La formation d’un squelette de CaCO3 par les coraux Scléractiniaires est à la base de l’édification des récifs coralliens. Nombre de ces coraux constructeurs de récif vivent en symbiose avec des Dinoflagellés photosynthétiques. Ces deux processus reposent sur le transport et l’utilisation de carbone inorganique (Ci) provenant de l’eau de mer pour la photosynthèse, et du métabolisme animal pour la calcification. Cette thèse s’est intéressée à l’étude moléculaire et physiologique des mécanismes, permettant l’utilisation de ce carbone inorganique.Malgré l’importance des transports de HCO3-, aucun transporteur n’a été caractérisé à cejour et leur implication dans la physiologie des coraux n’est que suggérée par la pharmacologie. Durant cette thèse nous avons cloné un gène codant pour un transporteur deHCO3- chez le corail Acropora sp. La conversion de ce HCO3- en CO2 pour la photosynthèse est facilitée par l’acidification de l’environnement proche du Dinoflagellé dans la cellule animale. Cette acidification est causée par une H+-ATPase de type P que nous avons caractérisée. Ce gène est le premier à montrer une expression dépendante de la vie en symbiose chez le symbiote.Nous avons aussi cloné et localisé deux anhydrases carboniques (AC). L’une impliquée dans la calcification et l’autre dans la régulation du pH intracellulaire et l’équilibre entre leCO2 et HCO3-. Une étude pharmacologique de ces deux AC, a identifié des molécules inhibitrices et activatrices qui ont permis des expériences de physiologie in vivo. Celles-ci permettent une analyse plus discriminante du rôle des AC dans la calcification. / Coral reefs edification is based on the formation of a calcium carbonate skeleton byscleractinian corals. Many of these reef-building corals establish a symbiotic association with photosynthetic Dinoflagellates. Both processes involve the transport and utilization of inorganic carbon (Ci) coming from seawater for photosynthesis, and from animal metabolismfor calcification. This work focused on the molecular and physiological study of poorlyknown mechanisms that allow the utilization of Ci.Despite the importance of bicarbonate transport, no transporter has been characterized and their role in coral physiology is only suggested by pharmacological experiments. We have cloned a gene encoding a bicarbonate transporter in the coral Acropora sp. The conversion of this bicarbonate into CO2 for photosynthesis is mediated by the acidification of the are asurrounding the Dinoflagellate in the animal cell. This is performed by a P type H+-ATPasethat we characterized here. This is the first gene with a symbiosis-dependent expression in the symbiont.This work also allowed the cloning and the localization of two carbonic anhydrases (CA).The first one is involved in calcification, the second one plays a role in the intracellular pHregulation and the CO2 / HCO3- equilibrium. A pharmacological study of these two enzymes identified inhibitor and activator compounds that have been then used in physiology experiments. This last approach represents a more accurate study of the role of CAs incalcification.

Kieselalgen (Diatomee) sind eukaryotische einzellige Algen die hochspezifische Proteine (sogenannte Silaffine) erzeugen, um ‘nanopatterned’ Silica-Zellwände herzustellen. Diese Proteine zeigen geringe oder gar keine Homologie innerhalb der Diatomeen Gattung und sind ausgiebig (extensiv) posttranslatorisch modifiziert. Zum Unterschied zu konventioneller Modifikation (z.B. Phosphorylierung und Glykosylierung) weisen Lysinreste von Silaffinen einige Polyaminketten mit sehr heterogenen molekularen Strukturen auf. Diese Modifikationen sind spezifisch für Kieselalgen und spielen somit hypothetisch eine Rolle in der Biosilica-Synthese. Allerdings sind Lysin Polyamin Modifikationen, modifizierte Proteine und modifizierte Stellen kaum charakterisiert. Um diese Frage zu beantworten entwickelten wir eine Methode Polyamine zu quantifizieren und die Position von Polyamin-Modifikationen in engverwandte Proteine zu identifizieren (in morphologisch unterschiedliche Diatomeen Thalassiosira pseudonana, T. oceanica und Cyclotella cryptica). Wir zeigten, dass das Gesamtmuster von Polyaminender phylogenetischen Nähe dieser Kieselalgenarten folgt und dass diese Polyaminmodifikationen an Konsensusstellen sogar in Proteinen auftraten, die keine Sequenzähnlichkeit zeigten.:CONTENTS Summary Zusammenfassung List of figures List of tables Abbreviations 1 Introduction 1.1 Diatoms 1.2 Diatom biosilica 1.2.1 Biosilicification in nature 1.2.2 Diatom biosilica structure and cell cycle 1.2.3 The cell biology of biosilica morphogenesis 1.3 The role of polyamine PTMs in diatom biosilicification 1.3.1 Identifying biomolecules associated with diatom biosilica 1.3.2 PTM complexity of biosilica-associated proteins 1.3.3 Lysine ε-polyamine PTMs in biosilica-associated proteins 1.4 Mass spectrometry in PTM discovery 1.4.1 Modification-specific proteomics 1.4.2 Analysis of polyamine-modified lysines by MS 1.4.3 Fractionation of proteins and peptides prior to MS 1.4.4 MS/MS analysis in modification-specific proteomics 1.4.5 Bioinformatics tools for modification-specific proteomics 1.5 Rationale of the thesis 2 Aim of the thesis 3 Results and discussion 3.1 A method for analysis of ε-polyamine PTMs 3.1.1 Establishing a method to analyse ε-polyamines 3.1.2 Method applicability for lysine PTM profiling 3.1.3 Profiling of lysine PTMs in silaffin-3 3.2 Profiling lysine PTMs in biosilica extracts 3.2.1 Lysine PTM profile and characteristic fragments 3.2.2 Elucidation of phosphopolyamine structures 3.2.3 LysinePTMprofilesofAFSMextracts 3.2.4 Comparison of AFIM and AFSM profiles in T. pseudonana 3.2.5 Phylogenetic relationship across three diatom species 3.3 PTM localization and discovery of consensus motifs 3.3.1 Multiple protease strategy for mapping lysine PTMs 3.3.2 Selection of deprotection technique 3.3.3 Mapping lysine PTMs on tpSil3 using iterative search strategy 3.3.4 Deconvolution of raw MS/MS spectra 3.3.5 PTM mapping by polyamine-specific fragments 3.3.6 Identification of consensus motifs harboring lysine PTMs 4 Conclusions and Outlook 5.1 Synthesis of polyamine standards 5.2 Isolation of biosilica-associated proteins 5.3 Expression of tpSil3 from synthetic gene 5.4 HCl hydrolysis 5.5 AQC-derivatization of amino acids and polyamines 5.6 LC-MS/MS analysis of QAC-derivatives 5.7 Amino acid measurement using UV-detection 5.8 Direct infusion MS/MS analysis 5.9 Acetylation of phosphopolyamines 5.10 31P-NMR measurements 5.11 Deglycosylation with TFMS 5.12 Treatment with HF·pyridine soluble complex 5.13 Anhydrous HF-treatment 5.14 Protein analysis by GeLC-MS/MS 5.15 Proteomics data processing A Appendix B Bibliography Acknowledgments Publications Declaration / Erklärung / Diatoms are eukaryotic unicellular algae that employ highly specialized proteins called silaffins for making nanopatterned silica-based cell walls. These proteins share little or no homology across diatom species and are extensively post-translationally modified. Apart from conventional modifications (e. g., phosphorylation and glycosylation) lysine residues of silaffins bear polyamine chains with highly heterogeneous molecular structure. The latter appear to be specific for silicifying organisms and therefore hypothesized to play a key role in biosilica synthesis. However, polyamine modifications of lysines, modified proteins, and modification sites remain poorly characterized. To address these questions, we developed a method to quantify polyamines and identify sites of polyamine modifications in proteins from phylogenetically closely related, yet morphologically distinct diatoms Thalassiosira pseudonana, T. oceanica, and Cyclotella cryptica. We demonstrated that the overall pattern of polyamines followed the phylogenetic proximity across these diatom species and showed that polyamine modifications occurred at consensus sites even in proteins showing no sequence similarity.:CONTENTS Summary Zusammenfassung List of figures List of tables Abbreviations 1 Introduction 1.1 Diatoms 1.2 Diatom biosilica 1.2.1 Biosilicification in nature 1.2.2 Diatom biosilica structure and cell cycle 1.2.3 The cell biology of biosilica morphogenesis 1.3 The role of polyamine PTMs in diatom biosilicification 1.3.1 Identifying biomolecules associated with diatom biosilica 1.3.2 PTM complexity of biosilica-associated proteins 1.3.3 Lysine ε-polyamine PTMs in biosilica-associated proteins 1.4 Mass spectrometry in PTM discovery 1.4.1 Modification-specific proteomics 1.4.2 Analysis of polyamine-modified lysines by MS 1.4.3 Fractionation of proteins and peptides prior to MS 1.4.4 MS/MS analysis in modification-specific proteomics 1.4.5 Bioinformatics tools for modification-specific proteomics 1.5 Rationale of the thesis 2 Aim of the thesis 3 Results and discussion 3.1 A method for analysis of ε-polyamine PTMs 3.1.1 Establishing a method to analyse ε-polyamines 3.1.2 Method applicability for lysine PTM profiling 3.1.3 Profiling of lysine PTMs in silaffin-3 3.2 Profiling lysine PTMs in biosilica extracts 3.2.1 Lysine PTM profile and characteristic fragments 3.2.2 Elucidation of phosphopolyamine structures 3.2.3 LysinePTMprofilesofAFSMextracts 3.2.4 Comparison of AFIM and AFSM profiles in T. pseudonana 3.2.5 Phylogenetic relationship across three diatom species 3.3 PTM localization and discovery of consensus motifs 3.3.1 Multiple protease strategy for mapping lysine PTMs 3.3.2 Selection of deprotection technique 3.3.3 Mapping lysine PTMs on tpSil3 using iterative search strategy 3.3.4 Deconvolution of raw MS/MS spectra 3.3.5 PTM mapping by polyamine-specific fragments 3.3.6 Identification of consensus motifs harboring lysine PTMs 4 Conclusions and Outlook 5.1 Synthesis of polyamine standards 5.2 Isolation of biosilica-associated proteins 5.3 Expression of tpSil3 from synthetic gene 5.4 HCl hydrolysis 5.5 AQC-derivatization of amino acids and polyamines 5.6 LC-MS/MS analysis of QAC-derivatives 5.7 Amino acid measurement using UV-detection 5.8 Direct infusion MS/MS analysis 5.9 Acetylation of phosphopolyamines 5.10 31P-NMR measurements 5.11 Deglycosylation with TFMS 5.12 Treatment with HF·pyridine soluble complex 5.13 Anhydrous HF-treatment 5.14 Protein analysis by GeLC-MS/MS 5.15 Proteomics data processing A Appendix B Bibliography Acknowledgments Publications Declaration / Erklärung

Kieselalgen – auch Diatomeen genannt – verfügen über die einzigartige Fähigkeit, nanostrukturierte, hierarchisch aufgebaute Zellwände aus Siliziumdioxid – auch als Biosilica bekannt – mit beispielloser Genauigkeit und Reproduzierbarkeit zu bilden. Ein tieferes Verständnis für diesen Prozess, der als “Biomineralisation“ bekannt ist, ist nicht nur auf dem Gebiet der Grundlagenforschung zu Kieselalgen sehr bedeutsam, sondern auch für die Nutzung dieser Nanostrukturierung in den Materialwissenschaften oder der Nanobiotechnologie. Nach dem derzeitigem Stand der Wissenschaft wird diese Strukturierung durch die Selbstorganisation von Proteinmustern, an denen sich das Siliziumdioxid bildet, erreicht. Um die Funktion und das Zusammenspiel einzelner Proteine, die an diesem Biomineralisationsprozess beteiligt sind, entschlüsseln zu können, ist es essentiell ihre strukturelle Organisation aufzuklären und diese mit den morphologischen Zellwandmerkmalen zu korrelieren. Die Größenordnung dieser Merkmale ist im Bereich von Nanometern angesiedelt. Mit Hilfe der Elektronenmikroskopie können diese Biosilicastrukturen aufgelöst werden, jedoch ist keine proteinspezifische Information verfügbar. Ziel dieser Arbeit war es daher, eine Technik zu etablieren, die in der Lage ist, einzelne Biosilica-assozierte Proteine mit Nanometer-Präzision zu lokalisieren. Um dieses Ziel zu erreichen, wurde Einzelmoleküllokalisationsmikroskopie (single-molecule localization microscopy, kurz: SMLM) beispielhaft in der Kieselalge Thalassiosira pseudonana etabliert. Die Position verschiedener Biosilica-assoziierte Proteine innerhalb des Biosilicas und nach dessen chemischer Auflösung wurde mit einer hohen räumlichen Auflösung bestimmt. Um quantitative Ergebnisse zu erhalten, wurde ein Analyse-Workflow entwickelt, der grafische Benutzeroberflächen und Skripte für die Visualisierung, das Clustering und die Kolokalisation von SMLM Daten beinhaltet. Um optimale Markierungen für SMLM an Biosilica-eingebetteten Proteinen zu finden, wurde ein umfassendes Screening von photo-schaltbaren fluoreszierenden Proteinen durchgeführt. Diese wurden als Fusionsproteine mit Silaffin3, einem Protein, welches eng mit der Biosilica-Zellwand assoziiert ist, exprimiert. Es konnte gezeigt werden, dass nur drei von sechs Kandidaten funktional sind, wenn sie in Biosilica eingebettet sind. Silaffin3 konnte indirekt mittels SMLM mit einer Lokalisationsgenauigkeit von 25 nm detektiert werden. Dies erlaubte es, seine strukturelle Organisation aufzulösen und Silaffin3 als eine Hauptkomponente in der Basalkammer der Fultoportulae zu identifizieren.:1 INTRODUCTION 1 1.1 Diatoms – a model system for biomineralization 3 1.2 Imaging of biosilica and associated organic components 8 1.3 Single-molecule localization microscopy (SMLM) 10 2 METHODS & METHOD DEVELOPMENT FOR SMLM DATASETS 17 2.1 Super-resolution reconstruction 19 2.2 Tools for SMLM resolution estimates 21 2.3 Voronoi tessellation for noise-removal and cluster estimation 25 2.4 Tools for SMLM cluster analysis 27 2.5 Coordinate-based co-localization 32 2.6 PairRice – A novel algorithm to extract distances between cluster pairs 33 2.7 SiMoNa – A new GUI for exploring SMLM datasets 35 3 RESOLUTION OF THE SMLM SETUP TESTED WITH DNA ORIGAMI NANOSTRUCTURES 41 3.1 DNA origami as a length standard 42 3.2 Global resolution estimates 44 3.3 Local resolution estimates 47 3.4 Conclusion 53 4 EVALUATION OF PHOTO-CONTROLLABLE FLUORESCENT PROTEINS FOR PALM IN DIATOMS 55 4.1 Selecting PCFPs to minimize interference with the diatom autofluorescence 56 4.2 Screening results for cytosolic and biosilica-embedded PCFPs 58 4.3 The underlying conversion mechanism 61 4.4 Conclusion 63 5 IMAGING THE SIL3 MESHWORK 65 5.1 Analyzing protein layer thickness using tpSil3-Dendra2 65 5.2 Imaging the valve region using tpSil3 68 5.3 Resolution and localization parameters of tpSil3 70 5.4 Conclusion 72 6 DECIPHERING CINGULIN PATTERNS WITH CO LOCALIZATION STUDIES 73 6.1 A two-color cingulin construct for PALM-STORM 73 6.2 Steps towards PALM-STORM: screening, alignment, and imaging routine 76 6.3 Co-localization studies: quantification, clustering, and correlations 83 6.4 Conclusion 91 7 OUTLOOK 93 8 MATERIALS & METHODS 97 8.1 Microscope specifications 97 8.2 DNA origami annealing and AFM measurements 99 8.3 Diatom sample preparations 100 8.4 Fluorescence imaging conditions 102 8.5 Buffer systems 103 9 APPENDICES 105 9.1 Tables and Protocols 105 9.2 Satellite projects 112 9.2.1 Quantitative fluorescence intensity analysis of 3D time-lapse confocal microscopy data in diatoms 112 9.2.2 Applying neural networks to filter SMLM localizations 118 9.2.3 In vivo imaging at super-resolution conditions using SOFI 121 9.2.4 Quantifying chromatic aberrations in the microscope using fiducials 123 10 REFERENCES 127 / Diatoms feature the unique ability to form nanopatterned hierarchical silica cell walls with unprecedented accuracy and reproducibility. Gathering a deeper understanding of this process that is known as “biomineralization” is vitally important not only in the field of diatom research. In fact, the nanopatterning can also be exploited in the fields of material sciences or nanobiotechnology. According to the current understanding, the self-assembly of protein patterns along which biosilica is formed is key to this nanopatterning. Thus, in order to unravel the function of individual proteins that are involved in this biomineralization process, their structural organization has to be deciphered and correlated to morphological cell wall features that are in the order of tens of nanometer. Electron microscopy is able to resolve these features but does not provide protein-specific information. Therefore, a technique has to be established that is able to localize individual biosilica-associated proteins with nanometer precision. To achieve this objective, single-molecule localization microscopy (SMLM) for the diatom Thalassiosira pseudonana has been pioneered and exploited to localize different biosilica associated proteins inside silica and after silica removal. To obtain quantitative data, an analysis workflow was developed including graphical user interfaces and scripts for SMLM visualization, clustering, and co-localization. In order to find optimal labels for SMLM to target biosilica-embedded proteins, a comprehensive screening of photo-controllable fluorescent proteins has been carried out. Only three of six candidates were functional when embedded inside biosilica and fused to Silaffin3 – a protein that is tightly associated with the biosilica cell wall. Silaffin3 could be localized using SMLM with a localization precision of 25 nm. This allowed to resolve its structural organization and therefore identified Silaffin3 as a major component in the basal chamber of the fultoportulae. Additionally, co-localization studies on cingulins – a protein family hypothesized to be involved in silica formation – have been performed to decipher their pattern-function relationship. Towards this end, novel imaging strategies, co-localization calculations and pattern quantifications have been established. With the help of these results, the spatial arrangement of cingulins W2 and Y2 could be compared with unprecedented resolution. In summary, this work has laid ground for quantitative SMLM studies of proteins in diatoms in general and contributed insights into the spatial organization of proteins involved in biomineralization in the diatom T. pseudonana.:1 INTRODUCTION 1 1.1 Diatoms – a model system for biomineralization 3 1.2 Imaging of biosilica and associated organic components 8 1.3 Single-molecule localization microscopy (SMLM) 10 2 METHODS & METHOD DEVELOPMENT FOR SMLM DATASETS 17 2.1 Super-resolution reconstruction 19 2.2 Tools for SMLM resolution estimates 21 2.3 Voronoi tessellation for noise-removal and cluster estimation 25 2.4 Tools for SMLM cluster analysis 27 2.5 Coordinate-based co-localization 32 2.6 PairRice – A novel algorithm to extract distances between cluster pairs 33 2.7 SiMoNa – A new GUI for exploring SMLM datasets 35 3 RESOLUTION OF THE SMLM SETUP TESTED WITH DNA ORIGAMI NANOSTRUCTURES 41 3.1 DNA origami as a length standard 42 3.2 Global resolution estimates 44 3.3 Local resolution estimates 47 3.4 Conclusion 53 4 EVALUATION OF PHOTO-CONTROLLABLE FLUORESCENT PROTEINS FOR PALM IN DIATOMS 55 4.1 Selecting PCFPs to minimize interference with the diatom autofluorescence 56 4.2 Screening results for cytosolic and biosilica-embedded PCFPs 58 4.3 The underlying conversion mechanism 61 4.4 Conclusion 63 5 IMAGING THE SIL3 MESHWORK 65 5.1 Analyzing protein layer thickness using tpSil3-Dendra2 65 5.2 Imaging the valve region using tpSil3 68 5.3 Resolution and localization parameters of tpSil3 70 5.4 Conclusion 72 6 DECIPHERING CINGULIN PATTERNS WITH CO LOCALIZATION STUDIES 73 6.1 A two-color cingulin construct for PALM-STORM 73 6.2 Steps towards PALM-STORM: screening, alignment, and imaging routine 76 6.3 Co-localization studies: quantification, clustering, and correlations 83 6.4 Conclusion 91 7 OUTLOOK 93 8 MATERIALS & METHODS 97 8.1 Microscope specifications 97 8.2 DNA origami annealing and AFM measurements 99 8.3 Diatom sample preparations 100 8.4 Fluorescence imaging conditions 102 8.5 Buffer systems 103 9 APPENDICES 105 9.1 Tables and Protocols 105 9.2 Satellite projects 112 9.2.1 Quantitative fluorescence intensity analysis of 3D time-lapse confocal microscopy data in diatoms 112 9.2.2 Applying neural networks to filter SMLM localizations 118 9.2.3 In vivo imaging at super-resolution conditions using SOFI 121 9.2.4 Quantifying chromatic aberrations in the microscope using fiducials 123 10 REFERENCES 127

Pour bien comprendre comment les espèces actuelles ont évolué, il est important d’étudier certains groupes clés. Ces groupes clés sont parfois bien négligés au profit d’autres groupes apparentés. L’embranchement Hemichordata forme, avec Echinodermata, le clade Ambulacraria. Ce dernier, avec l’embranchement Chordata, forme le super-embranchement Deuterostomia. Parmi les deutérostomes, la classe d’hémichordé Enteropneusta est souvent considérée comme étant la plus ressemblante au dernier ancêtre commun des deutérostomes. Les entéropneustes partagent en effet plusieurs caractéristiques avec Chordata et Ambulacraria et en étudiant celles-ci on peut reconstruire et comprendre leurs états ancestraux. Dans le chapitre d’introduction, j’aborde la morphologie générale des hémichordés et leurs relations évolutives avec les deux autres embranchement deutérostomes. Je présente aussi les caractéristiques qu’ils partagent avec les échinodermes et les cordés. J’aborde ensuite les formes que prennent les parties dures chez les animaux et en particulier chez les deutérostomes. Dans le chapitre deux, j’examine et décris la forme et la composition chimique des osselets chez huit espèces d’hémichordé. Cette étude représente un énorme bond dans nos connaissances sur la biominéralisation chez les hémichordés, car jusqu’à présent les osselets n’avaient été décrits que chez deux espèces, et la composition chimique déterminée chez une seule d’entre elle. J’interprète également ces données dans un contexte évolutif, car les osselets d’hémichordé sont probablement homologues au squelette des échinodermes. Ce chapitre est important, car il nous donne une hypothèse sur l’origine des osselets chez le dernier ancêtre commun des ambulacraires. Dans le chapitre trois, je quantifie l’asymétrie dans les fentes pharyngiennes de populations de deux espèces d’entéropneustes et d’une espèce de cordé non-vertébré. En mettant ces différents nivaux de symétrie en parallèle avec leur comportement alimentaire, les résultats supportent l’hypothèse de l’alimentation par filtration comme rôle initial des fentes pharyngiennes chez les deutérostomes et que la perte de cette fonction induit du bruit développemental, une vestigialisation ou une perte des fentes branchiales. Dans le chapitre quatre, J’utilise la micro-tomographie aux rayons-X pour décrire une espèce d’hémichordé qui était jusqu’à présent un numen nudum. Cette nouvelle technique est comparée avec l’histologie traditionnelle afin de prouver qu’elle pourrait être utilisée dans les futures études taxonomiques sur les hémichordés. Dans le chapitre cinq, je présente quelques expériences qui ont dû être exclues des chapitres précédents car elles ont donné des résultats négatifs non-publiables. Je discute des raisons pour lesquelles ces expériences ont échoué ainsi que quelques pistes de solutions possibles pour qui voudrait tenter de les refaire. Ensuite je récapitule les résultats des chapitres précédents pour montrer comment étudier les hémichordés peut encore nous apprendre beaucoup sur d’autres groupes pourtant déjà très étudiés. / The phylum Hemichordata forms, with Echinodermata, the group Ambulacraria that in turns forms with Chordata, the Deuterostomia. Among deuterostomes, the hemichordate class Enteropneusta is often viewed as the group that most closely resembles the last common ancestor of deuterostomes. Enteropneusts indeed share many traits with the other two deuterostome phyla and by studying them, we can infer the ancestral states of those traits. In the first chapter, I present the general morphology of hemichordates and their relationships with the other two deuterostome phyla. I also discuss the shared traits between the hemichordates, the echinoderms and the chordates. Last, I present the varied shapes that hard parts can take in animals, with a focus on deuterostomes. In chapter two, I describe the shape and mineral composition of ossicles in eight enteropneust species. This study is a major leap in our understanding of biomineralization in Hemichordata since up to this point ossicles were only described in two species and the mineral composition determined for only one. I discuss these results in an evolutionary context since hemichordate ossicles are probably homologous with echinoderm skeletal ossicles. This chapter is significant because it provides a hypothesis on the origin of ossicles in the last common ancestor of ambulacrarians. In chapter three, I quantify the level of asymmetry of the pharyngeal slits in populations of two species of enteropneusts and the invertebrate cephalochordate Branchiostoma floridae. We found that adults of these species display fluctuating asymmetry in the gills and that this asymmetry is lower in filter feeding. This is significant because it supports the hypothesis that filter feeding is an ancestral feature of deuterostomes and that the loss of this function increases developmental noise, vestigiality, or loss of the gills. In chapter four, I use X-ray microtomography to describe the enteropneust Balanoglossus occidentalis that was heretofore a nomen nudum. This new technique is compared with traditional histology to show that it is a viable tool in hemichordate taxonomical studies. In chapter five, I present a few experiments that had to be excluded from the other chapters because they gave negative, unpublishable results. I discuss the probable causes of their failures and potential ways to solve these issues for those who would want to pursue them further. Finally, I summarise the results of the previous chapters to show how studying hemichordates can still teach us a lot about the origin and evolution of the better studied deuterostome phyla.

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La gazéification est aujourd'hui l'une des stratégies les plus prometteuses pour valoriser les déchets en énergie. Cette technologie thermo-chimique permet une réduction de 95 % de la masse des intrants et génère des cendres inertes ainsi que du gaz de synthèse (syngaz). Le syngaz est un combustible gazeux composé principalement de monoxyde de carbone (CO), d'hydrogène (H2) et de dioxyde de carbone (CO2). Le syngaz peut être utilisé pour produire de la chaleur et de l'électricité. Il est également la pierre angulaire d'un grand nombre de produits à haute valeur ajoutée, allant de l'éthanol à l'ammoniac et l'hydrogène pur. Les applications en aval de la production de syngaz sont dictées par son pouvoir calorifique, lui-même dépendant de la teneur du gaz en H2. L’augmentation du contenu du syngaz en H2 est rendu possible par la conversion catalytique à la vapeur d’eau, largement répandu dans le cadre du reformage du méthane pour la production d'hydrogène. Au cours de cette réaction, le CO est converti en H2 et CO2 selon : CO + H2O → CO2 + H2. Ce processus est possible grâce à des catalyseurs métalliques mis en contact avec le CO et de la vapeur. La conversion catalytique à la vapeur d’eau a jusqu'ici été réservé pour de grandes installations industrielles car elle nécessite un capital et des charges d’exploitations très importantes. Par conséquent, les installations de plus petite échelle et traitant des intrants de faible qualité (biomasse, déchets, boues ...), n'ont pas accès à cette technologie. Ainsi, la seule utilisation de leur syngaz à faible pouvoir calorifique, est limitée à la génération de chaleur ou, tout au plus, d'électricité. Afin de permettre à ces installations une gamme d’application plus vaste de leurs syngaz, une alternative économique à base de catalyseur biologique est proposée par l’utilisation de bactéries hyperthermophiles hydrogénogènes. L'objectif de cette thèse est d'utiliser Carboxydothermus hydrogenoformans, une bactérie thermophile carboxydotrophe hydrogénogène comme catalyseur biologique pour la conversion du monoxyde de carbone en hydrogène. Pour cela, l’impact d'un phénomène de biominéralisation sur la production d’H2 a été étudié. Ensuite, la faisabilité et les limites de l’utilisation de la souche dans un bioréacteur ont été évaluées. Tout d'abord, la caractérisation de la phase inorganique prédominante lorsque C. hydrogenoformans est inoculé dans le milieu DSMZ, a révélé une biominéralisation de phosphate de calcium (CaP) cristallin en deux phases. L’analyse par diffraction des rayons X et spectrométrie infrarouge à transformée de Fourier de ce matériau biphasique indique une signature caractéristique de la Mg-whitlockite, alors que les images obtenues par microscopie électronique à transmission ont montré l'existence de nanotiges cristallines s’apparentant à de l’hydroxyapatite. Dans les deux cas, le mode de biominéralisation semble être biologiquement induit plutôt que contrôlé. L'impact du précipité de CaP endogène sur le transfert de masse du CO et la production d’H2 a ensuite été étudié. Les résultats ont été comparés aux valeurs obtenues dans un milieu où aucune précipitation n'est observée. Dans le milieu DSMZ, le KLa apparent (0.22 ± 0.005 min-1) et le rendement de production d’H2 (89.11 ± 6.69 %) étaient plus élevés que ceux obtenus avec le milieu modifié (0.19 ± 0.015 min-1 et 82.60 ± 3.62% respectivement). La présence du précipité n'a eu aucune incidence sur l'activité microbienne. En somme, le précipité de CaP offre une nouvelle stratégie pour améliorer les performances de transfert de masse du CO en utilisant les propriétés hydrophobes de gaz. En second lieu, la conversion du CO en H2 par la souche Carboxydothermus hydrogenoformans fut étudiée et optimisée dans un réacteur gazosiphon de 35 L. Parmi toutes les conditions opérationnelles, le paramètre majeur fut le ratio du débit de recirculation du gaz sur le débit d'alimentation en CO (QR:Qin). Ce ratio impacte à la fois l'activité biologique et le taux de transfert de masse gaz-liquide. En effet, au dessus d’un ratio de 40, les performances de conversion du CO en H2 sont limitées par l’activité biologique alors qu’en dessous, elles sont limitées par le transfert de masse. Cela se concrétise par une efficacité de conversion maximale de 90.4 ± 0.3 % et une activité spécifique de 2.7 ± 0.4 molCO·g–1VSS·d–1. Malgré des résultats prometteurs, les performances du bioréacteur ont été limitées par une faible densité cellulaire, typique de la croissance planctonique de C. hydrogenoformans. Cette limite est le facteur le plus contraignant pour des taux de charge de CO plus élevés. Ces performances ont été comparées à celles obtenues dans un réacteur à fibres creuses (BRFC) inoculé par la souche. En dépit d’une densité cellulaire et d’une activité volumétrique plus élevées, les performances du BRFC à tout le moins cinétiquement limitées quand elles n’étaient pas impactées par le transfert de masse, l'encrassement et le vieillissement de la membrane. Afin de parer à la dégénérescence de C. hydrogenoformans en cas de pénurie de CO, la croissance de la bactérie sur pyruvate en tant que seule source de carbone a été également caractérisée. Fait intéressant, en présence simultanée de pyruvate et de CO, C. hydrogenoformans n’a amorcé la consommation de pyruvate qu’une fois le CO épuisé. Cela a été attribué à un mécanisme d'inhibition du métabolisme du pyruvate par le CO, faisant ainsi du pyruvate le candidat idéal pour un système in situ de secours. / Gasification is today one of the most promising strategies to recover energy from waste. This thermo-chemical technology allows a 95% weight reduction of the input and generates inorganic inert ashes as well as a synthesis gas (syngas). Syngas is a gaseous fuel mainly composed of carbon monoxide (CO), hydrogen (H2) and carbon dioxide (CO2). Syngas can be burned to produce heat and electricity. It is also the building block of many high added- value products ranging from ethanol to ammonia and pure hydrogen. Downstream applications of syngas production will depend on its heating value, which is determined by its content in H2. Upgrading the H2 content in syngas is performed by the water-gas shift (WGS) reaction, widely utilized during methane reforming for hydrogen production. During the WGS reaction CO is converted to H2 and CO2 according to: CO + H2O → CO2 + H2. This process is achieved using a metallic catalyst in a heterogeneous gas-phase reaction with CO and steam. The WGS reaction has so far been reserved for large-scale gasification plants and requires high capital and operational expenditures. Hence, smaller scale plants that process low-grade materials (biomass, waste, sludge...), would not have access to such technology. The only possible outcome with the synthesis gas (syngas) produced and which generally has a poor heating value, is to generate heat or at best, electricity. In order to offer small plants access to the WGS reaction and to a higher range of products from their syngas, an alternative to the expensive and energy-intensive established catalyst-based WGS is here considered, such as extreme-thermophilic microbial processes carried out by hydrogenogens. The goal of this thesis was to use Carboxydothermus hydrogenoformans, a thermophilic carboxydotrophic hydrogenogenic bacterium as a biological catalyst for the WGS reaction. This was done by characterizing the impact of a growth-associated biomineralization phenomenon on H2 production and assessing the feasibility and limitations of using the strain in a bioreactor. First, characterization of the predominant inorganic phase when Carboxydothermus hydrogenoformans was inoculated in the DSMZ medium revealed the biomineralization of two crystalline CaP phases. The X-ray diffractometry peaks and Fourier transform infrared spectroscopy spectrum of this biphasic material consistently showed features characteristic of Mg-whitlockite, whereas transmission electron microscopy analysis showed the existence of hydroxyapatite-like nanorods crystals. In both cases, the mode of biomineralization appears to be biologically induced rather than biologically controlled. The impact of the endogenous CaP precipitate on CO mass transfer and H2 production was thus assessed and compared to a medium where no precipitation was observed. In the DSMZ medium, the apparent KLa (0.22 ±0.005 min-1) and H2 production yield (89.11 ±6.69%) were higher than the ones obtained in the modified medium (0.19 ±0.015 min-1 and 82.60 ±3.62% respectively). The presence of the precipitate had no impact on C. hydrogenoformans CO uptake. Overall, the CaP precipitate offers a novel strategy for gas-liquid mass transfer enhancement using CO hydrophobic properties. Second, the conversion of CO into H2 by C. hydrogenoformans was investigated and optimized in a 35 L gas-lift reactor. Upon all operational conditions, the ratio of gas recirculation over CO feed flow rates (QR:Qin) was the major parameter that impacted both biological activity and volumetric gas-liquid mass transfer. The CO conversion performance of the gas lift reactor was kinetically limited over a QR:Qin ratio of 40, and mass transfer limited below that ratio, resulting in a maximum conversion efficiency of 90.4±0.3% and a biological activity of 2.7±0.4 molCO· g–1VSS· day–1. Despite very promising results, CO conversion performance was limited by a low cell density, typical of C. hydrogenoformans planktonic growth. This limitation was found to be the most restrictive factor for higher CO loading rates. Results were compared to the performance of the strain inoculated in a hollow fiber membrane bioreactor where performance, despite the higher cell density and volumetric activity, was biokinetically limited, when not limited by gas–liquid mass transfer, membrane fouling and aging. To avoid any C. hydrogenoformans decay during potential CO shortages, growth of the bacterium on pyruvate as a sole carbon source was characterized. Interestingly, when grown simultaneously on pyruvate and CO, pyruvate consumption was initiated upon CO depletion. This was attributed to the inhibition of pyruvate oxidation by CO, making pyruvate the ideal candidate for an in-situ back-up system.

This study was conducted to determine how the structure and surface chemistry of bulk CaCO3 differs from that of nanometer-sized CaCO3 and then to determine rate, extent and mechanisms of As adsorption on various synthetic CaCO3 materials. Additionally, we sought to devise a chemical CaCO3 precipitate that approximates biogenic CaCO3. The bulk CaCO3 precipitation was performed by using a solution that was highly oversaturated so that large CaCO3 precipitates rapidly form. Two different methods were employed for the synthesis of nanometer size CaCO3 i) an in situ deposition technique and ii) an interfacial reaction (water in oil emulsion). Mineral characterization of all CaCO3 precipitates was done with Nitrogen Porosimetry (Brunauer Emmett Teller method), particle size analysis, X-ray diffraction and Fourier Transform Infrared/ Fourier Transform Raman spectroscopy. The principal objective of the research was to assess the overall reactivity of As(III) and As(V) with different synthetic CaCO3 minerals. This was accomplished by i) running adsorption isotherms (varying As concentration), ii) measuring pH envelopes (varying pH at a fixed concentration) and iii) kinetic experiments (varying reaction time). Also, electrophoretic mobility experiments were performed in the presence of As(III) and As(V), and these studies revealed that As(III) forms stronger inner-sphere complexes with CaCO3 than As(V). Also, it was found that nanometer-sized CaCO3 prepared via deposition formed stronger inner-sphere complexes with As oxyanions (q = 5.26 µmol/m2) compared to either nano-sized CaCO3 from interfacial reactions (q = 4.51 µmol/m2) or bulk CaCO3 (q = 4.39 µmol/m2).<p> The PEG-based nano CaCO3 prepared by an in-situ deposition technique presents a novel and readily available synthesis route that can be used as proxy for the biogenic CaCO3 known to be present in many different environmental conditions. The results of this study suggest that CaCO3 can be used as a sorbent for As in groundwater.

This study was conducted to determine how the structure and surface chemistry of bulk CaCO3 differs from that of nanometer-sized CaCO3 and then to determine rate, extent and mechanisms of As adsorption on various synthetic CaCO3 materials. Additionally, we sought to devise a chemical CaCO3 precipitate that approximates biogenic CaCO3. The bulk CaCO3 precipitation was performed by using a solution that was highly oversaturated so that large CaCO3 precipitates rapidly form. Two different methods were employed for the synthesis of nanometer size CaCO3 i) an in situ deposition technique and ii) an interfacial reaction (water in oil emulsion). Mineral characterization of all CaCO3 precipitates was done with Nitrogen Porosimetry (Brunauer Emmett Teller method), particle size analysis, X-ray diffraction and Fourier Transform Infrared/ Fourier Transform Raman spectroscopy. The principal objective of the research was to assess the overall reactivity of As(III) and As(V) with different synthetic CaCO3 minerals. This was accomplished by i) running adsorption isotherms (varying As concentration), ii) measuring pH envelopes (varying pH at a fixed concentration) and iii) kinetic experiments (varying reaction time). Also, electrophoretic mobility experiments were performed in the presence of As(III) and As(V), and these studies revealed that As(III) forms stronger inner-sphere complexes with CaCO3 than As(V). Also, it was found that nanometer-sized CaCO3 prepared via deposition formed stronger inner-sphere complexes with As oxyanions (q = 5.26 µmol/m2) compared to either nano-sized CaCO3 from interfacial reactions (q = 4.51 µmol/m2) or bulk CaCO3 (q = 4.39 µmol/m2).<p> The PEG-based nano CaCO3 prepared by an in-situ deposition technique presents a novel and readily available synthesis route that can be used as proxy for the biogenic CaCO3 known to be present in many different environmental conditions. The results of this study suggest that CaCO3 can be used as a sorbent for As in groundwater.

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