BIODEGRADATION OF N-HETEROCYCLIC AROMATIC HYDROCARBONS IN CONTAMINATED SOIL Zhe Wang A thesis submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Master of Science in the Department of Environmental Sciences and Engineering. Chapel Hill 2017 Approved by: David R. Singleton Michael D. Aitken Jill R. Stewart © 2017 Zhe Wang ALL RIGHTS RESERVED ii ABSTRACT Zhe Wang: Biodegradation of N-Heterocyclic Aromatic Hydrocarbons in Contaminated Soil (Under the direction of David R. Singleton) While progress has been made understanding the biodegradation of polycyclic aromatic hydrocarbons (PAHs) in contaminated soil, the fate of co-existing contaminants, such as nitrogen-containing heterocyclic aromatic hydrocarbons (azaarenes) is rarely reported. Compared to their PAH analogues, azaarenes have higher toxicity and mobility in the environment, which might pose significant risk to public health. In this research, the fate of three azaarenes (carbazole, acridine, and phenanthridine) in contaminated soil was investigated. Carbazole was degraded in both untreated soil and soil treated in a lab-scale aerobic bioreactor. For phenanthridine, co-metabolism with phenanthrene was a major mechanism for its removal from untreated soil. Both acridine and phenanthridine were not significantly removed in untreated soil when these compounds were examined individually, but were removed in the bioreactor. Overall, this study of the fate and biodegradation of three azaarenes in contaminated soil provided insights and potential for future research on bioremediation of azaarenes- contaminated sites. iii ACKNOWLEDGMENTS I am extremely fortunate to be able to complete thesis under the support and guidance of Dr. David Singleton. He really set a model of scholarly research for me and the impacts from these valuable advice on me would beyond the boundary of science research. Surely, if it had not been for his valuable advice and stoic patience, nothing of this project can be achieved. In addition, I would like to express my gratitude to Dr. Michael Aitken who always supports me and provides me insightful advice in many aspects. Special thanks to Dr. Joaquim Vila Grajales for the sincere advice and guidance on my lab work. I am also grateful to Dr. Jill Stewart for serving in my committee and providing useful support on my project. Finally, my deepest thanks would also go to my beloved family, other group members, my friends, and professors of the ESE department for supporting me and helping me out of any difficulties. iv TABLE OF CONTENTS LIST OF TABLES .................................................................................................................... vii LIST OF FIGURES ................................................................................................................. viii CHAPTER 1: INTRODUCTION ............................................................................................... 1 CHAPTER 2: LITERATURE REVIEW .................................................................................... 2 A. Soil Contamination and Sites of Manufactured Gas Plants ............................................. 2 B. Polycyclic Aromatic Hydrocarbons ................................................................................. 3 1. Introduction of Polycyclic Aromatic Hydrocarbons .................................................... 3 2. Biodegradation of Phenanthrene and Anthracene ....................................................... 9 C. N-Heterocyclic Aromatic Hydrocarbons ....................................................................... 12 1. Carbazole.................................................................................................................... 13 a. Introduction of Carbazole ....................................................................................... 13 b. Carbazole-Degrading Microorganisms ................................................................... 14 c. Metabolic Pathways of Carbazole Degradation ...................................................... 21 2. Acridine ...................................................................................................................... 26 a. Introduction of Acridine ......................................................................................... 26 b. Acridine-Degrading Microorganisms ..................................................................... 27 c. Metabolic Pathways of Acridine Degradation ........................................................ 29 3. Phenanthridine ........................................................................................................... 31 a. Introduction of Phenanthridine ............................................................................... 31 b. Phenanthridine-Degrading Microorganisms ........................................................... 32 c. Metabolic Pathways of Phenanthridine Degradation .............................................. 33 v CHAPTER 3: METHODS ........................................................................................................ 36 A. Chemicals ....................................................................................................................... 36 B. Soil Preparation .............................................................................................................. 36 C. Quantification of Azaarenes ........................................................................................... 37 D. Isolation of Putative Azaarene-Degrading Bacteria ....................................................... 38 E. Growth Tests .................................................................................................................. 41 F. Tests of Azaarene Concentration on Soil Microbial Activity ........................................ 43 G. Chemical Analysis of PAH and Azaarene Disappearance in Soils ................................ 44 H. Molecular Analysis ........................................................................................................ 45 I. Pure Culture Tests for Co-metabolism of Azaarene ...................................................... 46 CHAPTER 4: RESULTS .......................................................................................................... 48 A. HPLC Chromatogram for Identification of Three Azaarenes ........................................ 48 B. Effects of Azaarene Concentration on Soil Microbial Activity ..................................... 49 C. Co-Incubation of Azaarenes with Each Other and With PAHs ..................................... 53 D. Transformation of Azaarenes by Isolates ....................................................................... 57 E. Degradation Test of Azaarenes by Isolates .................................................................... 60 F. Community Analyses ..................................................................................................... 62 G. Tests of Pure Cultures for Co-metabolism of Phenanthridine ....................................... 67 1. Acidovorax sp. NA3 ................................................................................................... 67 2. Immundisolibacter cernigliae TR3.2T ........................................................................ 68 3. Sphingobium sp. PAP1 ............................................................................................... 69 CHAPTER 5: DISCUSSION .................................................................................................... 70 APPENDIX ............................................................................................................................... 80 REFERENCES .......................................................................................................................... 81 vi LIST OF TABLES Table 1. List of carbazole-degrading bacteria and fungi. ............................................................. 16 Table 2. Closest described relatives to 44 isolates based on partial 16S rRNA genes and the azaarene-transforming ability of those isolates. ......................................... 59 Table A1. Re-growth of select strains on a variety of (mostly) aromatic carbon sources in the 96-well plate assay. .................................................................................... 80 vii LIST OF FIGURES Figure 1. Numbered structures and nomenclatures of the 16 PAHs on the EPA priority pollutant list. .......................................................................................................... 5 Figure 2. Bay- and fjord-region of Dibenzo[a,l]pyrene. ................................................................. 7 Figure 3. Numbered structure of 3 azaarenes: carbazole, acridine and phenanthridine. .............. 12 Figure 4. A proposed metabolic pathway for the degradation of carbazole through Pseudomonas spp. Strain CA06, CA10 and Pseudomonas stutzeri OM1 ........................ 22 Figure 5. Other proposed metabolic pathways for the degradation of Carbazole. ....................... 25 Figure 6. Proposed metabolic pathways for the degradation of acridine. ..................................... 30 Figure 7. Proposed metabolic pathways for the degradation of phenanthridine. .......................... 35 Figure 8. List of carbon sources and distribution in 96-well plates. ............................................. 43 Figure 9. HPLC chromatograms