Reductive Dechlorination of Polychlorinated Dibenzo-P-Dioxins
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REDUCTIVE DECHLORINATION OF POLYCHLORINATED DIBENZO-P-DIOXINS BY DEHALOCOCCOIDES-ENRICHED CULTURES FROM CONTAMINATED SOILS AND SEDIMENTS By HANG THUY DAM A dissertation submitted to the Graduate School-New Brunswick Rutgers, The State University of New Jersey In partial fulfillment of the requirements For the degree of Doctor of Philosophy Graduate Program in Microbial Biology Written under the direction of Max M. Häggblom And approved by _________________________________ _________________________________ _________________________________ _________________________________ New Brunswick, New Jersey October, 2016 ABTRACT OF THE DISSERTATION Reductive Dechlorination of Polychlorinated Dibenzo-p-dioxins by Dehalococcoides-enriched Cultures from Contaminated Soils and Sediments By HANG THUY DAM Dissertation Director Max M. Häggblom Polychlorinated dibenzo-p-dioxins (PCDDs) are pervasive environmental pollutants and resistant in soil and sediment environments. Under anaerobic conditions, PCDDs can undergo reductive dechlorination which produces less chlorinated dibenzo-p-dioxin congeners, these compounds are more susceptible to further degradation. The process is mediated by dehalogenating bacteria, some of which gain energy from the process by using PCDDs as terminal electron acceptors. The objective of our study was to use a conventional enrichment approach to enrich for dehalogenating bacteria, to investigate the PCDD dechlorination activity and the microbial community of enrichment cultures, and to investigate genomes of the abundant dehalogenating bacteria in enrichment cultures from metagenomics data of the corresponding enrichment cultures. PCDD dechlorination was observed in five sets of enrichment cultures established from Hackensack River sediments collected along the tidally influenced river, in which dechlorination activity was negatively affected by high salinity and high sulfate concentrations. Dechlorination activity was also recorded in enrichment cultures established from freshwater sediments of the Kymijoki River (Findland), a water reservoir (Vietnam), as well as from rice paddy soil (Vietnam). Reductive dechlorination of PCDDs was mediated by organohalide-respiring bacteria. PCDD dechlorinating anaerobic bacteria appeared to be ubiquitous, and their activity was observed in ii almost all enrichment cultures established using soils and sediments collected from sites of different contamination backgrounds. Dehalococcoides spp. were identified as the main bacterial species that couple dechlorination of PCDDs to growth. Genomes of two Dehalococcoides spp. were reconstructed from metagenomes of the Hackensack and the Kymijoki River enrichment cultures in which they were present at relatively high abundance. Comparative genome analysis suggested that a reductive dehalogenase cbrA ortholog is responsible for reductive dechlorination of 1,2,3,4-Tetrachlorodibenzo-p-dioxin in a respiratory process. Genome analysis provided preliminary evidence for the function of reductive dehalogenases in PCDD dechlorination which have not been studied before due to the complex nutritional requirements and slow growth of Dehalococcoides spp. iii Acknowledgement First of all, I would like to express my deepest appreciation to my advisor Dr. Max Häggblom for his endless guidance, encouragement, and support throughout my study at Rutgers. I would also like to thank my committee members, Drs. Lee Kerkhof, Donna Fennell, and Lisa Rodenburg, for their insightful suggestions and comments on the draft of this dissertation. I am grateful to all the people without whom my dissertation project would have been impossible. Thank you to Drs. Hui Liu, Vien Duong, and Joong-Wook Park for the initial work they had done on dechlorination of chlorinated dibenzo-p-dioxins, which was the basis of my study. I thank Lora McGuinness for helping me with molecular work I conducted in the Kerkhof lab. She taught me many tricks to work with “stubborn” sediments and anaerobic cultures. I would also like to thank our collaborator at the Leibniz Instutute – DSMZ: Drs Anne-Kristin Kaster and John Vollmers for their generous contribution in sequencing and analyzing metagenomes of the Hackensack and Kymijoki River enrichment cultures. I would like to thank Drs. Ines Rauschenbach and Diane Davis for taking me in as a teaching assistant of the Microbiology Laboratory, especially Dr. Rauschenbach, for being a great mentor. I’ve learned a lot from her to be a better teacher. I would also like to express my appreciation to the administrative staff of the Department of Biochemistry and Microbiology. Over the past six years, I’m fortunate and proud to be a Bloomer. Thank you to all the current and past members of the Bloomer Lab, Tiffany, Seoyean, Tong, Jie, Preshita, Weimin, Isabel, Michelle, Aamani, … for all their help and friendship. Thanks to my friends I’ve met at Rutgers, Minh and Tuan for helping me when I first arrived in the US, and my other friends for keeping me company during my long journey here. Thanks to my brother and his family for cheering me up over Skype almost every traditional Vietnamese holidays when I could not be part of. iv I would like to thank the Vietnam International Education Development (Ministry and Education and Training, Vietnam) for providing me with a 2-year fellowship, and the Department of Biochemistry and Microbiology, Rutgers University, for providing support through the teaching assistantship for the last four years. This research was partly supported by New Jersey Water Resource Research Institute, TA/GA Professional Development Fund, and the New Jersey Agricultural Experiment Station. Finally, thank you, Mom and Dad, for your unconditional love and support for me to pursue my PhD study on the other side of the globe. This dissertation is dedicated to you. v Table of Contents ABTRACT OF THE DISSERTATION ............................................................................................ ii Acknowledgement ................................................................................................................ iv List of Tables ....................................................................................................................... viii List of Figures ........................................................................................................................ ix Chapter 1 Introduction .......................................................................................................................... 1 Abstract ........................................................................................................................................ 1 Chemistry and toxicology of PCDDs ............................................................................................. 2 PCDDs in the environments ......................................................................................................... 4 Microbially mediated transformation of PCDDs .......................................................................... 9 Reductive dechlorination as an electron accepting process ..................................................... 10 Reductive dehalogenase homologous genes (rdhAs) ................................................................ 12 Hypotheses ................................................................................................................................ 16 Overall Goals and Objectives ..................................................................................................... 16 Chapter 2 Impact of Estuarine Gradients on Reductive Dechlorination of 1,2,3,4-Tetrachlorodibenzo-p- dioxin in River Sediment Enrichment Cultures ....................................................................... 32 Abstract ...................................................................................................................................... 32 Introduction ............................................................................................................................... 33 Materials and Methods .............................................................................................................. 35 Results ........................................................................................................................................ 40 Discussion .................................................................................................................................. 45 Conclusions ................................................................................................................................ 48 Chapter 3 Identification of a Chlorodibenzo-p-dioxin Dechlorinating Dehalococcoides spp. by Stable Isotope Probing and Pyrosequencing of 16S rRNA ................................................................. 62 Abstract ...................................................................................................................................... 62 Introduction ............................................................................................................................... 63 Materials and methods .............................................................................................................. 65 Results ........................................................................................................................................ 69 Discussion .................................................................................................................................