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Identification and Cultivation of Exopolysaccharide-Degrading Bacteria in Two Soils

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Identification and Cultivation of Exopolysaccharide-Degrading Bacteria in Two Soils University of Calgary PRISM: University of Calgary's Digital Repository Graduate Studies The Vault: Electronic Theses and Dissertations 2013-09-24 Identification and Cultivation of Exopolysaccharide-Degrading Bacteria in Two Soils Wang, Xiaoqing Wang, X. (2013). Identification and Cultivation of Exopolysaccharide-Degrading Bacteria in Two Soils (Unpublished master's thesis). University of Calgary, Calgary, AB. doi:10.11575/PRISM/26419 http://hdl.handle.net/11023/1033 master thesis University of Calgary graduate students retain copyright ownership and moral rights for their thesis. You may use this material in any way that is permitted by the Copyright Act or through licensing that has been assigned to the document. For uses that are not allowable under copyright legislation or licensing, you are required to seek permission. Downloaded from PRISM: https://prism.ucalgary.ca UNIVERSITY OF CALGARY Identification and Cultivation of Exopolysaccharide-Degrading Bacteria in Two Soils by Xiaoqing Emily Wang A THESIS SUBMITTED TO THE FACULTY OF GRADUATE STUDIES IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF BIOLOGICAL SCIENCES CALGARY, ALBERTA SEPTEMBER, 2013 © Xiaoqing Emily Wang 2013 Abstract In this study, we hypothesized that bacterial exopolysaccharides (EPS) may serve as energy substrates for K-selected bacteria in soils, and we would be able to identify and culture previously uncultured species using various EPS as growth substrates. The exopolysaccharides gellan, indican_2, and cellulose were produced by the bacteria Sphingomonas elodea (ATCC 31461), Beijerinckia indica (ATCC 9039) and Gluconacetobacter xylinus (ATCC 53524) respectively. Two experiments were performed using these exopolysaccharides (EPS) as growth substrates. The first experiment involved detecting EPS- degrading bacteria by using a stable isotope probing technique. 13C labeled EPS were used as growth substrates for Big Hill Spring (BHS) and Paint Pots mound (PP) soil samples. EPS- degrading bacteria were identified using DNA density ultracentrifugation coupled with 454- pyrosequencing of the 16S rRNA genes recovered from 13C-labelled DNA fractions. Several uncultured Xanthomonadaceae sp. were enriched on cellulose (produced by G. xylinus) during incubation from BHS soil and several previously uncultured Planctomycetes were highly enriched on indican_2 (produced by B. indica) incubation from PP soil. The second experiment was to isolate novel bacteria by using gellan or indican_2 as sole carbon sources. Based on cultivation results, we found several previously uncultured bacteria could be identified and isolated from our gellan (produced by S. elodea) and indican_2 plates. For instance, we successfully isolated previous uncultured Acidobacteria by using gellan and indican_2 as our carbon source. All in all, in this study, we found some previously uncultured bacteria belonging to the groups of Acidobacteria, Verrucomicrobia, Planctomycetes, Chloroflexi, Actinobacteria, Cyanobacteria, Proteobacteria, Bacteroidetes, Armatimonadetes and Candidate division OD1, ii which are readily enriched or cultured using these EPS substrates. These poorly understood groups, especially some members of Planctomycetes, showed preference for our EPS indican_2. The research results indicate that using novel EPS as carbon sources might be a new way to improve current cultivation techniques. iii Acknowledgements I am grateful to the many people who helped and encouraged me to be successful. Without their support, this thesis would not be possible. In particular, I would like to express my deepest gratitude to my supervisor, Dr. Peter Dunfield, for providing me the opportunity to work on this interesting project. To Dr. Peter Dunfield, thanks for always believing in me and allowing me a chance to grow. Your knowledge and insights guide me through the project. Thanks to the members of my committee Dr. Lisa Gieg, Dr. Raymond Turner, and Dr. Kenneth Sanderson for taking the time to support and encourage my academic efforts. To Dr. Kenneth Sanderson, thanks to encourage me when I am feel lack of confidence at very beginning of my master study, your support and care means a lot to me. To Dr. Allyson Brady and Christine Sharp, without your assisting me with so many details of this research, this research would not have been possible. You were not only taught me many hands-on experimental skills, the knowledge of research, you also helped me to know Canada. You bring a lot of first time to me, first Camping, first ski, first make camp fire...thanks you guys to bring such wonderful experiences to me. I will be forever grateful! Thanks to Gareth Jones for all cellulose supports and all the grammar corrections, without his help this thesis could be done. Thanks all the members in the Dunfield lab Dr. Angela Smirnova, Dr. Joongjae Kim, AliReza Saidi-Mehrabad, Fauziah Rochman and Roshan Khadka, for sharing their experience and knowledge. Finally, I wish to show my appreciation to my family and many wonderful friends for their love and support. To my Dad and Mom, without your love and supports, I cannot finish this study. To my fiancé Mark Zhong, thanks for always be with me in those difficult time. This research would not have been possible without you all. Thank you! iv Table of Contents Abstract ............................................................................................................................... ii Acknowledgements ............................................................................................................ iv Table of Contents .................................................................................................................v List of Tables ................................................................................................................... viii List of Figures and Illustrations ...........................................................................................x CHAPTER ONE: THESIS INTRODUCTION ...................................................................1 1.1 Introduction ................................................................................................................1 1.2 Hypothesis .................................................................................................................4 1.3 Research Objectives ...................................................................................................4 1.4 Study Sites .................................................................................................................4 CHAPTER TWO: LITERATURE REVIEW ......................................................................8 2.1 Known and Unknown Microbial Diversity ...............................................................8 2.1.1 Culture-Independent Methods Overview ..........................................................8 2.1.1.1 Total Nucleic Acid Extraction from Soil .................................................8 2.1.1.2 PCR of the 16S rRNA Gene ....................................................................9 2.1.1.3 Fingerprinting Method ...........................................................................10 2.1.2 Abundance and Diversity of Soil Bacterial Communities ..............................13 2.2 r- and K-Selection Theory .......................................................................................18 2.3 Microbial cultivation methods .................................................................................19 CHAPTER THREE: CHARACTERIZATION OF SOIL MICROBIAL COMMUNITIES24 3.1 Introduction ..............................................................................................................24 3.2 Methods ...................................................................................................................24 3.2.1 Sampling ..........................................................................................................24 3.2.1.1 Paint Pots Site ........................................................................................24 3.2.1.2 Big Hill Spring Site ................................................................................24 3.2.2 Extraction of Total DNA .................................................................................25 3.2.3 PCR Amplification, Purification and Quantification ......................................27 3.2.4 Microbial Community Analyses ......................................................................28 3.3 Results and Discussion: ...........................................................................................28 3.3.1 Previous Paint Pots Microbial Community Research Review ........................28 3.3.2 Big Hill Spring Microbial Community............................................................29 CHAPTER FOUR: DETECTION OF AS-YET-UNCULTURED BACTERIAL GROUPS IN BHS AND PP SOIL USING STABLE ISOTOPE PROBING ................................32 4.1 Introduction ..............................................................................................................32 4.2 Materials and Methods .............................................................................................35 4.2.1 13C and 12C EPS Production .............................................................................35 4.2.1.1 Cellulose Production ..............................................................................35 4.2.1.2 Indican_2 Production .............................................................................36
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