METHYLATED AMINE-UTILISING BACTERIA AND MICROBIAL NITROGEN CYCLING IN MOVILE CAVE DANIELA WISCHER UNIVERSITY OF EAST ANGLIA PHD 2014 METHYLATED AMINE-UTILISING BACTERIA AND MICROBIAL NITROGEN CYCLING IN MOVILE CAVE A thesis submitted to the School of Environmental Sciences in fulfilment of the requirements for the degree of Doctor of Philosophy by DANIELA WISCHER in SEPTEMBER 2014 University of East Anglia, Norwich, UK This copy of the thesis has been supplied on condition that anyone who consults it is understood to recognise that its copyright rests with the author and that use of any information derived there from must be in accordance with current UK Copyright Law. In addition, any quotation or extract must include full attribution. i Table of Contents List of Figures ............................................................................................................... vii List of Tables .................................................................................................................. xi Declaration....................................................................................................................xiii Acknowledgements ...................................................................................................... xiv Abbreviations & Definitions ......................................................................................... xv Abstract........ ................................................................................................................ xix Chapter 1. Introduction ................................................................................................ 1 1.1 Cave ecosystems and Movile Cave ..................................................................... 2 1.1.1 Hypogenic caves ........................................................................................ 2 1.1.2 Movile Cave – a unique underground ecosystem ...................................... 3 1.1.3 Movile Cave – formation and features....................................................... 4 1.1.4 Conditions in Movile Cave ........................................................................ 6 1.1.5 Evidence for isolation of Movile Cave ...................................................... 7 1.2 Microbiology of Movile Cave ............................................................................. 8 1.2.1 Primary producers: sulfur, methane and ammonia oxidisers ..................... 8 1.2.2 Microorganisms of the sulfur cycle ........................................................... 9 1.2.3 C1 metabolism: methanotrophs and methylotrophs ................................. 10 1.2.4 Nitrogen cycling in Movile Cave ............................................................. 11 1.2.5 Other microbial processes in Movile Cave .............................................. 12 1.2.6 Proposed microbial food web of Movile Cave ........................................ 13 1.3 The microbial nitrogen cycle ............................................................................. 14 1.3.1 Overview of the nitrogen cycle ................................................................ 14 1.3.2 Organic nitrogen compounds in the N-cycle ........................................... 16 1.4 Methylotrophy in bacteria ................................................................................. 18 1.4.1 The concept of methylotrophy ................................................................. 18 1.4.2 Methanotrophs ......................................................................................... 19 1.4.3 Methylotrophs .......................................................................................... 22 1.4.4 Formaldehyde oxidation .......................................................................... 24 ii 1.4.5 Formaldehyde assimilation ...................................................................... 26 1.5 Methylated amines as carbon, energy and nitrogen sources ............................. 30 1.6 Aims and approach of this PhD project ............................................................ 33 1.6.1 Techniques used in this study .................................................................. 34 1.6.2 Key questions of the PhD thesis .............................................................. 34 Chapter 2. Materials and Methods ............................................................................ 38 2.1 Chemicals and reagents .................................................................................... 39 2.2 Bacterial strains ................................................................................................. 39 2.3 Collection and processing of sample material from Movile Cave .................... 39 2.4 DNA stable isotope probing (DNA-SIP) experiments ..................................... 40 2.5 Culture media and growth of control organisms .............................................. 42 2.5.1 Dilute basal salts (DBS) medium ............................................................ 43 2.5.2 Mixed carbon solution for DBS-C medium ............................................ 43 2.5.3 Nitrate mineral salts (NMS) medium ...................................................... 43 2.5.4 Mineral salts (MS) medium ..................................................................... 44 2.5.5 Growth of Methylosinus trichosporium OB3b ........................................ 44 2.5.6 Azotobacter medium and growth of Azotobacter vinelandii .................. 44 2.5.7 Luria-Bertani (LB) medium .................................................................... 45 2.5.8 Super optimal broth with catabolic repressor (SOC) medium ................ 45 2.5.9 R2A agar .................................................................................................. 45 2.5.10 Nutrient broth .......................................................................................... 45 2.5.11 Rose-Bengal chloramphenicol agar plates .............................................. 45 2.5.12 Medium for ammonia-oxidising bacteria (Medium 181) ........................ 45 2.5.13 Growth of Nitrosomonas europaea ......................................................... 46 2.5.14 Medium for nitrite-oxidising bacteria (Medium 182) ............................. 46 2.6 Enrichment, isolation and maintenance of methylated amine-utilising bacteria from Movile Cave ............................................................................................. 47 2.6.1 Methylotrophic methylated amine-utilising bacteria .............................. 47 2.6.2 Non-methylotrophic, methylated amine-utilising bacteria ...................... 47 2.6.3 Microscopy .............................................................................................. 48 iii 2.6.4 Preservation of cultures............................................................................ 48 2.6.5 Growth tests of new isolates with methanol and under anoxic conditions ................................................................................................. 48 2.7 DNA extraction, processing and storage ........................................................... 49 2.8 Agarose gel electrophoresis............................................................................... 49 2.9 Polymerase chain reaction (PCR)...................................................................... 50 2.10 Purification of PCR products for further applications....................................... 51 2.11 Design of PCR primers targeting gmaS ............................................................ 56 2.11.1 Identification of gmaS sequences and alignment ..................................... 56 2.11.2 Primer sequences and PCR conditions .................................................... 56 2.11.3 Validation of the new primers .................................................................. 57 2.12 Denaturing gradient gel electrophoresis (DGGE) ............................................. 59 2.13 Cloning of PCR products .................................................................................. 59 13 2.14 RFLP analysis of cloned sequences from C-MMA-SIP incubations ............. 60 2.15 DNA sequencing and phylogenetic analysis ..................................................... 60 2.15.1 Sequencing of PCR products ................................................................... 60 2.15.2 Metagenome analysis ............................................................................... 61 2.15.3 Nucleotide sequence accession numbers ................................................. 62 2.16 N2 fixation assay (acetylene reduction) ............................................................. 62 2.17 Colorimetric assays ........................................................................................... 63 2.17.1 Ammonium assay..................................................................................... 63 2.17.2 Nitrite assay ............................................................................................. 63 2.17.3 Nitrate assay ............................................................................................. 65 2.18 Measuring methylated amine concentrations .................................................... 65 2.19 Isotope pairing experiments for detection of denitrification and anammox ..... 65 Chapter 3. Isolation of methylated amine-utilising bacteria from Movile Cave ... 67 3.1 Introduction ......................................................................................................