PCR Optimisation for the Detection of a Functional Gene (Pmoa) Using the Taguchi Methods
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University of Warwick institutional repository: http://go.warwick.ac.uk/wrap A Thesis Submitted for the Degree of PhD at the University of Warwick http://go.warwick.ac.uk/wrap/49093 This thesis is made available online and is protected by original copyright. Please scroll down to view the document itself. Please refer to the repository record for this item for information to help you to cite it. Our policy information is available from the repository home page. Impact of land-use changes on the methanotrophic community structure Loïc Nazaries A thesis submitted to the Department of Life Sciences in fulfilment of the requirements for the degree of Doctor of Philosophy March 2011 University of Warwick Coventry, UK Table of contents Table of contents TABLE OF CONTENTS ................................................................................... I LIST OF TABLES ..........................................................................................VII LIST OF FIGURES ......................................................................................... IX ABBREVIATIONS.........................................................................................XII ACKNOWLEDGEMENTS ......................................................................... XVI DECLARATION..........................................................................................XVII ABSTRACT................................................................................................ XVIII CHAPTER 1 INTRODUCTION.......................................................................1 1. Methane in the context of global warming ................................................................1 2. The carbon and methane cycles..................................................................................3 2.1. Definition ....................................................................................................................3 2.2. Sources and sinks of CH4 on Earth.............................................................................4 2.3. Importance of terrestrial CH4 in relation to climate change .......................................6 2.4. Methane-producing Archaea (methanogens)..............................................................7 2.4.1. Definition and taxonomy..................................................................................7 2.4.2. Ecology of methanogens ..................................................................................9 2.4.3. CH4-production pathways ..............................................................................10 2.4.4. MCR, the key enzyme in methanogenesis .....................................................13 3. Methane-oxidising Bacteria (methanotrophs) .........................................................15 3.1. Definitions ................................................................................................................15 3.2. Taxonomy of aerobic methanotrophs .......................................................................18 3.3. Differences between aerobic methanotrophs............................................................20 3.3.1. Intracytoplasmic membrane (ICM) formation ...............................................20 3.3.2. Formaldehyde assimilation pathways ............................................................20 3.3.3. MMO activity.................................................................................................24 3.3.4. Phospholipids fatty acid (PLFA) signature ....................................................25 i Table of contents 3.3.5. N2 fixation ......................................................................................................25 3.4. Particularities to the type I/type II separation...........................................................26 3.4.1. The case of type X methanotrophs.................................................................26 3.4.2. The case of facultative methanotrophs...........................................................26 3.4.3. The case of Methylacidiphilum ......................................................................27 3.5. sMMO vs. pMMO.....................................................................................................28 3.5.1. Structure of sMMO and pMMO.....................................................................28 3.5.2. Gene copy number and variations..................................................................31 3.5.3. Regulation by copper and copper-uptake system...........................................32 4. Techniques used for identifying methanotrophs.....................................................35 4.1. Biochemical techniques ............................................................................................35 4.2. Biomolecular techniques...........................................................................................36 4.2.1. Terminal-restriction fragment length polymorphism (T-RFLP)....................36 4.2.2. Cloning and sequencing .................................................................................37 4.2.3. Stable isotope probing (SIP) ..........................................................................38 4.2.4. Diagnostic microarrays ..................................................................................39 5. Regulation of aerobic methanotrophy .....................................................................40 5.1. Effects of environmental factors on methanotrophs and CH4 oxidation rates..........40 5.2. Effects of change in land management and land use ................................................43 5.3. Importance of afforestation/reforestation in relation to climate change...................46 6. Aims of the study........................................................................................................48 CHAPTER 2 MATERIALS AND METHODS .............................................50 1. Field site description..................................................................................................50 1.1. In New Zealand.........................................................................................................50 1.2. In Scotland ................................................................................................................53 2. Soil sampling...............................................................................................................57 2.1. In New Zealand.........................................................................................................57 2.2. In Scotland ................................................................................................................58 3. Soil analyses................................................................................................................59 3.1. New Zealand soils.....................................................................................................59 3.2. Scottish soils .............................................................................................................59 3.2.1. Processing of soils..........................................................................................60 3.2.2. Chemical analyses..........................................................................................60 3.2.3. Physical analyses............................................................................................61 4. Gas flux measurements..............................................................................................62 4.1. Gas sampling.............................................................................................................62 ii Table of contents 4.2. Gas analysis ..............................................................................................................63 5. Methanotroph characterisation of using molecular methods................................66 5.1. DNA extraction.........................................................................................................66 5.2. PCR conditions for T-RFLP analysis .......................................................................66 5.2.1. Target preparation ..........................................................................................68 5.2.2. Target purification..........................................................................................68 5.3. Terminal-restriction fragment length polymorphism (T-RFLP) analyses................69 5.4. Cloning and sequencing analysis..............................................................................70 5.4.1. Target preparation ..........................................................................................70 5.4.2. Cloning reaction .............................................................................................70 5.4.3. Alignment and identification of clone sequences ..........................................71 5.4.4. Nucleotide sequence accession numbers........................................................72 5.5. Diagnostic pmoA microarray analysis ......................................................................72 5.5.1. Target preparation ..........................................................................................72 5.5.2. In vitro transcription.......................................................................................72 5.5.3. Hybridisation..................................................................................................73