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http://waikato.researchgateway.ac.nz/ Research Commons at the University of Waikato Copyright Statement: The digital copy of this thesis is protected by the Copyright Act 1994 (New Zealand). The thesis may be consulted by you, provided you comply with the provisions of the Act and the following conditions of use: Any use you make of these documents or images must be for research or private study purposes only, and you may not make them available to any other person. Authors control the copyright of their thesis. You will recognise the author’s right to be identified as the author of the thesis, and due acknowledgement will be made to the author where appropriate. You will obtain the author’s permission before publishing any material from the thesis. Microbial Biodiversity of Thermophilic Communities in Hot Mineral Soils of Tramway Ridge, Mt. Erebus, Antarctica A thesis submitted in partial fulfilment of the requirements for the Degree of Masters of Science in Biological Science at the University of Waikato by Rochelle Melissa Soo August 2007 II Abstract Only a few studies have looked at microbial biogeography in soils and whether microorganisms are endemic to an area is still debatable. Tramway Ridge, a geothermal area on Mount Erebus, Antarctica, provides a unique opportunity due to its isolation and extreme conditions to explore the possibilities of microbial endemism and to identify novel Bacteria and Archaea. This site was chosen for a culture-independent study with a preliminary culturing survey for bacterial communities along three temperature gradients (65°C – 2.5ºC). In addition, a physico-chemical analysis was undertaken to identify which environmental factors were driving the different diversity along the transects. An automated rRNA intergenic spacer analysis (ARISA) was used to assess the diversity across the transects using Bacteria and Cyanobacteria-specific primers and results showed that temperature and pH were the main drivers for these communities. Due to its unique physico-chemical and ARISA profile, a hot temperature site (T- 3A, 65ºC) was chosen for further investigation by bacterial and archaeal 16S rDNA clone libraries. Unique rDNA types among the 78 bacterial and 83 archaeal clones were identified by restriction fragment length polymophisms and 18 bacterial and 5 archaeal operational taxonomic units (>97% identity) were observed. All of the bacterial sequences were deeply branching and loosely affiliated with other recognised bacterial divisions, with 40% of the sequences not affiliated to any genus. The archaeal clones were found to be deep-branching and sequences clustered together within the Crenarcaheota. In addition, two strains of Bacilli were isolated. The novel microorganisms show that the Tramway Ridge communities are unique from organisms found in other environments and show that “Everything is (not) everywhere”. III Preface As part of writing this thesis, I wanted to write a journal article so that I could share my work with the wider scientific community. This thesis has been written in three parts with the first chapter as a literature review and a background on the research. The second chapter has been written as a journal article, which will be submitted to Environmental Micrbiology shortly after this thesis has been submitted. The third chapter is a general conclusion and discussion, which discribes the work that has been completed and future work that will provide us with a more comprehensive overview of the microbial communities at Tramway Ridge. The appendix includes a journal article that was submitted to Environmental Microbiolgy with Tom Niederberger as the first author. I have included it into the appendix as I was a co-author for the article, in which I worked with one sample, LV High 1-3 as part of a learning process for when I obtained my own samples to process from Mt. Erebus. In the study, I extracted DNA from the soil samples, ran denaturing gradient gel electrophoresis (DGGE) and performed PCR to produce products for cloning. I then cloned the PCR product, sequenced and edited them. IV Acknowledgements I’d like to thank my supervisor, Craig Cary for his support during the whole project. I couldn’t have asked for a better mentor. I’d also like to thank Amy Hacker for taking me under her wing when I first came to the lab and teaching me the basics. These two wonderful people have been like a second set of parents to me and I’ve always known that I have somebody to turn to if things go wrong. I’d like to thank my Mum and Dad and my brother, Gareth for listening to me complain and provide support during the many phone calls home. I’d like to thank Tom Niederberger for his knowledge and support whenever I got stuck. Also, Ian McDonald for helping me with ARB to produce the phylogenetic trees. I’d like to thank John Longmore and Arvina Ram in the Waikato Sequencing Unit, who have seen my face so many times, Steve Cameron at the Chemical Services unit and Anjana Rajendram at the Waikato Stable Isotope Unit. I’d also like to thank the staff in the earth sciences department, Jacinta Parenzee, Annette Rogers, Megan Balks, Louis Schipper and Roger Briggs for advice with the geochemistry. I’d like to thank the people in TRU, especially Susie Wood, who read over the manuscript many times and helped with the ARISA and statistics. Thank you to Johnathan Banks for reading over the manuscript and also helping with ARISA. Thanks to Julie Smith for manuscript reading and Charlie Lee for helping with the many computer problems. I’d also like to thank Joe Grzymski and Mike Barker for collecting the samples from Tramway Ridge and the support group at Antarctica New Zealand in Christchurch and Scott Base. A big thank you to my friends, Naomi Crawford, Tesha Tsai, Katie Glover in London and my flatmates who have been there to keep me sane. Thank you to David Clement and Julian Maffre, who have been there from the start and shown me that I can play hard and work hard. And lastly, I’d like to thank the National Geographic Society, Antarctica New Zealand and the University of Waikato with helping to fund the project. V Table of Contents ABSTRACT II PREFACE III ACKNOWLEDGEMENTS IV TABLE OF CONTENTS V LIST OF TABLES AND FIGURES VIII CHAPTER 1 : LITERATURE REVIEW 1 1.1 Extreme environments and microbes 1 1.2 Hot soils and microbial communities 2 1.2.1 Hot soils as environments for microbes 3 1.2.2 Thermophiles 4 1.2.3 The effect of geochemistry and pH and how it affects thermophillic microbial communities 5 1.3 Antarctica 6 1.3.1 Hot mineral soils on Antarctica as an environment and a habitat 7 1.3.2 Mount Erebus and Tramway Ridge 8 1.4 Approaches used to investigate the microbial ecology of geothermal habitats 11 1.4.1 Culture-dependent approaches 11 1.4.2 Culture-independent approaches 13 1.4.3 Culture-dependent and culture-independent approaches used to study microbial ecology and Phylogenetics 16 1.5 Aims, approach and outline of Thesis 18 References 19 VI CHAPTER 2 : MICROBIAL BIODIVERSITY OF THERMOPHILIC COMMUNITIES IN HOT MINERAL SOILS OF TRAMWAY RIDGE 33 2.1. Abstract 33 2.2 Introduction 34 2.3 Methods and Materials 36 2.3.1 Soil collection and physico-chemical analysis 36 2.3.2 Extraction of genomic DNA and DNA fingerprinting analysis 38 2.3.3 Statistical analysis of ARISA fingerprints 39 2.3.4 16S rRNA clone library construction and analysis 40 2.3.5 16S rRNA gene library analysis 41 2.3.6 Estimation of soil bacterial and archaeal richness 42 2.3.7 Aerobic enrichment cultures and isolation 42 2.4 Results 43 2.4.1 Physico-chemical analysis 43 2.4.2 DNA fingerprinting analysis 46 2.4.3 16S rRNA gene library analysis 50 2.4.4 Phylogenetic analysis 50 2.4.5 Analysis of cultures and isolates 56 2.5 Discussion 56 2.5.1 Physico-chemical analysis 57 2.5.2 DNA fingerprinting 58 2.5.3 Phylogeny 60 2.5.4 Culturing and isolation studies 63 2.5.5 Conclusion 64 References 65 CHAPTER 3 : GENERAL CONCLUSION 78 References 81 VII APPENDICES 83 Appendix A - Physico-chemical 83 Appendix B - Phylogeny 102 Appendix C - DGGE 117 Appendix D - Microbial community structure in soils of Northern Victoria Land, Antarctica 122 VIII List of Tables and Figures Table 2.1. Physico-chemical data of the soils from Tramway Ridge, Mt. Erebus, Anarctica sampled in January 2006 45 Table 2.2. 16S rRNA clones identified in a soil sample from site T-3A, Mt. Erebus, Antarctica, 2006 51 Figure 1.1. Tramway Ridge, Mt. Erebus, Antarctica 9 Figure 1.2. Tramway Ridge, ASPA 130, Mt. Erebus, Antartica 10 Figure 2.1. Temperature for 10 months beginning 20 February 2006 for sampling site T-3A 46 Figure 2.2. ARISA fragment lengths for Bacteria and Cyanobacteria 48 Figure 2.3. Nonmetric multidimensional scaling (MDS) for Bacteria and Cyanobacteria 49 Figure 2.4. Phylogenetic tree showing the placement of 16S rDNA gene clones from Tramway Ridge, T-3A within the Bacteria 53 Figure 2.5. Phylogenetic tree showing the placement of 16S rDNA gene clones from Tramway Ridge, T-3A within the Archaea 54 Figure 2.6. Rarefaction curve using partial 16S rRNA Bacteria and Archaea sequenes from Tramway Ridge (T-3A) 55 Chapter 1: Literature Review 1 Chapter 1 : Literature Review 1.1 Extreme environments and microbes An extremophile is an organism that thrives in extreme environments. Extremes include physical extremes (for example, temperature, radiation or pressure) and geochemical extremes (for example, desiccation, salinity, pH, oxygen species or redox potential).