Technische Universität München Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt Lehrstuhl für Bodenkunde Functional and phylogenetic diversity of cellulase genes in agricultural soil under two different tillage treatments Maria de Vries Vollständiger Abdruck der von der Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt der Technischen Universität München zur Erlangung des akademischen Grades eines Doktors der Naturwissenschaften genehmigten Dissertation. Vorsitzender: Prof. Dr. Siegfried Scherer Prüfer der Dissertation: 1. Hon. Prof. Dr. Michael Schloter 2. Prof. Dr. Wolfgang Liebl Die Dissertation wurde am 17.04.2018 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 27.07.2018 angenommen. Problems cannot be solved with the same mindset that created them. ― Albert Einstein Table of Contents Summary ................................................................................................................................................. 1 Zusammenfassung .................................................................................................................................. 3 1 - Introduction ....................................................................................................................................... 5 Soil degradation .................................................................................................................................. 5 Degradation of cellulose by soil microorganisms ............................................................................... 6 Cellulase domain families and substrate specificity ........................................................................... 8 Functional redundancy of cellulases ................................................................................................. 10 Aims and hypotheses of this thesis ................................................................................................... 12 2 - Materials and Methods .................................................................................................................... 15 Site description and soil sampling .................................................................................................... 15 Soil chemical and biological analysis................................................................................................. 16 Enzymatic activity assays .................................................................................................................. 18 DNA-extraction, library preparation and sequencing ....................................................................... 18 GH5-primer design, amplification and sequencing .......................................................................... 19 Quantitative real-time PCR assay...................................................................................................... 20 Analysis of metagenome data and prediction of cellulase genes .................................................... 21 Amplicon data analysis ..................................................................................................................... 24 Phylogenetic analysis of GH5 protein sequences ............................................................................. 25 3 - Results of the conventional farming experiment ............................................................................ 27 Soil description .................................................................................................................................. 27 Microbial community structure ........................................................................................................ 29 Cellulase enzymatic groups and taxonomic assignment .................................................................. 32 Annotation method of cellulase domain families ............................................................................. 34 Cellulase domain families ................................................................................................................. 35 Taxonomic assignment of cellulase domain families ........................................................................ 35 4 - Results of the organic farming experiment ..................................................................................... 41 Soil description .................................................................................................................................. 41 Microbial community structure ........................................................................................................ 43 Cellulase enzymatic groups and taxonomic assignment .................................................................. 49 Annotation method of cellulase domain families ............................................................................. 51 Cellulase domain families ................................................................................................................. 51 Taxonomic assignment of cellulase domain families ........................................................................ 52 Co-occurring microbial communities ................................................................................................ 58 Taxonomic characterisation of communities................................................................................ 59 Cellulose degradation potential within communities ................................................................... 60 5 - Results of phylogenetic analysis of amplified GH5-genes ............................................................... 63 GH5-primer design and quality analysis ........................................................................................... 63 GH5-cellulase identification by amplicon sequencing ...................................................................... 64 Taxonomic assignment ..................................................................................................................... 65 Phylogenetic tree of GH5-sequences from CAZy database .............................................................. 67 Phylogenetic tree of GH5-amplicon sequences ................................................................................ 69 Phylogenetic tree of top BLAST hits and complete database sequences ......................................... 71 6 - Discussion ........................................................................................................................................ 75 I - Assessment of the soil genetic diversity ....................................................................................... 75 Potentials and drawbacks of shotgun metagenomic datasets ..................................................... 75 Cellulase gene annotation methods ............................................................................................. 79 Phylogenetic diversity of GH5 cellulase genes in soil samples ..................................................... 81 II - Influence of tillage on microbial cellulose degraders .................................................................. 87 Responses of the soil microbiome to tillage in the conventional farming experiment ................ 87 Responses of the soil microbiome to tillage in the organic farming experiment ......................... 92 Comparison of metagenome-analysis results from both field experiments .............................. 101 7 - Conclusions and final remarks ....................................................................................................... 107 8 - Outlook .......................................................................................................................................... 109 Appendix ............................................................................................................................................. 110 Table A1 .......................................................................................................................................... 110 Table A2 .......................................................................................................................................... 111 Table A3 .......................................................................................................................................... 117 Table A4 .......................................................................................................................................... 118 Table A5 .......................................................................................................................................... 118 Table A6 .......................................................................................................................................... 119 Table A7 .......................................................................................................................................... 121 Table A8 .......................................................................................................................................... 121 Table A9 .......................................................................................................................................... 134 Table A10 .......................................................................................................................................