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Literature Review ABSTRACT DEVINE, ANTHONY ANDREW. Examining Complex Microbial Communities Using the Terminal Restriction Fragment Length Polymorphism Method and Dedicated TRFLP Analysis Software. (Under the direction of Amy Michele Grunden). The purpose of this research was to develop a robust methodology that could be used to determine the composition of complex microbial communities found in a variety of environments. In an effort to profile these communities, a 16S rDNA directed, sequencing independent method, Terminal Restriction Fragment Length Polymorphism (TRFLP) was used to generate fragment profiles from genomic DNA isolated from each sample. A custom designed software package, In Silico©, was then used to match these terminal restriction fragments in the sample to patterns of 16S rDNA fragments in a custom database of reference patterns. Identifying these patterns allowed for inferences to be made about the structure of the microbial populations in the environments sampled. TRFLP was chosen as the method of identification since it is a high throughput, cost effective method for community profiling. A combination of this method and the In Silico© software was used to examine microbial communities associated with open air swine waste lagoon systems, the large intestine of the Trichechus manatus latirostrus, Florida manatee, and the microbial populations found in rumen fed fermentors. The community analysis methodology we developed and employed was able to not only detect large and diverse microbial populations within each sample, but was also able to show differences between the detected microbial populations under the various experimental regimes used in the studies presented. Examining Complex Microbial Communities Using the Terminal Restriction Fragment Length Polymorphism Method and Dedicated TRFLP Analysis Software by Anthony Devine A dissertation to the Graduate Faculty of the North Carolina State University in partial fulfillment of the requirements for the Degree of Doctor of Philosophy Microbiology Raleigh, North Carolina 2009 APPROVED BY: ___________________________ __________________________ Vivek Fellner, Ph.D. Michael Sikes, Ph.D. __________________________ __________________________ James Brown, Ph.D. Amy Grunden, Ph.D. Chair of Advisory Committee DEDICATION I would like to dedicate this work to my wife Catherine Devine, and both our families. I would not have been able to finish this work without their love and support. Thank you all. ii BIOGRAPHY Drew was born in Greensboro, North Carolina, after graduating from Northwest Guilford High School he begun his college career. After a failed start as an engineer at Virginia Tech, Drew went to the University of North Carolina at Greensboro where he got his B.S. in Biology in 2002. Drew then came to North Carolina State University joining the Microbiology Department in 2003 to earn his PhD. iii ACKNOWLEDGEMENTS Like all science, this has been a collaborative effort and I would not have been able to accomplish all that I have done on my own. I would like to thank Amy Grunden for letting me do my research in her lab, she not only provided a mentally engaging and rigorous research environment, but was always supportive in my research endeavors. I would also like to thank the members of her lab, both current and former, for providing a fun working environment. I have had a number of excellent undergraduate students and their contributions cannot be overlooked. The rest of the Department of Microbiology also deserves thanks for providing various forms of support. I would also like to thank my dissertation committee for providing guidance during this whole time. I would also like to thank Mark Rice, in the Department of Biological and Agricultural Engineering, for his time and effort into helping me with the field component of my research. Without his knowledge I would not have been able to get onto the hog farms to complete my research. I would also like to thank my funding agencies, The North Carolina Pork Council, the United States Department of Agriculture, and DSM Nutritional Products, for all of their monetary support during my research. Thank you also to my friends and family, who have been there through it all. Every instance of help they have been there and without them I would not have been able to finish. iv TABLE OF CONTENTS LIST OF FIGURES ................................................................................................................. ix LIST OF TABLES.................................................................................................................. xii CHAPTER 1: LITERATURE REVIEW.................................................................................. 1 Brief Overview of the Importance of Microbial Communities ............................................ 1 Determination of Microbial Communities Using Direct Cultivation Techniques................ 2 Determination of Microbial Communities Using Culture-Independent Techniques: Selection of Molecular Targets for Analysis........................................................................ 4 The Influence of the Polymerase Chain Reaction on Cultivation-Independent Microbial Community Analysis Methods............................................................................................. 7 Determination of Microbial Communities Using Culture-Independent Techniques: 16S rDNA Clone Library Analysis ............................................................................................. 8 Determination of Microbial Communities Using Culture-Independent Techniques: Denaturing Gradient Gel Electropohoresis Analysis ......................................................... 17 Determination of Microbial Communities Using Culture-Independent Techniques: Restriction Fragment Length Polymorphism Analysis ...................................................... 21 Determination of Microbial Communities Using Culture-Independent Techniques: Terminal Restriction Fragment Length Polymorphism Analysis....................................... 25 Improving the TRFLP Method for Microbial Community Analysis by Using Software to Match Restriction Fragments to Candidate Microorganisms............................................. 36 LITERATURE CITED ....................................................................................................... 50 CHAPTER 2: DETERMINING THE MICROBIAL COMMUNITY OF HOG WASTE LAGOON SYSTEMS USING TERMINAL RESTRICTION FRAGMENT LENGTH POLYMORPHISM ANALYSIS............................................................................................ 66 ABSTRACT........................................................................................................................ 67 INTRODUCTION .............................................................................................................. 68 MATERIALS AND METHODS........................................................................................ 71 Genomic DNA isolation .................................................................................................. 71 PCR reactions ................................................................................................................. 71 Amplified DNA fragment purification and enzymatic digestion..................................... 72 TRFLP Analysis .............................................................................................................. 72 Genomic DNA spiking experiment.................................................................................. 73 RESULTS ........................................................................................................................... 74 DNA Spiking Experiments............................................................................................... 74 Sample collection and analysis....................................................................................... 75 DISCUSSION..................................................................................................................... 79 Use of TRFLP analysis for examination of complex microbial communities................. 79 In Silico© software package........................................................................................... 80 v Genomic DNA spiking and serial dilution...................................................................... 83 Microbial communities in hog lagoon systems as determined by TRFLP analysis using In Silico©........................................................................................................................ 83 Archaeal TRFLP ............................................................................................................. 87 CONCLUSIONS................................................................................................................. 89 LITERATURE CITED ....................................................................................................... 99 CHAPTER 2: APPENDIX .................................................................................................. 106 CHAPTER 3: TRACKING MICROBIAL COMMUNITIES IN EASTERN NORTH CAROLINA HOG WASTE LAGOON SYSTEMS USING TERMINAL RESTRICTION FRAGMENT LENGTH POLYMORPHISM ANALYSIS.................................................. 150 ABSTRACT...................................................................................................................... 151 INTRODUCTION ...........................................................................................................
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