From Nature to Nurture: Isolation, Physiology and Preservation of Methane-Oxidizing Bacteria Sven Hoefman Promotor Prof. Dr. Paul De Vos Co-Promotors Prof. Dr. Peter Vandamme Dr. Kim Heylen Dissertation submitted in fulfillment of the requirements for the degree of Doctor (Ph.D.) in Sciences, Biotechnology Sven Hoefman – From Nature to Nurture: Isolation, Physiology and Preservation of Methane-Oxidizing Bacteria Copyright ©2013 Sven Hoefman ISBN-number: 978-9-4619711-8-0 No part of this thesis protected by its copyright notice may be reproduced or utilized in any form, or by any means, electronic or mechanical, including photocopying, recording or by any information storage or retrieval system without written permission of the author and promotors. Printed by University Press | www.universitypress.be Ph.D. thesis, Faculty of Sciences, Ghent University, Ghent, Belgium. This Ph.D. work was financially supported by a project of the Geconcerteerde Onderzoeksacties of Ghent University (BOF09/GOA/005) Publicly defended in Ghent, Belgium, May 27th, 2013 Examination committee Prof. Dr. Savvas Savvides (Chairman) L-Probe: Laboratory for protein Biochemistry and Biomolecular Engineering, Faculty of Sciences, Ghent University, Belgium Prof. Dr. Paul De Vos (Promotor) LM-UGent: Laboratory of Microbiology, Faculty of Science, Ghent University, Belgium BCCM-LMG Bacteria Collection, Ghent, Belgium Prof. Dr. Peter Vandamme (Co-promotor) LM-UGent: Laboratory of Microbiology, Faculty of Science, Ghent University, Belgium Dr. Kim Heylen (Co-promotor) LM-UGent: Laboratory of Microbiology, Faculty of Science, Ghent University, Belgium Prof. Dr. Nico Boon LabMET: Laboratory of Microbial Ecology and Technology, Faculty of Bioscience Engineering, Ghent University, Belgium Dr. Huub Op den Camp Institute for Water and Wetland Research, Faculty of Science, Radboud University Nijmegen, The Netherlands Prof. Dr. Hendrikus Laanbroek NIOO-KNAW: Netherlands Institute of Ecology, Wageningen, The Netherlands Prof. Dr. Stéphane Declerck Faculty of Biological, Agricultural and Environmental Engineering, Université catholique de Louvain, Louvain-la-Neuve, Belgium i List of abbreviations AMO ammonia monooxygenase ANOVA analysis of variance AOB ammonia-oxidizing bacteria AUC area under the curve BLAST basic local alignment search tool CBB Calvin-Benson-Bassham cycle CFU colony forming units CPA cryoprotectant (d)AMS (diluted) ammonium mineral salts DGGE denaturing gradient gel electrophoresis DMSO dimethyl sulfoxide (d)NMS (diluted) nitrate mineral salts DO dissolved oxygen EM expectation maximization FCM flow cytometry GC gas chromatography GTR general time reversible HAO hydroxylamine oxidoreductase HRT hydraulic retention time ICM intracytoplasmic membrane LPA lyoprotectant MALDI TOF matrix assisted laser desorption ionisation time of flight MS mass spectrometry ML maximum likelihood MOB methane-oxidizing bacteria MOR methane oxidation rate MPN most probable number NOB nitrite-oxidizing bacteria iii OD optical density PAR photosynthetic active range PFP primary facultative pond PI propidium iodide PLFA phospholipid-derived fatty acids (P)PCA (probabilistic) principle component analysis (p/s)MMO (particulate/soluble) methane monooxygenase rep-PCR repetitive element sequence based polymerase chain reaction ROC receiver operating characteristic RuMP ribulose monophosphate (s)COD (soluble) chemical oxygen demand SRT sludge retention time T-RFLP terminal-restriction fragment length polymorphism TSA trypticase soy agar TSB trypticase soy broth UPGMA unweighted pair group method with arithmetic averages VBNC viable but non-culturable VSS volatile suspended solids WSP waste stabilization pond WWTP waste water treatment plant iv Table of contents Examination committee i List of abbreviations iii Table of contents v Part A: General Introduction 1 Background & Scope 3 An introduction to aerobic methane-oxidizing bacteria 5 Aims & Outline 29 References 33 Part B: Isolation of methane-oxidizing bacteria 41 Chapter I: Miniaturized extinction culturing for rapid isolation of methane-oxidizing bacteria 43 Chapter II: Facultative ponds harbor methane oxidizing communities supported by algae 69 Reflection & Discussion 89 Part C: Physiology of methane-oxidizing bacteria 95 Chapter III: Customized media based on miniaturized screening improve growth rate and cell yield of Methylomonas strains 97 Chapter IV: Versatile and strain-dependent nitrogen metabolism within the genus Methylomonas 121 Chapter V: Exploration and prediction of interactions between methanotrophs and heterotrophs 145 Chapter VI: Methyloparacoccus murrellii, a novel methanotroph isolated from pond water in South Africa and Japan 171 v Chapter VII: Methylomonas lenta, a novel methanotroph isolated from manure and a denitrification tank in Belgium 189 Reflection & Discussion 205 Part D: Preservation of methane-, ammonia- and nitrite-oxidizing bacteria 215 Chapter VIII: Survival or Revival: Long-term preservation induces a reversible viable but non-culturable state in methane-oxidizing bacteria 217 Chapter IX: Efficient cryopreservation protocol enables accessibility of a broad range of ammonia-oxidizing bacteria 241 Chapter X: Influence of carbon and DMSO concentration on the successful preservation of nitrite-oxidizing bacteria 253 Reflection & Discussion 269 Concluding remarks 273 Summary 275 Samenvatting 277 Dankwoord 281 Curriculum vitae 285 vi Part A: General Introduction Part A - General Introduction Background & Scope The surface of our planet is warming up. Iconic to this fact is the melting of the polar ice caps, but other direct major implications are observed that affect us all, including extreme drought, loss of biodiversity and an increase in weather extremities, to name a few. The temperature on Earth is governed by a balance of greenhouse gases, such as water vapor, carbon dioxide, methane and nitrous oxide, which absorb solar energy and reradiate it in the form of heat into the atmosphere. For ages, these gases are continuously being produced and broken down by a variety of chemical and biological processes. However, since the industrial revolution the atmospheric greenhouse gas concentration has gone up due to an increase in human-induced emissions. For example, extensive methane emissions are detected from rice paddies, animal husbandry and landfills. All around the world, microbes, termed methanotrophs, are found that can consume this greenhouse gas, and are thus viewed as gatekeepers preventing excessive methane emissions from escaping to the atmosphere. It is of vital importance to understand what factors may positively or negatively impact the methane consuming activity of these organisms. Therefore, tools are needed to efficiently bring and keep these organisms into culture and to catalog and safeguard the methane-oxidizing biodiversity. In the future, this might allow us to manage these human-related methane emissions, by stimulating these organisms to consume methane, which can potentially be coupled to the degradation of groundwater pollutants or the production of high-value biomass. The work in this thesis describes (i) the successful long-term preservation of methane- utilizing bacteria and confirms the broad applicability of the applied methodology for fastidious bacteria in general, (ii) advocates the use of miniaturized tools for rapid isolation, screening and characterization of these methanotrophs, (iii) discusses the large variability and its implications of important physiological features between closely related strains and (iv) validates the research by depositing all characterized strains in public culture collections and describing several novel methanotrophic taxa. 3 Part A - General Introduction The research was performed between May 2009 and March 2013 at the Laboratory of Microbiology (LM-UGent), Faculty of Science, Ghent University. The central research themes of this lab are diversity, classification and identification of a wide variety of bacteria, including food-associated, animal and human-associated and environmental strains. In 2009, a project of the Geconcerteerde Onderzoeksacties of Ghent University (BOF09/GOA/005) was initiated in collaboration with the Laboratory of Microbial Ecology and Technology (LabMET), Ghent University. The aim of this project, named “Sustainable methanotrophs: from ecology to microbial resource management”, is to develop the concept of managing microbial methanotrophic communities by (i) the transposition and selection of natural methane-oxidizing inocula in diverse conditions, (ii) identifying, isolating and preserving the key microbial partners in these consortia and (iii) exploring methanotroph applications relevant to the society. As LM-UGent had no prior experience or expertise in working with these organisms, several tools had to be implemented initially in order to perform the experiments described throughout the thesis and to initiate this exciting new research line at the lab. 4 Part A - General Introduction An introduction to aerobic methane-oxidizing bacteria METHANE-OXIDIZING BACTERIA Definition Methanotrophs are microorganisms defined by their ability to generate energy via the oxidation of the greenhouse gas methane (Semrau et al., 2010). In this way, all methanotrophs fully oxidize CH4 to CO2 (Chistoserdova, 2011; Joye, 2012). The organisms that fulfill this definition are remarkably diverse. In anaerobic environments, both archaea and bacteria can oxidize methane. Anaerobic methanotrophic archaea couple methane oxidation