AN ABSTRACT OF THE THESIS OF

Michael Richard Niemet for the degree of Master of Science in Civil Engineering presented on May 18. 1995.

Title: Continuous-Flow and Batch Column Studies of Anaerobic Carbon Tetrachloride Biotransformation on Hanford Soil. Redacted for Privacy

Abstract approved: L is Semprini

Continuous-flow and batch experiments were conducted with a column reactor system containing Hanford aquifer material in order to evaluate the potential of in-situ bioremediation of carbon tetrachloride (CT) at Hanford. The effectiveness of benzoate and acetate as primary substrates was considered. Nitrate and sulfate were potential electron acceptors. Transport experiments indicated the following characteristics: porosity, 27%; longitudinal dispersivity, 11 cm; and CT retardation factor, 3.9. Denitrification and CT transformation occurred during periods of benzoate and acetate addition. Chloroform (CF) was detected as a product of CT transformation in all cases. Benzoate generally induced the fastest rates of CT transformation. However, acetate was much better at inducing denitrification. Sulfate reduction was never observed, even during extended absences of nitrate and nitrite. The continuous-flow experiments showed more rapid transformation near the point of injection; however, residence times were not long enough to completely degrade CT. In batch experiments CT transformation appeared to follow pseudo-first-order kinetics, with rates decreasing from the point of injection. Switching from continuous-flow to batch experiments appeared to be an effective means of determining spatial differences in microbial activity within the column. Overall, these results indicate that the microbial population at Hanford is capable of transforming CT in the subsurface. However, methods to control the production of CF may be necessary before this technology can be successfully employed. Continuous-Flow and Batch Column Studies of Anaerobic Carbon Tetrachloride Biotransformation on Hanford Soil

by

Michael Richard Niemet

A THESIS

submitted to

Oregon State University

in partial fulfillment of the requirements for the degree of

Master of Science

Completed May 18, 1995 Commencement June 1996 Master of Science thesis of Michael Richard Niemet presented on May 18, 1995

APPROVED: Redacted for Privacy

ate Professor of 'evil E gineering in charge of major Redacted for Privacy

Head of Department of Civil Engineering

Redacted for Privacy Dean of Graduate chool

I understand that my thesis will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my thesis to any reader upon request. Redacted for Privacy ichael iemet, Author i

ACKNOWLEDGMENTS

Many people have contributed to the completion of this thesis and the numerous other challenges culminating in the associated degree; unfortunately, I can only recognize a few select individuals here. Of those, I am most grateful to my wife, Bronwyn Evans, who has provided support without fail and has sacrificed considerably in order for me to focus on my studies. I will never forget her thoughtfulness. I am also thankful to have had the privilege to work under Dr. Lewis Semprini, my major professor. In addition to his kind and generous personality, Lew has provided opportunities and guidance beyond all expectations. My parents, Norman and Sally Niemet, have always allowed me the freedom to make my own career decisions while providing their full support, for which I will always be grateful. Also, it was my father who sparked my interest in engineering at an early age. Finally, I would like to mention that Ann Evans and Doug Barbe, who in addition to their encouragement and support, have helped to enlighten and open my mind over the years. Their inspiration undoubtedly played a major role in the circumstances leading to my decision to pursue a graduate degree. Thanks again, to everybody that helped me along the way. This research was jointly funded by the U.S. Department of Energy and the U.S. Environmental Protection Agency-sponsored Western Region Hazardous Substance Research Center under agreement R-815738. ii

TABLE OF CONTENTS

1. INTRODUCTION 1

2. BACKGROUND 4 Chemical Properties 4 Solute Transport 5 Transformations of Carbon Tetrachloride 7 Substitution Reactions 7 Oxidation and Reduction Reactions 8 Biological Considerations 12 Literature Review 13 Batch Studies 14 Mixed Culture Systems 14 Pure Culture Systems 14 Cell-Free Metallo-Porphyrin Systems 17 Continuous-Flow Column Studies 17 Studies Involving Systems Containing Aquifer Material 18 Studies in Support of the Hanford Demonstration 19 Summary 20

3. MATERIALS AND METHODS 22 Column Reactor 22 Media 24 Aquifer Material 24 Groundwater 24 Amendments 26 Supply System 26 Experimental Procedure 28 Sampling Procedure 30 Analytical 31 Chemical Sources 31 CAHs 31 Anions 32 111

TABLE OF CONTENTS (CONTINUED)

Other Analyses 33

4. RESULTS & DISCUSSION 34 Transport Studies 34 Transformations 37 Pre-Substrate 37 Benzoate as the Primary Substrate 42 First Benzoate Addition 42 First Batch Test with Benzoate 43 Second Benzoate Addition (with and without Nitrate) 44 Second Batch Test with Benzoate (without Nitrate) 48 Acetate as the Primary Substrate 54 First Acetate Addition 54 Yeast Extract Addition with Acetate 56 Second Acetate Addition (without Nitrate) 58 First Batch Test with Acetate (without Nitrate) 61 Third Acetate Addition (Nitrate Replaced) 66 Second Batch Test with Acetate (with Nitrate) 69

5. SUMMARY AND CONCLUSIONS 75 Summary 75 Comparison of Primary Substrates 76 Comparison of Continuous-Flow to Batch Experiments 81 Conclusions 82 Future Research 83

BIBLIOGRAPHY 85

APPENDICES 91 APPENDIX A: Timing of Experimental Events 92

APPENDIX B: Summary of Physical Parameters 93

APPENDIX C: Cumulative Data Summary 94

APPENDIX D: Preliminary Control Experiments 115 iv

TABLE OF CONTENTS (CONTINUED)

APPENDIX E: Stoichiometric Balances 119

APPENDIX F: Basic Concepts 121

APPENDIX G: Protocol for Anaerobic Column Packing 126

APPENDIX H: Protocol for Preparing Saturated Aqueous Stock Solutions 128

APPENDIX I: GC Standard Curve Protocol for CAHs & Sample Chromatograms 129

APPENDIX J: IC Standard Curve Protocol for Anions & Sample Chromatograms 135

APPENDIX K: Column and Supply Network Parts List 140 V

LIST OF FIGURES

Figure 2.1. Comparison of redox potentials, in Volts, for selected half- reactions, assuming unit activity and corrected for pH = 7. 9 Figure 2.2. Known abiotic and biotic transformations of CT. 11 Figure 3.1. Column configuration 23 Figure 3.2. Column supply system 27 Figure 4.1. Bromide breakthrough data; flow rate = 340 mL/day 34 Figure 4.2. Comparison of breakthrough data to equilibrium Cfitm model; flow rate = 340 mL/day. 36 Figure 4.3. Nitrate and nitrite concentrations prior to substrate addition; retention time = 0.5 days. 37 Figure 4.4. Amount of benzoate, expressed as carbon, required to denitrify the amounts shown in Figure 4.3 38 Figure 4.5. Influent and effluent CAH concentrations prior to substrate addition; retention time = 0.5 days 39 Figure 4.6. Column profiles (day 27 anions and day 29 for CAHs) prior to substrate addition; retention time = 0.5 days. 41 Figure 4.7. Column profiles 6 days after flow reduction; primary substrate = benzoate, retention time = 2 days 45 Figure 4.8. Column profiles 1 week after acetate was added at 40 mg/L to complete denitrification within the pre-mix chamber; primary substrate (within soil column) = benzoate, retention time = 2 days. 47 Figure 4.9. Concentrations at port 3 during the second batch experiment with benzoate, acetate was used to remove nitrate prior to entry into soil column. 50 Figure 4.10. First-order check on CAHs for the second batch experiment with benzoate. 51 Figure 4.11. CAH concentrations throughout the column at the end of the second batch experimen