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Investigation of a Sulfur-Utilizing Perchlorate-Reducing Bacterial Consortium University of Massachusetts Amherst ScholarWorks@UMass Amherst Open Access Dissertations 5-13-2011 Investigation of a Sulfur-Utilizing Perchlorate-Reducing Bacterial Consortium Teresa Anne Conneely University of Massachusetts Amherst Follow this and additional works at: https://scholarworks.umass.edu/open_access_dissertations Part of the Bacteriology Commons Recommended Citation Conneely, Teresa Anne, "Investigation of a Sulfur-Utilizing Perchlorate-Reducing Bacterial Consortium" (2011). Open Access Dissertations. 372. https://scholarworks.umass.edu/open_access_dissertations/372 This Open Access Dissertation is brought to you for free and open access by ScholarWorks@UMass Amherst. It has been accepted for inclusion in Open Access Dissertations by an authorized administrator of ScholarWorks@UMass Amherst. For more information, please contact [email protected]. INVESTIGATION OF A SULFUR-UTILIZING PERCHLORATE-REDUCING BACTERIAL CONSORTIUM A Dissertation Presented by TERESA ANNE CONNEELY Submitted to the Graduate School of the University of Massachusetts Amherst in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY May 2011 Department of Microbiology © Copyright by Teresa Anne Conneely 2011 All Rights Reserved INVESTIGATION OF A SULFUR-UTILIZING PERCHLORATE-REDUCING BACTERIAL CONSORTIUM A Dissertation Presented by TERESA ANNE CONNEELY Approved as to style and content by: ____________________________________ Klaus Nüsslein, Chair ____________________________________ Jeffery Blanchard, Member ____________________________________ James F. Holden, Member ____________________________________ Sarina Ergas, Member __________________________________________ John Lopes, Department Head Department of Microbiology DEDICATION Lé grá dó mo chlann ACKNOWLEDGMENTS I would like to thank my advisor Klaus Nüsslein for inviting me to join his lab, introducing me to microbial ecology and guiding me on my research path and goal of providing clean water through microbiology. Thank you to all my committee members throughout my Ph.D. program: Jim Holden, Jeff Blanchard, Sue Leschine, and Pat Schloss, from the Department of Microbiology, for adding their microbiology expertise to my projects, and to Sarina Ergas, from the Department of Civil and Environmental Engineering, for introducing me to environmental engineering. Thank you to the members of the Department of Microbiology for varied help, support, and friendship throughout my Ph.D. program. In particular, thank you to all the members of the Nüsslein lab that I had the pleasure of knowing. Special thanks to Caryl Becerra for being a good friend and colleague. Thank you also to Professors Steve Petsch, Dave Reckhow, and Chul Park for use of equipment and helpful discussions. Thank you to the environmental engineers, in particular Ashish Sahu, for their help integrating engineering and microbiology. Thank you to NSF, EPA, AWWARF, and the Department of Microbiology for funding. v ABSTRACT INVESTIGATION OF A SULFUR-UTILIZING PERCHLORATE-REDUCING BACTERIAL CONSORTIUM MAY 2011 TERESA ANNE CONNEELY, B.A., SUFFOLK UNIVERSITY Ph.D., UNIVERSITY OF MASSACHUSETTS AMHERST Directed by: Professor Klaus Nüsslein We present research investigating how, with in depth knowledge of the community, microbial communities may be harnessed for bioremediation of hazardous water contaminants. We focused on the bacterial reduction of perchlorate, a common water contaminant. For this we studied the structure and capabilities of a novel sulfur- utilizing, perchlorate-reducing bacterial (SUPeRB) consortium. Initially, we characterized the minimal consortium that retained functional capabilities, using 16S rRNA and functional gene analysis. A diverse functional consortium dominated by Beta-Proteobacteria of the family Rhodocyclaceae and sulfur-oxidizing Epsilon- Proteobacteria was found. We also examined the optimal growth conditions under which perchlorate degradation occurred and uncovered the upper limits of this function. Bacterial isolates were screened for function and the presence of functional genes. We expanded to bioreactor studies at bench- and pilot-scale, and first used a perchlorate-reducing, bench-scale bioreactor to probe the stability of the microbial ecosystem. During stable reactor function, a core consortium of Beta- and Epsilon- Proteobacteria reduced perchlorate and the co-contaminant nitrate. A disturbance of the vi consortium led to a failure in function and to higher system diversity. This suggests that the SUPeRB consortium was not metabolically flexible and high population diversity was necessary for a return to stable function. In a pilot-scale bioreactor we determined that the SUPeRB consortium could stably degrade low levels of perchlorate to below the EPA maximum recommended limit. Field conditions, such as temperature extremes and intermittent perchlorate feed, did not negatively impact overall function. When all reactor consortia were compared we observed that the volume of the reactor and the initial inoculum were not as important to stable reactor function as the acclimatization of the consortium to the system and maintenance of favorable conditions within the reactor. In summary we found that the SUPeRB consortium successfully degraded perchlorate in multiple systems. The study of this novel consortium expands our knowledge of the metabolic capabilities of perchlorate-reducing bacteria and suggests potential evolutionary pathways for perchlorate-reduction by microorganisms. The SUPeRB consortium may be used to establish bioremediation systems for perchlorate and other environmental contaminants. vii TABLE OF CONTENTS ACKNOWLEDGMENTS ................................................................................................v ABSTRACT.....................................................................................................................vi LIST OF TABLES............................................................................................................x LIST OF FIGURES .........................................................................................................xi CHAPTER 1. INTRODUCTION ........................................................................................................1 Perchlorate as an Environmental Contaminant.............................................................1 Perchlorate Bioremediation ..........................................................................................2 Perchlorate-Reducing Bacteria (PRB) ..........................................................................3 Sulfur as an Electron Donor..........................................................................................5 Perchlorate Remediation using Bioreactors..................................................................6 Microbial Ecology within Bioreactors..........................................................................7 Hypotheses and Research Objectives ...........................................................................8 Significance ................................................................................................................10 References...................................................................................................................12 2. CHARACTERIZATION OF THE MICROBIAL CONSORTIUM COUPLING PERCHLORATE-REDUCTION TO SULFUR-UTILIZATION ..................................25 Abstract.......................................................................................................................25 Introduction.................................................................................................................25 Materials and Methods................................................................................................27 Results.........................................................................................................................34 Discussion...................................................................................................................39 References...................................................................................................................50 3. PHYLOGENETIC STRUCTURE AND FUNCTIONAL RELATIONSHIPS IN A BENCH-SCALE BIOREACTOR ..................................................................................64 Abstract.......................................................................................................................64 Introduction.................................................................................................................65 Materials and Methods................................................................................................67 Results.........................................................................................................................73 Discussion...................................................................................................................77 References...................................................................................................................87 4. MICROBIOLOGICAL INVESTIGATION OF THE SUPeRB CONSORTIUM FROM THE PILOT-SCALE BIOREACTOR .............................................................104 viii Abstract.....................................................................................................................104 Introduction...............................................................................................................104
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