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A Role for Dehalobacter Spp

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A Role for Dehalobacter Spp REDUCTIVE DEHALOGENATION OF CHLORINATED BENZENES: A ROLE FOR DEHALOBACTER SPP. A Dissertation Presented to the Faculty of the Graduate School of Cornell University in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy by Jennifer Lynn Nelson August 2012 REDUCTIVE DEHALOGENATION OF CHLORINATED BENZENES: A ROLE FOR DEHALOBACTER SPP. Jennifer Nelson Cornell University 2012 Chlorinated benzenes have been extensively used in industry and agriculture and are common groundwater pollutants. Chlorobenzenes readily migrate to anaerobic zones in sediment and groundwater, thus understanding the fate of these compounds and anaerobic remediation are important goals. In previous studies in this laboratory, sediment microcosms were developed that reductively dehalogenated dichlorobenzene (DCB) isomers to monochlorobenzene (MCB) and MCB to benzene at high rates. Enrichment cultures that dehalogenated either 1,2-DCB, 1,3-DCB, or 1,4-DCB to MCB were derived from these microcosms, and Dehalobacter spp. were identified in an enrichment using 1,2-DCB. In this study, quantitative PCR (qPCR) of 16S rRNA genes indicated Dehalobacter spp. were responsible for dehalogenation in all DCB cultures, and 16S rRNA clone library analysis indicated 1,2- and 1,4-DCB dehalogenating Dehalobacter spp. were closely related while the Dehalobacter sp. in 1,3-DCB cultures was more divergent. In the course of purification of these cultures, methanogens and acetogens were eliminated, and culture conditions for growth of the dehalogenators were optimized. Extracts from a Sedimentibacter sp. isolated from a 1,2-DCB enrichment culture were added to 10-8 dilution cultures, allowing the isolation of 1,2-DCB and 1,3-DCB dehalogenating Dehalobacter sp. strains 12DCB1 and 13DCB1, although the extracts were not required for growth in subsequent transfers. Dehalobacter sp. strain 14DCB1 grew more slowly and was characterized in a highly enriched 1,4-DCB dehalogenating culture. Dehalogenation capabilities of each strain were tested with all chlorobenzene isomers, dichlorotoluenes, and tetrachloroethene (PCE), and each strain dehalogenated different combinations of these compounds. Strain 12DCB1 dehalogenated predominantly singly flanked chlorines on aromatic compounds and PCE, and did not dehalogenate compounds with only doubly flanked chlorines or those without any flanked chlorines. Strain 13DCB1 had the widest dehalogenation range of compounds tested, utilizing singly flanked chlorines, doubly flanked chlorines, meta substituted unflanked chlorines, and PCE, while strain 14DCB1 had the narrowest substrate range, dehalogenating para substituted chlorines and slowly dehalogenating some singly flanked chlorines. MCB dehalogenation to benzene was investigated in microcosms constructed with sediment from two different contaminated areas within the Chambers Works site. Attempts to transfer dehalogenation activity to sediment-free enrichment culture were not successful, though transfers into medium containing a commercial potting mix were achieved, which reduced the reliance upon limited supplies of sediment for perpetuation of MCB dehalogenation activity. A threshold below which MCB was not dehalogenated was investigated, and results suggested MCB was not utilized at concentrations below ca. 15 µM in microcosms despite the thermodynamic favorability of the reaction. Lastly, qPCR confirmed Dehalobacter spp. played a role in MCB dehalogenation, and different Dehalobacter spp. were detected in 16S rRNA clone libraries from the two sediment types which, together with DCB culture sequences, suggested 16S rRNA gene sequence is not a good predictor of Dehalobacter spp. dehalogenation spectra. These studies have established a role for Dehalobacter spp. in the dehalogenation of chlorobenzenes, and this genus should be considered when determining the fate of diverse halogenated organic compounds. BIOGRAPHICAL SKETCH Jennifer Lynn Nelson was born on March 6th, 1985, in Helena, MT, to Sam and Arlene Nelson. At age nine months, Jenny learned to walk due to an intense desire to follow her big brother wherever he went. Jenny spent countless days with her brother and cousins at their grandparents’ house in the Montana mountains which instilled a love of flowers, animals, and the outdoors. While attending Helena High School, Jenny lettered in academics, choir, and softball, and worked part time as a delivery driver for Papa John’s Pizza. She graduated as valedictorian in 2003 and enrolled at Morningside College in Sioux City, IA, where she received an athletic scholarship to play fastpitch softball. As a college sophomore in 2005, Jenny was the Morningside Mustangs’ starting right fielder and received a Research Experience for Undergraduates at the University of Nebraska-Lincoln in the lab of Dr. Greg Somerville, where she continued doing research each summer through 2007. After graduating from Morningside with a B.S. in biology, Jenny began graduate school in the Department of Microbiology at Cornell University in the fall of 2007. She joined Dr. Stephen Zinder’s laboratory in March of 2008 and enjoyed four years of graduate research. During her time at Cornell, Jenny continued to play softball, survived a bout of thyroid cancer, learned to play ice hockey, and could be found enjoying Zinder Lab Wine Hour with good friends almost every late Friday afternoon. iii ACKNOWLEDGMENTS I would like to thank my advisor, Steve Zinder. In addition to being a fantastic teacher and mentor, Steve’s support and understanding made some very difficult situations easier to handle and his lab was a great place to be a graduate student. Dr. Greg Somerville supervised my formative undergraduate research years at the University of Nebraska-Lincoln and was the first person to suggest I think about attending graduate school. I thank him for his support, advice, and friendship throughout the years. Thanks to the laboratories of Elizabeth Edwards and Barbara Sherwood-Lollar at the University of Toronto, specifically Xiaoming Liang and Shuiquan Tang, for fruitful collaborations; and thanks to the DuPont Corporation for funding the entirety of my graduate research projects. Dr. Jennifer Fung was my research mentor during my Zinder Lab rotation and she handed her project off to me when she graduated. Her patience while teaching me anaerobic techniques, willingness to answer my frequent questions, and great friendship has been invaluable. Xu Cheng visited the lab for only six months in 2009, but he never fails to wish me a happy birthday or to offer unsolicited words of encouragement when I need them the most. Thanks to other Zinder Lab members past and present: Heather Fullerton, Jiandong Jiang, Michael Schwartz, John Mariano, Garrett Graham, Zach Zanini, Harrison Specht, Mytien Nguyen, and Yanting Wang; the second floor Wing Hall girls: Julie Brown, Armanda Roco, and Erin Eggleston; Tony Kingston; Kevin Posman; Greg Smaldone; the Department of Microbiology, and Shirley Cramer, Patti Butler, and Cathy Shappell for keeping Wing Hall running like a well-oiled machine. iv I acknowledge the various sports teams that occasionally distracted me from lab work: Lansing League slowpitch softball; Ithaca Sirens women’s ice hockey; Cornell Engineering Graduate Student Association intramural softball; the Buffalo Sabres; the Washington Nationals; the Chicago White Sox; and the five-time champion Cornell intramural bowling dynasty, E. Bowling Virus. Ryan Seipke was my FoMS-assigned graduate student mentor and took me under his wing the first day I arrived in Ithaca. Adam Lowe, Brian Forster, Jay Worley, and Cresten Mansfeldt could always be counted on to meet up at the Chapter House after a long day. Sarah Perdue, Marie Donnelly, and Julia Crane were the founding members of Girl’s Night and were always up for a laugh, a baseball game, or a romantic comedy. Dr. Matthew Kennedy, steadfast in his belief that eating dinner is always more important than working, has been my emotional support and sounding board for the past five years and is the best boyfriend a girl could ask for. Thanks especially to my parents Sam and Arlene, my grandmother Mary, and my big brother Bob for being my unconditional super-fans. v TABLE OF CONTENTS BIOGRAPHICAL SKETCH ....................................................................................................... iii ACKNOWLEDGMENTS ............................................................................................................ iv CHAPTER ONE: Reductive Dehalogenation of Chlorobenzenes: An Introduction to Dehalobacter and Dehalococcoides spp. ....................................................................................... 1 CHAPTER TWO: A Role for Dehalobacter spp. in the Reductive Dehalogenation of Dichlorobenzenes and Monochlorobenzene .............................................................................. 19 CHAPTER THREE: Characterization of Three Dehalobacter spp. Cultures that Reductively Dehalogenate Different Dichlorobenzene Isomers ...................................................................... 53 CHAPTER FOUR: Dehalogenation of Chlorobenzenes, Chloroethenes, and Chlorotoluenes by Three Dehalobacter spp. ......................................................................................................... 83 CHAPTER FIVE: Diverse Dehalobacter spp. are Involved in Reductive Dehalogenation of Monochlorobenzene to Benzene in Microcosms ....................................................................... 106 CHAPTER SIX: Reductive
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