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MICROBIAL Cr(VI) REDUCTION: ROLE of ELECTRON DONORS, ACCEPTORS, and MECHANISMS, with SPECIAL EMPHASIS on CLOSTRIDIUM Spp

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MICROBIAL Cr(VI) REDUCTION: ROLE of ELECTRON DONORS, ACCEPTORS, and MECHANISMS, with SPECIAL EMPHASIS on CLOSTRIDIUM Spp MICROBIAL Cr(VI) REDUCTION: ROLE OF ELECTRON DONORS, ACCEPTORS, AND MECHANISMS, WITH SPECIAL EMPHASIS ON CLOSTRIDIUM spp. By KANIKA SHARMA A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 2002 Copyright 2002 by Kanika Sharma For my parents, whose support and understanding has helped culminate my dream into a reality. ACKNOWLEDGMENTS I am grateful to my mentor, Dr. Andrew V. Ogram, for excellent supervision during the course of this dissertation. He has truly been a great source of inspiration, insight, and input. The knowledge he has imparted, and the patience he has displayed was vital to completing this study. I am thankful for the immense encouragement and financial support that he graciously provided during this study. I would like to express my sincere gratitude to the committee members, Drs. K. Hatfield, L. O. Ingram, K. R. Reddy, and R. D. Rhue, for each contributing in special and meaningful ways to my personal development and academic success. I also thank Drs. W. Harris, L.T. Ou, and H. Aldrich for all of their advice and help. A special word of thanks is due to Dr. Derek Lovley, and members of his lab at the University of Massachusetts, Amherst, for extending their lab facilities so that I might learn various anaerobic microbial techniques. At this time, I would also like to thank Dr. John Thomas, Bill Reve, and Irene Poyer for all their help with the analytical equipment. I am especially grateful to T. Van Pham and Lisa Stanley who provided me the extra pair of hands when work demanded. I thank Ilker Uz and Hector Castro for being wonderful friends and for being by my side through all happy and tough times. I sincerely thank members of my lab, past and present, Viji Ramakrishnan, Weiwei Chen, Drs. Yong Ping Duan, Milind A. Chavan, and Ashvini Chauhan. Without the advice and friendship of these colleagues, surviving the last 5 years would have been impossible. iv I acknowledge the support of the faculty, staff, and all the graduate students in the Soil and Water Science Department. I owe much of my academic and personal success to my parents and my brother, who, by example, provided me with the motivation and courage to pursue a Ph.D. degree. Special thanks go to my friends, near and far, for their love and support that made my stay at University of Florida so memorable. v TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................................................................................. iv LIST OF TABLES........................................................................................................... viii LIST OF FIGURES ........................................................................................................... ix LIST OF ABBREVIATIONS AND ACRONYMS .......................................................... xi ABSTRACT...................................................................................................................... xii CHAPTER 1 GENERAL INTRODUCTION........................................................................................1 Chromium in Environment ............................................................................................. 1 Nutrition and Toxicity: Risks to Human Health............................................................. 2 Animals .................................................................................................................... 2 Plants and Algae....................................................................................................... 3 Microorganisms ....................................................................................................... 3 Environmental Chemistry ............................................................................................... 5 Reduction of Cr(VI) ................................................................................................. 6 Oxidation of Cr(III) in Soils..................................................................................... 7 Cr(VI) Remediation Strategies ....................................................................................... 7 Chromium Resistance in Bacteria................................................................................... 9 Pathways for Chromium(VI) Reduction....................................................................... 11 Direct Enzymatic Reduction of Cr(VI).................................................................. 11 Bacterially Mediated Indirect Reduction of Cr(VI)............................................... 13 Factors Affecting Microbial Chromium Reduction...................................................... 15 Outline of Dissertation.................................................................................................. 18 2 ENRICHMENT, ISOLATION, AND CHARACTERIZATION OF Cr(VI)- REDUCING BACTERIA .............................................................................................27 Introduction................................................................................................................... 27 Materials and Methods.................................................................................................. 29 Results and Discussion ................................................................................................. 33 Effect of Electron Donors and Acceptors on Cr(VI) Reduction............................ 33 Phylogenetic Analysis of Cr(VI) Reducing Bacteria............................................. 36 vi 3 Cr(VI) REDUCTION BY A CONSORTIUM OF GRAM POSITIVE FERMENTATIVE BACTERIA ...................................................................................42 Introduction................................................................................................................... 42 Materials and Methods.................................................................................................. 43 Results and Discussion ................................................................................................. 45 Composition of Fermentative Consortium GCAF ................................................. 45 Biotic versus Abiotic Reduction of Cr(VI) ............................................................ 45 Kinetics of Cr(VI) Reduction................................................................................. 46 Effect of Electron Donor on Cr(VI) Reduction ..................................................... 47 Effect of Cr(VI) Reduction on Cell Growth in Consortium GCAF....................... 47 Effect of Cr(VI) on Metabolites............................................................................. 47 4 IDENTIFICATION AND CHARACTERIZATION OF THE CHROMIUM REDUCING ISOLATE CLOSTRIDIUM sp. GCAF1 ..................................................58 Introduction................................................................................................................... 58 Material and Methods ................................................................................................... 59 Results and Discussion ................................................................................................. 62 Chemotaxonomic Data........................................................................................... 63 Phylogeny of Strain GCAF-1................................................................................. 63 5 ELECTRON SHUTTLE-MEDIATED CHROMIUM REDUCTION BY CLOSTRIDIUM sp GCAF-1 .........................................................................................79 Introduction................................................................................................................... 79 Materials and Methods.................................................................................................. 80 Results and Discussion ................................................................................................. 82 Effect of Cr(VI) on Production of Metabolic Products of Strain GCAF-1............ 85 Proposed Mechanism of Cr(VI) Reduction by Strain GCAF-1............................. 86 Environmental Relevance of Cr(VI) Reduction by Gram Positive Spore-forming Fermentative Species.............................................................. 87 6 SUMMARY AND CONCLUSIONS ............................................................................97 APPENDIX MEDIA USED FOR ENRICHMENT STUDIES......................................103 LIST OF REFERENCES.................................................................................................104 BIOGRAPHICAL SKETCH ...........................................................................................119 vii LIST OF TABLES Table page 1-1. Cr(VI) reducing bacteria described in literature.........................................................24 2-1. Combination of electron acceptors and donors supplemented in the media for anaerobic enrichment studies.................................................................................38 2-2. Accession numbers for 16S rDNA sequences used in this study...............................39 3-1. Cell growth in the presence of different electron acceptors .......................................56 3-2. Cr(VI) reduction by consortium GCAF in presence of varying concentrations of electron donor ........................................................................................................56
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