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TAXONOMIC AND FUNCTIONAL CHARACTERIZATION OF MICROBIAL COMMUNITIES LINKED TO CHLORINATED SOLVENT, 1,4-DIOXANE AND RDX BIODEGRADATION IN GROUNDWATER AND SOIL MICROCOSMS By Hongyu Dang A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of Environmental Engineering—Doctor of Philosophy 2021 ABSTRACT TAXONOMIC AND FUNCTIONAL CHARACTERIZATION OF MICROBIAL COMMUNITIES LINKED TO CHLORINATED SOLVENT, 1,4-DIOXANE AND RDX BIODEGRADATION IN GROUNDWATER AND SOIL MICROCOSMS By Hongyu Dang Bioremediation is becoming increasing popular for the remediation of sites contaminated with a range of different contaminants. Molecular methods such as 16S rRNA gene amplicon sequencing, shotgun sequencing, and high throughput quantitative PCR offer much potential for examining the microorganisms and functional genes associated with contaminant biodegradation, which can provide critical additional lines of evidence for effective site remediation. In this work, the first project examined the taxonomic and functional biomarkers associated with chlorinated solvent and 1,4-dioxane biodegradation in groundwater from five contaminated sites. Each site had previously been bioaugmented with the commercially available dechlorinating mixed culture, SDC-9. The results highlighted the occurrence of numerous genera previously linked to chlorinated solvent biodegradation. The functional gene analysis indicated two reductive dehalogenase genes (vcrA and tceA) from Dehalococcoides mccartyi were abundant. Additionally, aerobic and anaerobic biomarkers for the biodegradation of various chlorinated compounds were observed across all sites. The approach used (shotgun sequencing) is advantageous over many other methods because an unlimited number of functional genes can be examined and a more complete picture of the functional abilities of microbial communities can be depicted. Another research project evaluated the functional genes and species associated with RDX biodegradation at a RDX contaminated Navy site where biostimulation had been adopted. For this, DNA samples extracted from groundwater samples pre- and post- biostimulation were subject to shotgun sequencing and high throughput qPCR. DNA sequences from thirty-one RDX biodegraders were detected, with the most abundant species being Variovorax sp. JS1663. Further, nine RDX biodegrading species significantly (p<0.05) increased in abundance following biostimulation. Both the sequencing data and qPCR indicated xenA and xenB exhibited the highest relative abundance among the six functional genes examined. Four genes, diaA, nsfI, xenA and pnrB, exhibited higher relative abundance values in some wells following biostimulation. The study provides a comprehensive approach for assessing biomarkers during RDX bioremediation and provides evidence that biostimulation generated a positive impact on a set of key species and genes. A third study examined laboratory microcosms to determine the phylotypes and functional genes associated with the biodegradation of cis-dichloroethene (cDCE) and 1,4- dioxane. The impact of amending with lactate on cDCE and 1,4-dioxane biodegradation was also investigated. Stable isotope probing (SIP) was then used to determine which phylotypes were actively involved in biodegradation. The most enriched phylotypes for 13C assimilation from 1,4- dioxane included Rhodopseudomonas and Rhodanobacter. The dominant enriched phylotypes for 13C assimilation from cDCE included Bacteriovorax, Pseudomonas and Sphingomonas. The functional genes associated with the degradation of these contaminants was predicted using PICRUSt2. The results suggest aerobic concurrent biodegradation of cDCE and 1,4-dioxane should be considered for chlorinated solvent site remediation. Overall, the data generated and approaches utilized in all three projects have the potential to be incorporated into diagnostic molecular tools for assessing biodegradation potential and for evaluating bioremediation performance at contaminated sites. Copyright by HONGYU DANG 2021 To my Mom and Dad, for encouraging me to go on every adventure, nurturing me to cherish the value of knowledge and everything else. v ACKNOWLEDGEMENTS I would like to express my deepest appreciation to Dr. Alison Cupples, who continually supported and guided me in the past four years. Without her help and exceptional scientific guidance, the work in this thesis would not have been possible. I would like to thank my committee members, Dr. Syed Hashsham, Dr. Irene Xagoraraki and Dr. Brian Teppen, for their valuable research advice. I would like to thank the current and past members in our laboratory and Dr. Hashsham’s laboratory, particularly, Yogendra Kanitkar, Robert Stedtfeld and Maggie Williams, for their willingness to teach me many methods during the early stages of my doctoral program. I am also grateful to the research efforts of Jenny Collier and Vidhya Ramalingam, as their work built foundations for my research. Also, thanks Dr. Benli Chai for introducing me to bioinformatic tools, which had lasting effects across this thesis. In addition, thanks to my friends, Jianzhou He and Xun Qian for enhancing my life and providing constructive advice in my research during my time at MSU. Lastly, I would like to thank my family for their endless love and support. vi TABLE OF CONTENTS LIST OF TABLES ....................................................................................................................................... ix LIST OF FIGURES ...................................................................................................................................... x KEY TO ABBREVIATIONS .................................................................................................................... xvi CHAPTER 1 INTRODUCTION ............................................................................................................... 1 1. Chlorinated Solvent, RDX and 1,4-Dioxane Contamination ............................................................... 1 2. Biodegradation of Chlorinated Solvents, 1,4-Dioxane and RDX ......................................................... 2 3. High Throughput Sequencing for Monitoring Biodegradation ............................................................ 4 4. Dissertation Outline and Objectives ..................................................................................................... 5 REFERENCES ............................................................................................................................................. 7 CHAPTER 2 ABUNDANCE OF CHLORINATED SOLVENT AND 1,4-DIOXANE DEGRADING MICROORGANISMS AT FIVE CHLORINATED SOLVENT CONTAMINATED SITES DETERMINED VIA SHOTGUN SEQUENCING .................................................................................... 13 Abstract ................................................................................................................................................... 13 1. Introduction ........................................................................................................................................ 14 2. Methods .............................................................................................................................................. 16 2.1 DNA Extraction from Groundwater and SDC-9 .......................................................................... 16 2.2 Sequencing and Taxonomic Analysis .......................................................................................... 16 2.3 Reference Sequences Collection, Functional Gene Analysis, qPCR ........................................... 17 3. Results ................................................................................................................................................ 17 3.1 Sequencing and Taxonomic Analysis .......................................................................................... 17 3.2 Occurrence of Chlorinated Solvent Degrading Microorganisms in SDC-9 and In Situ............... 18 3.3 Functional Gene Analysis ............................................................................................................ 21 4. Discussion ........................................................................................................................................... 30 Acknowledgements ................................................................................................................................ 35 APPENDIX ................................................................................................................................................. 36 REFERENCES ........................................................................................................................................... 84 CHAPTER 3 DIVERSITY AND ABUNDANCE OF THE FUNCTIONAL GENES AND BACTERIA ASSOCIATED WITH RDX DEGRADATION AT A CONTAMINATED SITE PRE- AND POST- BIOSTIMULATION .................................................................................................................................. 94 Abstract ................................................................................................................................................... 94 1. Introduction ........................................................................................................................................ 95 2. Materials and Methods ......................................................................................................................
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