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Oregon Health & Science University School Of OREGON HEALTH & SCIENCE UNIVERSITY SCHOOL OF MEDICINE APPLICATION OF MOLECULAR BIOLOGICAL TOOLS TO THE STUDY OF BIOREMEDIATION IN CONTAMINATED SEDIMENTS By Christina N. Brow A DISSERTATION Presented to the Division of Environmental & Biomolecular Systems and the Oregon Health & Science University School of Medicine in partial fulfillment of the requirements for the degree of Doctor of Philosophy April 2011 I DIVISION OF ENVIRONMENTAL AND RIOMOLECI JLAR SYSTEMS INSTITUTE OF ENVIRONMENTAL I-II'AI,TH OREGON HEALTI-I & SCIENCE UNIVERSITY CEIITTFICA'T'E OF AI'PTIOVAL --- --.----.-.----...-. - -- ..... .. This is to ccitifL Illat thc Pl3.D. dissc~tatioi~of . Christina N. RI-c~w has bccli appr-ovccl - -- - - -36 -- -------- Dl-. HOIGM. Sjnio~i,Assistant i'mfcssc~r Reseal-cli Co-Ad visor --. ..- --,-.- -.*-- -t-.- s. Richar i . .1 I~nson,P~.oli.ssos ---r~TdResearch Co-Advisor - --- v----- Dr. Paul G. Tralriyck, Professor ----.. w..3- -- .- -.--- , , Dl-. ewis Sc~~~psi~ii.cgor atc linivcr-sity -3 ExternalE-xamine~- - Table of Contents TABLE OF CONTENTS..............................................................................................................................I ACKNOWLEDGEMENTS.......................................................................................................................III ABSTRACT ................................................................................................................................................IV 1. INTRODUCTION ................................................................................................................................... 1 1.1. BIODEGRADATION .........................................................................................................................1 1.2. METHODS OF ASSESSING BIODEGRADATION ..................................................................................1 1.2.1. Hydrogeochemical methods ....................................................................................................2 1.2.2. Microbial and molecular methods...........................................................................................3 1.3. SAMPLING APPROACHES FOR MBT ANALYSIS ...............................................................................5 1.4. TOLUENE AS A MODEL CONTAMINANT ..........................................................................................6 1.4.1 BTEX .......................................................................................................................................6 1.4.2. Biodegradation of toluene .......................................................................................................7 1.5. AIM OF THIS THESIS .......................................................................................................................7 1.6. ORGANIZATION OF THIS THESIS .....................................................................................................8 2. GENERAL METHODS ........................................................................................................................ 11 2.1. CHEMICAL ANALYSIS OF MODEL AQUIFER PORE WATER ..............................................................11 2.2. BSS A AND TMO A Q PCR PRIMER DESIGN .......................................................................................11 2.3. CONSTRUCTION OF PLASMID STANDARDS FOR Q PCR..................................................................12 2.4. CLONE LIBRARY ANALYSIS OF ARCHAEAL ENRICHMENT CULTURE .............................................13 3. BENCH SCALE EVALUATION OF CRYOGENIC PRESERVATION AND STORAGE FOR THE MOLECULAR CHARACTERIZATION OF MICROORGANISMS IN SEDIMENT............................................................................................... 20 3.1. ABSTRACT ..................................................................................................................................20 3.2. INTRODUCTION ...........................................................................................................................21 3.3. METHODS ....................................................................................................................................23 3.3.1. Laboratory-generated samples..............................................................................................23 3.3.2. DNA and RNA extraction ......................................................................................................24 3.3.3. Effects of freezing on quantification of genes and gene transcripts ......................................25 3.3.4 Effects of storage on quantification of genes and gene transcripts .......................................26 3.3.5. Effects of freezing and storage on relative phylotype abundance .........................................26 3.4. RESULTS AND DISCUSSION ..........................................................................................................28 3.4.1 Effects of freezing and storage on quantification of genes and gene transcripts ..................28 3.4.2 Effects of freezing and storage on the relative abundance of bacterial phylotypes...............30 3.5. CONCLUSION ...............................................................................................................................31 4. MESOCOSM SCALE EVALUATION OF CRYOGENIC PRESERVATION AND STORAGE, AND HIGH-RESOLUTION CRYOGENIC CORE SAMPLING FOR THE MOLECULAR CHARACTERIZATION OF MICROORGANISMS IN SEDIMENT............................................................................................... 49 4.1. ABSTRACT ..................................................................................................................................49 4.2. INTRODUCTION ...........................................................................................................................50 i 4.3. METHODS ....................................................................................................................................52 4.3.1. Model aquifer ........................................................................................................................52 4.3.2 Sample collection for MBT analysis......................................................................................52 4.3.3 Nucleic acid extraction and qPCR ........................................................................................53 4.3.4 Quantitative PCR...................................................................................................................54 4.3.5 Microbial community analysis...............................................................................................54 4.4. RESULTS AND DISCUSSION ..........................................................................................................55 4.4.1 Chemical analysis of pore water ...........................................................................................55 4.4.2 Cryogenic sample preservation and storage .........................................................................56 4.4.3 Cryogenic core sampling.......................................................................................................57 4.5. CONCLUSIONS .............................................................................................................................59 5. EFFECTS OF SAMPLING BIAS REVEALED BY HIGH-RESOLUTION MOLECULAR BIOLOGICAL ANALYSIS OF PAIRED SEDIMENT AND WATER SAMPLES FROM A MODEL AQUIFER .......................................................................... 73 5.1. ABSTRACT ..................................................................................................................................73 5.2. INTRODUCTION ...........................................................................................................................74 5.3. METHODS ....................................................................................................................................75 5.3.1. Model Aquifer........................................................................................................................75 5.3.2 Sample collection for MBT analysis......................................................................................75 5.3.3. DNA extraction and quantitative PCR ..................................................................................75 5.3.4. Microbial community analysis...............................................................................................76 5.4. RESULTS AND DISCUSSION ..........................................................................................................77 5.4.1 Toluene plume .......................................................................................................................77 5.4.2 DNA yields.............................................................................................................................78 5.4.3. Microbial community analysis...............................................................................................78 5.4.4 Depth distribution of functional genes ..................................................................................78 5.5. CONCLUSIONS .............................................................................................................................81 6. MICROBIAL TRANSPORT HINDERS USE OF FUNCTIONAL GENE ABUNDANCE IN PORE WATER TO CHARACTERIZE ZONES OF ACTIVE BIODEGRADATION...........................................................................................................................
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