Microbial Mercury Methylation and Demethylation
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©2011 Riqing Yu ALL RIGHTS RESERVED MICROBIAL MERCURY METHYLATION AND DEMETHYLATION: BIOGEOCHEMICAL MECHANISMS AND METAGENOMIC PERSPECTIVES IN FRESHWATER ECOSYSTEMS by RIQING YU 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 Environmental Sciences Written under the direction of Professor Tamar Barkay and approved by __________________________________ __________________________________ __________________________________ __________________________________ New Brunswick, New Jersey May 2011 ABSTRACT OF THE DISSERTATION Microbial Mercury Methylation and Demethylation: Biogeochemical Mechanisms and Metagenomic Perspectives in Freshwater Ecosystems by RIQING YU Dissertation Director: Professor Tamar Barkay Elevated concentration of methylmercury (MeHg) in fish is a worldwide concern due to its detrimental effects on human health. Although Hg methylation is a key issue regarding MeHg contamination, neither abiotic nor microbial methylation mechanisms are well understood. The overall objective of this study was to link the potential for microbial methylation and demethylation to the molecular characterization of microbial communities in two typical freshwater ecosystems and to gain in-depth understanding of Hg methylation mechanisms by syntrophy. Sunday Lake is a remote and “pristine” forest lake exposed to Hg mostly through atmospheric deposition in the Adirondack Mountains, New York. This study demonstrated that floating Sphagnum moss mats near the lake water front were hot spots for MeHg accumulation and microbial methylation, and sub-habitats where sulfate reducing bacteria (SRB) community was highly developed. SRB were identified as a major group of Hg methylators, as sulfate addition to the mat samples doubled the potential Hg methylation rates while molybdate significantly inhibited them. The dominant distribution of Syntrophobacter spp. in the Sphagnum mats led to the ii investigation of syntrophy in Hg methylation. By incubating mono- or co-cultures of Syntrophobacter spp., with Desulfovibrio spp., this study was the first to demonstrate that a Syntrophobacter-Desulfovibrio coculture significantly increased growth of both syntrophic partners and stimulated MeHg synthesis compared to activities of Desulfovibro spp. monocultures. Syntrophy could stimulate MeHg synthesis by two pathways: Desulfovibrio growing with methanogens in sulfate-free environments, and Desulfovibrio growing with Syntrophobacter in sulfate-limited environments where sources of energy and carbon are limited. In the South River, an industrially Hg-contaminated site in Virginia, high Hg methylation rates and low demethylation activities were observed in nine sites downstream from the contaminating source, partially explaining why fish in this river have high MeHg levels. 16S rRNA sequencing from sediment cDNA showed that at least three groups of SRB and one group of Geobacter-like iron reducing bacteria (IRB) that were closely affiliated to known Hg methylators, were active in the sediments. Further metabolic inhibition and stimulation experiments confirmed that both SRB and IRB were involved in the microbial methylation in South River sediments. iii DEDICATION To my father, in memory of his anticipation. iv ACKNOWLEDGEMENTS I wish to express my sincere gratitude to my dissertation advisor, Dr. Tamar Barkay, for her constructive suggestions, patient guidance, and continuous encouragement and motivation through the entire course of study. Special thanks are also given to Dr. John Reinfelder and Dr. Max M. Häggblom, members of my advisory committee for their inspiration and valuable suggestions in the planning of this study, and important comments on my studies. I am grateful to all the help from Dr. Mark E. Hines, my outside advisory committee member. Without his guide, I could not complete my Hg methylation and demethylation experiments. Thanks to the full supports and illuminating discussions from my labmates and colleagues, Jeffra Schaefer, Jonna Coombs, Yanping Wang, Chu-ching Lin, Heather Wiatrowski, Kritee, Sharron Crane, Zachary Freedman, Melitza Crespo-Medina, Rachel Ward, Aspa Chatziefthimiou and Kim Cruz. Thanks to all my friends, especially to Ruyan Han, Gavin Swiatek, Hui Liu, and Yun Li for their always supporting and help. I am indebted to my mother and my wife. Their support and encouragement have motivated and helped me to complete this study. I would also like to appreciate the financial aid from the National Science Foundation Biocomplexity Grant, the E. I. Du Pont DE Nemours & Co., New Jersey Water Resources Research Institute, and the Department of Biochemistry and Microbiology during the course of my dissertation study. v TABLE OF CONTENTS Title Page Title Page Abstract of the Dissertation ii Dedication iv Acknowledgements v Table of Contents vi List of Tables ix List of Figures x Chapter 1 − General Introduction 1 1.1. Mercury Chemistry – Overview 1 1.2. Biogeochemical Cycling of Mercury 1 1.3. Mercury Methylation and Methylating Microorganisms 4 1.4. Microbial Demethylation of Methylmercury 8 1.5. Purpose of Study 9 References 14 Chapter 2 − Mercury Methylation in Sphagnum Moss Mats and Its 19 Association with Sulfate-Reducing Bacteria in an Acidic Adirondack Forest Lake Wetland Abstract 19 Introduction 21 Materials and Methods 23 Results 31 Disscussion 38 vi References 46 Chapter 3 − Presence of Syntrophobacter spp. in Hg Methylating Sphagnum 69 Moss Mats and Two Proposed Pathways for the Stimulation of Methylation by Syntrophic Interactions Abstract 69 Introduction 71 Materials and Methods 73 Results 82 Disscussion 93 References 106 Chapter 4 − Potentials of Microbial Methylmercury Production and 131 Degradation and Molecular Characterization of Active Microbial Communities in Sediments from the South River, VA Abstract 131 Introduction 133 Materials and Methods 135 Results 144 Disscussion 155 References 165 Chapter 5 − Conclusion 186 References 191 Appendix A 192 vii Fig.A-1. Sampling sites in Sunday Lake, Adirondack Mountains, New 192 York. Fig.A-2. Phylogenetic tree of Syntrophobacteraceae-like clone 16S rRNA 193 genes from the MAT enrichment with propionate-sulfate medium Appendix B 194 Fig. B-1. mcrA genes in the native samples of Sunday Lake 194 Appendix C 195 Table C-1. Sequences similarity of 16S rRNA genes from the clone 195 library of RRD 10.0, the South River in May 2008 Table C-2. Sequences similarity of 16S rRNA genes from the clone 198 library of RRD 14.0, the South River in May 2008 Table C-3. Sequences similarity of 16S rRNA genes from the clone 201 library of RRD 10.0, the South River in August 2008 Table C-4. Sequences similarity of 16S rRNA genes from the clone 204 library of RRD 20.6, the South River in Aug. 2008 viii LIST OF TABLES Table Title Page 2.1 Physical and chemical characteristics of study sites in Sunday Lake 56 2.2 Distributions of SRB groups and effects of sulfate in Sunday Lake 58 2.3 MPN estimates of SRB and PO-SRB in the MAT in Sunday Lake 59 S2.1 Primers and PCR conditions that were used in Chapter 2 66 3.1 Methylation activities by mono- & co-cultures of syntrophy 111 S3.1 Coculture medium for M. hungatei with S. wolinii or S. 119 Sulfatireducens, and for S. sulfatireducens with D. desulfuricans ND132 or D. africanus S3.2 Coculture medium for S. fumaroxidans with M. hungatei 121 S3.3 Coculture medium for M. hungatei with D. desulfuricans ND132 or 122 D. africanus S3.4 Sequence similarity of selected excised DGGE fragments of 16S 123 rRNA genes amplified from the MAT sample before and after sulfate enrichment by using SRB group-specific primer sets S3.5 Sequence similarity of 16S rRNA genes obtained with 125 Syntrophobacter-specific primers from DNA extract of MAT SRB- MPN incubations and Cheesequake mat 4.1 Physical and chemical characteristics of sediments from the South 171 River, VA in 2008 and 2010 S4.1 Sequences similarity of 16S rRNA genes from the clones with 184 affiliation with the Deltaproteobacteria in the South River sediments ix LIST OF FIGURES Figure Title Page 1.1 The biogeochemical cycling of mercury in the environment 12 1.2 Model of a typical Gram-negative mercury resistance (mer) operon 13 2.1 Potential methylation rates of Sunday Lake samples and the impact of 60 various treatments on these rates 2.2 dsrAB genes in the native and sulfate-enriched samples, Sunday Lake 61 2.3 Phylogenetic analysis of dsrB genes in the native MAT, Sunday Lake 62 2.4 DGGE of bacterial dsrB in MPN cultures from MAT samples 64 2.5 Phylogenetic analysis of dsrB-based DGGE fragments of SRB and 65 syntrophic propionate-oxidizing SRB in the MAT enrichments S2.1 PCR amplification products indicating presence of 16S rRNA genes 68 homologous to those of SRB5 in Sunday Lake samples 3.1 Phylogenetic tree of 16S rRNA genes obtained by DGGE of PCR 112 products using four group-specific SRB primer sets from DNA Extracts of floating Sphagnum moss mats in Sunday Lake 3.2 Phylogenetic tree of 16S rRNA gene sequences obtained using PCR 113 with Syntrophobacteraceae-specific primers from DNA extracts of SRB medium enrichments with Sphagnum mat samples, Sunday Lake 3.3 Synthesis of CH3Hg and change in protein contents of monocultures 114 of S. wolinii, S. sulfatireducens, S. fumaroxidans, M. hungatei, and D. Africanus 3.4 Synthesis of CH3Hg and change in protein contents of mono- and 115 cocultures of M. hungatei with three Syntrophobacter spp. x in sulfate-free propionate media 3.5 Synthesis of CH3Hg and change in protein contents of mono- and 116 cocultures of M. hungatei with D. desulfuricans ND 132 or D. africanus in a sulfate-free lactate medium 3.6 CH3Hg synthesis, change in cell numbers, and change of sulfate in 117 mono- and cocultures of S. sulfatireducens with D. desulfuricans ND132 or D. africanus in a propionate-sulfate (3.94 mM) medium 3.7 Effect of sulfate concentration on Hg methylation during syntrophic 118 growth of S. sulfatireducens with D. africanus or D. desulfuricans S3.1 Light scatter distributions of pure strains and co-cultures in the 127 associations of S.