MOLECULAR CHARACTERIZATION OF MICROBIAL COMMUNITIES IN LAKE ERIE SEDIMENTS Torey Looft A Thesis Submitted to the Graduate College of Bowling Green State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE December 2005 Committee: Juan L. Bouzat, Advisor George Bullerjahn Michael McKay ii ABSTRACT Juan L. Bouzat, Advisor Microorganisms perform important roles in elemental cycling and organic decomposition, which are vital for ecosystems to function. Lake Erie offers a unique opportunity to study microbial communities across a large environmental gradient. Lake Erie consists of three basins and is affected by allochthonous inputs of dissolved organic matter (DOM) that increase to the west of the lake. In addition, the Central Basin of Lake Erie is characterized by a large area dominated by a Dead Zone, which experiences periodic hypoxic events. To evaluate patterns of microbial diversity, environmental samples from eleven sites were selected for PCR amplification, cloning and sequencing of 16S ribosomal DNA genes from microbial species. Samples included inshore sites from the Western, Central and Eastern Basins as well as from the Dead Zone of the Central Basin. DNA representing the microbial community was extracted directly from sediment samples and universal primers were designed to amplify a 370 bp region of the small subunit of the 16S rDNA gene. Characterization of DNA sequences was performed through sequence database searches and phylogenetic analyses of environmental DNA sequences, the latter using reference DNA sequences from Archaea and all major bacterial groups. These analyses were used to assign environmental sequences to specific taxonomic groups. Biodiversity indices (Berger-Parker number and Bray-Curtis cluster analysis) were calculated and measures of sequence diversity were obtained from inshore sites of the three basins and the Dead Zone of Lake Erie. Results from this study demonstrated considerable levels of spatial variability of microbial communities throughout Lake Erie. Characterized species included bacterial groups with diverse metabolic capabilities and key members involved in the iii cycling of nutrients. The relative preponderance of Gammaproteobacteria in the Western and Central Basins, but not in the Eastern Basin, may reflect the presumably widespread carbon substrate range found in the Western and Central Basins due to the greater number of allochthonous inputs of DOM. East and Central Basins showed similarities by the cluster analysis for species diversity, while the Dead Zone represented the most distinct group. These results are consistent with the idea that the Dead Zone’s unique conditions may lead to a unique microbial community structure. Although the presence/absence of some taxonomic groups revealed some patterns of spatial structuring, genetic distance calculations showed limited sequence differences between sites and groups. iv ACKNOWLEDGMENTS Many people made contributions to the completion of this project. I would like to thank Juan L. Bouzat for his advising, and Michael McKay and George Bullerjahn for completing my thesis committee. I would like to thank David Porta for collecting the Lake Erie sediments used in this study. Matt Hoostal assisted with data analysis and provided helpful suggestions during the writing process. I would like to thank my other MOLECON cohorts: Jeremy Ross, Brian Roller, Bethany Swanson and Tim Herman for many helpful discussions. Thanks to Sandra Marcu for her grammatical edits; her English is better than mine. I would like to thank my parents, Terry and Carole Looft support. My parents’ encouragement through my long college career was much appreciated and they never let me forget that there is life outside college. Finally, I would like to thank the discipline of science and scientific principles to help explain how and why things happen. v TABLE OF CONTENTS Page ABSTRACT………………………………………………………………… ii ACKNOWLEDGEMENTS ………………………………………………… iv TABLE OF CONTENTS…………………………………………………… v LIST OF TABLES………………………………………………………….. vi LIST OF FIGURES………………………………………………………….. vii INTRODUCTION…………………………………………………………... 1 MATERIALS AND METHODS…………………………………………… 7 RESULTS…………………………………………………………………… 15 DISCUSSION……………………………………………………………….. 21 TABLES AND FIGURES…………………………………………………… 31 APPENDIX………………………………………………………………….. 49 REFERENCES………………………………………………………………. 58 vi LIST OF TABLES Page Table 1. Characterization of environmental DNA sequences based on the Ribosome Database Project Classifier…………………………………………….…... 32 Table 2. Rank Abundance table based on characterizations of environmental DNA sequences using the Ribosome Database Project Sequence Match…..…… 33 Table 3. Berger-Parker Indexes of species dominance for the Western, Central, and Eastern Basins as well as the Dead Zone and sites associated with industrial inputs in Lake Erie……….………………………………………………… 34 Table 4. Characterization of unclassified sequences, cross-searched with the RDP Sequence Match.………………………………………….………………… 35 Table 5. Characterization of DNA sequences based on phylogenetic analysis. …..... 36 Table 6. Neighbor-Joining distances between sampling sites estimated using the Kimura-2 Parameter model of evolution and estimated standard errors based on 1050 bootstrap resampling of the data………..……..…………… 37 Table 7. Genetic distances within sampling sites and group averages estimated using the Kimura-2 Parameter model of evolution……….…………….…… 38 Table 8. Genetic distances between sampling group averages estimated using the Kimura-2 Parameter model of evolution…………….………………….…… 39 vii LIST OF FIGURES Page Figure 1. Map and cross-section of Lake Erie……………………………………… 40 Figure 2. Map of Lake Erie showing location of sampling stations. Pie charts represent results from the Ribosome Database Project Sequence Classifier for each sample group………………………………………….. 41 Figure 3. Bray-Curtis Cluster analysis trees based on diversity between sampled groups…..………………………………………………………...…….…. 42 Figure 4. Bray-Curtis Cluster analysis tree, based on species diversity between sampled groups………………………..……………………………..……. 43 Figure 5. Neighbor-Joining phylogenetic tree of all 16S rDNA sequences obtained in this study……….…….......……………………………..…….. 44 Figure 6. Neighbor-Joining phylogenetic tree of the 16S rDNA sequences obtained from the Western Basin..………………………………….….….. 45 Figure 7. Neighbor-Joining phylogenetic tree of the 16S rDNA sequences obtained from the Central Basin…….……………………………………… 46 Figure 8. Neighbor-Joining phylogenetic tree of the 16S rDNA sequences obtained from the Eastern Basin……………………………………..……... 47 Figure 9. Neighbor-Joining phylogenetic tree of the 16S rDNA sequences obtained from the Dead Zone………………………………………..…..….. 48 1 MOLECULAR CHARACTERIZATION OF MICROBIAL COMMUNITIES IN LAKE ERIE SEDIMENTS INTRODUCTION Microorganisms are important components of every ecosystem because they perform significant roles such as elemental cycling and organic decomposition. Microbial community processes include photosynthesis, N2 fixation, denitrification, sulfate reduction, methanogenesis, and metal reduction reactions (Paerl and Pickney, 1996). Many of these processes result in the detoxification of pesticides and other harmful contaminants that pose health risks to humans. This wide range of metabolic plasticity allows microorganisms to inhabit many habitats, including habitats under extreme environmental conditions (Head et al., 1998). Microorganisms play an indispensable role in the environment and many organisms are dependent on their relationships with microorganisms (Berman-Frank and Parker, 2003). An assessment of microbial community diversity and structure is therefore an important step for understanding the role of microbial communities in ecosystem function. One of the longstanding problems in environmental microbiology is determining what species are present in a given environment (Kirk et al., 2004). The extent of microbial diversity is unknown since it is much greater than what is observed from studies that obtain microorganisms in culture from the environment under laboratory conditions. Given the limitations of culture-based methods to identify non-culturable organisms (Cho and Tiedje et al., 2000), it has been estimated that up to 99% of soil microorganisms are unidentified (Borneman et al., 1996). Microorganisms are not easy to characterize on the basis of morphology because few morphological traits are widespread enough to allow meaningful comparisons between 2 distantly related microorganisms (Edwards, 2000). Many microbial species are characterized biochemically on the basis of their metabolic function. Microbial metabolism varies with regard to the sources of energy microbial species use for assembling macromolecules and other cellular components as seen, for example, with sulfur-oxidizers (Dexter-Dyer, 2003). However, biochemical assays also have limitations since these often require laboratory culturing, which for most taxa is not applicable. Other factors affecting microbial diversity, such as interactions between microbes, dispersal rates, changing conditions, and evolutionary change, cannot be taken into account in the laboratory setting Travisano and Rainey, 2000). An alternative approach to the biochemical characterization of microbial species and communities has been the use of DNA molecular techniques (Kirk et al., 2004). Examination of 16S ribosomal DNA sequences can be used to identify organisms and establish evolutionary relationships among species (Gray and