The methanotrophic interactome: Microbial partnerships for sustainable methane cycling ir. Frederiek-Maarten Kerckhof 1 Notation index Promotors: Prof. dr. ir. Nico Boon Department of Biochemical and Microbial Technology, Faculty of Bioscience Engineering, Ghent University, Ghent, Belgium. Dr. Kim Heylen Department of biochemistry and microbiology, Faculty of Sciences, Ghent University, Ghent, Belgium. Members of the examination committee: Prof. dr. ir. Koen Dewettinck (Chairman) Laboratory of food technology and engineering, Faculty of bioscience engineering, Ghent University, Gent, Belgium Prof. dr. ir. Diederik Rousseau (Secretary) Laboratory of industrial water- and ecotechnology, Faculty of bioscience engineering, Ghent University, Kortrijk, Belgium Prof. dr. ir. Pascal Boeckx ISOFYS, Faculty of bioscience engineering, Ghent University, Gent, Belgium Prof. dr. Paul De Vos Laboratory of microbiology (LM-Ugent), Faculty of sciences, Ghent University, Ghent, Belgium Dr. Paul Bodelier Department of microbial ecology, Netherlands institute of ecology (NIOO-KNAW), Wageningen, The Netherlands Dr. Hannah Marchant Department of biogeochemistry, Max-Planck Institute (MPI) for Marine Microbiology, Bremen, Germany Dean Faculty of Bioscience Engineering Prof. dr. ir. Marc van Meirvenne Rector Ghent University Prof. dr. Anne De Paepe 2 The methanotrophic interactome: Microbial partnerships for sustainable methane cycling ir. Frederiek-Maarten Kerckhof Thesis submitted in fulfilment of the requirements for the degree of Doctor (Ph.D.) in Applied Biological Sciences 3 Notation index Titel van het doctoraat in het Nederlands: Het methanotroof interactoom: microbiële relaties voor een duurzame methaancyclus. Cover illustration by Tim Lacoere (www.timternet.be) “Sustainable interactomes” Chapter illustrations by Maarten Van Praet (www.maartenisdemax.be) Please refer to this work as: Kerckhof F.M. (2016). The methanotrophic interactome: microbial partnerships for sustainable methane cycling. PhD thesis, Ghent University, Belgium. ISBN: 978-90-5989-892-9 This work was supported by the Ghent University BOF-GOA project on sustainable methanotrophs (BOF/GOA05/009) and the Belgian Federal Government Belspo inter- university attraction program (IAP) µ-manager http://www.mrm.ugent.be/ The author and promotors give the authorization to consult and to copy parts of this work for personal use only. Every other use is subject to the copyright laws. Permission to reproduce any material contained in this work should be obtained from the author. 4 Opgedragen aan: Dr. Jozef Kerckhof 5 NOTATION INDEX A Adenin BLAST Basic Local Alignment Search Tool Bp Base pares C Cytosin CFU Colony Forming Unit Co Community Organization DGGE Denaturating Gradient Gel Electrophoresis DMSO Dimethyl Sulfoxide DNA Deoxyribonucleic Acid dNMS diluted Nitrate Mineral Salts dNTP Deoxyribonucleotide triphosphate EDTA Ethylenediamine Tetracetic Acid EPS Extracellular polymeric substances FADH Formaldehyde dehydrogenase FDH Formate dehydrogenase FID Flame Ionization Detector FWER Family-wise error rate G Guanin G+ Gram-positive G- Gram-negative GC Gas Chromatography GHG Greenhouse gas IPCC Intergovernmental Panel on Climate Change LCA Last Common Ancestor LOD Limit of detection LOQ Limit of quantification MDH Methanol dehydrogenase MOB Methanotrophic bacteria, methanotrophs, aerobic MOB I Notation index MOR Methane oxidation rate MRM Microbial Resource Management N Total count NA Nutrient Agar NMS Nitrate Mineral Salts Nt Nucleotide NCBI National Center for Biotechnology Information OD Optical Density OTU Operational Taxonomic Unit Ppmv Volumetric parts per million PC Principal Component PCA Principal Component Analysis PCR Polymerase Chain Reaction pMMO Particulate Methane Mono-oxygenase RDP Ribosomal Database Project Rr Range-weighted Richness RNA Ribonucleic Acid SCP Single-cell protein SG SYBR Green SIP Stable isotope probing sMMO Soluble Methane Mono-oxygenase SRA Sequence Read Archive T Thymin TAE Tris-Acetate-EDTA TSA Trypticase Soy Agar QQ Quantile-Quantile plot qPCR Quantitative (real-time) polymerase chain reaction II TABLE OF CONTENTS CHAPTER 1: INTRODUCTION ........................................................................... 3 1. Global methane cycling: sources and sinks .................................................... 3 1.1. Methane as a greenhouse gas (GHG) ................................................................. 3 1.2. Methane sources ................................................................................................. 4 1.3. Methane sinks and reservoirs ............................................................................. 6 2. Methanotrophic bacteria ................................................................................. 7 2.1. Phylogeny, taxonomy and classification ............................................................ 8 2.2. Physiology ........................................................................................................ 11 2.2.1. Aerobic methylotrophs and methanotrophs ...................................................... 11 2.2.2. Anaerobic methane oxidation ........................................................................... 17 2.2.3. Facultative and high-affinity MOB .................................................................. 18 2.2.4. Methane fermentation under hypoxia ............................................................... 19 2.3. Ecology and ecophysiology .............................................................................. 19 3. The methanotrophic interactome ................................................................... 21 3.1. The methanotrophic food web .......................................................................... 21 3.2. Preferential partnerships in methanotrophic microbial ecosystems ................. 22 4. Applications of methanotrophic interactomes .............................................. 25 5. Research outline and objectives .................................................................... 28 CHAPTER 2: THE METHANOTROPHIC ‘INTERACTOME’: CARBON FLOW THROUGH AN INTERACTING METHANE-OXIDIZING COMMUNITY ................................................................................................. 35 1. Introduction .................................................................................................... 36 2. Material and methods .................................................................................... 42 2.1. Mixed culture growth conditions and MOR calculation .................................. 42 2.2. DNA extraction and DNA-SIP ......................................................................... 42 2.3. Abundance of MOB .......................................................................................... 43 III Table of contents 2.4. PCR-DGGE and 454 amplicon sequencing ...................................................... 44 2.4.1. PCR and DGGE analysis .................................................................................. 44 2.4.2. PCR and 454 amplicon pyrosequencing ........................................................... 44 2.5. Data analysis ..................................................................................................... 45 3. Results and discussion .................................................................................... 46 3.1. Initial community composition, MOB activity and MOB abundance .............. 46 3.2. Community structure dynamics and the selection of sampling points ............. 49 3.3. Non-MOB partners intricately associated with the methanotrophic interactome. .......................................................................................................................... 51 3.4. Primary consumers, secondary consumers and the possible mode of interaction in the methanotrophic interactome ................................................................... 55 4. Conclusion and perspectives .......................................................................... 56 5. Acknowledgments and author contributions ............................................... 57 CHAPTER 3: OPTIMAL PARTNERSHIPS IN THE METHANOTROPHIC INTERACTOME: SUPERVISED SELECTION ................................... 63 1. Introduction .................................................................................................... 65 2. Material and methods .................................................................................... 66 2.1. Bacterial strains, growth conditions and experimental design ......................... 66 2.2. Analytical methods ........................................................................................... 70 2.3. DNA extraction, primer design, qPCR and data analyses ................................ 70 3. Results .............................................................................................................. 73 3.1. Phase I: establishing initial 1:1 partner compatibility ...................................... 73 3.2. Phase II: challenging the 1:1 interactome ........................................................ 79 3.2.1. Impact on functionality: methane oxidation rates ............................................ 79 3.2.2. Impact on the 1:1 interactome .......................................................................... 80 4. Discussion ........................................................................................................ 83 4.1. Phase I: establishing compatibility