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Microbial Community Dynamics Within Frost Boils of Browning Peninsula, Antarctica

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Microbial Community Dynamics Within Frost Boils of Browning Peninsula, Antarctica Microbial Community Dynamics within Frost Boils of Browning Peninsula, Antarctica Sarita Pudasaini A thesis submitted in fulfillment of the requirements for the degree of Master of Philosophy (Research) School of Biotechnology and Biomolecular Sciences University of New South Wales August, 2014 TABLE OF CONTENTS TABLE OF CONTENTS ....................................................................................... ii ABSTRACT ..........................................................................................................vii ACKNOWLEDGMENT ........................................................................................ x LIST OF FIGURES ............................................................................................. xi LIST OF TABLES.............................................................................................xviii ABBREVIATIONS ............................................................................................. xix 1 INTRODUCTION.......................................................................................... 1 1.1 Background ..................................................................................................... 1 1.2 Microbial diversity of Antarctica ................................................................... 2 1.2.1 Bacterial diversity of Antarctica................................................................................ 2 1.2.2 Relationship of bacterial abundance with environmental parameters in Antarctica ..... 3 1.2.3 Fungal diversity in terrestrial Antarctica ................................................................... 4 1.2.4 Relationship of fungal abundance with environmental parameters in Antarctica ......... 5 1.3 Culture- independent approach ..................................................................... 6 1.3.1 Qualitative PCR (qPCR) ........................................................................................... 6 1.3.2 Molecular tools for identification of bacteria and fungi.............................................. 6 1.3.3 Pyrosequencing: a powerful tool of next generation sequencing(NGS) ...................... 9 1.3.4 Illumina metabarcoding .......................................................................................... 11 1.4 Available culture-dependent approach ........................................................ 11 1.4.1 Novel approach to cultivation ................................................................................. 11 1.4.2 Novel approach to cultivate fungi ........................................................................... 13 ii 1.5 Windmill Islands of Eastern Antarctica ....................................................... 15 1.6 Browning Peninsula (BP) .............................................................................. 15 1.6.1 Frost boils formation .............................................................................................. 17 1.7 Microbiology of Browning Peninsula (BP) ................................................... 19 1.7.1 Bacterial community of Browning Peninsula........................................................... 19 1.7.2 Bryophyte and fungal community of Browning Peninsula ....................................... 20 1.8 Aim ................................................................................................................ 21 2 MATERIALS AND METHODS ...................................................................22 2.1 Site description .............................................................................................. 22 2.2 Sampling design and collection ..................................................................... 22 2.3 Physical and chemical analysis of soil samples............................................. 24 2.4 Culture independent techniques ................................................................... 25 2.4.1 DNA extraction from Soil ....................................................................................... 25 2.4.2 Quantification of genomic DNA using Picogreen assay ........................................... 26 2.4.3 Multiplexed pyrosequencing targeting bacterial and fungal genes ............................ 26 2.4.4 Raw data processing pipeline for pyrosequencing data ............................................ 27 2.4.5 Quantitative PCR (qPCR) ....................................................................................... 30 2.5 Culture dependent techniques ...................................................................... 31 2.5.1 Soil substrate membrane system (SSMS) cultivation ............................................... 31 2.5.2 Cultivation of bacteria and fungi on artificial media ................................................ 38 2.6 DNA extraction from pure cultures.............................................................. 40 2.6.1 DNA extraction of pure cultures by boiling ............................................................. 40 2.6.2 DNA extraction of pure cultures using bead beating ................................................ 40 2.6.3 DNA extraction of fungal cultures .......................................................................... 41 iii 2.7 Bacterial 16S rDNA and fungal internal transcribed spacer (ITS) gene PCR amplification 41 2.8 Gel electrophoresis ........................................................................................ 43 2.9 Restriction fragment length polymorphism (RFLP) .................................... 43 2.10 PCR product purification ............................................................................. 44 2.11 Cloning ITS region of fungi into T vector .................................................... 45 2.11.1 Ligation of DNA .................................................................................................... 45 2.11.2 Optimisation of insert : vector molar ratios ............................................................. 45 2.11.3 Transformation using the pGEM®-T ligation reaction ............................................. 45 2.12 Sanger sequencing of PCR products ............................................................ 46 2.13 Identification of 16S and ITS sequences ....................................................... 47 3 MICROBIAL COMMUNITY DYNAMYCS .................................................48 3.1 Bacterial and fungal community diversity ................................................... 48 3.2 Phylogenetic distribution of bacteria in BP.................................................. 49 3.2.1 Distribution of bacterial diversity across individual polygons .................................. 50 1.1.2 Distribution of genus level diversity across polygons .............................................. 52 3.3 Phylogenetic distribution of fungi in Browning Peninsula .......................... 55 3.3.1 Distribution of fungi phyla level fungal diversity across individual polygons ........... 56 3.3.2 Distribution of fungi genus level diversity across individual polygons ..................... 57 3.4 Bacterial community dissimilarity across Browning Peninsula .................. 58 3.5 Fungal community dissimilarity across Browning Peninsula ...................... 61 3.6 Relationships between environmental variables and bacterial community structure 64 iv 3.7 Genes distribution across polygons of Browning Peninsula ........................ 67 3.7.1 .................................................................................................................................... 69 4 CULTURE BASED CHARACTERISATION OF MICROBIAL COMMUNITY DYNAMICS ............................................................................................70 4.1 Soil substrate membrane system cultivation diversity ................................. 70 4.1.1 Bacterial phylum level diversity following SSMS enrichment ................................. 71 1.1.3 Distribution of bacterial genus level diversity on SSMS enrichment ........................ 72 4.1.2 Distribution of fungi phylum and genus level diversity on SSMS enrichments ......... 74 4.1.3 Bacterial and fungal community dissimilarity on SSMS enrichments ....................... 75 4.2 Bacterial and fungal diversity recovered from artificial media ................... 78 4.2.1 Sub-culturing ......................................................................................................... 78 1.1.4 Molecular identification of bacterial isolates ........................................................... 78 4.2.2 Sub-culturing of fungi ............................................................................................ 81 1.1.5 Molecular identification of fungal isolates............................................................... 84 4.2.3 Comparison between molecular and cultivation approaches to microbial characterisation 87 5 DISCUSSION ...............................................................................................91 5.1 Microbial Diversity of Browning Peninsula ................................................. 91 5.1.1 Bacterial diversity in soils ....................................................................................... 92 5.1.2 Fungal diversity in soils .......................................................................................... 95 5.1.3 Pure bacterial and fungal cultures ........................................................................... 97 5.2 Environmental drivers of community composition.....................................
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