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Factors Influencing the Fate of Chromium in Soils: Microbial

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Factors Influencing the Fate of Chromium in Soils: Microbial FACTORS INFLUENCING THE FATE OF CHROMIUM IN SOILS: MICROBIAL ECOLOGY, PHYSIOLOGY AND METAL TRANSFORMATION STUDIES by Erin Kirby Field A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Microbiology MONTANA STATE UNIVERSITY Bozeman, Montana April, 2011 ©COPYRIGHT by Erin Kirby Field 2011 All Rights Reserved ii APPROVAL of a dissertation submitted by Erin Kirby Field This dissertation has been read by each member of the dissertation committee and has been found to be satisfactory regarding content, English usage, format, citation, bibliographic style, and consistency, and is ready for submission to the Division of Graduate Education. Dr. Robin Gerlach Approved for the Department of Microbiology Dr. Mark A. Jutila Approved for The Graduate School Dr. Carl A. Fox iii STATEMENT OF PERMISSION TO USE In presenting this dissertation in partial fulfillment of the requirements for a doctoral degree at Montana State University, I agree that the Library shall make it available to borrowers under rules of the Library. I further agree that copying of this dissertation is allowable only for scholarly purposes, consistent with “fair use” as prescribed in the U.S. Copyright Law. Requests for extensive copying or reproduction of this dissertation should be referred to ProQuest Information and Learning, 300 North Zeeb Road, Ann Arbor, Michigan 48106, to whom I have granted “the exclusive right to reproduce and distribute my dissertation in and from microform along with the non- exclusive right to reproduce and distribute my abstract in any format in whole or in part.” Erin Kirby Field April, 2011 iv ACKNOWLEDGEMENTS First and foremost, I would like to thank my advisor, Dr. Robin Gerlach for all the guidance, knowledge and support he has provided both professionally and personally throughout my graduate program here. I am deeply grateful for everything he has done for me. Thank you to the additional members of my committee Drs. Matthew Fields, Anne Camper, Brent Peyton, Gill Geesey and Isaac Klapper for everything they have done for me that far exceeded their obligations as my committee. Thank you to everyone at the CBE and especially my lab group for their help with my research and making the CBE such a great place to work. Additional thanks to John Blaskovich for all of his hard work and dedication in the laboratory. Most importantly, I would like to thank my family, especially my father, for their never-ending moral support of which I could not have made it without. Similarly, thank you to Dr. Abigail Richards for everything, especially for being my biggest support system through these last five years. Thank you to my fellow students, both past and present, through which this experience would not have been the same without. Thanks to the Inland Northwest Research Alliance, the Microbiology Department and U.S. Department of Energy Subsurface Biogeochemical Research Program for financial support through my graduate career. v TABLE OF CONTENTS 1. INTRODUCTION ......................................................................................................... 1 Chromium in Soils ......................................................................................................... 1 Chromium at Department of Energy Sites ..................................................................... 2 Idaho National Laboratory Site ............................................................................... 3 Hanford Site ............................................................................................................ 5 Chromium Remediation in Soils .................................................................................... 6 Microorganisms and Chromium .................................................................................... 7 Chromium Toxicity to Microorganisms ................................................................. 7 Chromate Resistance by Microorganisms .............................................................. 7 Chromate Reduction by Microorganisms ............................................................. 11 Cellulose in the Environment ....................................................................................... 13 Cellulose Degradation by Microorganisms .................................................................. 13 Cellulolytic Enzymes ............................................................................................ 15 Research Goals and Objectives .................................................................................... 17 References .................................................................................................................... 19 2. ISOLATION AND CHARACTERIZATION OF POTENTIAL CELLULLOSE-DEGRADING MICROORGANISMS FROM A SIMULATED LOW-LEVEL WASTE SITE .............................................................. 26 Introduction .................................................................................................................. 26 Materials and Methods ................................................................................................. 27 Isolation Methods ................................................................................................. 27 Sample Preparation ........................................................................................ 27 Direct Isolation Plates ................................................................................... 27 Liquid Enrichments ....................................................................................... 28 Column Studies ............................................................................................. 29 Sequencing and Identification .............................................................................. 32 Bacterial Isolates ........................................................................................... 32 Fungal Isolate ................................................................................................ 32 Characterization Methods ..................................................................................... 33 Media and Culture Inoculum ......................................................................... 33 Carboxymethylcellulose Plates ..................................................................... 33 Pure Culture Studies ...................................................................................... 34 Cellobiose Utilization .................................................................................... 34 Carbon Source Utilization Studies ................................................................ 35 Cr(VI) Reduction Studies .............................................................................. 35 Cr(VI) Quantification .................................................................................... 36 Results and Discussion ................................................................................................. 37 vi TABLE OF CONTENTS - CONTINUED Isolation and Identification ................................................................................... 37 Direct Isolation .............................................................................................. 37 Liquid Enrichments ....................................................................................... 37 Column Studies ............................................................................................. 38 Phylogeny and Potential Cellulose Degradation Capabilities ....................... 40 Characterization Studies ....................................................................................... 41 Cellulose Degradation Capabilities ............................................................... 41 Carbon Source Utilization Capabilities ......................................................... 46 Cr(VI) Reduction Capabilities ....................................................................... 47 Conclusions .................................................................................................................. 52 References .................................................................................................................... 54 3. APPLICATION OF MOLECULAR TECHNIQUES TO ELUCIDATE THE INFLUENCE OF CELLULOSIC WASTE ON THE BACTERIAL COMMUNITY STRUCTURE AT A SIMULATED LOW-LEVEL-RADIOACTIVE-WASTE SITE ......................................................... 56 Contribution of Authors and Co-Authors .................................................................... 56 Manuscript Information Page ....................................................................................... 57 Abstract ........................................................................................................................ 58 Introduction .................................................................................................................. 59 Materials and Methods ................................................................................................. 61 Site Description ..................................................................................................... 61 CTPS Sampling ..................................................................................................... 62 Sampling Extraction and 16S rRNA Gene Amplification .................................... 63 Cloning and Sequencing ......................................................................................
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