Microbial degradation of organic micropollutants in hyporheic zone sediments Dissertation To obtain the Academic Degree Doctor rerum naturalium (Dr. rer. nat.) Submitted to the Faculty of Biology, Chemistry, and Geosciences of the University of Bayreuth by Cyrus Rutere Bayreuth, May 2020 This doctoral thesis was prepared at the Department of Ecological Microbiology – University of Bayreuth and AG Horn – Institute of Microbiology, Leibniz University Hannover, from August 2015 until April 2020, and was supervised by Prof. Dr. Marcus. A. Horn. This is a full reprint of the dissertation submitted to obtain the academic degree of Doctor of Natural Sciences (Dr. rer. nat.) and approved by the Faculty of Biology, Chemistry, and Geosciences of the University of Bayreuth. Date of submission: 11. May 2020 Date of defense: 23. July 2020 Acting dean: Prof. Dr. Matthias Breuning Doctoral committee: Prof. Dr. Marcus. A. Horn (reviewer) Prof. Harold L. Drake, PhD (reviewer) Prof. Dr. Gerhard Rambold (chairman) Prof. Dr. Stefan Peiffer In the battle between the stream and the rock, the stream always wins, not through strength but by perseverance. Harriett Jackson Brown Jr. CONTENTS CONTENTS CONTENTS ............................................................................................................................ i FIGURES.............................................................................................................................. vi TABLES ............................................................................................................................... ix EQUATIONS ........................................................................................................................ xi ABBREVIATIONS ............................................................................................................... xii 1. SUMMARY ................................................................................................................. 1 2. ZUSAMMENFASSUNG .............................................................................................. 5 3. INTRODUCTION ......................................................................................................... 9 3.1. Emerging pollutants in the environment.............................................................. 9 3.1.1. Occurrence ................................................................................................................ 9 3.1.2. Importance of organic pollutants in the environment ........................................... 11 3.1.3. The fate of TrOCs in the environment ................................................................... 12 3.2. Hyporheic zone .................................................................................................... 17 3.3. Removal of TrOCs in the hyporheic zone .......................................................... 18 3.4. Drivers and processes influencing TrOC removal in the hyporheic zone ....... 19 3.4.1. Hyporheic exchange flow and subsurface residence time .................................. 19 3.4.2. Temperature gradients ........................................................................................... 20 3.4.3. Redox gradients ...................................................................................................... 22 3.4.4. Organic carbon gradients ....................................................................................... 25 3.4.5. Microbial community structure ............................................................................... 27 3.5. Hypotheses and objectives ................................................................................. 30 4. MATERIALS AND METHODS .................................................................................. 32 4.1. Site description .................................................................................................... 32 4.2. Sampling procedure ............................................................................................ 37 4.3. Experimental setups ............................................................................................ 38 4.3.1. TrOC removal potential of hyporheic zone sediments ......................................... 38 4.3.2. Effect of redox conditions on the TrOC removal potential ................................... 40 4.3.3. TrOC removal under contrasting organic carbon conditions ............................... 42 4.3.4. Influence of HEF and microbial diversity on TrOC removal ............................... 43 4.4. Analytical methods .............................................................................................. 47 4.4.1. High performance liquid chromatography (HPLC) ............................................... 47 4.4.1.1. Preparation of liquid samples ....................................................................... 47 4.4.1.2 Ibuprofen ................................................................................................... 47 4.4.1.3. Metoprolol ................................................................................................. 47 i CONTENTS 4.4.2. Mass spectrometry .................................................................................................. 48 4.4.2.1. Sample preparation ..................................................................................... 48 4.4.2.2. Targeted quantification of parent compounds and TPs ................................. 49 4.4.2.3. Non-target/suspect screening of TPs ............................................................ 53 4.4.3. Quantification of TOC ............................................................................................. 54 4.4.4. Quantification of Nitrate .......................................................................................... 54 4.4.5. pH ............................................................................................................................. 55 4.4.6. Salt tracer dilution test /Electrical conductivity ..................................................... 55 4.5. Microbiological methods ..................................................................................... 57 4.5.1. Solutions and media ............................................................................................... 57 4.5.1.1 Defined mineral medium DM 1 ..................................................................... 57 4.5.1.2. Defined mineral medium DM 2 .................................................................... 59 4.5.1.3. LB agar medium ........................................................................................... 60 4.5.1.4. LB agar with ampicillin/IPTG/X-Gal ............................................................... 61 4.5.1.5. SOC medium ................................................................................................ 61 4.5.2. Enrichment and isolation procedures .................................................................... 62 4.5.3. Growth measurments ............................................................................................. 63 4.5.4. Microscopy............................................................................................................... 63 4.6. Molecular techniques .......................................................................................... 63 4.6.1. Extraction of nucleic acids ...................................................................................... 63 4.6.2. Separation of DNA and RNA ................................................................................. 64 4.6.3. Quantification of nucleic acids ............................................................................... 64 4.6.4. Reverse transcription of RNA ................................................................................ 65 4.6.5. Polymerase chain reaction (PCR) ......................................................................... 66 4.6.5.1. ‘Control’ PCR................................................................................................ 67 4.6.5.2. Isolate full-length 16S rRNA PCR ................................................................. 67 4.6.5.3. PCR for representative enriched OTUs (4.3.1) ............................................. 68 4.6.5.4. PCR for Illumina sequencing ........................................................................ 68 4.6.6. Quantitative PCR (qPCR)....................................................................................... 73 4.6.7. Cloning ..................................................................................................................... 76 4.6.1. Agarose gel electrophoresis................................................................................... 77 4.6.2. Purification of PCR products .................................................................................. 78 4.7. Bioinformatics ..................................................................................................... 79 4.7.1. Primer design and specificity assessment ............................................................ 79 4.7.2. Isolate sequence processing and phylotype assignment .................................... 79 4.7.3. Processing of Illumina amplicon sequence data .................................................. 80 ii CONTENTS 4.7.4. Accession numbers................................................................................................. 82 4.8. Calculations