Phylogenetic and functional diversity of soil prokaryotic communities in temperate deciduous forests with different tree species Dissertation for the award of the degree "Doctor of Philosophy" Ph.D. Division of Mathematics and Natural Sciences of the Georg-August-Universität Göttingen within the doctoral program in Biology of the Georg-August University School of Science (GAUSS) submitted by Amélie Dukunde from Nyamyumba (Rwanda) Göttingen, 2018 Thesis committee Prof. Dr. Rolf Daniel, Department of Genomics and Applied Microbiology, Institute of Microbiology and Genetics, Georg-August Universität Göttingen PD. Dr. Michael Hoppert, Department of General Microbiology, Institute of Microbiology and Genetics, Georg-August Universität Göttingen Members of the examination board Reviewer: Prof. Dr. Rolf Daniel, Department of Genomics and Applied Microbiology, Institute of Microbiology and Genetics, Georg-August Universität Göttingen Second Reviewer: PD. Dr. Michael Hoppert, Department of General Microbiology, Institute of Microbiology and Genetics, Georg-August Universität Göttingen Further members of the examination board: PD. Dr. Fabian Commichau, Department of General Microbiology, Institute of Microbiology and Genetics, Georg-August Universität Göttingen Prof. Dr. Kai Heimel, Department of Molecular Microbiology and Genetics, Institute of Microbiology and Genetics, Georg-August Universität Göttingen PD. Dr. Ina Meier, Plant Ecology and Ecosystem Research, Albrecht von Haller Institute of Plant Sciences Prof. Dr. Stefanie Pöggeler, Department of Genetics of Eukaryotic Microorganisms, Institute of Microbiology and Genetics, Georg-August Universität Göttingen Date of the oral examination: 17.05.2018 2 Table of contents Chapter 1: Literature review .................................................................................. 5 1.1 Overview of forest biomes .................................................................................................................... 5 1.2 Temperate deciduous forests ................................................................................................................ 7 1.2.1 Structure and vegetation type ........................................................................................................ 7 1.2.2 Forest microbial diversity ............................................................................................................... 9 1.3 Research overview and design ............................................................................................................ 13 1.3.1 The Hainich National Park .......................................................................................................... 13 1.3.2 Objective of thesis ......................................................................................................................... 14 Chapter 2: Tree species shape soil bacterial community structure and function in temperate deciduous forests ........................................................................... 15 2.1 Abstract .................................................................................................................................................. 16 2.2 Introduction ........................................................................................................................................... 17 2.3 Methods and materials ......................................................................................................................... 19 2.3.1 Sample site description ................................................................................................................. 19 2.3.2 Sampling and environmental nucleic acid isolation ................................................................. 20 2.3.3 Processing of 16S rRNA gene sequence data and statistical analyses ................................... 21 2.3.4 Accession numbers ....................................................................................................................... 22 2.4 Results and Discussion ......................................................................................................................... 23 2.4.1 Edaphic properties reflect stand-related variations .................................................................. 23 2.4.2 Stand-specific patterns in bacterial richness and diversity ...................................................... 24 2.4.3 Tree species effect of bacterial community structure .............................................................. 24 2.4.4 General patterns in bacterial community composition and structure ................................... 26 2.4.5 Taxa-habitat association patterns ................................................................................................ 30 2.4.6 Bacterial functional profiles across forest stands ...................................................................... 32 2.5 Conclusion ............................................................................................................................................. 36 Acknowledgments ....................................................................................................................................... 36 2.6 Supplementary information ................................................................................................................. 37 Chapter 3: Bioprospecting for novel biocatalysts ............................................... 49 3.1 Harvesting the soil metagenome ......................................................................................................... 49 3.2 Functional screening for lignocellulolytic enzymes.......................................................................... 52 3.2.1 Esterases ......................................................................................................................................... 52 3.2.2 Laccases .......................................................................................................................................... 54 3 3.2.3 Lignocellulases ............................................................................................................................... 54 3.2.4 Objectives: ...................................................................................................................................... 55 3.3 Methods and materials ......................................................................................................................... 56 3.3.1 General molecular cloning techniques ....................................................................................... 56 3.3.2 Function-based screening............................................................................................................. 57 3.4 Results and discussion .......................................................................................................................... 59 3.4.1 Construction metagenomic plasmid libraries ............................................................................ 59 3.4.2 Screening for lignocellulolytic enzymes ..................................................................................... 59 Chapter 4: A novel, versatile family IV carboxylesterase exhibits high stability and activity in a broad pH spectrum .................................................................. 65 4.1 Publication ............................................................................................................................................. 65 4.2 Supporting material ............................................................................................................................... 77 Chapter 5: Summary ............................................................................................ 82 References ........................................................................................................... 84 Acknowledgements ........................................................................................... 102 Appendices ........................................................................................................ 104 List of non-standard abbreviations ........................................................................................................ 104 Academic curriculum vitae ...................................................................................................................... 105 Thesis Declaration .................................................................................................................................... 106 4 Chapter 1 Chapter 1: Literature review 1.1 Overview of forest biomes The forest biome is the largest terrestrial ecosystem and covers approximately 4 billion hectares or 30% of Earth’s land area (FAO, 2006). It has global significance as the largest habitat of the of Earth’s terrestrial biodiversity and the greatest source of plant biomass (80%), which contributes as much as 75% to gross primary productivity (Baldrian, 2017; Pan et al., 2013). As such, forests act as major sinks of stored carbon and facilitates biogeochemical cycling of other elements and regulating the global climate (Lladó et al., 2017). Characterized by the dominance of trees and layered vegetation, forests are distributed across several biogeographic and climate zones (Figure 1.1). Forests also have numerous benefits for humans, by offering goods and services such as timber, biofuels, clean water, agricultural