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Photographs included in the original manuscript have been reproduced xerographically in this copy. Higher quality 6" x 9" black and white photographic prints are available for any photographs or illustrations appearing in this copy for an additional charge. Contact UMI directly to order. Bell & Howell Information and Learning 300 North Zeeb Road, Ann Arbor, Ml 48106-1346 USA 800-521-0000 UMÏ LTsfTVERSITY OF OKLAHOMA GRADUATE COLLEGE THE MICROBIAL METABOLISM OF BENZENE AND CRUDE OIL UNDER DIVERSE ANAEROBIC CONDITIONS A Dissertation SUBMITTED TO THE GRADUATE FACULTY in partial fulfillment of the requirements for the degree of Doctor of Philosophy By Matthew Edward Caldwell Norman, Oklahoma 2000 UMI Number. 9980454 UMI UMI Microform 9980454 Copyright 2000 by Bell & Howell information and Learning Company. All rights reserved. This microform edition is protected against unauthorized copying under Title 17, United States Code. Bell & Howell Information and Leaming Company 300 North Zeeb Road P.O. Box 1346 Ann Arbor, Ml 48106-1346 THE MICROBIAL METABOLISM OF BENZENE AND CRUDE OIL UNDER DIVERSE ANAEROBIC CONDITIONS A Dissertation APPROVED FOR THE DEPARTMENT OF BOTANY AND MICROBIOLOGY BY © Copyright by Matthew Edward Caldwell 2000 All Rights Reserved. ACKNOWLEDGEMENTS This work was supported by research grants from the Department of Defense (DOD). the OfBce of Naval Research, in particular, the Augmentation Awards for Science and Engineering Research Training (AASERT) Grants and in part, by the American Petroleum Institute (API). Above and beyond, 1 would like to especially thank Dr. Joseph M. Suflita. His patience and support during my graduate career was instrumental for its completion. Further, his guidance and advice have made a lasting impression on me. Did 1 mention "patience”? I would also like to thank the members of my committee. Dr. Ralph Tanner, Dr. Michael Mclnemey, Dr. John Fletcher and Dr. David Sabatini. Particular thanks goes out to Drs. Mclnemey and Tanner for their openness to answer queries and discuss topics whether they were related to my research or to life experiences. In addition, appreciation goes out to Dr. Mark Nanny for his help and expertise in the '^C-NMR work with toluene. I would like to acknowledge several researchers not associated with the University of Oklahoma who have contributed to my research goals. Special thanks goes out to Dr. Roger Prince of Exxon Research and Engineering Company for his contributions, as well as Dr. Robert Garrett for his analysis of Alaskan North Slope oil, as well as others I have collaborated with in the past. I would also like to thank the members and staff of the Department of Botany and Microbiology that I have gotten to know for o’ these many years. I would also like to put forth a special recognition to Dr. John Skvarla, Dr. Ed Vezey, and Dr. Neil Adrian who’s time and patience in teaching anaerobic technique will not be forgotten. IV In addition, this work would not be possible without the support of my family. I dedicate this dissertation to my parents, Robert and Jean Caldwell. Without their unending support I would not have reached my goal. Also, the continued support of my brothers and sisters needs to be recognized, as well. I would also like to say thanks to so many people that have affected me for the entire length of my graduate school tenure including, but certainly not limited to, Laura and Jim Cornell for their friendship and support, as well as a special recognition to Keith and Jermi Allen, and my good friends. Dr. Vishvesh Bhupathiraju, Spencer Philo, Dr. Paul Olson, and John Adkins. TABLE OF CONTENTS Page Acknowledgements ............................................................................................................ iv List of Tables ......................................................................................................................viii List of Illustrations ..............................................................................................................ix Preface................................................................................................................................. xi Abstract............................................................................................................................... xiv Chapter 1. Anaerobic Degradation of Benzene in Diverse Anoxic Environments Abstract.................................................................................................................................1 Introduction .......................................................................................................................... 3 Materials and Methods ........................................................................................................ 6 Results and Discussion ........................................................................................................ 12 References ............................................................................................................................ 24 Chapter 2. Microbial Metabolism of Benzene and the Oxidation of Ferrous Iron under Anaerobic Conditions: Implications for Bioremediation Abstract.................................................................................................................................28 Introduction ..........................................................................................................................29 Materials and Methods ........................................................................................................ 31 Results.................................................................................................................................. 35 Discussion ............................................................................................................................ 44 References ............................................................................................................................ 50 Chapter 3. Detection of Phenol and Benzoate as Intermediates of Anaerobic Benzene Biodégradation under Different Terminal Electron-Accepting Conditions Abstract................................................................................................................................ 59 Introduction ..........................................................................................................................60 Materials and Methods ........................................................................................................62 Results..................................................................................................................................64 VI Page Discussion .......................................................................................................................... 71 References .......................................................................................................................... 77 Chapter 4. Anaerobic Biodégradation of Long-Chain n-Alkanes under Sulfate-Reducing Conditions. Abstract...............................................................................................................................80 Introduction ........................................................................................................................ 81 Materials and Methods ......................................................................................................84 Results and Discussion ......................................................................................................86 References .......................................................................................................................... 96 vu U ST OF TABLES Page Table I. Characteristics of samples assayed for presence of microorganisms 7 capable of anaerobic benzene degradation and reference to procedures used in assay protocol Table 2. Evidence for anaerobic biodégradation of benzene in samples from 13 diverse environments under different redox conditions Table 3. Amount of [I4C-UL]benzene detected as gaseous end products in 14 incubations of aquifer sediments from M3 and CA Table 4. Consumption of S 04~’ during degradation o f benzene in New York/ 19 New Jersey Harbor sediments Table 5. Recovery of ‘‘‘C-labeled products of ^'^C-ring-toluene biodégradation 37 by Ponca City sediment microorganisms under different electron accepting conditions Table 6 . Fe(II) oxidizing capacities of various samples sssayed under nitrate 43 reducing conditions Table 7. Recovery of *'*C^