TECHNISCHE UNIVERSITÄT MÜ NCHEN Lehrstuhl für Grundwasserökologie Spatio-temporal patterns of suspended and attached bacterial communities in a hydrologically dynamic aquifer (Mittenwald, Germany) Yuxiang Zhou 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. R.F. Vogel Prüfer der Dissertation: 1. Univ. - Prof. Dr. R.U. Meckenstock 2. Univ. - Prof. Dr. J. Geist Die Dissertation wurde am 03.04.2013 bei der Technischen Universität München eingereicht und durch die Fakultät Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt am 02.07.2013 angenommen. 天行健，君子以自强不息 地势坤，君子以厚德载物 ——《周易.乾》 TABLE OF CONTENT I. TABLE OF CONTENT I TABLE OF CONTENT………………………………………………………………………………………….Ⅰ II ABSTRACT…………………………………………………………………………………………………….....Ⅳ 1 INTRODUCTION .......................................................................................................................... 1 1.1 Groundwater ecosystems ................................................................................................................................. 1 1.1.1 Basic features ........................................................................................................................................................................ 1 1.1.2 Environmental factors controlling the spatio-temporal distribution of microbes ................................ 2 1.2 Biomass, activity and composition of microbial communities ............................................................. 4 1.2.1 Microbial biomass and activity ..................................................................................................................................... 4 1.2.2 Microbial composition and diversity .......................................................................................................................... 7 1.2.2.1 Microbial community composition .................................................................................................................... 7 1.2.2.2 Diversity of microbial communities ................................................................................................................... 7 1.2.2.3 Protozoa in groundwater ........................................................................................................................................ 9 1.3 Viruses in groundwater .................................................................................................................................. 10 1.3.1 Characterization, distribution and diversity of viruses .................................................................................. 10 1.3.2 Role of viruses in groundwater .................................................................................................................................. 11 1.4 Concept and objectives of the thesis .......................................................................................................... 13 1.4.1 Spatio-temporal patterns of bacterial communities in a hydrologically dynamic pristine porous aquifer ................................................................................................................................................................................................. 13 1.4.2 Bacterial colonization in groundwater explored by 454 pyrosequencing ............................................. 15 1.4.3 Microbial food web structure and carbon cycling in a pristine porous aquifer (Mittenwald, Germany) ............................................................................................................................................................................................. 17 2 MATERIALS AND METHODS ................................................................................................ 20 2.1 Study area ........................................................................................................................................................... 20 2.2 Sampling .............................................................................................................................................................. 21 2.3 Physical–chemical conditions ...................................................................................................................... 22 2.4 Water stable isotope analysis ....................................................................................................................... 23 2.5 Microbiological variables .............................................................................................................................. 23 2.5.1 Counts of bacterial cells, protozoa and virus-like particles ........................................................................... 23 2.5.2 Bacterial carbon production ........................................................................................................................................ 25 2.5.3 Assimilable organic carbon .......................................................................................................................................... 25 2.5.4 ATP concentration ........................................................................................................................................................... 25 2.6 Microbiological molecular analysis ............................................................................................................ 26 2.6.1 DNA extraction and amplification ............................................................................................................................. 26 i TABLE OF CONTENT 2.6.2 Terminal restriction fragment length polymorphism analysis ................................................................... 26 2.6.3 Clone libraries .................................................................................................................................................................... 26 2.6.4 Tag sequencing .................................................................................................................................................................. 27 2.6.5 Sequence data analysis .................................................................................................................................................. 28 2.6.6 Eukaryotic communities profiling by T-RFLP, cloning and sequencing .................................................. 28 2.7 Statistical analysis ............................................................................................................................................ 29 2.8 Estimation of bacterial growth rates and mortality ............................................................................. 29 2.8.1 Estimation of growth rates .......................................................................................................................................... 29 2.8.2 Estimation of bacterial mortality .............................................................................................................................. 29 3 RESULTS ..................................................................................................................................... 31 3.1 Physical-chemical characteristics ............................................................................................................... 31 3.2 Spatio-temporal patterns of bacterial communities ............................................................................ 34 3.2.1 Suspended bacterial communities ............................................................................................................................ 34 3.2.2 Attached bacterial communities ................................................................................................................................ 40 3.3 Colonization of bacterial communities revealed by 454 pyrosequencing .................................... 42 3.3.1 Estimation of microbial diversity and richness .................................................................................................. 42 3.3.2 Colonization patterns of bacterial communities on sterile sediments ..................................................... 45 3.3.2.1 Colonization patterns of relative abundances of dominant groups.................................................. 45 3.3.2.2 Colonization patterns of cell abundances of dominant groups .......................................................... 48 3.3.2.3 Growth patterns of dominant groups ............................................................................................................ 50 3.3.3 Comparison of bacterial community composition from groundwater and sediments ..................... 54 3.3.3.1 Comparison of suspended and attached bacterial communities ....................................................... 54 3.3.3.2 Comparison of bacterial communities on initially river bed sediments and initially sterile sediments .................................................................................................................................................................................... 58 3.3.3.3 Multi-dimensional scaling analysis of all samples for the genus level data .................................. 61 3.4 Microbial food web structure