the Dead Sea………………………………………………………. …………………………………………... 20 2.2.1 Introduction …………………………………………………………………………………………………………… 21

2.2.2 Materials and methods…………………………………………………………………………………………….. 22 2.2.3 Results ……………………………………………………………………………………………………………………..28 2.2.4 Discussion ………………………………………………………………………………………………………………. 32

2.2.4.1 Sediments as archives of environmental changes in the paleo Dead Sea …………….…... 33 2.2.4.2 Water column artifact or in situ communities ………………………………………………………. 35

2.2.4.3 Key parameters for the Dead Sea sediment subsurface ecology …………………………...... 36

2.2.4.4 Microbial community of halite-gypsum samples …………………………………………………… 39 2.2.4.5 Microbial community of aragonitic samples ………………………………………………………..... 40

2.2.4.6 Microbial communities in continental sediments ………………………………………………….. 42

2.2.5 Conclusion ………………………………………………………………………………………………………………. 43

2.3 Fluid inclusions as time capsules of chemistry and life ………………………………………………….. 44

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III. Metabolic potential of microbial communities in the subsurface of the Dead Sea ………………………………………………………………………………………………….. 59 3.1 Metagenomic studies in the Dead Sea subsurface…………………………………………………………. 61 3.2 Archaeal populations in two distinct sedimentary facies of the subsurface of the

Dead Sea……………………………………………………………………………………………………………………………. 61

3.2.1 Introduction …………………………………………………………………………………………………………... 62

3.2.2 Methods ………………………………………………………………………………………………………………… 64

3.2.3 Results …………………………………………………………………………………………………………………... 66

3.2.4 Discussion ……………………………………………………………………………………………………………... 71 3.2.4.1 Metagenomics in poorly characterized environments …………………………………………. 71

3.2.4.2 Coping with harsh Dead Sea conditions ……………………………………………………………… 75

3.2.4.2.1 Salinity …………………………………………………………………………………………………………. 75

3.2.4.2.2 Toxicity of metals ………………………………………………………………………………………….. 76

3.2.4.3 Coping with deep sedimentary environments …………………………………………………….. 77

3.2.4.3.1 Fermentation ………………………………………………………………………………………………... 77

3.2.4.3.2 Methanogenesis …………………………………………………………………………………………….. 78

3.2.4.3.3 Sulfur reduction …………………………………………………………………………………………….. 79 3.2.4.3.4 Nitrogen cycle ……………………………………………………………………………………………….. 81

3.2.5 Conclusion …………………………………………………………………………………………………………….. 81

IV. Influence of microbes on the sedimentary record of the Dead Sea Basin………………………………………………………………………………………………………….97 4.1 Introduction …………………………………………………………………………………………………………….… 99

4.2 Geological setting ………………………………………………………..……………………………………………. 101

4.3 Material ……………………………………………………………………………………………………………………. 102

4.4 Methods ……………………………………………………………………………………………………………………. 105

4.5 Results ……………………………………………………………………………………………………………………… 105 4.5.1 X-ray fluorescence scanning ………………………………………………………………………………...... 105

4.5.2 Scanning electron microscopy ……………………………………………………………………………….. 107

4.6 Discussion …………………………………………………………………………………………………………….….. 111

4.6.1 Occurrence of traces of microbial activity in the Dead Sea sediment .……………………...... 111

4.6.2 Organic matter production and sulfate reduction .…………………………………………………… 115

4.6.3 Microbial effect of early diagenesis in the deep Dead Sea sediment…………………………... 118

4.7 Conclusion ………………………………………………………………………………………………………..……… . 122

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V. Influence of life in the Dead Sea geochemical record ………………………… 129 5.1 General influence of the biosphere on the carbon cycle of the Dead Sea (and its

precursor lakes) …………………………………………………………………………………………………………….. 131 5.1.1 Introduction to the Dead Sea carbon cycle …………………………………………………………… 131

5.1.2 Carbon isotopes in the Dead Sea realm ………………………………………………………………… 132

5.1.3 Aragonite precipitation and its δ13C signature in the core ……………………………………... 135

5.2 Anaerobic oxidation of methane disturbed organic proxies in the Early Holocene

Dead Sea ……………………………………………………………………………………………………………………….. 138 5.2.1 Introduction ………………………………………………………………………………………………………. 139

5.2.2 Geological setting ……………………………………………………………………………………………….. 140

5.2.3 Material ………………………………………………………………………………………………………………140

5.2.4 Methods …………………………………………………………………………………………………………….. 141 5.2.5 Results ………………………………………………………………………………………………………………. 143

5.2.6 Discussion …………………………………………………………………………………………………………. 147

5.2.6.1 Decoupling of isotopes with the precipitation-evaporation ratio in P6 ……………... 147

5.2.6.2 Information carried by biomarkers in the Dead Sea framework …..…………………… 149

5.2.6.3 Anaerobic oxidation of methane ……………………………………………………………………… 151

5.2.6.4 Diachronous δ13CDIC and δ34Ssulfate extrema ……………………………………………………….. 152 5.2.6.5 Tentative model for enhanced microbial activity ……………………………………………... 153

5.2.7 Conclusion …………………………………………………………………………………………………………. 154

VI. Conclusion: the subsurface biosphere of the Dead Sea and lacustrine geomicrobiology …………………………………………….………………………………….….163 6.1 Towards the building of a unified model for geomicrobiology in the Dead Sea

subsurface……………………………………………………………………………………………………………….…… 165 6.2 Reactions of microbial ecosystems to a changing environment: can the past of

lakes be a key to the future …………………………………………………………………………………….…….. 168 6.2.1 From surface to bottom: microbial action in a changing environment ………….……... 168 6.2.2 When subsurface microbiology reflects climatic variations: geomicrobiology

of lacustrine environments ………………………………………………………………………………………….. 170 6.2.3 Collaborative approach for understanding future changes in the aquatic

biosphere of lakes and oceans ……………………………………………………………………………………… 171

Appendix ……………………………………………………………………………………………. 175 xiv

LIST OF TABLES Chapter II.

Table 2.1 Sample description and principal geochemical characteristics ………………………………. 25

Table 2.2 Summary of primers for PCR amplification …………………………………………………………… 28

Table 2.3 Summary of PCR conditions …………………………………………………………………………………. 28

Table 2.4 Summary of number of clones picked and obtained OTUs ……………………………………… 32

Table 2.5 Sequences recovered from fluid inclusions …………………………………………………………….49

Chapter III. Table 3.1 Sequence information and diversity indexes for metagenomic samples …………………. 67

Table 3.2 M5RNA annotations for Archaea of sample AD ……………………………………………………… 68

Chapter V. Table 5.1 Principal chemical characteristics of lipid biomarker samples ………...…………………… 143

Appendix Table A1 OTU definition and presence/absence in each sample ………………………………………….. 177

Table A2 OTU distribution and phylotypes definition …………………………………………………………. 180 Table A3 Calibration values for microthermometry analysis ………………………………………………. 180

Table A4 Temperatures of homogenization for 4 samples of the Holocene part of the core ...... 181

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LIST OF FIGURES Chapter I.

1.1 Prokaryotic cell concentration and distribution in the sediment …………………………………… … 5

1.2 Idealized biogeochemical zone scheme in the marine sediment ……………………………………….. 6 1.3 ICDP drilled and planned lacustrine sites ………………………………………………………………………... 6 1.4 Location, elevation and geology of the Dead Sea Basin …………………………………………………….. 8 1.5 Unconformity between the Samra and Lisan Formations ………………………………………………… 9

1.6 Earthquake-related wavy structure within AAD laminations …………………………………………..10

1.7 Overview of DSDDP drilling sites ………………………………………………………………………………….. 11

Chapter II. 2.1 Overview of the sampling sites ……………………………………………………………………………………… 23

2.2 Detailed workflow for 16S rRNA gene sequence analysis ……………………………………………….. 27

2.3 DNA extraction samples and TOC and C/N profiles along the core ………………………………….. 29 2.4 CARD-FISH images of samples CT1 and CT3 ………………………………………………………………….. 31

2.5 Identified archaeal and bacterial OTUs ………………………………………………………………………….. 34

2.6 16S rRNA gene based phylogenetic tree of obtained Archaea sequences ……………………….... 37

2.7 Cluster analysis of identified phylotypes in the Dead Sea samples ………………………………….. 39

2.8 Halite rafts formation in the Dead Sea in December 2010 ………………………………………………. 45

2.9 Fluid inclusions and trapped organic matter in drilled halite ………………………………………….. 48

2.10 Putative Dunaliella cell enclosed in a fluid inclusion……………………………………………………… 48

Chapter III. 3.1 Archaeal classes from AD and GY …………………………………………………………………………………. 69

3.2 Heatmap of level 1 subsystems ……………………………………………………………………………………. 71

3.3 Heatmap of subsystems for osmotic adaptation and heavy metal tolerance …………………... 72

3.4 Heatmap of subsystems for methanogenesis, fermentation, nitrogen and sulfur

metabolisms …………………………………………………………………………………………………………………… 74

3.5 Indications if active S cycle in the sediment core ………………………………………………………….. 77

3.6 SEM pictures of active organic matter and potential sulfate reduction ………………………….. 80

xvi

Chapter IV. 4.1 Overview of sampling sites and material ……………………………………………………………………... 103

4.2 Aragonite needles, Dunaliella algae and EPS in the microbial mat………………………………….. 104

4.3 µ-XRF mapping of the microbial mat ……………………………………………………………………………. 107 4.4 Lithology and Fe-S content of the core …………………………………………………………………………. 108

4.5 Fe-S mineralization morphologies in the Dead Sea realm ……………………………………………… 110

4.6 EDX composition of Fe-S mineralizations ……………………………………………………………………... 112

4.7 Photographs of the cores exhibiting sulfur mineralizations…………………………………................114

4.8 SEM photographs of EPS morphologies in the core ……………………………………………………….. 117

4.9. Photographs of EPS structures in the mat and the core ………………………………………………… 119

Chapter V. 5.1 Carbon isotopes of DIC in the Dead Sea between 1963 and 1994 …………………………………….136

5.2 Carbon isotopes of aragonite in the core ………………………………………………………………………. 137

5.3 Location of the Dead Sea and the DSDDP drill sites ……………………………………………………….. 138

5.4 Lithological and geochemical profiles of the first 120 m of the core ……………………………….. 144 5.5 Lipid profiles of samples S17, S18, S19 and S21 ……………………………………………………………. 146

5.6 Proposed structures of mGD and macrocyclic archaeol……………………………..…………………… 147

5.7 Euhedral morphologies of Fe-S mineralizations in sample S17 ……………………………………... 148

Chapter VI.

6.1 Summary of the analysis carried out on the core core material within the PhD project…….168

6.2 Model for microbial assemblage settlement in the Dead Sea sediment …………………………... 169

6.3 Study of ecosystems evolution and reaction in a changing environment ……………………….. 172

Appendix Fig. A1 Non-specific binding of tyramide on Dead Sea sediment …………………………………………. 182

Fig. A2 16S rRNA gene based phylogenetic tree of obtained Bacteria sequences …………………. 182

Fig. A3 Comparison of microbial diversity between clones amplicons and metagenomes……... 183

Fig. A4 Distribution of temperatures of homogenization from Holocene fluid inclusions ……… 183

Fig. A5 Halite block used for calibration of microthermometry …………………………………………… 183

Fig. A6 Carbon isotopes of AAD laminae of the microbial mat……………………………………………....184

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Fig. A7 Mass spectra of pentamethylicosene…………………………. …………………………………………... 184

Fig. A8 Partial chromatogram of fraction F3 for samples S17 to S21…………………………………….. 185 Fig. A9 Mass spectra of mGD and macrocyclic archaeol………..……………………………………………... 186

Fig. A10 SEM picture of greigite in P6 zone …………………………………………………….………………….. 187

Fig. A11 SEM picture of framboidal pyrite in P6 zone ………………………………………….……………… 187

Fig. A12 Linear relationship between δ18O and δ34S of pore water sulfate ……………………...……. 188

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xix

xx

 

Chapter  I.   Introduction:  geomicrobiology  and  the   Dead  Sea  Deep  Drilling  Project                                                                       “2+2  =  5”   Hail  to  the  Thief,  Radiohead         1

Chapter  I.  Introduction  

                                                                  2

Chapter  I.  Introduction  

1.1  Geomicrobiology  and  the  importance  of  the  microbial  world  to  geology     The   study   of   small   life   in   the   earth.   The   ancient   Greek   roots   for   the   word   “geomicrobiology”   defines   the   purpose   of   this   scientific   domain.   It   encompasses   the   study   of   microscopic   organisms,   either   prokaryotes   or   eukaryotes,   in   a   geological  realm.   This   geological   realm   may   well   be   a   mineral,   a   rock,   soil,   sediment,   or   any   other   geological   object.   The   definition   is   actually   wider,   as   it   encompasses   any   microbial   process   influencing   a   geological   object.   Therefore,   geomicrobiology   deals   with   the   degradation   of   organic   matter   and   the   synthesis   of   fossil   fuels,   with   bioremediation   in   contaminated   sites   and   bioleaching   in   ore   deposits,   with   management   of   aquifers   and   drinking   water   quality.   Obviously,   it   is   a   key   scientific   domain   that   has   numerous   applications   in   our   modern   society.   It   also   covers   purely   theoretical   scientific   questions,   in   the   domain   of   the   origin,   development   and   limits   of   life,   on   Earth   and   in   the   universe.   The   establishment   of   chemical   or   physical   biologically   derived   signatures   defines   the   search   for   the   first   traces   of   life   on   Earth.   The   understanding   of   the   appearance   of   oxidized   conditions   on   our   planet,   or   the   evolution   of   first   eukaryotic   cells   requires   knowledge  of  interactions  of  life  with  its  immediate  biological  and  mineral  environment.   The  search  for  life  on  other  planets  demands  the  establishment  of  conditions  and  limits   to   its   development,   through   for   instance   the   study   of   extreme   environments.   The   expansion  of  such  science  has  however  been  restrained  by  the  complexity  of  observing   microscopic  to  nanoscopic  features  in  the  environments.  In  the  last  decades,  advances  in   techniques   of   observation   and   DNA   analysis,   mostly   guided   by   the   field   of   medical   sciences   has   allowed   to   establish   geomicrobiology   as   a   relatively   common   branch   of   geology,   consolidating   the   already   existing   bridge   between   the   living   and   the   mineral   worlds.       There   are   very   few   environments   where   microbes   are   not   present.   A   general   rule   has   arisen   from   this:   wherever   there   is   liquid   water,   there   is   life.   May   the   water   be   to   the   extremes  of  pH,  temperature,  radiation,  pressure  or  activity.  Archaea  Pyrolobus  fumarii   can  grow  at  121°C  (Blöchl  et  al.,  1997).  A  barophilic  bacterial  strain  close  to  the  genus   Moritella   has   optimum   growth   at   80   MPa   (Kato   et   al.,   1998).   Deinococcus   radiodurans   can   withstand   up   to   20   kGy   of   gamma   radiation   (Battista,   1997).   The   iron-­‐oxidizing   Archaea  Ferroplasma  acidarmanus  grows  at  pH  0  (Edwards  et  al.,  2000).  Many  archaeal   species   live   at   salt-­‐saturation   levels.   As   a   result,   the   extensive   colonization   of   media,  

  3

Chapter  I.  Introduction  

almost  regardless  of  their  physic-­‐chemical  characteristics,  has  shown  that  the  potential   for   microbial   influence   on   geology   is   everywhere.   This   is   of   major   importance   when   it   comes   to   the   study   of   paleo-­‐climates   and   paleo-­‐environments.   In   most   cases,   such   studies   use   the   sedimentary   record   to   tackle   issues   such   as   climate   change,   or   mass   extinction  events.  This  is  mostly  done  by  combining  different  parameters  influenced  by   the   environment   that   are   called   “proxy”.   These   proxies   may   be   elemental   concentrations,   microfossil   appearances,   isotopic   ratios   or   physical   properties   of   the   rock.   Some   of   these   proxies   are   likely   to   be   influenced   by   biological   activity.   Hence,   they   necessitate   a   very   good   control   over   their   formation   and   preservation.   Projects   involving   paleoclimatic   and   paleoenvironmental   reconstructions   thus   need   a   good   understanding   of   the   microbiological   processes   at   stake   in   the   environment   they   are   studying.  One  of  the  major  sources  for  such  studies  is  the  ocean  floor.  It  is  also  where  the   field   of   geomicrobiology   has   taken   a   major   turn.   Since   1986,   the   International   Ocean   Discovery   Program   (formerly   Integrated   Ocean   Drilling   Program)   has   been   a   major   actor  in  the  development  of  the  field  of  geomicrobiology.  Through  the  unique  recovery   of  deep  sediment,  and  well-­‐adapted  sampling  and  analysis  methods,  the  notion  of  deep   life   has   emerged,   and   with   it,   models   for   the   distribution   of   microbes   in   the   marine   sediment  (Fig.  1.1)  and  the  suite  of  metabolic  processes  without  light  and  oxygen  (Fig.   1.2).   Therefore,   studies   have   allowed   estimating   that   the   sedimentary   prokaryotic   biosphere  accounts  for  one  tenth  of  the  global  biomass,  and  contributes  almost  equally   as  plants  to  the  total  fixed  carbon  (58    to  92  %  of  the  plant-­‐fixed  carbon  mass)  making   them   key   actors   in   the   global   carbon   cycle   (Whitman   et   al.,   1998).   Such   studies   have   pushed  the  limits  of  life  by  for  example  emphasizing    incredibly  slow  growth  rates  in  the   deep   ocean   sediment   (Røy   et   al.,   2012)   and   allowed   to   define   new   widespread   phylogenetic  groups  (e.g.  Miscellaneous  Crenarchaeotic  Group  ,  Inagaki  et  al.,  2003).  The   biomass  has  been  tentatively  quantified  (Fig.  1.1A  and  B)  and  its  tremendous  influence   on   the   global   carbon   cycle   has   been   discussed   (D’Hondt   et   al.,   2004;   Schippers   et   al.,   2005;   Kallmeyer   et   al.,   2012).   Among   others,   the   role   played   by   microbes   in   the   formation  and  decomposition  of  methane  hydrates  (see  AOM  and  methanogenesis  in  Fig.   1.2)   is   deeply   investigated   (e.g.   Kvenvolden,   1995;   Orphan   et   al.,   2002;   Inagaki   et   al.,   2006;  Knittel  and  Boetius,  2009)  as  several  gigatons  of  methane  carbon  are  estimated  to   be  stored  in  continental  margins  (Milkov,  2004).  These  methane  hydrates  form         4

Chapter  I.  Introduction  

    Fig.  1.1:  (A)  Cell  concentration  along  depth  for  different  ocean  sites  and  (B)  geographic   distribution  model  for  cell  number  in  the  sedimentary  column.  Dots  are  calculated  cell   numbers  at  given  sites.  Figures  taken  from  Kallmeyer  et  al.  (2012).     enormous   sources   of   greenhouse   gases   in   case   of   destabilization,   and   have   already   been   evoked   as   the   primary   cause   of   mass   extinction   and   global   climate   change   in   the   past   (e.g.  during  the  Paleocene-­‐Eocene  Thermal  Maximum  (Dickens  et  al.,  1995;  Sluijs  et  al.,   2007)).    Thereafter,  methane  seep  sites  or  gas-­‐hydrate  hosting  shelves  are  prime  target   for  the  investigation  of  the  ocean  floor  subsurface  communities,  and  permit  to  link  two   main   facets   of   IODP   scientific   objectives:   the   deep   biosphere   and   environmental   changes.   Onto   the   continent,   carbon   cycles   are   less   constrained.   For   example,   Whitman   (1998)   estimated   that   the   continental   subsurface   was   7   to   70   %   as   important   as   the   ocean   sediment  one.  The  immense  diversity  of  sites  will  possibly  prevent  any  good  prediction   of  microbial  cell  number,  but  the  specificity  of  some  of  these  environments  have  raised   interest  in  the  scientific  community.  Among  them,  lake  sediments  are  prevailing  target   as  they  form   excellent   archive   of   past   environments   and   are   present   in   very   varied   size,   geological   settings   and   climate   zones.   Geomicrobiology   of   deep   lacustrine   sediment   is   only  at  its  premises,   and   only   very   few   lake   sediments   have   been   drilled   with   a   planned   microbiological  investigation  component  in  the  ICDP  framework  (Fig.  1.3).  However,  the   recognition   of   the   importance   of   integrating   such   studies   and   a   subsequent   sampling   protocol   within   the   standard   tools   of   scientific   drilling   has   now   emerged.   This   is   highlighted   by   the   new   2013   ICDP   Science   Plan,   which   encompasses   for   the   first   time   subsurface  microbiology  as  a  main  focus  point  of  its  program.  

  5

Chapter  I.  Introduction  

 

Fig.   1.2:   Idealized   biogeochemical   zone   scheme   in   the   marine   sediment   modified   from   Konhauser   (2007);   *Anaerobic   Oxidation   of   Methane   (AOM)   can   work   with   other   electron  donor  than  sulfate  reduced  by  sulfate  reducers.      

Lake El’gygytgyn Lake Baikal

Great Salt Lake

Bear Lake

Lake Petén-Itzá

Lake Ohrid

Lake Van Dead Sea

Lake Bosumtwi

Lake Qinghai Lake Towuti

Lake Malawi

Lake Titicaca

 

Laguna Potrok Aike

Fig.   1.3:   Map   showing   already   drilled   and   planned   ICDP   sites   with   different   levels   of  subsurface   biosphere   studies   (larger   dark   gray   and   white   dots,   respectively).   The   smaller   dots   indicate   sites   already   drilled   without   special   sampling   for   subsurface   Figure 3w – hite   Ariztegui, Thomas & Vuillemin biosphere   studies.   Notice   that   only   ca.   30%   of   the   so   far   studied   sites   have   included   geomicrobiological   and   subsurface   biosphere   sampling   and   subsequent   investigations.   From  Ariztegui,  Thomas  &  Vuillemin  (2015).         6

Chapter  I.  Introduction  

The   first   main   results,   mainly   available   for   Laguna   Potrok   Aike   (Vuillemin,   2013)   and   few  from  Lake  Van  (Glombitza  et  al.,  2013)  suggest  important  and  varied  communities,   influenced   not   only   by   the   geology   and   geochemistry   of   the   sediment   (as   it   is   widely   observed   in   the   ocean   sediment),   but   also   by   environmental   changes   and   climatic   variations,  as  lakes  are  more  prone  to  these  fluctuations  than  oceans.  Peculiar  responses   of  lakes  subsurface  biospheres  seem  inherent  to  the  lake  specificity,  through  diversities   of  location,  response  time  to  global  changes,  type  and  rates  of  sedimentation,  and  so  on.     As   the   turn   towards   continental   subsurface   microbiology   is   currently   being   taken,   we   intend  to  show  through  our  research  in  the  Dead  Sea  Basin,  that  even  in  environments   hostile  for  life,  it  is  relevant  to  undergo  geomicrobiological  study,  and  to  integrate  it  into   its  geological  history.         1.2  The  Dead  Sea  Deep  Drilling  Project   Introduction   to   the   geomicrobiology   specific   issues   of   the   Dead   Sea   realm   will   be   addressed  in  each  of  the  following  chapters.  Here,  I  will  rather  give  a  broad  view  of  the   Dead  Sea  Basin  geology  and  of  the  stakes  and  goals  of  a  scientific  drilling  project  in  the   Dead  Sea.       The   Dead   Sea   is   a   terminal   lake   of   the   Levantine   region   (Fig.   1.4a).   It   now   lies   at   the   lowest  point  on  Earth,  at  427  below  mean  sea  level  (Fig.  1.4b).  It  is  situated  in  the  Dead   Sea  Basin,  a  pull-­‐apart  structure  located  within  the  Arava-­‐Jordan  Rift  valley,  between  the   Jordan  Plateau  and  the  Judean  Mountains,  and  extending  south  towards  the  Red  Sea  in   the  Arava  Valley,  and  North  up  to  the  Sea  of  Galilee  (fig.  1.4c).  In  the  past,  difference  in   precipitation   patterns   and   evaporation   intensities   have   triggered   extension   and   shrinking   of   the   ancestor   lakes   of   the   Dead   Sea   (namely   Lake   Lisan,   Lake   Samra   and   Lake   Amora,   chronologically)   within   this   basin.   Such   lake   level   variations   have   led   to   sediment   deposition   in   areas   that   are   today   exposed   (see   Lisan   Lake   sediment   key   on   Fig.   1.4c).   These   extensive   variations   have   subsequently   allowed   the   deposition   and   exposure  of   sediments.   These   easily   reachable  lake  sediments  constitute  high  resolution   archives   for   paleoenvironmental,   paleoseismic   and   paleoclimatic   studies.   Additionally,   due   to   the   nature   of   freshwater   inputs,   often   occurring   as   flash   floods,   transport   or   organic   debris   is   common   and   allows   radiocarbon   measurements.   Moreover,   uranium-­‐ rich   aragonitic   sediment   dominating   the   Lisan   and   Samra   Formation   outcrops   (Stein,   2001;  Waldmann  et  al.,  2007)  extends  dating  capacities     7

Chapter  I.  Introduction  

   

Fig.   1.4:   (A)   location   of   the   Levantine   region   within   the   Mediterranean   basin   (NASA   picture).     (B)   Elevation   map   of   the   Levantine   region.   Note   that   the   Dead   Sea   Basin   lies   below   sea   level,   and   that   the   Dead   Sea   northern   and   southern   basins   were   actually   connected   at   the  period   of   map   design   (1950’s).   Red   lines   mark   borders   as   defined   by   the   UN   Palestine   Partition   Plan   for   Palestine   and   the   Armistice   Demarcation   Lines   of   1949.  Map  taken  from  http://en.wikipedia.org/wiki/Geography_of_Israel  (C)  Geological   map  of  the  Dead  Sea  tributaries  catchment  area,  taken  from  Neugebauer  et  al.  (2014).     (Schramm  et  al.,  2000;  Torfstein  et  al.,  2009)  and  permits  good  correlation  with  global   climatic  events,  beyond  two  hundred  thousand  years.  However,  the  halitic  nature  of  the   sediment   dominating   during   interglacial   periods   prevents   good   preservation,   as   freshwater   inputs   readily   dissolve   the   sediment.   As   a   result,   important   hiatuses   have     been  recorded  in  the  outcropping  record  of  the  Dead  Sea  Basin  (Stein,  2001  ;  Fig.  1.5).    

  8

Chapter  I.  Introduction   275

  Fig.  1.5:  Unconformity  between  the  Samra  Formation  and  the  Lisan  Formation,  with  U-­‐ series  ages  of    ca  80  kyr  and  70  kyr,  respectively.  Taken  from  Stein  (2001).     Lake Lisan water, and of clastic material transported by floods. The Figure 3. The exposure of the Samra and Lisan Formations at Perazim Valley (west of Mt. Sedom). The Samra-Lisan depositional unconformity appears between U-series ages of ~ 80 and 70 kyr. It is marked by sand and gravel. The upper part of the section, above the unconformity consist mainly of alternating laminae of aragonite and detritus, and hard benches that are made of gypsum.

aragonite appears in thin (~ 0.5–1 mm thick) laminae

Additionally,   the   geographical   and   location   of   the   Dead   makes   it   highly   Lake Lisan existed between ~ 70 and 15 kyr.tectonic   During the alternating with detrital laminae of similarSea   thickness. time of highest water level (> 180 m b.s.l.) the lake

The detrital laminae are comprised mainly of quartz

tion, which was deposited from Lake Lisan and its

rocks. The gypsum appears in thin laminae, thicker lay-

aragonite and gypsum, which precipitated from the lake

clastic layers are composed of sand, gravel and clay.

from the Sea of Galilee in the north to the probably of wind-blown origin, dolomite, calcite, susceptible   to  extended seismic   activity.   This   area   is   gains indeed   known   to   be   active   since   ancient   Hazeva area in the south (Figure 2). The Lisan Formaand clay minerals derived from the Cretaceous wallsurroundingbiblical   fan deltas consists mainly of chemical ers (up to 70 cm thick), and the   in disseminated form. Thick times.   Particularly,   episodes,   such   as   the   fall   of   Jericho   wall,   have   often  

tentatively   been   explained   by   scientists   by   earthquake   events   (Bentor,   1989;   Ambraseys,   2006).   Due   to   its   high   sedimentation   rate   and   fine   grained   nature,   the   sediments   have   recorded   this   paleoseismic   activity   through   wavy   structures   seismites   notably  observed  in  the  highly  laminated  sediment  of  the  Masada  canyons  (Fig.  1.6).     The  Dead  Sea  Deep  Drilling  Project  (DSDDP)  aims  to  obtain  a  continuous  record  of  the   Quaternary  sediments  of  the  Dead  Sea  Basin.  This  archive  will  help  reconstructing  past   environments   and   climatic   variation   in   a   region   at   the   crossroads   of   early   hominid   expansion,  and  that  is  considered  as  the  religious  and  cultural  cradle  of  society.     In   this   way,   the   ICDP-­‐sponsored   DSDDP   has   drilled   the   lake   floor   at   two   key   locations  (Fig.  1.7a):  the  first  one  (5017-­‐1)  at  297,46  m  in  one  of  the  deepest  point  of  the   lake   (N31°30’28.98”,   E   35°28’15.6”),   and   two   other   ones   (5017-­‐2   and   5017-­‐3)   on   the   shore   of   the   lake,   near   the   Ein   Gedi   spa   (N   31°25’13.998”,   E   35°23’38.4”   and   N   31°25’22.74”,  E35°23’39.58”  respectively).  The  drilling  operations  debuted  in  December   2010  and  ended  in  March  2011,  and  obtained  in  total  722.65  m  of  sedimentary  core,       9

Chapter  I.  Introduction  

1m     Fig.   1.6:   photograph   of   a   seismite   within   the   AAD   facies   of   the   Mishmar   outcrop,   near   Masada.   It   is   interpreted   as   the   result   of   earthquake   disturbance   of   unconsolidated   laminae.     with   a   recovery   of   78.34   %.   More   details   regarding   recovery   for   each   location   can   be   found  in  Neugebauer  et  al.  (2014).  Such  depth  had  never  been  attained  in  the  Dead  Sea   sediment.   The   sedimentary   archive   obtained   from   the   DSDDP   is   therefore   unique   and   gives   access   to   pristine   material,   highly   valuable   for   geobiologists.   Furthermore,   the   hypersalinity  of  its  subsurface  is  for  geomicrobiologists  a  unique  feature  as  the  Dead  Sea   constitutes  an  end  member  of  hypersaline  lakes  on  Earth,  and  as  the  study  of  subsurface   communities  in  deep  continental  setting  is  very  limited.  Given  the  harshness  of  the  lake   water   chemistry,   and   probably   of   its   sedimentary   pore   water,   the   almost   constant   precipitation   of   evaporitic   minerals,   and   the   desire   to   use   proxies   derived   from   the   drilled   sediment   for   paleoclimatic   reconstructions,   several   questions   that   will   be   discussed  along  this  manuscript,  arise.   (1)   Given   the   limited   total   number   of   cells   in   the   present   Dead   Sea,   and   the   extreme   salinity  of  the  water:  is  there  life  currently  developing  and/or  surviving  in  the  sediment?   It  is  known  that  life  exists  in  the  surface  of  the  sediments,  thanks  to  freshwater  springs   that   locally   decrease   salinity   of   the   environment   and   allow   microbial   mats   to   develop   (Ionescu   et   al.,   2012;   Häusler   et   al.,   2014).   Is   this   limited   to   the   springs,   do   we   see   similar  processes  at  depths,  or  is  there  another  living  community  currently  unknown?    

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Chapter  I.  Introduction  

  Fig.   1.7:   Overview   of   drilling   sites.   (A)   elevation   map   of   the   Dead   Sea   with   names   and   location  of  ICDP  drilled  sites  (taken  from  Neugebauer  et  al.,  2014)  (B)  aerial  photograph   of   the   drilling   platform   on   location   5017-­‐1   (center   of   the   lake).   (C)   photograph   of   the   drilling  platform  at  the  coastal  sites.  Photo  credit  :  ICDP      (2)   If   there   is   life,   what   are   the   sources   for   its   development:   how   do   communities   manage  to  survive  in  those  allegedly  extreme  salinities  and  low  nutrient  concentrations   (phosphorus),   and   what   kind   of   metabolism   can   be   sustained   in   this   hypersaline   subsurface.  Some  thermodynamics  limits  have  been  set  for  the  functioning  of  the  main   metabolic  pathways  at  high  salinities  (Oren,  2010),  are  they  validated  in  situ  ?   (3)  Are  potential  microbes  currently  active  in  the  sediment,  or  are  they  only  dormant?  If   active,   what   kind   of   effect   would   they   have   on   their   environment   and   is   it   possible   to   find  physical  traces  of  their  hypothetic  activity?  If  traces  of  past  microbial  activity  have   existed,   is   it   also   possible   to   trace   them,   and   would   they   have   an   influence   on   the   sedimentary   process   ongoing   in   the   subsurface.   Among   others,   questions   regarding   precipitation/dissolution  of  mineral  phases,  and  preservation  or  consumption  of  organic   matter   are   at   stake.   Tackling   these   issues   would   allow   constraining   the   carbon   and   sulfur   cycles   of   the   Dead   Sea,   and   understand   the   impact   microbial   communities   can   have  on  the  early  diagenesis  of  hypersaline  sediments.   (4)   Is   microbial   activity   susceptible   of   disturbing   proxies   used   for   paleoenvironmental   and   paleoclimatic   reconstructions?   Sulfur   isotopic   ratios   of   gypsum   from   lake   Lisan   outcrops  have  been  interpreted  to  bear  signatures  of  bacterial  sulfate  reducing  activity     11

Chapter  I.  Introduction  

(Torfstein   et   al.,   2005).   Within   the   framework   of   the   DSDDP   project,   it   is   highly   relevant   to  set  limits  towards  the  use  of  some  proxies  potentially  affected  by  life,  albeit  in  settings   hostile  for  life.  If  microbial  disturbance  exists,  can  one  assess  its  trigger,  its  limits  and  its   consequences?   (5)   Finally,  wider   questions  bounded  to  the  field  of  geomicrobiology  could  possibly  be   addressed:  first,  how  do  communities  develop  in  the  sediment?  Do  they  originate  from   other  environments,  have  they  been  transported  from  underground  sources  or  do  they   relocate   along   the   pore   water   of   the   sedimentary   column?   What   is   the   actual   control   over   their   distribution?   The   precedent   questions   can   be   addressed   within   the   specific   framework   of   the   Dead   Sea   subsurface,   and   possibly   extended   towards   subsurface   continental  settings,  when  compared  to  the  ocean  realm,  for  example.         The   following   chapters   will   combine   geological,   microbiological,   geochemical   and   biochemical   datasets,   in   order   to   address   these   questions.   Chapter   II   will   focus   on   the   currently   living   Dead   Sea   community   and   the   information   given   by   the   community   distribution,   with   16S   rRNA   gene   sequences   as   main   tool.   Chapter   III   will   use   metagenomic   information   to   further   understand   the   metabolic   potential   of   the   principal   communities   described   in   the   sediment.   In   chapter   IV,   methods   for   the   search   for   microbial   traces   will   be   discussed,   as   well   as   their   meaning   while   the   effect   of   past   microbial  activity  will  be  questioned  in  chapter  V,  under  the  form  of  a  multidisciplinary   case  study  of  a  peculiar  interval  of  the  core.       References     Ambraseys  NN  (2006)  Earthquakes  and  archaeology.  J.  Archaeol.  Sci.,  33,  1008–1016.   Ariztegui  D,  Thomas  C,  Vuillemin  A  (2015)  Present  and  future  of  subsurface  biosphere   studies  in  lacustrine  sediments  through  scientific  drilling,  Int.  Journal  of  Earth   Sciences   Battista  JR  (1997)  Against  all  odds:  the  survival  strategies  of  Deinococcus  radiodurans.   Annu.  Rev.  Microbiol.,  51,  203–224.   Bentor  YK  (1989)  Geological  events  in  the  bible.  Terra  Nov.,  1,  326–338.   Blöchl  E,  Rachel  R,  Burggraf  S,  Hafenbradl  D,  Jannasch  HW  and  Stetter  KO  (1997)   Pyrolobus  fumarii,  gen.  and  sp.  nov.,  represents  a  novel  group  of  archaea,  extending   the  upper  temperature  limit  for  life  to  113  degrees  C.  Extremophiles,  1,  14–21.     12

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D’Hondt  S,  Jørgensen  BB,  Miller  DJ,  Batzke  A,  Blake  R,  Cragg  B  a,  Cypionka  H,  Dickens  GR,   Ferdelman  T,  Hinrichs  K-­‐U,  Holm  NG,  Mitterer  R,  Spivack  A,  Wang  G,  Bekins  B,   Engelen  B,  Ford  K,  Gettemy  G,  Rutherford  SD,  Sass  H,  Skilbeck  CG,  Aiello  IW,  Guèrin   G,  House  CH,  Inagaki  F,  Meister  P,  Naehr  T,  Niitsuma  S,  Parkes  RJ,  Schippers  A,  Smith   DC,  Teske  A,  Wiegel  J,  Padilla  CN  and  Acosta  JLS  (2004)  Distributions  of  microbial   activities  in  deep  subseafloor  sediments.  Science,  306,  2216–2221.   Dickens  G,  O’Neil  J,  Rea  D  and  Owen  R  (1995)  Dissociation  of  ocean  methane  hydrate  as   a  cause  of  the  carbon  isotope  excursion  at  the  end  of  the  Paleocene.   Paleoceanography,  10,  965–971.   Edwards  KJ,  Bond  PL,  Gihring  T  and  Banfield  JF  (2000)  An  Archaeal  Iron-­‐Oxidizing   Extreme  Acidophile  Important  in  Acid  Mine  Drainage.  Science,  287,  1796–1799.   Glombitza  C,  Stockhecke  M,  Schubert  CJ,  Vetter  A  and  Kallmeyer  J  (2013)  Sulfate   reduction  controlled  by  organic  matter  availability  in  deep  sediment  cores  from  the   saline,  alkaline  Lake  Van  (Eastern  Anatolia,  Turkey).  Front.  Microbiol.,  4,  1–12.   Häusler  S,  Noriega-­‐Ortega  BE,  Polerecky  L,  Meyer  V,  de  Beer  D  and  Ionescu  D  (2014)   Microenvironments  of  reduced  salinity  harbour  biofilms  in  Dead  Sea  underwater   springs.  Environ.  Microbiol.  Rep.,  6,  152–8.   Inagaki  F,  Nunoura  T,  Nakagawa  S,  Teske  A,  Lever  M,  Lauer  A,  Suzuki  M,  Takai  K,   Delwiche  M,  Colwell  FS,  Nealson  KH,  Horikoshi  K,  D’Hondt  S  and  Jørgensen  BB   (2006)  Biogeographical  distribution  and  diversity  of  microbes  in  methane  hydrate-­‐ bearing  deep  marine  sediments  on  the  Pacific  Ocean  Margin.  Proc.  Natl.  Acad.  Sci.,   103,  2815–20.   Inagaki  F,  Suzuki  M,  Takai  K,  Oida  H,  Sakamoto  T,  Aoki  K,  Nealson  KH  and  Horikoshi  K   (2003)  Microbial  communities  associated  with  geological  horizons  in  coastal   subseafloor  sediments  from  the  Sea  of  Okhotsk.  Appl.  Environ.  Microbiol.,  69,  7224– 7235.   Ionescu  D,  Siebert  C,  Polerecky  L,  Munwes  YY,  Lott  C,  Häusler  S,  Bižić-­‐Ionescu  M,  Quast  C,   Peplies  J,  Glöckner  FO,  Ramette  A,  Rödiger  T,  Dittmar  T,  Oren  A,  Geyer  S,  Stärk  H-­‐J,   Sauter  M,  Licha  T,  Laronne  JB  and  de  Beer  D  (2012)  Microbial  and  chemical   characterization  of  underwater  fresh  water  springs  in  the  Dead  Sea.  PLoS  One,  7.   Kallmeyer  J,  Pockalny  R,  Adhikari  RR,  Smith  DC  and  D’Hondt  S  (2012)  Global   distribution  of  microbial  abundance  and  biomass  in  subseafloor  sediment.  Proc.   Natl.  Acad.  Sci.,  109,  16213–16216.   Kato  C,  Li  L,  Nogi  Y,  Nakamura  Y,  Tamaoka  J  and  Horikoshi  K  (1998)  Extremely   barophilic  bacteria  isolated  from  the  Mariana  Trench,  Challenger  Deep,  at  a  depth  of   11,000  meters.  Appl.  Environ.  Microbiol.,  64,  1510–1513.   Knittel  K  and  Boetius  A  (2009)  Anaerobic  oxidation  of  methane:  progress  with  an   unknown  process.  Annu.  Rev.  Microbiol.,  63,  311–334.  

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Konhauser  KO  (2007)  Introduction  to  geomicrobiology.,  Blackwell  Publishing  Ltd.   Kvenvolden  A  (1995)  A  review  of  the  geochemistry  of  methane  in  natural  gas  hydrate.   Org.  Geochem.,  23,  997–1008.   Milkov  A  V  (2004)  Global  estimates  of  hydrate-­‐bound  gas  in  marine  sediments:  how   much  is  really  out  there?  Earth-­‐Science  Rev.,  66,  183–197.   Neugebauer  I,  Brauer  A,  Schwab  M,  Waldmann  N,  Enzel  Y,  Kitagawa  H,  Torfstein  A,  Frank   U,  Dulski  P,  Agnon  A,  Ariztegui  D,  Ben-­‐Avraham  Z,  Goldstein  SL,  Stein  M  and  the   DSDDP  Scientific  Party  (2014)  Lithology  of  the  long  sediment  record  recovered  by   the  ICDP  Dead  Sea  Deep  Drilling  Project  (DSDDP).  Quat.  Sci.  Rev.,  102,  149–165.   Oren  A  (2010)  Thermodynamic  limits  to  microbial  life  at  high  salt  concentrations.   Environ.  Microbiol.,  13,  1908–1923.   Orphan  VJ,  House  CH,  Hinrichs  K-­‐U,  McKeegan  KD  and  DeLong  EF  (2002)  Multiple   archaeal  groups  mediate  methane  oxidation  in  anoxic  cold  seep  sediments.  Proc.   Natl.  Acad.  Sci.,  99,  7663–7668.   Røy  H,  Kallmeyer  J,  Adhikari  RR,  Pockalny  R,  Jørgensen  BB  and  D’Hondt  S  (2012)   Aerobic  microbial  respiration  in  86-­‐million-­‐year-­‐old  deep-­‐sea  red  clay.  Science,   336,  922–925.   Schippers  A,  Neretin  LN,  Kallmeyer  J,  Ferdelman  TG,  Cragg  BA,  Parkes  RJ  and  Jørgensen   BB  (2005)  Prokaryotic  cells  of  the  deep  sub-­‐seafloor  biosphere  identified  as  living   bacteria.  Nature,  61,  861–864.   Schramm  A,  Stein  M  and  Goldstein  SL  (2000)  Calibration  of  the  14C  time  scale  to  >40  ka   by  234U–230Th  dating  of  Lake  Lisan  sediments  (last  glacial  Dead  Sea).  Earth  Planet.   Sci.  Lett.,  175,  27–40.   Sluijs  A,  Brinkhuis  H,  Schouten  S,  Bohaty  SM,  John  CM,  Zachos  JC,  Reichart  G-­‐J,  Sinninghe   Damsté  JS,  Crouch  EM  and  Dickens  GR  (2007)  Environmental  precursors  to  rapid   light  carbon  injection  at  the  Palaeocene/Eocene  boundary.  Nature,  450,  1218– 1221.   Stein  M  (2001)  The  sedimentary  and  geochemical  record  of  Neogene-­‐  Quaternary  water   bodies  in  the  Dead  Sea  Basin  –  inferences  for  the  regional  paleoclimatic  history  *.  J.   Paleolimnol.,  26,  271–282.   Torfstein  A,  Gavrieli  I  and  Stein  M  (2005)  The  sources  and  evolution  of  sulfur  in  the   hypersaline  Lake  Lisan  (paleo-­‐Dead  Sea).  Earth  Planet.  Sci.  Lett.,  236,  61–77.   Torfstein  A,  Haase-­‐Schramm  A,  Waldmann  N,  Kolodny  Y  and  Stein  M  (2009)  U-­‐series  and   oxygen  isotope  chronology  of  the  mid-­‐Pleistocene  Lake  Amora  (Dead  Sea  basin).   Geochim.  Cosmochim.  Acta,  73,  2603–2630.  

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Chapter  I.  Introduction  

Vuillemin  A  (2013)  Characterizing  the  Subsurface  Biosphere  in  Laguna  Potrok  Aike   Sediments  (Argentina):  A  Case  Study.  Thesis,  University  of  Geneva.   Waldmann  N,  Starinsky  A  and  Stein  M  (2007)  Primary  carbonates  and  Ca-­‐chloride   brines  as  monitors  of  a  paleo-­‐hydrological  regime  in  the  Dead  Sea  basin.  Quat.  Sci.   Rev.,  26,  2219–2228.   Whitman  WB,  Coleman  DC  and  Wiebe  WJ  (1998)  Prokaryotes :  The  unseen  majority.   Proc.  Natl.  Acad.  Sci.,  95,  6578–6583.                                          

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Chapter  I.  Introduction  

         

  16

Chapter II. Current microbial life in the Dead Sea sediment

“Is there anybody out there?” The Wall, Pink Floyd

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Chapter II. Current microbial life in the Dead Sea sediment

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Chapter II. Current microbial life in the Dead Sea sediment 2.1 Introduction Determination of microbial presence using a simple microscope is complicated. Within sedimentary environments, this issue is even more complex as communities are often attached to particles and difficult to observe. Methods excluding visual identifications have thus been generally used in the sediments, and have been associated to fluorescent imaging to highlight the presence of microorganisms. Carried by pioneered work from Carl Woese (e.g. Woese, 1987), the identification of 16S rRNA gene sequences for determining microbial diversity is used routinely. Sanger sequencing, time and money consuming is currently being replaced by the higher-throughput sequencing methods, which allow not only the sequencing of targeted genes, but that of complete genomes present in a given environment. These techniques evolve very quickly and are constantly enhanced and tuned for better yields, time and money-wise. Reviews of these methods are available, and give a good view of pros and cons for the main ones (e.g. Thomas et al., 2012; Loman et al., 2012). In addition to the non-visual identification of DNA in a given samples, the need for understanding the spatial association of communities in a sample has arisen. With it, methods mostly developed from clinical investigations, have quickly established and are now used routinely. This is the case for 4',6-diamidino-2-phenylindole (DAPI) staining and fluorescent in-situ hybridization (FISH). They allow qualifying the presence of dead versus living organisms, and can target specific groups, either taxonomic or metabolic, using a sequence signature. FISH methods, which are based on the hybridization of a fluorescing probe to a targeted RNA sequence allows detecting RNA within a sample using a fluorescent microscope. With various probes, and combined with DAPI that binds to cellular material, the counting of microbial communities living in a given sample is made possible and gives more precise insight in a specific environment (e.g. Amann et al., 1995; Amann et al., 2001; Pernthaler et al., 2002; Pernthaler and Amann, 2004). These methods are routinely used in marine sediments and have become a corner stone of IODP investigations in microbial ecology (e.g. D’Hondt et al., 2004; Schippers et al., 2005; Inagaki et al., 2006; Biddle et al., 2008). It is with flow cytometry, a very efficient way of deciphering and counting microorganisms (McFeters et al., 1995; Porter et al., 1996). For such reasons, fixation of cells in order to allow the use of such dyes has been realized in the on-shore geobiology lab of the DSDDP, in Ein Gedi during the drilling campaign. Additional sampling for DNA extraction has been realized in the same lab. This material will be used as base material

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Chapter II. Current microbial life in the Dead Sea sediment for the geomicrobiological investigation of the Dead Sea subsurface, addressed in the following part. 2.2 Impact of paleoclimate on the distribution of microbial communities in the subsurface sediment of the Dead Sea A modified version of this chapter has been submitted to Geobiology as Thomas C., Ionescu D., Ariztegui D., and the DSDDP scientific party, Impact of paleoclimate on the distribution of microbial communities in the subsurface sediment of the Dead Sea Abstract A long sedimentary core has been recently retrieved from the Dead Sea Basin (DSB) within the framework of the ICDP sponsored Dead Sea Deep Drilling Project. Contrasting climatic intervals were evident by distinctive lithological facies such as laminated aragonitic muds and evaporites. A geomicrobiological investigation was conducted in representative sediments of this core. To identify the microbial assemblages present in the sediments and their evolution with changing depositional environments through time, the diversity of the 16S rRNA gene was analyzed in gypsum, aragonitic laminae and halite samples. The subsurface microbial community was largely dominated by the Euryarcheota phylum (Archaea). Within the latter, Halobacteriaceae members were ubiquitous, probably favored by their “high salt-in” osmotic adaptation which also makes them one of the rare inhabitants of the modern Dead Sea. Bacterial community members were scarce, emphasizing that the “low salt-in” strategy is less suitable in this environment. Substantial differences in assemblages are observed between aragonitic sediments and gypsum-halite ones, independently of the depth and salinity. The aragonite sample, deposited during humid periods when the lake was stratified, consists mostly of the archaeal MSBL1 and bacterial KB1 Candidate Divisions. This consortium probably relies on compatible solutes supplied from the lake by halotolerant species present in these more favorable periods. In contrast, members of the Halobacteriaceae were the sole habitants of the gypsum-halite sediments which result from a holomictic lake. Although the biomass is low, these variations in the observed subsurface microbial populations appear to be controlled by biological conditions in the water column at the time of sedimentation, and subsequently by the presence or absence of stratification and dilution in the lake. Since the latter are controlled by climatic changes, our data suggests

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Chapter II. Current microbial life in the Dead Sea sediment a relationship between local lacustrine subsurface microbial assemblages and largescale climatic variations over the Dead Sea Basin. 2.2.1 Introduction The present Dead Sea is located in the Dead Sea Basin at the border between Israel, Jordan and the Palestinian Authority, (Fig. 1a). Its sediment record spans several hundred thousand years of climatic variations (Torfstein et al., 2009), making it a prime target for geological investigations. To better understand the climatic response of this currently water stressed region, over the last glacial-interglacial cycles (Goldstein et al., 2011; Neugebauer et al., 2014), the ICDP sponsored Dead Sea Deep Drilling Project (DSDDP) retrieved the longest sedimentary core to date from the deepest point in the lake. Since more than 50 years now, the Dead Sea water budget has been largely negative, resulting in a massive drop of the shoreline at an increasing rate which in the last decade exceeded one meter per year. Since 1979 the lake has been fully mixed and oxic with the exception of short periods of stratification. Halite is almost constantly precipitating and the total dissolved salt concentration has reached one of the highest levels on Earth with up to 348 g. L-1 (Oren and Gunde-Cimerman, 2012). Interestingly, the Dead Sea chemistry is characterized by a high concentration of divalent cations (~2 M Mg2+ and ~0.5 M Ca2+), which make it a specifically harsh environment even for microorganisms (Oren, 2001). Indeed, except for some oases of life located at the outflow of groundwater springs, the microbial cell number is extremely low in the present Dead Sea water (