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
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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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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).
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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?
10
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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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 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 (