TECHNISCHE UNIVERSITÄT MÜNCHEN Lehrstuhl für Bodenökologie Microbial community composition during degradation of organic matter Stefanie Elisabeth Wallisch 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: Univ.-Prof. Dr. A. Göttlein Prüfer der Dissertation: 1. Hon.-Prof. Dr. M. Schloter 2. Univ.-Prof. Dr. S. Scherer Die Dissertation wurde am 14.04.2015 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 03.08.2015 angenommen. Table of contents List of figures .................................................................................................................... iv List of tables ..................................................................................................................... vi Abbreviations .................................................................................................................. vii List of publications and contributions .............................................................................. viii Publications in peer-reviewed journals .................................................................................... viii My contributions to the publications ....................................................................................... viii Abstract ............................................................................................................................ 1 Zusammenfassung ............................................................................................................ 3 1 Introduction......................................................................................................... 5 1.1 Soil functions and soil organic matter dynamics .............................................................. 5 1.2 Microbial contribution to organic matter degradation ..................................................... 6 1.3 Anthropogenic influences affecting leaf litter degradation in soil .................................... 8 1.4 Aims and hypotheses .................................................................................................... 10 My contribution to this work ................................................................................................... 12 2 Material and methods ....................................................................................... 13 2.1 Experimental setup ....................................................................................................... 13 2.1.1 Experiment A: Monitoring the dynamics of microbial communities during leaf litter degradation of Z. mays under different tillage management practices .......................................... 13 2.1.2 Experiment B: Monitoring the dynamics of bacterial communities during leaf litter degradation of F. sylvatica incubated in different soil types........................................................... 18 2.1.3 Experiment C: Monitoring of alkB-harboring bacterial communities during compost degradation in petroleum-contaminated soil ................................................................................. 21 2.2 Methods ....................................................................................................................... 23 2.2.1 Measurement of the degradation rate ............................................................................................ 23 2.2.2 Nucleic acid extraction from leaf material ....................................................................................... 23 2.2.3 Amplification of 16S rRNA gene fragments ...................................................................................... 25 2.2.4 Randomized PCR .............................................................................................................................. 26 2.2.5 Terminal restriction fragment length polymorphisms ..................................................................... 26 2.2.6 Amplicon sequencing and data analysis ........................................................................................... 28 ii 2.2.7 Generating phylogenetic trees ......................................................................................................... 31 2.3 Statistical analyses ........................................................................................................ 31 3 Results ............................................................................................................... 33 3.1 Nucleic acid extraction from leaf material ...................................................................... 33 3.2 Microbial diversity during different stages of Z. mays leaf litter degradation .................. 35 3.2.1 Degradation of Z. mays leaf material ............................................................................................... 35 3.2.2 Bacterial community composition on Z. mays leaf material ............................................................ 36 3.2.3 Comparison of ecological indices ..................................................................................................... 39 3.2.4 Phylogenetic classification ............................................................................................................... 50 3.3 Microbial diversity during different stages of F. sylvatica leaf litter degradation ............. 54 3.3.1 Degradation of F. sylvatica leaf material .......................................................................................... 54 3.3.2 Bacterial community composition on F. sylvatica leaf material ...................................................... 55 3.3.3 Phylogenetic classification ............................................................................................................... 59 3.4 Influence of compost amendments on the diversity of alkane degrading bacteria in petroleum-contaminated soil ........................................................................................ 65 3.4.1 Diversity of alkB-harboring bacteria in soil ...................................................................................... 65 3.4.2 Phylogenetic classification ............................................................................................................... 68 4 Discussion .......................................................................................................... 72 4.1 Comparison of methods for determining changes in bacterial community composition... 72 4.2 Bacterial key players involved in Z. mays leaf litter degradation ..................................... 76 4.3 Bacterial key players involved in F. sylvatica leaf litter degradation ................................ 78 4.4 Bacterial key players occurring independently from nutrient input source ...................... 80 4.5 Bacterial community composition in petroleum contaminated soils enriched with organic compost............................................................................................................ 80 5 Conclusion and outlook ...................................................................................... 83 6 References ......................................................................................................... 84 7 Acknowledgement ............................................................................................ 101 8 Appendix .......................................................................................................... 102 iii List of figures Figure 1: Soil food web .............................................................................................................................7 Figure 2: Map of the research farm Scheyern ....................................................................................... 14 Figure 3: Placement of litter bags filled with Z. mays leaf material. ..................................................... 15 Figure 4: Methodological approach for experiment A with Z. mays leaf litter ..................................... 16 Figure 5: Soluble sugars and starch in leaf material of Z. mays ............................................................ 17 Figure 6: Microcosms with F. sylvatica leaf litter .................................................................................. 19 Figure 7: Methodological approach for experiment B with F. sylvatica leaf litter ................................ 19 Figure 8: Soluble sugars and starch in leaf material of F. sylvatica ....................................................... 20 Figure 9: Methodological approach for experiment C with petroleum-contaminated Technosol ....... 23 Figure 10: Scheme of fusion primers for 454 multiplex sequencing ..................................................... 28 Figure 11: Nucleic acids extracted from Z. mays ................................................................................... 33 Figure 12: Nucleic acids extracted from F. sylvatica ............................................................................. 34 Figure 13: Purity of nucleic acids extracted from F. sylatica leaf litter ................................................. 35 Figure 14: Dry mass of Z. mays leaf material .......................................................................................