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Atrazine Mineralization Potential and Catabolic Gene Detection In ATRAZINE MINERALIZATION POTENTIAL AND CATABOLIC GENE DETECTION IN AGRICULTURAL AND WETLAND SITES DISSERTATION Presented in Partial Fulfillment of the Requirements of the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Kristen Lynn Anderson, B. S. * * * * * The Ohio State University 2003 Dissertation Committee: Approved by Dr. Olli H. Tuovinen, Advisor Dr. Michael J. Boehm _____________________________ Dr. Mark Morrison Advisor Dr. Samuel J. Traina Department of Microbiology ABSTRACT Atrazine (2-chloro-4-ethylamine-6-isopropylamino-1,3,5 triazine) is a commonly applied herbicide in corn fields. Although the fate of atrazine in agricultural systems has been well studied, the environmental fate of atrazine in wetland systems is less well characterized. The majority of research in this area has focused on aerobic mineralization of atrazine, although anaerobic conditions are commonly found in wetland sediments and bulk soils associated with agricultural fields. The hypothesis for this work was that atrazine would be actively mineralized in agricultural and wetland sites. It was further hypothesized that active mineralization in soils could be correlated with the presence of selected genes involved in atrazine metabolism. Soil, sediment, and water samples were obtained from three sites in Ohio. Atrazine mineralization was investigated under aerobic and anaerobic conditions in these samples using a biometer system in 14 which CO2 evolution was correlated with atrazine mineralization. All samples mineralized atrazine under aerobic conditions. Under anaerobic conditions, some external electron acceptor amendments inhibited mineralization, while others enhanced it. The effect on mineralization varied with the sample and season. Attempts were made to amplify some of the genes involved in atrazine mineralization. Community DNA was ii isolated from each sample and PCR amplification using three primer sets specific for atzA, trzD, and trzN was performed. Although PCR inhibitors were present in many of the samples, trzN and trzD could be successfully amplified from some DNA samples. All Defiance samples tested positive for trzN with the exception of field B and reservoir water samples. An analog of trzD was amplified from DNA extracted from Defiance and OSU wetland samples. The atzA gene was not detected in any of the samples. An atrazine mineralizing bacterium was isolated from Piketon soil samples and another was obtained from Defiance corn soil. Characterization of these organisms by 16S rDNA sequencing and FAME analysis indicated that both organisms were Arthrobacter spp. From these results, it is clear that the potential for atrazine mineralization was identified in agricultural soils and wetland sediments. No correlation could be made between atrazine mineralization and the presence of any of the tested genes involved in atrazine mineralization. iii To Matt - for supporting me through all the good times, but especially for being there through all the bad times. iv ACKNOWLEDGMENTS I would first like to thank my advisor, Dr. Olli Tuovinen, for encouraging me and helping me to keep on track. I would also like to thank him for giving me the freedom to pursue avenues that we hadn’t initially considered in this project. I would also like to thank my committee members, Dr. Sam Traina, Dr. Mike Boehm, Dr. Mark Morrison, and Dr. Michelle Rondon for their insights into my project and their helpful suggestions. I would like to thank Mr. Peter Bierman, former extension agent, and Mr. Wayne Lewis, farm manager, at the Piketon Research and Extension Center for helping me gather the crop and herbicide application information on the Piketon sites. Dr. Kent Harrison graciously allowed me to use his biological oxidizer and Dr. Xiaoyu Yang instructed me on how to operate the oxidizer and answered my questions on how it actually worked. Dr. J. S. Karns generously provided the Escherichia coli construct containing the trzD gene and Dr. M. de Souza provided the E. coli construct containing the atzA gene. Dr. W. Mulbry provided the pWM221011 plasmid containing the trzN gene and also helped me optimize the PCR reaction to amplify this gene. v Kevin Wheeler, formerly of the University of Dayton, performed the atzA and trzD PCR amplification reactions on the OSU sediment samples. I would like to thank Dr. Mark Radosevich for the FAME analysis on the soil bacterial isolates. The Defiance Agricultural Research Association, especially Bruce Clevenger, provided me with information on the Defiance site. I would also extend my thanks to past and present members of the Tuovinen lab for their encouragement and discussions on my project. I would especially like to thank Dr. Ellen Duffy (nee Ostrofsky) who helped me get started on this project and also provided the MPN calculation program used to determine MPN estimates. Dr. Duongruitai Nicomrat helped me with the 16 rDNA sequencing and provided many helpful suggestions on DNA extraction and PCR amplification from soil samples. This research was supported by the United States Department of Agriculture, National Research Initiative Program (Grant No. 98-35107-6388) vi VITA October 23, 1973……………………………Born – Sewickley, PA 1996…………………………………………..B. S. Molecular biology/biotechnology, Clarion University of Pennsylvania 1997 – present……………………………….Graduate Teaching Associate, The Ohio State University PUBLICATIONS Research Publications 1. Kwak, J., L. A. McCue, K. Trzcianka, and K. E. Kendrick. 2001. Identification and characterization of a developmentally regulated protein, EshA, required for sporogenic hyphal branches in Streptomyces griseus. J. Bacteriol.183:3004- 3015. 2. Anderson, K. L., K. A. Wheeler, J. B. Robinson, and O. H. Tuovinen. 2002. Atrazine mineralization in two wetlands. Wat. Res. 36:4785-4794. vii FIELDS OF STUDY Major field: Microbiology viii TABLE OF CONTENTS Page Abstract ............................................................................................................................. ii Dedication ........................................................................................................................ iv Acknowledgements ........................................................................................................... v Vita ...................................................................................................................................vii List of Tables....................................................................................................................xii List of Figures..................................................................................................................xiii Chapters 1. Introduction.................................................................................................................. 1 1.1 Atrazine mineralization in agricultural soils......................................................... 2 1.1.1 Aerobic mineralization in surface and subsurface soils .......................... 2 1.1.2 Effect of the plant rhizosphere ................................................................ 4 1.1.3 Anaerobic mineralization of atrazine in bulk soil..................................... 5 1.1.4 Effect of amendments on atrazine mineralization................................... 6 1.2 Atrazine mineralization in wetland sediments..................................................... 7 1.2.1 Aerobic mineralization in wetland sediments.......................................... 7 1.2.2 Anaerobic mineralization in wetland sediments...................................... 9 1.3 Abiotic dechlorination of atrazine...................................................................... 10 1.4 Genetic basis of atrazine degradation.............................................................. 11 1.4.1 The thc genes....................................................................................... 11 1.4.2 The atrA gene....................................................................................... 11 1.4.3 The trz genes: Pseudomonas str. NRRLB-12227 ................................ 14 1.4.4 The trz genes: Nocardiodes str. C190 .................................................. 14 1.4.5 The atz genes....................................................................................... 15 1.5 Statement of purpose.......................................................................................... 17 2. Evaluation of the atrazine mineralization potential at an agricultural research site by soil biometer studies and PCR amplification of catabolic genes ..................................... 19 2.1 Introduction...................................................................................................... 19 2.2 Materials and methods..................................................................................... 22 2.2.1 Site description..................................................................................... 22 2.2.2 Sampling............................................................................................... 22 2.2.3 Mineralization studies........................................................................... 25 2.2.4 Data analysis........................................................................................ 27 2.2.5 Mass balances...................................................................................... 27 2.2.6 DNA isolation........................................................................................ 28
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