THESIS THERMALLY ENHANCED BIOREMEDIATION OF LNAPL Submitted by Natalie Rae Zeman Department of Civil and Environmental Engineering In partial fulfillment of the requirements For the Degree of Master of Science Colorado State University Fort Collins, Colorado Spring 2013 Master’s Committee: Advisor: Susan K. De Long Co-Advisor: Thomas Sale Mary Stromberger ABSTRACT THERMALLY ENHANCED BIODEGRADATION OF LNAPL Inadvertent releases of petroleum liquids into the environment have led to widespread soil and groundwater contamination. Petroleum liquids, referred to as Light Non-Aqueous Phase Liquid (LNAPL), pose a threat to the environment and human health. The purpose of the research described herein was to evaluate thermally enhanced bioremediation as a sustainable remediation technology for rapid cleanup of LNAPL zones. Thermally enhanced LNAPL attenuation was investigated via a thermal microcosm study that considered six different temperatures: 4°C, 9°C, 22°C, 30°C, 35°C, and 40°C. Microcosms were run for a period of 188 days using soil, water and LNAPL from a decommissioned refinery in Evansville, WY. The soil microcosms simulated anaerobic subsurface conditions where sulfate reduction and methanogenesis were the pathways for biodegradation. To determine the optimal temperature range for thermal stimulation and provide guidance for design of field-scale application, both contaminant degradation and soil microbiology were monitored. CH4 and CO2 generation occurred in microcosms at 22°C, 30°C, 35°C and 40°C but was not observed at 4°C and 9°C. The total volume of biogas generated after 188 days of incubation was 19 times higher in microcosms at 22°C and 30°C compared to the microcosms at 35°C. When compared to microcosms at 40°C, the total biogas generated was 3 times higher at both 22°C and 30°C. The onset of CH4 and CO2 production occurred first within the microcosms held at 30°C beginning after 28 days of incubation, and second within microcosms at 22°C beginning after 58 days of incubation. A delay in CH4 and CO2 production was observed within microcosms held at 35°C and 40°C (beginning after 173 and 138 days after incubation) ii contributing to lower cumulative biogas generation at these temperatures relative to 22ºC and 30ºC. Microcosms incubated at 4ºC, 22°C, 30ºC, 35 ºC and 40 ºC showed statistically significant biological removal of gasoline range organics (GRO) at the α=0.05 level over the course of the 188-day incubation period. Biotic removal of GRO was significantly higher at 22ºC and 30ºC when compared to 4ºC and 9ºC. The observed removals at 22 ºC, 30 ºC, 35 ºC and 40ºC were not statistically different from each other. The biological removal of DRO compounds was found to be statistically significant at 22ºC, 30 ºC, 35 ºC and 40 ºC and was significantly higher at 22ºC when compared to 4ºC and 9ºC. The percent biological removal of DRO compounds at these temperatures was not statistically different from each other and ranged from 18-22%. Statistically significant biological degradation of all the BTEX compounds only occurred in microcosms at 22°C and 30°C. Benzene, toluene, and xylene biodegradation was observed to be statistically significant in microcosms at 35°C. Phylogenetic analysis of the microbial communities present at 22°C via a 16S rRNA gene archaeal clone library and pyrosequencing revealed the presence of both hydrogenotrophic and acetoclastic methanogens. Pyrosequencing data from microcosms at 4°C, 22°C and 35°C revealed that temperature impacted both archaeal and bacterial community structure. A potential requirement for changes in microbial community structure at the higher temperatures (35°C and 40°C) could explain the observed delay in biogas production. The results of the thermal microcosm study led to the development of a pilot study to investigate the efficacy of Sustainable Thermally Enhanced LNAPL Attenuation STELA at the site in Evansville, WY. The thermal pilot study approach and preliminary data is included in this iii thesis. The outcome and results of the pilot study will be presented in the masters theses of other students. iv ACKNOWLEDGEMENTS I would like to thank my advisors Dr. Tom Sale and Dr. Susan De Long for their support and guidance throughout the course of this work. Their passion for science and knowledge is inspiring. Through this experience I have come to admire and respect them immensely. I would also like to thank my committee member Dr. Mary Stromberger for her interest in my research topic and willingness to provide assistance. Furthermore, I would like to thank Julio Zimbron and Mitchell Olson for their input and enthusiasm in teaching me new techniques. I would also like to thank Gary Dick, Kevin McCoy, Paige Griffin, Emilie Lefèvre and others in my lab group who helped me with the thermal microcosm study. Furthermore, I would like to thank Maria Irianni Renno and Daria Akhbari for their help in the field application. Finally, I would like to thank my family and loved ones for their constant love and encouragement. It was only with their support that I was able to take on this challenge and succeed. v TABLE OF CONTENTS Abstract ........................................................................................................................................... ii Acknowledgments .......................................................................................................................... v Table of Contents ........................................................................................................................... vi List of Tables ............................................................................................................................... viii List of Figures ................................................................................................................................ ix 1 Introduction .................................................................................................................................. 1 1.1 Research Motivation ............................................................................................................. 2 1.2 Research Objectives .............................................................................................................. 3 1.3 Thesis Overview ................................................................................................................... 3 2 Background and Literature Review ............................................................................................. 4 2.1 Petroleum LNAPL Composition........................................................................................... 4 2.2 LNAPL Fate and Transport .................................................................................................. 5 2.3 Microbiological Processes .................................................................................................... 7 2.4 Microorganisms Involved in Anaerobic LNAPL Degradation ............................................. 9 2.5 Bioremediation of Petroleum Hydrocarbons ...................................................................... 13 2.6 Biological Natural Attenuation ........................................................................................... 14 2.7 Thermally Enhanced Bioremediation ................................................................................. 16 2.8 Molecular Tools to Assess Bioremediation Technologies.................................................. 20 3 LNAPL Thermal Microcosm Study .......................................................................................... 23 3.1 Overview ............................................................................................................................. 23 3.2 Introduction ......................................................................................................................... 24 3.3 Methods............................................................................................................................... 27 3.3.1 Site Background ......................................................................................................... 27 3.3.2 Soil Collection and Storage ....................................................................................... 28 3.3.3 Water Collection and Storage .................................................................................... 28 3.3.4 Microcosm Set Up ..................................................................................................... 28 3.3.5 Analytical Techniques ............................................................................................... 30 3.4 Results and Discussion ....................................................................................................... 36 3.4.1 Biogas Data ................................................................................................................ 36 3.4.2 Hydrocarbon Data ...................................................................................................... 38 3.4.3 Molecular Data........................................................................................................... 44 3.5 Conclusion .......................................................................................................................... 53 4 Sustainable Thermally Enhanced LNAPL Attenuation Pilot Study .......................................... 55 4.1 Introduction ........................................................................................................................