ALKALINE HYDROLYSIS OF EXPLOSIVES by Catherine Elizabeth VanEngelen A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Engineering MONTANA STATE UNIVERSITY Bozeman, Montana January 2010 ©COPYRIGHT by Catherine Elizabeth VanEngelen 2010 All Rights Reserved ii APPROVAL of a dissertation submitted by Catherine Elizabeth VanEngelen 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. Brent M. Peyton Approved for the Department of Chemical and Biological Engineering Dr. Ron W. Larsen Approved for the Division of Graduate Education 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.” Catherine Elizabeth VanEngelen January 2010 iv DEDICATION This work is dedicated to my parents, Patricia and Daniel Albaugh, and to my husband, Michael VanEngelen. Only their loving support and encouragement has made this possible. v ACKNOWLEDGEMENTS Financial support for this research was provided by: the Thermal Biology Institute at MSU (TBI grant #NNG04GR46G), the Inland Northwest Research Alliance (INRA Student Fellowship), the Center for Biofilm Engineering at MSU, the College of Engineering at MSU (Benjamin Fellowship), and the Radford Army Ammunition Plant, Radford, VA. I would also like to gratefully acknowledge the following people for their help and guidance throughout my time at Montana State: Shelley Thomas, Jill Newman, John Neuman, Ann Willis, John and Flower Aston, Melinda Clark, Angela and Dennis O’Brien, Chris and Angie Albaugh, Mary and Dean Lutz, Patrick Albaugh, John and Susan VanEngelen, Tamara and Rich Cullen, Jen and Tim Traynor, Kristen and Chip George, members of the Peyton Research Group, Ron Larsen, and my committee: Ross Carlson, Robin Gerlach, Anne Camper, and Brent Peyton. vi TABLE OF CONTENTS 1. INTRODUCTION .................................................................................................... 1 2,4,6-Trinitrotoluene (TNT)...................................................................................... 1 Abiotic Treatments............................................................................................... 2 Biological Treatments .......................................................................................... 5 Nitrocellulose (NC) ................................................................................................. 11 Abiotic Treatments ............................................................................................ 13 Biological Treatments ........................................................................................ 15 Combined Hydrolysis and Biological Treatments ....................................... 16 Summary ................................................................................................................. 18 References ............................................................................................................... 19 2. ENRICHMENT FOR A TNT-DEGRADING MICROORGANISM .................... 24 Background ............................................................................................................. 24 Materials and Methods ............................................................................................ 24 Results and Discussion ........................................................................................... 26 Conclusions ............................................................................................................. 32 References ............................................................................................................... 33 3. ALKALINE HYDROLYSIS OF TNT ................................................................... 39 Introduction ............................................................................................................. 39 Materials and Methods ............................................................................................ 41 Chemicals ........................................................................................................... 41 Hydrolysis Experiments ..................................................................................... 41 Sampling and Storage ........................................................................................ 42 Analyses ............................................................................................................. 42 Radiolabeled TNT Experiments ........................................................................ 43 Biological Screening .......................................................................................... 43 Results and Discussion ........................................................................................... 44 Kinetics of Hydrolysis ....................................................................................... 47 Hydrolysis Products ........................................................................................... 53 Radiolabeled 14C-TNT Experiments .................................................................. 55 Biological Degradation of TNT Hydrolysate .................................................... 58 Conclusions ............................................................................................................. 60 References ............................................................................................................... 62 vii TABLE OF CONTENTS – CONTINUED 4. NC TREATABILITY STUDY ............................................................................... 68 Background ............................................................................................................. 68 Materials and Methods ............................................................................................ 69 Results and Discussion ........................................................................................... 71 Conclusions ............................................................................................................. 79 Recommendations ................................................................................................... 81 References ............................................................................................................... 83 5. ALKALINE HYDROLYSIS OF NC ..................................................................... 85 Introduction ............................................................................................................. 85 Materials and Methods ............................................................................................ 87 Results and Discussion ........................................................................................... 89 Hydrolysis of NC ............................................................................................... 89 Biological Degradation of NC Hydrolysate ....................................................... 95 Conclusions ............................................................................................................. 98 References ............................................................................................................... 99 6. SUMMARY AND FUTURE WORK .................................................................. 101 TNT ....................................................................................................................... 101 NC ......................................................................................................................... 103 APPENDICES ............................................................................................................ 104 APPENDIX A: Consensus Sequence for Anoxybacillus kualowohkensis ............ 105 APPENDIX B: Raw Data for TNT/TNB Experiments ........................................ 107 APPENDIX C: Raw Data for 14C-TNT Experiments ........................................... 112 APPENDIX D: Raw Data for Biological Degradation of TNT Hydrolysate ....... 119 APPENDIX E: Raw Data for TNT Hydrolysis Experiments ............................... 121 APPENDIX F: HPLC 1090 Chromatogram: “10 Mix” Standards ....................... 134 APPENDIX G: HPLC 1090 Chromatogram: “14 Mix” Standards ...................... 136 APPENDIX H: Raw Data for NC Treatability Study ........................................... 138 APPENDIX I: Raw Data for NC Hydrolysis Experiments .................................. 142 APPENDIX J: Raw Data for NC Hydrolysate Biological Screening .................. 147 viii LIST OF TABLES Table ............................................................................................................................... Page 1. YNP spring samples ........................................................................................................ 25 2. Comparison of molar TNT and hydroxide concentrations at various pH values ....................................................................................................................... 45 3. Comparison of pseudo-first-order kinetic rate constants, k’ (h-1) ................................... 50 4. Composition for NC experiments at both room temperature (RT) and 60°C .......................................................................................................................... 69 5. Denitrification rates (mg NO / h · mg biomass) for x WWTP culture spiked with NC hydrolysate .................................................................. 97 ix LIST OF FIGURES Figure .............................................................................................................................. Page 1. Chemical structure of TNT ............................................................................................... 1 2. Pathways of biological TNT transformation..................................................................... 7 3. Chemical structure of NC ............................................................................................... 12 4. Biological treatment and controls for 16mg/L TNT degradation with 1g/L yeast extract at 60°C and pH 9 ....................................................................... 27 5. Protein production for 16mg/L TNT degradation and 1g/L yeast extract at 60°C and pH 9 ................................................................................................. 28 6. Occurrence of TNB as the abiotic product of 16mg/L TNT degradation at 60°C and pH 9 ......................................................................................... 29 7. Chemical structure of TNB ............................................................................................. 29 8. Effect of pH and temperature on TNT concentration ..................................................... 46 9. TNT degradation with production of nitrite and nitrate at pH 9 ..................................... 47 10. TNT degradation with production of nitrite and nitrate at pH 10 ................................. 48 11. TNT degradation with production of nitrite and nitrate at pH 11 ................................. 48 12. Linearized Arrhenius plot for determination of activation energy of the alkaline hydrolysis of TNT ................................................................................. 52 13. Initial distribution of 14C -TNT and corresponding HPLC chromatogram ............................................................................................................... 56 14. Day 1 distribution of 14C from TNT treated at pH 10 and 60°C and corresponding HPLC chromatogram ..................................................................... 57 15. Day 9 distribution of 14C from TNT treated at pH 10 and 60°C and corresponding HPLC chromatogram ..................................................................... 58 16. Growth of WWTP denitrifying culture on TNT hydrolysate ....................................... 59