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by Copyright Annette Summers Engel 2004 The Dissertation Committee for Annette Summers Engel Certifies that this is the approved version of the following dissertation: Geomicrobiology of Sulfuric Acid Speleogenesis: Microbial Diversity, Nutrient Cycling, and Controls on Cave Formation Committee: Philip C. Bennett, Supervisor Libby A. Stern John M. Sharp Barbara J. Mahler Katrina Edwards Geomicrobiology of Sulfuric Acid Speleogenesis: Microbial Diversity, Nutrient Cycling, and Controls on Cave Formation by Annette Summers Engel, B.A., M.Sc. Dissertation Presented to the Faculty of the Graduate School of The University of Texas at Austin in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy The University of Texas at Austin May, 2004 Acknowledgements First and foremost, it is because of my family and friends who supported me throughout this long process, and it has been with their encouragement that I was able to complete this work. I honestly think that many of them thought this day would never come.... Now, a new stage of my life begins and I feel fortunate in knowing they champion me. I am greatly indebted to Scott for his love, patience, and strength. Without his devotion and never-ending sacrifices, finishing this work would have been much more difficult. How many days of your vacations have you surrendered in search of one last stinky spring, or for collecting slime in some godforsaken place? To Astrid and Jason, I am grateful for our friendship and will fondly remember all the margaritas we shared together. I extend very special thanks to Megan Porter, a truly wonderful person and valued friend. This research would have suffered if not for her collaboration, companionship, and intellectual banter. I owe much to Phil Bennett and Libby Stern, both caring and supportive individuals, for all their creativity and guidance. They honed my crazed thoughts and refined many ideas. It is still unbelievable to me how these unsuspecting souls quickly embraced the research, donned gas masks with passion, and forcibly slithered into a smelly hole in the ground. Together, after logging some 1665 hours (!!!) in the caves with friends and colleagues, we unburied quite an unforgettable treasure in Lower Kane Cave. This work was funded in part by the National Science Foundation's Life in Extreme Environments (LExEN) program, the Geology Foundation of the iv University of Texas at Austin, and the National Speleological Society. Sample acquisition would not have been possible without the cooperation of the Bureau of Land Management, Cody Office, Wyoming. Finally, I am especially grateful to my countless friends who took precious time from their lives to help with work in Lower Kane Cave. Their interest and enthusiasm for the research will not be forgotten, and I hope that someday I can repay their kindness. The purpose and heart of a caver, of my friends and companions, are described in the poem In Praise of Limestone by W.H. Auden. It is with these words that I end this dedication and begin the story: If it form the one landscape that we, the inconstant ones, Are consistently homesick for, this is chiefly Because it dissolves in water. Mark these rounded slopes With their surface fragrance of thyme and, beneath, A secret system of caves and conduits; hear the springs That spurt out everywhere with a chuckle, Each filling a private pool...; when I try to imagine a faultless love Or the life to come, what I hear is the murmur Of underground streams, what I see is a limestone landscape. W.H. Auden, excerpt from In Praise of Limestone v Geomicrobiology of Sulfuric Acid Speleogenesis: Microbial Diversity, Nutrient Cycling, and Controls on Cave Formation Publication No. _________ Annette Summers Engel, Ph.D. The University of Texas at Austin, 2004 Supervisor: Philip C. Bennett Much of the terrestrial subsurface is inaccessible for study, but caves represent distinctive shallow subsurface habitats where biogeochemical processes can be easily examined. Previously defined speleogenesis models are almost entirely based on abiotic chemical and hydrologic controls, as biological controls on cave formation have not been considered significant. Hydrogen sulfide-rich groundwater discharges from springs into Lower Kane Cave, Wyoming, and the sulfuric acid speleogenesis model was introduced in the early 1970s as a cave enlargement process resulting primarily from hydrogen sulfide autoxidation to sulfuric acid and replacement of carbonate by gypsum on subaerially exposed surfaces. The reduced sulfur compounds serve as rich energy sources for microorganisms that colonize the cave in both subaqueous and subaerial vi environments. Several evolutionary lineages of the class "Epsilonproteobacteria" dominate the microbial diversity of subaqueous mats, and these microbes support the cave ecosystem through sulfur cycling and chemolithoautotrophic carbon fixation. The "Epsilonproteobacteria" occupy microbial mats in additional sulfidic cave and spring habitats, expanding the evolutionary and ecological diversity of these previously unknown organisms. The interior of the Lower Kane Cave microbial mats is devoid of oxygen and this provides habitat for anaerobic metabolic guilds, dominated by sulfate-reducing and fermenting bacteria. These anaerobic groups are responsible for autochthonous hydrogen sulfide and volatile organosulfur gas production. Cycling of carbon and sulfur compounds by the subaqueous microbial communities affects sulfuric acid speleogenesis. Compared to the total flux of sulfide into the cave, little hydrogen sulfide volatilizes into the cave atmosphere or oxidizes abiotically. Instead, the primary loss mechanism is from subaqueous microbial sulfur oxidation. Consequently, despite the cave waters being slightly supersaturated with respect to calcite, the "Epsilonproteobacteria" generate sulfuric acid as a byproduct of their metabolism, locally depress pH, and focus carbonate dissolution. The hydrogen sulfide that volatilizes into the cave air is oxidized at the cave walls where interactions between cave-wall biological and physicochemical factors influence subaerial speleogenesis and low temperature authigenic quartz precipitation. The recognition of the geomicrobiological contributions to subaqueous and subaerial carbonate dissolution fundamentally changes the model for sulfuric acid speleogenesis and the mechanisms for subsurface porosity development. vii Table of Contents List of Tables........................................................................................................ xvi List of Figures .....................................................................................................xvii Chapter 1: Introduction ........................................................................................... 1 Microorganisms is Sulfidic Caves ................................................................. 3 Sulfuric Acid Speleogenesis .......................................................................... 4 Primary Field Site: Lower Kane Cave, Wyoming ........................................ 6 The Bighorn Basin ................................................................................ 6 Lower Kane Cave.................................................................................. 9 General Cave Description ............................................................ 9 A Note Regarding Safety in Lower Kane Cave ......................... 12 A Note Regarding Sample Collection........................................ 13 Research Questions ...................................................................................... 14 Hypotheses and Research Approach ............................................................ 15 Figure 1-1: Bighorn Basin, Wyoming, showing major cities and physiographic features............................................................................... 18 Figure 1-2: Plan-view map of Lower Kane Cave, Wyoming ...................... 19 Figure1-3: Topography associated with Little Sheep Mountain anticline and the Bighorn River in the vicinity of the Kane Cave, Wyoming ......... 20 Figure 1-4: Photographs of the back of Lower Kane Cave.......................... 21 Figure 1-5: Photographs of the Fissure Spring, Lower Kane Cave ............. 22 Figure 1-6: Photographs of the Upper Spring orifice, Lower Kane Cave.... 23 Figure 1-7: Photographs of the Upper Spring outflow channel ................... 24 Figure 1-8: Photographs of the red mats, Lower Kane Cave ....................... 25 Figure 1-9: Photographs of the Lower Spring, Lower Kane Cave............... 26 Figure 1-10: Peak discharge and peak stage data for the Bighorn River, Wyoming ................................................................................................... 27 Figure 1-11: -, -, -radiation in Lower Kane Cave .................................. 28 Chapter 2: Bacterial Diversity and Ecosystem Function of Filamentous Microbial Mats from Aphotic (cave) Sulfidic Springs Dominated by Chemolithoautotrophic "Epsilonproteobacteria.......................................... 29 Abstract ........................................................................................................ 29 Introduction .................................................................................................. 30 Materials and Methods................................................................................. 33 viii Sample Acquisitions............................................................................ 33 Geochemical Analysis......................................................................... 34 Scanning Electron Microscopy ........................................................... 35 Carbon, Nitrogen, and Sulfur Content ................................................ 35 Carbon Isotope Methods ..................................................................... 36 Sulfur Isotope Methods ....................................................................... 37 DNA Extraction and PCR Amplification of 16S rRNA Gene Sequences ............................................................................................ 37 16S rRNA Gene Clone Library Construction ..................................... 38 Sequencing of 16S rRNA Genes and Phylogenetic Analysis ............. 39 Statistical Analysis and Sequence Population Density ....................... 41 Nucleotide Sequence Accession Numbers .......................................... 42 Results .......................................................................................................... 43 Spring and Stream Geochemistry........................................................ 43 Morphologic Description of the Microbial Mats ................................ 43 C:N Ratios, Sulfur Content, and Biomass Estimates .......................... 45 Carbon Isotope Systematics ................................................................ 46 Sulfur Isotope Systematics .................................................................. 47 Clone Library Coverage, Species Richness, and Diversity................. 47 Phylogenetic Analysis of 16S rRNA Gene Clone Libraries ............... 49 The "Epsilonproteobacteria" class ............................................ 50 The Gammaproteobacteria class ............................................... 52 The Betaproteobacteria class..................................................... 54 The Deltaproteobacteria class ................................................... 54 The Acidobacterium division ..................................................... 55 The Bacteroidetes/Chlorobi division ......................................... 55 Discussion .................................................................................................... 56 "Epsilonproteobacteria" Diversity and Ecophysiology...................... 56 Geochemical Controls on Community Structure and Ecosystem Function............................................................................................... 60 Chemolithoautotrophy in the Subsurface ............................................ 65 Nutrient Spiraling................................................................................ 69 Conclusions .................................................................................................. 70 Table 2-1: Geochemical parameters from representative water samples..... 72 ix Table 2-2: Elemental analysis, C:N ratios, and stable carbon isotopes ....... 73 Table 2-3: Distribution of bacterial 16S rRNA clones................................. 74 Table 2-4: Bacterial clone library coverage and ecological indices ............ 75 Table 2-5: Percentage of 16S rRNA sequence similarity for "Epsilonproteobacteria" ........................................................................... 75 Figure 2-1: Dissolved hydrogen sulfide and oxygen profiles ...................... 76 Figure 2-2: Photographs of microbial mat sampling locations .................... 77 Figure 2-3: Scanning electron photomicrographs of white microbial mats . 79 Figure 2-4: Scanning electron photomicrographs of gray sediment ............ 80 Figure 2-5: Carbon isotope composition of microbial mats......................... 81 Figure 2-6: Sulfur isotope compositions of microbial mats and dissolved sulfide ....................................................................................................... 82 Figure 2-7: Rarefaction curves of the 16S rRNA clone library diversity .... 83 Figure 2-8: 16S rRNA gene-based phylogenetic tree of Lower Kane Cave belonging to the "Epsilonproteobacteria" ............................................... 84 Figure 2-9: 16S rRNA gene-based phylogenetic tree of Lower Kane Cave belonging to the Gammaproteobacteria ................................................... 86 Figure 2-10: 16S rRNA gene-based phylogenetic tree of Lower Kane Cave belonging to the Betaproteobacteria......................................................... 87 Figure 2-11: 16S rRNA gene-based phylogenetic tree of Lower Kane Cave belonging to the Deltaproteobacteria ....................................................... 88 Figure 2-12: 16S rRNA gene-based phylogenetic tree of Lower Kane Cave belonging to the Bacteriodetes/Chlorobi divisions ................................... 89 Chapter 3: Prevalence of Novel "Epsilonproteobacteria" from Filamentous Microbial Mats in Sulfidic Caves and Springs ............................................ 90 Abstract ........................................................................................................ 90 Introduction .................................................................................................. 91 Material and Methods .................................................................................. 94 Sample Acquisition from Lower Kane Cave, Wyoming .................... 94 Sample Acquisition from Other Sulfidic Caves and Springs .............. 94 DNA Extraction................................................................................... 95 PCR Amplification.............................................................................. 96 Cloning, Sequencing, and Genes and Phylogenetic Analysis 16S rRNA Genes ..................................................................................... 97 16S rRNA Oligonucleotide Probes ..................................................... 98 x Fluorescence In Situ Hybridization (FISH), Microscopy, and Quantification...................................................................................... 99 Nucleotide Sequence Accession Numbers ........................................ 101 Results ........................................................................................................ 101 Phylogenetic Analysis of 16S rRNA Clone Sequences from Lower Kane Cave ...................................................................................... 101 Fluorescence In Situ Hybridization of Lower Kane Cave Microbial Mats................................................................................................ 103 Distribution of "Epsilonproteobacteria" in Other Caves and Springs............................................................................................ 106 Discussion .................................................................................................. 108 Table 3-1: Geographic and physicochemical information for additional sampling sites .......................................................................................... 114 Table 3-2: FISH probe sequences used to screen cave microbial mats ..... 115 Table 3-3: Difference alignment of the target regions of the 16S rRNA... 116 Table 3-4: Quantification of epsilonproteobacterial filament groups ........ 117 Table 3-5: Difference alignment of the LKC-specific PCR primers ......... 118 Table 3-6: PCR results for epsilonproteobacterial screening of microbial mats from additional sampling sites........................................................ 118 Figure 3-1: 16S rRNA gene-based phylogenetic tree of LKC clones belonging to the "Epsilonproteobacteria" .............................................. 119 Figure 3-2: FISH formamide optimization series ...................................... 121 Figure 3-3: FISH results for Gammaproteobacteria and Betaproteobacteria.................................................................................. 122 Figure 3-4: FISH results for microbial mat samples using epsilonproteobacterial probes.................................................................. 123 Chapter 4: Diversity of Anaerobic Microorganisms in Cave Microbial Mats: Using a Culture-based Approach to Understand Carbon and Sulfur Cycling ....................................................................................................... 124 Abstract ...................................................................................................... 124 Introduction ................................................................................................ 125 Material and Methods................................................................................. 127 Sampling Strategy and Protocol ......................................................... 127 xi Anaerobic Biomass Estimates from Enrichment Cultures ................. 128 Fermenting Bacteria ................................................................. 128 Sulfate-reducing Bacteria ......................................................... 129 Sulfur-reducing Bacteria .......................................................... 131 Iron-reducing Bacteria.............................................................. 131 Methanogens ............................................................................ 132 Denitrifying Bacteria................................................................ 132 Results ........................................................................................................ 133 Diversity and Biomass of Anaerobic Enrichments Cultures.......... 133 Fermentation Diversity................................................................... 135 Discussion .................................................................................................. 135 Diversity and Ecology of Anaerobic Microorganisms................... 135 Implications for Carbon and Sulfur Cycling .................................. 141 Table 4-1: Culture groups, sampling sites, and microbial mats ................. 145 Table 4-2: Results from isolation and screening of fermenting bacteria ... 146 Figure 4-1: Most probable number estimates for Lower Kane Cave ......... 147 Figure 4-2: Most probable number estimates for Hellspont Cave ............. 149 Chapter 5: Production and Consumption of Hydrogen Sulfide and Volatile Organosulfur Compounds in a Sulfidic Cave System................................ 150 Abstract ...................................................................................................... 150 Introduction ................................................................................................ 151 Materials and Methods ............................................................................... 154 Cave Gas Sampling and Flux Measurements................................. 154 Enumeration of Anaerobic Enrichment Cultures ........................... 155 Isolation of Fermenting Bacteria and Metabolism of Sulfur Compounds.................................................................................. 155 Gas Cycling by Native Microbial Mat Communities..................... 156 H2S and VOSC Production from Homogenized Mat Samples 156 Inhibition of H2S and VOSC Production from Homogenized Mat Samples ............................................................................. 157 VOSC Consumption by Homogenized Microbial Mats .......... 158 Results ........................................................................................................ 158 Hydrogen Sulfide Dynamics in the Cave ....................................... 158 Field Description........................................................................ 158 xii Field Flux Experiments and Theoretical Volatilization ........... 160 Abiotic Autoxidation Rates...................................................... 161 Microbial Mat Sulfur Gas Production and Consumption Experiments ................................................................................ 163 Enrichment Cultures of Anaerobic Microorganisms .............. 163 Sulfur Gas Production from Fermenting Bacteria ................... 164 Native Mat Incubation.............................................................. 164 Homogenized Mat Incubations and Inhibition Experiments.... 165 VOSC Consumption by Homogenized Mats ........................... 167 Discussion .................................................................................................. 167 Table 5-1: Geochemical and gas flux data for the Upper Spring............... 174 Table 5-2: Volatile organosulfur gases and H2S from cultures.................. 176 Figure 5-1: Gas chromatography set-up in Lower Kane Cave................... 177 Figure 5-2: Dissolved sulfide and H2S gas in cave atmosphere................. 178 Figure 5-3: CTS= and dissolved oxygen, with volatilization loss............... 179 Figure 5-4: Fermenting bacterial strain isolates gas production ................ 180 Figure 5-5: M-series gas production incubations ....................................... 181 Figure 5-6: A-series gas production incubations........................................ 182 Figure 5-7: B-series gas production incubations........................................ 183 Figure 5-8: C-series gas production incubations........................................ 184 Figure 5-9: S-series gas production incubations ........................................ 185 Chapter 6: Microbial Contributions to Cave Formation: New Insights into Sulfuric Acid Speleogenesis ...................................................................... 186 Abstract ...................................................................................................... 186 Introduction ................................................................................................ 187 Materials and Methods ............................................................................... 189 Aqueous Geochemistry ..................................................................... 189 Atmosphere Gases............................................................................. 190 Calcite Field Chambers and Microcosms.......................................... 190 Electron Microscopy ......................................................................... 191 Fluorescence In Situ Hybridization................................................... 192 Results and Discussion ............................................................................. 193 Hydrogen Sulfide Transport and Reaction........................................ 193 xiii Subaqueous Microbial Carbonate Dissolution.................................. 195 A New Model for Microbial Sulfuric Acid Speleogenesis ............... 198 Conclusions............................................................................................... 199 Table 6-1: Aqueous geochemistry and saturation indices ........................ 201 Figure 6-1: Examples of field chamber and buried slide locations .......... 202 Figure 6-2: Deeply corroded limestone cobbles in stream ...................... 203 Figure 6-3: Environmental scanning electron photomicrographs of surfaces from native limestone in the cave stream............................ 204 Figure 6-4: Scanning electron photomicrographs of microcosm Iceland spar surfaces and filaments .................................................................. 205 Figure 6-5: Environmental scanning electron photomicrographs of filaments with and without intracellular sulfur ................................. 206 Figure 6-6: FISH images of filaments attached to experimental limestone surfaces.................................................................................. 207 Chapter 7: Geochemistry and Interfacial Phenomena of Acidic Condensation Droplets of Cave-wall Surfaces: Implications for Authigenic Quartz Precipitation and Sulfuric Acid Speleogenesis .......................................... 208 Abstract ...................................................................................................... 208 Introduction ................................................................................................ 209 Materials and Methods ............................................................................... 212 Study Site .......................................................................................... 212 Crust and Gypsum Characterization ................................................. 212 Droplet Collection and Characterization........................................... 214 Quartz Separation and Cathodoluminescence Microscopy............... 216 Results ........................................................................................................ 217 Cave-wall Crusts and Condensate Morphology................................ 217 Condensate Geochemistry................................................................. 218 Quartz ................................................................................................ 219 Brown Crust Microbiology ............................................................... 220 Discussion .................................................................................................. 221 Sulfuric Acid Speleogenesis and the Role of Cave-wall Surfaces.... 221 Speleogenetic Quartz Formation....................................................... 222 Sources of Silica................................................................................ 223 Influx of quartz-saturated meteoric water ................................ 223 Silicates dissolution.................................................................. 224 xiv Weathering of insoluble (residual) clays.................................. 225 Significance of Microorganisms to Cave Formation and Quartz Precipitation .................................................................................. 226 Conclusions ................................................................................................ 227 Table 7-1: Oligonucleotide probes used to screen brown crusts................ 229 Table 7-2: Contact angles of condensation droplets on crust and gypsum 230 Table 7-3: Major geochemistry of condensation droplets.......................... 231 Table 7-4: Calculated mineral saturation indices ....................................... 232 Figure 7-1: Gypsum crystals and brown crust ........................................... 233 Figure 7-2: ESEM photomicrographs and elemental map of brown crust. 234 Figure 7-3: Condensation droplets on brown crust .................................... 235 Figure 7-4: Condensation droplet sulfate chemistry and gypsum saturation indices versus pH .................................................................................... 236 Figure 7-5: Silica concentration and Al-sulfate complexes ....................... 237 Figure 7-6: SEM photomicrographs of quartz ........................................... 238 Figure 7-7: Quartz crystal lengths measured using ESEM ........................ 239 Figure 7-8: Cathodoluminescence image of polished quartz crystal ......... 240 Figure 7-9: DAPI stained brown crust and microbial cell morphotypes.... 241 Chapter 8: Conclusions ....................................................................................... 242 Appendix A: Chapter 2 Supplement ................................................................... 246 Appendix B: Chapter 3 Supplement.................................................................... 276 Appendix C: Chapter 4 Supplement.................................................................... 304 Appendix D: Chapter 5 Supplement ................................................................... 312 Appendix E: Chapter 6 Supplement.................................................................... 335 Appendix F: Chapter 7 Supplement .................................................................... 339 Bibliography........................................................................................................ 343 Vita ..................................................................................................................... 375 xv List of Tables Table 2-1: Geochemical parameters from representative water samples.............. 72 Table 2-2: Elemental analyses, C:N ratios, and stable carbon isotopes ................ 73 Table 2-3: Distribution of bacterial 16S rRNA clones.......................................... 74 Table 2-4: Bacterial clone library coverage and ecological indices...................... 75 Table 2-5: Percentage of 16S rRNA sequence similarity for "Epsilonproteobacteria"..................................................................................... 75 Table 3-1: Geographic and physicochemical information for additional sampling sites ................................................................................................... 114 Table 3-2: FISH probe sequences used to screen cave microbial mat samples 115 Table 3-3: Difference alignment of the target regions of the 16S rRNA ............ 116 Table 3-4: Quantification of epsilonproteobacterial filament groups ................. 117 Table 3-5: Difference alignment of the LKC-specific PCR primers .................. 118 Table 3-6: PCR results for epsilonprotobacterial screening of microbial mats from additional sampling sites ......................................................................... 118 Table 4-1: Culture groups, sampling sites, and microbal mats ........................... 145 Table 4-2: Results from isolation and screening of fermenting bacterial strains using TSI agar .................................................................................................. 146 Table 5-1: Geochemical and gas flux data per meter for the Upper Spring........ 174 Table 5-2: Volatile organosulfur gases and H2S from cultures........................... 176 Table 6-1: Aqueous geochemistry and saturation indices................................... 201 Table 7-1: Oligonucleotide probes used to screen brown crusts......................... 229 Table 7-2: Contact angles of condensation droplets on crust and gypsum ......... 230 Table 7-3: Major geochemistry of condensation droplets................................... 231 Table 7-4: Calculated mineral saturation indices ................................................ 232 xvi List of Figures Figure 1-1: Map of Bighorn Basin, Wyoming ...................................................... 18 Figure 1-2: Map of Lower Kane Cave, Wyoming ................................................ 19 Figure 1-3: Topographic map of Little Sheep Mountain anticline........................ 20 Figure 1-4: Photographs of the back of Lower Kane Cave................................... 21 Figure 1-5: Photographs of the Fissure Spring area .............................................. 22 Figure 1-6: Photographs of the Upper Spring orifice area .................................... 23 Figure 1-7: Photographs of the Upper Spring outflow channel ............................ 24 Figure 1-8: Photogarphs of the red mats ............................................................... 25 Figure 1-9: Photographs of the Lower Spring area ............................................... 26 Figure 1-10: Peak discharge and peak stage of the Bighorn River ....................... 27 Figure 1-11: Radioactive measurements from Lower Kane Cave ........................ 28 Figure 2-1: Dissolved hydrogen sulfide and oxygen profiles ............................... 76 Figure 2-2: Photographs of micorbial mat sampling locations ............................. 77 Figure 2-3: Scanning electron photomicrographs of white microbial mats .......... 79 Figure 2-4: Scanning electron photomicrographs of gray sediment ..................... 80 Figure 2-5: Carbon isotope composition of microbial mats.................................. 81 Figure 2-6: Sulfur isotope compositions of mirobial mats and dissolved sulfide 82 Figure 2-7: Rarefaction curves of the 16S rRNA clone library diversity ............. 83 Figure 2-8: 16S rRNA gene-based phylogenetic tree showing positions of Lower Kane Cave clones within the "Epsilonproteobateria" ............................ 84 Figure 2-9: 16S rRNA gene-based phylogenetic tree showing positions of Lower Kane Cave clones within the Gammaproteobacteria ............................. 86 Figure 2-10: 16S rRNA gene-based phylogenetic tree showing positions of Lower Kane Cave clones within the Betaprotoebacteria .................................. 87 Figure 2-11: 16S rRNA gene-based phylogenetic tree showing positions of Lower Kane Cave clones within the Deltaproteobacteria .................................. 88 Figure 2-12: 16S rRNA gene-based phylogenetic tree showing positions of Lower Kane Cave clones within the Bacteriodetes/Chlorobi divisions............. 89 Figure 3-1: 16S rRNA gene-based phylogenetic tree of LKC clones belonging to the "Epsilonproteobacteria"......................................................................... 119 Figure 3-2: FISH formamide optimization series ............................................... 121 Figure 3-3: FISH results for Gammaproteobacteria and Betaproteobacteria... 122 Figure 3-4: FISH results for microbial mat samples using epsilonproteobacterial probes ................................................................................................ 123 xvii Figure 4-1: Most probable number estimates for Lower Kane Cave mats ......... 147 Figure 4-2: Most probable number estimates for Hellspont Cave ...................... 149 Figure 5-1: Gas chromatography set-up in Lower Kane Cave............................ 177 Figure 5-2: Dissolved sulfide and H2S gas in cave atmosphere.......................... 178 Figure 5-3 CTS= and dissolved oxygen, with volatilization loss ......................... 179 Figure 5-4: Time course series for fermenting bacteria strains........................... 180 Figure 5-5: Time series gas production for M-series incubations....................... 181 Figure 5-6: Time course gas production for A-series incubations ...................... 182 Figure 5-7: Time course gas production for B-series incubations ...................... 183 Figure 5-8: Time course gas production for C-series incubations ...................... 184 Figure 5-9: VOSC sparge spring water and S-series gas cycling ....................... 185 Figure 6-1: Examples of field chamber and buried slide locations..................... 202 Figure 6-2: Deeply corroded limestone cobbles in stream.................................. 203 Figure 6-3: Environmental scanning electron photomicrographs of surfaces from native limesotne in the cave stream......................................................... 204 Figure 6-4: Scanning electron photomicrographs of microcosm Iceland spar surfaces and filaments ...................................................................................... 205 Figure 6-5: Environmental scanning electron photomicrographs of filaments with and without intracellular sulfur ................................................................ 206 Figure 6-6: FISH images of filaments attached to experimental limestone surfaces............................................................................................................. 207 Figure 7-1: Gypsum cystals and brown crust...................................................... 233 Figure 7-2: ESEM photomicrographs and elemental maps of brown crust ........ 234 Figure 7-3: Condensation droplets on brown crust ............................................. 235 Figure 7-4: Condensation droplet sulfate chemistry and gypsum saturation indices versus pH ............................................................................................. 236 Figure 7-5: Silica concentration and Al-sulfate complexed................................ 237 Figure 7-6: SEM photomicrographs of quartz .................................................... 238 Figure 7-7: Quartz crystal lengths measured using ESEM ................................. 239 Figure 7-8: Cathodoluminescence image of polished quartz crystal .................. 240 Figure 7-9: DAPI stained brown crust and microbial cell morphotypes............. 241 xviii Chapter 1: Introduction Microbial processes occurring in the absence of light have generally been considered insufficient to support ecosystem-level processes. Until recently, the dogma has been that life in the subsurface, if present and metabolically active, is dominated by heterotrophic consumption of surface-derived carbon. But the absence of light energy does not preclude life, as chemosynthesis also provides sustainable energy. Reactive rock surfaces and mineral-rich groundwater provide an assortment of potential energy sources for specialized microorganisms, defined as chemolithoautotrophs (literally a `self-feeding rock-eater'), that gain cellular energy from the chemical oxidation of inorganic compounds and fix inorganic carbon. Consequently, chemolithoautotrophs play important roles in global chemical and ecosystem processes, as they serve as catalysts for reactions that would not otherwise occur or would proceed slowly over geological time. While soil and shallow bedrock (<200 m depth) are major habitats for subsurface organisms, viable microbial communities are now known to be ubiquitous in the deep subsurface, including sedimentary basins, basaltic and granitic aquifers, geothermal systems, and caves. For the most part, this is attributable to chemolithoautotrophy. These environments are often limiting in organic carbon and couple relatively constant physicochemical conditions with protection from surface hazards, such as solar radiation or cosmological events. Some researchers have suggested that modern subsurface habitats are analogous to environments where life may have originated and existed on primitive Earth, or 1 even possibly in extraterrestrial systems (e.g., Schopf, 1983; Stevens, 1997; Pedersen, 2001). Consequently, it has been hypothesized that total subsurface primary productivity may even surpass the activity of surface photosynthetic organisms (Stevens, 1997; Gold, 1999). With this, the functional role of active microorganisms in subsurface systems remains poorly understood, although the potential for their impact on the surrounding geologic framework has recently been suggested (e.g., Ehrlich, 1996; Bachofen et al., 1998; Ben-Ari, 2002; Newman and Banfield, 2002). Because most subsurface habitats are relatively difficult to access, little is known about the biodiversity, community structure, ecosystem functioning, or nutrient cycling of terrestrial chemolithoautotrophically-based microbial ecosystems, or how these specialized microbes have impacted geochemical and geological processes through time. In this study I investigate the interrelationships between microbial ecosystems and the surrounding geologic and hydrologic framework in aphotic, sulfidic habitats in the shallow subsurface. Groundwater bearing dissolved hydrogen sulfide, but negligible allochthonous carbon, discharges as springs into cave passages of several known systems, including the Lower Kane Cave, Wyoming. Hydrogen sulfide is an energy-yielding substrate for microorganisms, and consequently sulfidic caves are frequently colonized by thick subaqueous microbial mats dominated by sulfur-oxidizing bacteria. Although these caves are <50 m below the Earth's surface, they offer a valuable opportunity to examine biologic-geologic interactions and microbial processes occurring in the absence of sunlight. The microbial ecology, geochemistry, and nutrient cycling investigated in 2 Lower Kane Cave can be used as proxies for similar processes occurring in deeper subsurface settings, such as carbonate aquifers, that can otherwise only be accessed though wells. MICROORGANISMS IN SULFIDIC CAVES Caves represent distinctive habitats with complete darkness, relatively constant air and water temperatures, and a limited supply of easily degradable organic matter. Since darkness precludes photosynthetic activity, the minimal organic matter found in caves is usually derived from dead photosynthetic material that has been carried into the subsurface system via air currents, speleothem dripwaters, stream drainage, or as guano from cave-dwelling organisms (Poulson and Lavoie, 2000). Early biospeleologists assumed that microorganisms in caves were secondary degraders and were food sources for higher organisms because chemosynthetic activity was not evident or was limited in most cave systems (Caumartin, 1963; Northup and Lavoie, 2001). However, more recent research in caves, including those with sulfidic water, demonstrate that bacteria generate energy as primary producers and sustain complex cave ecosystems (Sarbu et al., 1996; Porter, 1999; Sarbu et al., 2000; Vlasceanu et al., 2000; Engel et al., 2001). For instance, chemolithoautotrophy, at the base of the Movile Cave, Romania, ecosystem, supports the most diverse cave ecosystem known, with 33 new caveadapted taxa identified from 30 terrestrial invertebrate species (24 are endemic) and 18 species of aquatic animals (9 endemic) (Culver and Sket, 2000). Essentially, the chemolithoautotrophs utilize chemical energy that would otherwise be lost to the system. 3 Since the 1986 discovery of the chemolithoautotrophically-based ecosystem in Movile Cave (Sarbu et al., 1996), studies of sulfur-based, subaqueous cave microbial communities (i.e., living in microbial mats) have included geochemical and microscopy surveys (Hubbard et al., 1986; Olson and Thompson, 1988; Hubbard et al., 1990; Stoessell et al., 1993; Brigmon et al., 1994; Mattison et al., 1998; Garman, 2002), phylogenetic investigations based on 16S ribosomal RNA gene (rDNA)sequence analyses (Angert et al., 1998; Engel et al., 2001; Brigmon et al., 2003), stable isotope ratio analyses (Sarbu et al., 1996; Vlasceanu et al., 1997; Sarbu et al., 2000), and cultivation studies (Brigmon et al., 1994; Vlasceanu et al., 1997; Engel et al., 2001; Brigmon et al., 2003). Moreover, chemolithoautotrophic communities from subaerial cave-wall surfaces, associated with a variety of mineral deposits, have also been described (Maltsev et al., 1997; Hose et al., 2000; Vlasceanu et al., 2000; Northup and Lavoie, 2001; Northup et al., 2003; Barton et al., 2004). Sulfidic caves often have cave-wall biofilms that develop on gypsum and elemental sulfur surfaces, with exceedingly acidic condensation droplets, ranging from pH 0 to 4. SULFURIC ACID SPELEOGENESIS Karst landscapes form where soluble rocks dissolve, resulting in numerous geomorphic features including caves and conduit drainage systems (e.g., White, 1988; Ford and Williams, 1989; Palmer, 1991). The classic model for karst development (speleogenesis) involves carbonic acid dissolution, usually at shallow depths rarely far below the water table. More recently, sulfuric acid speleogenesis was proposed by S.J. Egemeier from work in Lower Kane Cave (Egemeier, 1981; 4 Hill, 1990; Jagnow et al., 2000). Based on observations of H2S-bearing thermal springs, extensive gypsum deposits, and gypsum-replaced limestone cave walls, Egemeier (1981) proposed the original sulfuric acid speleogenesis model to include the volatilization of H2S from the groundwater to the cave atmosphere and H2S oxidation to sulfuric acid on moist subaerial surfaces, where the acid reacts with and replaces carbonate with gypsum: H2SO4 + CaCO3 + H2O CaSO4 2H2O + CO2 (1-1). Gypsum, which spalls from the cave walls, easily dissolves into groundwater, and the net result is mass removal and an increase in void volume. Cave formation due to sulfuric acid is now recognized in several active sulfidic caves in the United States, Romania, Italy, and Mexico (van Everdingen et al., 1985; Hubbard et al., 1990; Galdenzi and Menichetti, 1995; Sarbu et al., 1996; Hose et al., 2000; Sarbu et al., 2000), as well as in ancient hypogene caves, e.g. Carlsbad Cavern, New Mexico (Hill, 1990; Polyak et al., 1998). In addition to the subaerial processes, cave formation due to sulfuric acid in the Guadalupe Mountains has also been attributed to sulfuric acid dissolution at or just below the water table (Hill, 1990; Palmer, 1991; Jagnow et al., 2000). Nearly all of the studies regarding sulfuric acid speleogenesis assumed that H2S was oxidized, and consequently sulfuric acid produced, by abiotic processes (e.g., Egemeier, 1981). The role of sulfuric acid-generating bacteria in cave formation, while previously alluded to, has never been substantiated (Symk and Drzal, 1964; Hubbard et al., 1990; Palmer, 1991; Brigmon et al., 1994; Hill, 1995; Lowe and Gunn, 1995; Sarbu et al., 1996; Vlasceanu et al., 1997; Angert et al., 5 1998; Hill, 2000; Hose et al., 2000; Palmer and Palmer, 2000; Sarbu et al., 2000; Vlasceanu et al., 2000; Engel et al., 2001). This study reexamines Lower Kane Cave, focusing not only on the microbial diversity of the cave, but also on the role microorganisms play in cave formation. PRIMARY FIELD SITE: LOWER KANE CAVE, WYOMING The Bighorn Basin The Kane Caves are located in the Bighorn Basin near Lovell, Wyoming, on the western margin of the Bighorn and Pryor Mountain Belts (Figure 1-1). The mountains are Precambrian-cored anticlinal structures of Laramide age (late Cretaceous to Eocene). The Bighorn Basin is a large asymmetric structural basin that contains Cambrian to Quaternary sedimentary rocks. Along the flanks of the basin, near the cities of Thermopolis, Lovell, and Cody, there are structural anticlines associated with deep thrust faults and mountain uplift. The basin is well known for its extensive Paleozoic oil fields, including the Spence Dome field (Madison Limestone, Mississippian) south of the Kane Caves on the western side of the Bighorn River, the Crystal Creek field (Madison Limestone) to the east of the Bighorn River, and the Byron, Garland, Frannie, Alkali Flats, and Goose Egg fields (Pennsylvanian through Cretaceous strata) west and northwest of the Kane Caves (Stone, 1967). Many of the Tensleep (Pennsylvanian) and Phosphoria (Permian) fields contain high hydrogen sulfide gas (Biggs and Espach, 1960). Thermal and non-thermal springs discharge along the flanks of the basin, commonly where faults are exposed at the surface (e.g., Five Springs at the Five Spring Fault) or where a river dissects an anticline (e.g., Spence Spring at the 6 Bighorn River level in the Sheep Mountain anticline) (Figure 1-1) (Egemeier, 1973, 1981; Heasler and Hinckley, 1985). Anomalous thermal gradients and groundwater temperatures have been recorded from sulfidic waters in the following areas: 1) southeast of the Little Sheep Mountain anticline south of Lovell, 2) within the Sheep Mountain anticline north of Greybull, 3) the region surrounding Thermopolis and the Thermopolis anticline, and 4) near Cody in the vicinity of the Shoshone River (Heasler and Hinckley, 1985). Groundwater circulation within the Paleozoic aquifers of the basin is controlled by flow along extensional fractures in fault-cored anticlinal or monoclinal folds (Jarvis, 1986; Spencer, 1986). Karst topography is rarely exposed within the Bighorn Basin, or in the Bighorn or Pryor Mountains. The main cave-forming unit in the region is the Madison Limestone, although small caves and pits also form within the Bighorn Dolomite (Ordovician) at higher elevations within the Bighorn Mountains (Hill et al., 1976). There are numerous pits and small caves in the Bighorn Mountains where these units are exposed, and where Mississippian-Triassic strata are exposed in Little Sheep and Sheep Mountains due to dissection of the anticlines by the Bighorn River (Figure 1-1). Most caves north of these anticlines and within the Bighorn Mountains are epigenic systems (Hill et al., 1976), while several cave systems, especially larger ones, formed or were modified by hypogenic processes involving sulfuric acid (Hill et al., 1976; Egemeier, 1981). Lower Kane Cave and Upper Kane Cave both formed from sulfuric acid speleogenesis in the upper member of the Madison Limestone at the apex of the Little Sheep Mountain anticline along the Bighorn River (Figure 1-1). Lower Kane Cave has 325 m of 7 mapped horizontal length, of which 180 m is stream passage (Figure 1-2). There is only one known entrance into Lower Kane Cave. Upper Kane Cave is a dry cave 30 m above Lower Kane Cave, with 290 m of total horizontal length. Upper Kane Cave has extensive gypsum deposits. Several other caves and springs are located along the Bighorn River in the vicinity of the Kane Caves (Figures 1-1 and 1-3). PBS spring, which is slightly thermal and sulfidic, discharges from the upper member limestone/dolostone of the Phosphoria Formation at the edge of Little Sheep Mountain along the Little Sheep Mountain fault (Figure 1-3). Hellspont Cave (Egemeier, 1981), or Coon Smell Cave (Hill et al., 1976), is 20 m long and has a thermal, sulfidic spring that discharges at the back of the cave. Salamander Cave is < 3 m long and has a large non-thermal, non-sulfidic spring (Figure 1-3). Spence Cave, in the Sheep Mountain anticline, also formed from sulfuric acid speleogenesis within the Madison Limestone along the Bighorn River (Figure 1-1) (Egemeier, 1973; 1981). Spence Cave is large, has several passage levels, and is similar to Upper Kane Cave in that it has dry passages and gypsum throughout (for a cave map and a complete description, refer to Egemeier, 1973 and 1981). Thermal, sulfidic springs discharge at river level within the Sheep Mountain anticline. The Bighorn-Horsethief Cave system is located 30 km north of the Kane Caves on the Wyoming-Montana border in the Bighorn Canyon (Hill et al., 1976) (Figure 1-1). This system is similar to Spence and Upper Kane Caves. Across the Bighorn Basin near Cody are the Spirit Mountain Caverns, with gypsum mineralization throughout the cave and reportedly high concentrations of H2S at lower elevation passage levels (Hill et al., 1976). 8 Although not in the Bighorn Basin, Stinkpot Cave, near Jackson Hole along the Hoback River, also has sulfidic water and microbial mats (Hill et al., 1976). Lower Kane Cave General Cave Description During this study (1999-2003) there were four springs discharging into the Lower Kane Cave passage (Figure 1-2). The Fissure, Upper, and Lower Springs issue from a fissure in the cave floor and Hidden Spring emerges from a hole in mud. The fissure in the cave floor, which corresponds to a fracture in the cave ceiling, is continuous throughout the entire length of the cave. There appears to be minimal offset of this fracture (<10 cm). The fissure is disrupted in places by secondary calcite that has precipitated over the open fracture; ~10 to 30 cm-wide holes through the calcite reveal the water-filled fissure underneath. In some places in the back of the cave the fracture is filled with sediment and/or gypsum. Faults with measurable offset are perpendicular to this continuous fracture (Figure 1-2), and these faults correspond to the springs outlets. Additionally, a resistant breccia composed of chert and limestone clasts with dolomitized cement protrudes into the passage at these fault zones. Hydrology of the cave springs has a complex history, evident from recent exploration of the cave. During the study there were two open water-filled fissures near the back of the cave (Figure 1-4); no flow was observed, although Egemeier reports that the Terminal Spring flowed in 1969 (Egemeier, 1973). In 1999, the back of the cave near the Fissure Spring and Iron Pool was dry, and only the Iron Pool (Figure 1-4B) had water and red-pigmented biological material and 9 mineralization in the orifice. In 2000, sulfidic water discharged from the Fissure Spring (Figure 1-5A) and the back of the cave had 1-5 cm of water in the passage and thick orange- to red-colored microbial mats. As a consequence, a stream flowed from the Fissure Spring, along the north side of the cave wall and under gypsum mounds, into the Lower Spring orifice where it mixed with sulfidic water. During the late spring 2003 sampling trip, there was no discharge from the Fissure Spring, but flow reinitiated several days later. White microbial filament bundles are found below the Fissure Spring orifice (Figure 1-5B and 1-5C). The Upper Spring discharges ~6 L s-1 from a sediment-filled hole in a large pool, as estimated from salt-dilution traces (Figure 1-6A, 1-6B). The Upper Spring pool is ~2.5 m wide and up to 0.5 m deep, with white filament bundles on the pool edge and sediment bottom (Figure 1-6C). A stream channel, ranging from 5 to 10 cm in depth, flows from the orifice pool toward the cave entrance, and white filamentous mats fill the channel (Figures 1-7A, 1-7B). Because of underflow from the Fissure Spring, oxygenated water seeps into the Upper Spring pool and mixes with the incoming sulfidic water, allowing for the oxidation of iron sulfides. An area halfway between the Upper and Lower Spring orifices has a seep in which water emerges from under a gypsum pile (Figure 1-8A). A thick (~15 cm) red mat forms 5 cm from the gypsum pile (Figure 1-8B). The Lower Spring has the deepest measurable orifice opening (2 m), and the orifice walls are coated with gray sediment/biological material and long white microbial filament bundles (Figure 1-9A). The water spills over the limestone lip, and a thick white mat develops downstream (Figure 1-9B). In early May 1999 the 10 Lower Spring microbial mat extended for > 2 m from the orifice, but following back-flooding of the Bighorn River June 1999, the mats were destroyed and consequently a mat <1 m length was present until 2003 when the mat again reached ~2 m. In 2001 through 2003, a slight trickle of sulfidic water issued from a small (~20 cm) hole in river mud at Hidden Spring (Figure 1-2), and sparse white filaments have been observed. The Bighorn River infrequently floods Lower Kane Cave (Figure 1-10). The cave was flooded in May 1999, corresponding to a peak stage of 7.57 ft above the gaging station, approximately 1 km downstream from the cave (United States Geological Station (USGS) station #06279500 at Kane, Wyoming; 3660 ft above msl). The river stage and discharge records are maintained by the USGS (http://waterdata.usgs.gov/nwis/sw). Based on the available data, the minimum river stage required to flood the cave entrance is at least 7.57 ft. Consequently, there have been at least seven times since 1956 when the cave may have flooded (Figure 1-10). The consequences of flooding are believed to be moderately significant to the Lower Spring microbial mats. Because of a slight floor gradient, a river stage of 7.57 ft would backflood river water to approximately the end of the Upper Spring microbial mats, but the back of the cave and the Fissure Spring area would not be impacted. There have not been any direct measurements to determine the age of Lower Kane Cave, but the timing of cave formation is loosely constrained by Bighorn River terraces. There is a small remnant terrace 24-m above the present river level near the entrance of Upper Kane Cave and a small terrace at the Lower Kane Cave 11 entrance (approximately 6 m above present river level). The 24-m terrace corresponds to a larger erosional terrace across the river, referred to as the PowellEmblem or Sanatarium bench (Reheis, 1984). Egemeier (1981) suggests that this terrace may correspond to the Bull Lake glaciation, from 40 to 130 ka, but Reheis (1984) postulates that the 24-m terrace is much older, from 92 to 161 ka. Depending on the timing of emplacement of the upper terrace, the river level terrace may correlate to 38 ka, but also to the Pinedale glaciation at 10 ka. Additional research is needed to resolve this issue and to help constrain the age of the caves along the Bighorn River. A Note Regarding Safety in Lower Kane Cave Hydrogen sulfide (CAS:7783-06-4) is a colorless flammable gas (STEL = 15 ppm, IDLH = 100 ppm) that can cause headaches and nausea with prolonged low-level exposure (Beauchamp et al., 1984). Above 20 ppm, H2S causes eye and mucous membrane irritation, and prolonged exposure can result in pulmonary edema. At all times while researchers were present, Lower Kane Cave air was monitored for H2S, CO, and PO2 using an MSHA approved multigas monitor (PhD Ultra Atmospheric Monitor, Biosystems, Middleton, CT). While the concentration was typically less than STEL, all researchers worked under Level-C respiratory protection using a half-face air-purifying gas mask with organic/acid vapor cartridges (H2S escape). These masks are effective for SO2, organosulfur gases, and radon, but have only short term protection against high H2S. What initially attracted Egemeier (1973) to the Kane Caves was the prospect of economic deposits of ore minerals, and in particular uranium and 12 vanadium in the region (Wilson, 1960); Egemeier found the cave sediments were highly radioactive. Radioactivity (as total counts of combined -, -, and -radiation) was measured using a Geiger counter in December 2001 (Figure 1-11). The highest concentrations were near the springs and along the anaerobic stream channels, as well as at the cave floor. The area around 215 m, associated with red microbial mats (Figure 1-8B), also had high radioactivity. Radioactivity on gypsum piles was low and considered to be at background levels. A Note Regarding Sample Collection Caves are valuable resources, both scientifically and recreationally, and karst regions are sensitive environments. Guidelines established by the National Speleological Society and the Conservation and Cave Management Organization were followed during the research; these guidelines suggest that researchers be `professional, selective, and minimalistic'. During field work, all researchers attempted to alter the pre-existing features, water, and biota as little as possible. At all times, conservative sample quantities were collected and removal of material from the caves was limited in order to preserve the integrity of the mineralogic, hydrologic, and ecological systems. Permission to conduct research and to sample in Lower Kane Cave, Upper Kane Cave, and Hellspont Cave was granted by the Bureau of Land Management, Cody Office. 13 RESEARCH QUESTIONS The primary research questions for this work are in five areas: 1. Relationships between microbial diversity and habitat geochemistry: What are the geochemical differences between the subaqueous (cave springs and stream) and subaerial (cave-wall) habitats? Is microbial community structure controlled by habitat geochemistry and geologic framework? 2. Microbial diversity and endemism: Are the microbes found in Lower Kane Cave unique to the cave, or are they widespread in other sulfidic systems, photic or aphotic? Can the presence of these microorganisms define any of the biogeochemical processes occurring in other systems? 3. Nutrient acquisition and cycling: What are the sources and availability of nutrients to the microbial cave ecosystem? What role do allochthonous and autochthonous substrates play in the ecosystem? Do microorganisms benefit or are they adversely affected by colonization of certain geologic materials? 4. Microbial role in speleogenesis: Can abiotic H2S autoxidation be distinguished from biotic (microbial) oxidation? What are the consequences of microbial metabolism on the geologic/hydrologic surroundings? Is carbonate dissolution and speleogenesis affected depending on how or where sulfide is oxidized? 5. Process biosignatures: Are there diagnostic geologic, chemical, or isotopic characteristics of the habitats that can be used to fingerprint microbial activity in ancient systems? 14 HYPOTHESES AND RESEARCH APPROACH I used an interdisciplinary approach to examine the cave microbial communities, nutrient sources and transfers, and the potential impact of the microorganisms to cave formation and modification during sulfuric acid speleogenesis. Chapter 2: I hypothesized that the structure of microbial communities in the subaqueous microbial mats would reflect habitat geochemistry and substrate availability, and that shifts in community composition were controlled by dissolved oxygen and hydrogen sulfide. Additionally, I postulated that chemolithoautotrophy serves as the basis of energy production in the Lower Kane Cave ecosystem. As it is often difficult to ascertain the metabolism of certain organisms based only on 16S rDNA gene sequence phylogenies (e.g., Gray and Head, 2001), elemental composition (carbon to nitrogen ratios and sulfur content) and stable carbon and sulfur isotope ratio analyses of microbial mat morphotypes were combined with the 16S rDNA phylogenies to link hypotheses of ecosystem functionality with genetic identity (e.g., Boschker and Middelburg, 2002; Chesson et al., 2002). Chapter 3: While many different types of sulfur-oxidizing bacteria have been found in sulfidic cave microbial mats, members of the "Epsilonproteobacteria" dominate the subaqueous mats from Lower Kane Cave. I hypothesized that these bacteria, which are successful colonizers of marine habitats (e.g., Alain et al., 2004), were also prevalent in terrestrial sulfidic caves and springs. I employed the full-cycle rRNA approach, including the construction of 16S rDNA clone libraries and application of 16S rRNA-targeted oligonucleotide 15 probes for fluorescence in situ hybridization (FISH), to characterize the microbial mats from Lower Kane Cave. I also applied the newly designed probes and polymerase chain reaction (PCR) primers to identify novel epsilonproteobacterial groups in microbial mats collected from other field locations. Chapter 4: Molecular evidence indicated that the subaqueous microbial mats in Lower Kane Cave were much more complex than previously thought, and geochemical evidence revealed that the microbial mats were physically and chemically stratified. I hypothesized that molecular methods overlooked anaerobic microbial community diversity because populations were less abundant than the aerobes (i.e., "Epsilonproteobacteria"). I employed culture-based approaches, including the most probable number method and enrichment cultures using specific media, to characterize the diversity of anaerobic microbial communities. Chapter 5: Inorganic and organic sulfur-containing compounds pass through multiple components of a microbial ecosystem and volatile components are released into the atmosphere. Although production and consumption of volatile sulfur gases (including hydrogen sulfide, methanethiol, dimethyl sulfide, and carbonyl sulfide) have been studied from photic marine and freshwater environments, virtually nothing is known about the flux of these gases from subsurface settings. I hypothesized that sulfur gases were produced and consumed by various metabolic guilds within the Lower Kane Cave microbial ecosystem, and potentially impact the local or global sulfur budgets. I employed culture-based approaches with time-series incubation experiments and gas chromatography (GC) to examine abiotic and biotic sulfur gas production and consumption. Hydrogen 16 sulfide flux and gas dynamics were characterized by direct-inject field GC inside Lower Kane Cave. Chapter 6: The basic mechanism for sulfuric acid speleogenesis has been debated (Jagnow et al., 2000). While it was clear that specialized microbial communities take advantage of reduced sulfur compounds as energy sources, it was not clear that biotic pathways influence karstification. I hypothesized that microbial sulfur oxidation dominated over abiotic autoxidation, and microbially-derived sulfuric acid controlled speleogenesis. I combined the interdisciplinary results from 1) H2S gas flux data (described in Chapter 5), 2) aqueous geochemistry and mineralogy, 3) the deployment of field microcosms, and 4) fluorescence in situ hybridization of colonized carbonate surfaces to examine the subaqueous sulfide loss mechanisms and the roles of sulfur-oxidizing bacteria on carbonate dissolution. Chapter 7: The sulfuric acid speleogenesis model of Egemeier (1981) requires volatilization and oxidation of H2S to sulfuric acid on cave-wall surfaces, which causes aggressive carbonate rock dissolution and replacement of limestone by gypsum. I hypothesized that the chemistry of condensation on subaerial cavewall surfaces influenced dissolution and precipitation reactions occurring on the walls, and that gypsum replacement of the walls was an episodic process. I characterized the mineralogy of the cave walls, the microbiology of cave-wall biofilms, and the chemical and physical nature of condensation droplets in Lower Kane Cave. Moreover, microcrystals of authigenic quartz that form in the cavewalls biofilms and gypsum potentially represent a biogeochemical marker of cavewall processes. 17 Montana Beartooth Mountains Axis of Bighorn Basin Shoshone River Little Sheep Mountain Wyoming Lovell BIGHORN CAVERNS KANE CAVES East Yellowstone Cody Sheep Mountain SPENCE CAVE SPIRIT MOUNTAIN CAVE Basin Bighorn River Axis of Bighorn Basin Bighorn Mounatin Range 18 Absaroka Mountains 0 kilometers 25 50 Owl Creek Mountains N Worland WYOMING Thermopolis Axis of Bighorn Basin city cave anticline Figure 1-1: Bighorn Basin, Wyoming, showing major cities and physiographic features. Map modified from Egemeier (1981). FISSURE SPRING UPPER SPRING LOWER SPRING dry cavewall biofilms white filaments gray river mud sulfur HIDDEN SPRING gray river mud gypsum needles Iron Pool thin white filaments & bubbles Black Hole pool white Red mats filamentous from gypsum mats seep sulfur white filaments at waterfall cave-wall biofilms and acid droplets dry red mats Terminal Spring Dry Spring breakdown pile ENTRANCE 50 m water cave passage, with gypsum cave passage, with sediment N red mats fault trace and breccia Figure 1-2: Plan-view map of Lower Kane Cave, Wyoming, showing major spring features. Map modified from Egemeier (1981) and annotated based on this current work. Bighorn River gray river mud 3 small soda straws 19 PBS Spring t Lit le Sh pM ee Hellspont Cave Salamander Spring Lower Kane Cave n ou in ta ul Fa t 0.5 km Bighorn River Figure 1-3: Topography associated with Little Sheep Mountain anticline and Bighorn River in the vicinity of the Kane Caves, Wyoming. Three springs discharge into the Bighorn River. The Upper Kane Cave passage (not shown) is 30 m directly above Lower Kane Cave. 20 A B A.S. Engel Figure 1-4: (A) Looking toward the back of the cave, and into a small fissure in the floor filled with water. The cave walls are covered with gypsum and a brown crust. (B) Looking into the Iron Pool. A.S. Engel 21 Dry Spring A B Fissure Spring orifice C flow A.S. Engel Figure 1-5: (A) Fissure Spring area, looking to the back of the cave. (B) Fissure Spring orifice. (C) White filaments and webs approximately 3 m from orifice. A.S. Engel A.S. Engel flow 22 A B C flow A.S. Engel A.S. Engel Upper Spring orifice Figure 1-6: (A) Upper Spring area, looking to the back of the cave. (B) Upper Spring orifice pool. (C) White filaments on side of the orifice pool in ~5 cm deep water. A.S. Engel 23 A B Figure 1-7: (A) Out-flow stream channel from Upper Spring orifice pool. (B) White filamentous microbial mats filling 0.5 to 1 m wide out-flow channel, looking downstream. A.S. Engel A.S. Engel 24 flow flow A B Figure 1-8: (A) Non-sulfidic water seep and downstream red mats under gypsum pile between Upper and Lower Spring. (B) Thick red mats; pen for scale in lower left corner. A.S. Engel A.S. Engel 25 A Lower Spring orifice B H.H.Hobbs III flow from Fissure Spg flow Figure 1-9: (A) Lower Spring orifice, with anaerobic water discharging from gray side and non-sulfidic water from upstream coming into the orifice in the foreground. (B) White filamentous microbial mats, ~1.5 m long, below spring orifice. A.S. Engel 26 Lower Spring orifice 30000 discharge stage 12 25000 10 20000 8 peak discharge (cfs) 5000 2 Figure 1-10: Peak discharge and peak stage data for the Bighorn River (USGS station # 06279500, Kane, Wyoming). Data from 1956 can be accessed at http://www.water.usgs.gov/ nwis/. 0 4 8 4 8 2 8 2 0 0 6 6 0 6 4 8 4 6 8 6 2 72 00 9 7 /1 9 19 7 9 7 19 8 19 8 19 8 198 198 19 9 19 9 19 9 19 9 199 /20 97 9 5 19 5 19 6 19 6 196 96 96 / / / / / / / /1 0/1 /1 / /1 9/1 3/1 / / / / / 9 8 0/ 16/ 5 8 1 17 6/9 /1 24 14 6/9 11 11 22 /2 27 26 /26 23 6/ 6/2 29 16 3/3 7/ 5/ 5/ 5 6/ 6 5/ 2/ 6/ 6/ 6/ 5/ 5/ 6/ 5/ 6/ 6/ 10 0 0 peak stage (ft above datum, 3660ft) 10000 4 minimum stage for cave to backflood 15000 6 27 300 250 200 150 100 Maximum 8 hr exposure Rankins. hr-1 Background radiation 50 0 0 Dry Spring Fissure Spring Upper Spring Lower Spring Hidden Spring 50 100 150 200 250 300 350 Distance (meters) Figure 1-11: -, -, and -radiation measured as total counts in Lower Kane Cave, Wymoing, July 2002. 28 Chapter 2: Bacterial Diversity and Ecosystem Function of Filamentous Microbial Mats from Aphotic (cave) Sulfidic Springs Dominated by Chemolithoautotrophic "Epsilonproteobacteria" ABSTRACT1 Filamentous microbial mats from three aphotic sulfidic springs in Lower Kane Cave, Wyoming, were assessed with regard to bacterial diversity, community structure influenced by geochemical changes, and ecosystem function using a 16S rDNA-based phylogenetic approach combined with elemental content and carbon and sulfur stable isotope ratio analyses. The bulk of the microbial mats had carbon isotope values (mean 13C = -34.7, 1 = 3.6) consistent with chemolithoautotrophic carbon fixation from a dissolved inorganic carbon reservoir (cave water, mean 13C = -7.4; n = 8). The most widespread mat morphotype consisted of white filament bundles, with low C:N ratios (3.5 5.4) and high sulfur content (16.1-51.2%). Bacterial diversity was low overall, and the most prevalent taxonomic group was affiliated with the "Epsilonproteobacteria" (68%); six genetically distinct epsilonproteobacterial groups were identified. Other bacterial sequences were affiliated with Gammaproteobacteria (12.2%), Betaproteobacteria (11.7%), Deltaproteobacteria (0.8%), and the Acidobacterium (5.6%) and Bacteriodetes/Chlorobi (1.7%) divisions. Epsilonproteobacterial and bacterial group abundances and overall community structure within the microbial mats A portion of this chapter was used for the publication A.S. Engel, M.L. Porter, L.A. Stern, S. Quinlan, and P.C. Bennett, 2004, Bacterial diversity and ecosystem function of filamentous microbial mats from aphotic (cave) springs dominated by chemolithoautotrophic "Epsilonproteobacteria", FEMS Microbiology Ecology, accepted. 1 29 shifted from the spring orifices downstream, corresponding to changes in habitat dissolved sulfide and oxygen concentrations and metabolic requirements of certain bacterial groups. Most of the epsilonproteobacterial groups were identified from high sulfide and low oxygen concentrations were measured, whereas Thiothrix spp. and Thiobacillus spp. had higher abundances where conditions of low sulfide and high oxygen concentrations were observed. Genetic and metabolic diversity among the "Epsilonproteobacteria" maximizes overall cave ecosystem function, and these organisms play a significant role in providing chemolithoautotrophic energy to the otherwise nutrient-poor cave habitat. These results expand the evolutionary and ecological views of "Epsilonproteobacteria" in terrestrial habitats and demonstrate that sulfur cycling supports this subsurface ecosystem through chemolithoautotrophy. INTRODUCTION Microbial processes occurring in the absence of light have generally been considered insufficient to support ecosystem-level processes, and until recently, the dogma has been that life in the subsurface, if present and metabolically active, is dominated by heterotrophic consumption of surface-derived carbon (Caumartin, 1963; Pedersen, 2001; Naeem, 2002; Alfreider et al., 2003). But the absence of light does not preclude life, as reactive mineral surfaces and solute-rich groundwater provide energy sources sufficient for chemolithoautotrophic growth in the subsurface (e.g., Stevens, 1997; Kinkle and Kane, 2000). Chemolithoautotrophy is now recognized as an important ecosystem-level process in aphotic 30 environments, including deep terrestrial aquifers (Stevens and McKinley, 1995; Pedersen, 2001) and caves (Sarbu et al., 1996; Vlasceanu et al., 2000; SchabereiterGurtner et al., 2002). Because subsurface habitats are relatively difficult to access, however, little is known about the biodiversity, community structure, ecosystem functioning, or carbon cycling of terrestrial chemolithoautotrophically-based microbial ecosystems. Caves represent distinctive habitats with complete darkness, relatively constant air and water temperatures, and a poor supply of easily degradable organic matter. Consequently, most cave ecosystems depend on allochthonous organic material for energy (Poulson and Lavoie, 2000; Simon et al., 2003). Previous investigations describing microorganisms in caves (e.g., Caumartin, 1963; Dickson and Kirk, 1976; Mikell et al., 1996), including from sediments and aquatic habitats, suggest that the microbes are similar to those found in surface environments and are only active under optimal growth conditions (Northup and Lavoie, 2001). However, groundwater bearing dissolved hydrogen sulfide and negligible allochthonous carbon discharges as springs into the passages of several caves (e.g., Egemeier, 1981; Sarbu et al., 1996; Angert et al., 1998; Hose et al., 2000); hydrogen sulfide is an energy-yielding substrate for some microorganisms, and areas of these sulfidic caves are colonized by thick subaqueous microbial mats (Brigmon et al., 1994; Angert et al., 1998), as well as extensive cave-wall biofilms (Hose et al., 2000). Molecular phylogenetic studies based on 16S rRNA gene sequences (rDNA) have expanded our understanding of the microbial diversity in caves (e.g., 31 Northup and Lavoie, 2001), including those with hydrogen sulfide-rich groundwater (Angert et al., 1998; Vlasceanu et al., 2000; Engel et al., 2001) and those without (Holmes et al., 2001; Schabereiter-Gurtner et al., 2002; Northup et al., 2003; Schabereiter-Gurtner et al., 2003; Barton et al., 2004). In sulfidic caves, the dominant bacterial groups from some subaqueous microbial mat communities belong to the "Epsilonproteobacteria" (Angert et al., 1998; Engel et al., 2001; Barton et al., 2002). Stable carbon isotope measurements and 14 C-radiolabeled substrate experiments suggest that chemolithoautotrophy is the base of these ecosystems (Sarbu et al., 1996; Porter, 1999; Sarbu et al., 2000). There has been relatively little study, however, of the biogeochemistry and ecological roles of the dominant groups involved with energy and nutrient transfers within these ecosystems, or on the physical or chemical controls that govern community structure and dynamics. This purpose of this chapter was to characterize microbial ecosystems and nutrient cycling in sulfidic cave habitats, using Lower Kane Cave as a proxy for deeper subsurface environments. The objectives were to describe genetic and functional diversity, and to define how habitat geochemistry controls community structure. I hypothesized that community composition and structure reflect substrate availability, and specifically that community composition shifts with changes in the concentrations of dissolved oxygen and hydrogen sulfide. As it is often difficult to ascertain the metabolism of many organisms based on 16S rDNAbased phylogenies (e.g., Gray and Head, 2001), elemental composition and stable carbon and sulfur isotope ratio analyses were combined with 16S rDNA 32 phylogenies to link hypotheses of ecosystem functionality with genetic identity for the as yet uncultured microorganisms (Boschker and Middelburg, 2002; Chesson et al., 2002; Naeem, 2002). The results of the isotopic and phylogenetic characterization of the Lower Kane Cave ecosystem expand the evolutionary and ecological views of "Epsilonproteobacteria" in terrestrial habitats and demonstrate that sulfur cycling supports this subsurface cave ecosystem through chemolithoautotrophy. MATERIALS AND METHODS Sample Acquisitions Samples of each microbial mat morphotype were aseptically collected from three of the sulfidic spring sites in Lower Kane Cave, Wyoming (Figure 1-2). Multiple mat samples were used for bulk biomass, elemental analysis, carbon isotope analysis, and DNA extraction and 16s rDNA clone library construction. To preserve the integrity of the sensitive ecological systems, conservative quantities of microbiological materials were collected. Sampling sites were numbered according to their location in meters from the back of the cave, with flow always toward the cave entrance (i.e. longer distances) (Figure 1-2): Fissure Spring (124-, and 127-m), Upper Spring (190-, 195-, 198-, and 203-m), and Lower Spring (one orifice and one mat sample from 248-m). Microbial mat morphotypes were collected separately and distinguished as white filaments (denoted as `f'), white webs (denoted as `w), yellowish-white mat (denoted as `y'), and whitish-gray filaments (denoted as `g'). White filament bundles in the water column or filaments from the surface of the mats were targeted for clone libraries; however, one library was 33 constructed with gray filaments ~2 cm below the top of the mat for comparison. Geochemical Analysis Geochemical data were acquired at the major microbiological sample locations, as well as throughout the cave, over a three year period. Unstable parameters (pH, EH, and dissolved oxygen) were measured using electrode methods. Dissolved hydrogen sulfide (as total dissolve sulfide, CTS=), ferrous iron (Fe2+), and low concentrations of dissolved oxygen were measured in the field using the Methylene Blue, Ferrozine and Rhodazine D colorimetric methods, respectively (CHEMetrics, Inc., Calverton, VA), using a MiniSpec 20 field spectrophotometer. Vertical profiles of dissolved oxygen through the mats were determined by fluorescence-quenching optical methods (Ocean Optics, Inc., Dunedin, FL). Unstable and reactive parameters (pH, oxygen, hydrogen sulfide, etc.) were also measured at several transects along and across the cave stream channels. Alkalinity (as total titratable bases, here dominated by bicarbonate) was determined in the field by titration to pH 4.5, and verified in the laboratory by endpoint seeking autotitration. Anions and acid-preserved metals were determined by ion chromatography and inductively coupled plasma mass spectrometry, respectively. Dissolved organic and inorganic carbon (DOC and DIC, respectively) were determined by Dorhman DC-180 wet-oxidation carbon analyzer. Dissolved gas species (e.g., methane, aromatic hydrocarbons, hydrogen sulfide, organosulfur gases) from the spring and stream water were analyzed by headspace gas chromatography. 34 Scanning Electron Microscopy Microbial mats were examined using scanning electron microscopy (SEM) by fixing biological material with a chemical critical-point drying method modified from Nation (1983). Briefly, samples were fixed with 25% gluteraldehyde, then freeze-fractured in liquid nitrogen, and freeze-dried. Samples were mounted on aluminum stubs, sputter-coated with gold, and examined using a JEOL JSMT330A SEM in the Jackson School of Geosciences at the University of Texas at Austin. Carbon, Nitrogen, and Sulfur Content Aliquots of each morphotype (~2 ml) was individually homogenized, acidified with dilute HCl and rinsed with dH2O (acidification and washing were repeated at least twice to ensure dissolution of carbonate mineral phases), followed by freeze-drying. Total organic carbon and nitrogen contents were determined by elemental analyzer interfaced with a FinniganMAT Delta Plus mass spectrometer at the University of Texas Marine Sciences Institute in Port Aransas, Texas, simultaneously with carbon isotope ratio analysis. Total sulfur content, as inorganic and organic sulfur compounds, was determined on a EuroEA3000 elemental analyzer (EuroVector, Milan, Italy). Minimum bulk mat biomass was determined from dry weight analysis of the mats followed by comparison of the percent carbon in each aliquot, using methods described in and modified from Bratbak and Dundas (1984). Briefly, ~ 1 ml replicates were individually homogenized, acidified with dilute HCl, weighed, freeze-dried, and re-weighed to obtain the dry weight. The percentage of carbon in 35 each dried aliquot was determined by elemental analyzer. Cell carbon content was calculated from the standard conversion factor of 350 fg C cell-1 (assuming an average cell size of 1 m3; Bratbak and Dundas, 1984) to determine the number of cells per ml. Carbon Isotope Methods For carbon isotope ratio (13C/12C) analysis, organic carbon of 1 to 2 ml mat samples was prepared by acidifying the sample in dilute HCl to ensure removal of carbonate mineral phases. Most measurements were made by elemental analyzer interfaced with a continuous flow FinniganMAT Delta Plus mass spectrometer, but some measurements were also made by sealed tube combustion, vacuum purification, and using a dual-inlet VG Prism II mass spectrometer at the Jackson School of Geosciences. Microbial mat carbon isotope values were compared to the values obtained from cave water dissolved inorganic carbon (DIC), including a composite of CO2(aq), HCO3-, and CO32- from the cave water. DIC was extracted for 13 C analysis by acidifying under vacuum with 100% phosphoric acid followed by cryogenic purification of the resulting CO2 (method modified from Hassan, 1982). At the pH and temperature of the cave water (pH ~7.3 at 21.5oC), the dominant DIC species was HCO3- (~90%) based on dissociation constants for H2CO3*, HCO3, and CO32- species (Stumm and Morgan, 1996). Carbon isotope values for the Madison Limestone hostrock were also measured by reaction with 100% phosphoric acid (McCrea, 1950). All carbon isotope values are expressed in delta () notation with respect to the international standard V-PDB, where 36 13 C/ 12 C sample -13 C/ 12 C standard 1000 C () = 13 C / 12 C standard 13 (4-1) Sulfur Isotope Methods Aqueous sulfide was collected by precipitating Ag2S from filtered spring water with 10% AgNO3 solution. Following sulfide removal, aqueous sulfate was collected by precipitating BaSO4 with BaCl2 (Carmody et al., 1998). Sulfur isotope ratios of sulfide, sulfate, and organic material were made with an elemental analyzer interfaced with a Micromass Optima mass spectrometer and ratios are expressed in delta notation with respect to the Ca on Diablo Troilite (CDT) standard. DNA Extraction and PCR Amplification of 16S rRNA Gene Sequences Approximately 0.2 to 0.5 ml mat morphotype material were aseptically collected and placed into tubes containing 0.5 to 1 ml sterilized DNA extraction buffer comprised of 10 mM Tris-HCl (pH 8), 100 mM EDTA, and 2% sodium dodecyl sulfate. Prior to DNA extraction and laboratory manipulation of the mat material, 9 l Proteinase K (20 mg/ml) was added to each sample. Samples were physically disrupted by a series of freeze-thaw (3 times, 80oC to 65oC) cycles, followed by incubation at 55oC overnight. RNase was added to the digests and incubated at 37oC for up to 1 hr, following manufacturer instructions. Proteins and other cellular debris were precipitated in 7.5 M ammonium acetate by centrifugation. The supernatant was transferred to cold 100% isopropanol and then nucleic acids were precipitated overnight at 20oC. DNA was pelleted by 37 centrifugation and pellets were washed in 70% ethanol, air-dried, and stored in either sterile water or TLE buffer. DNA quality and quantity from each extraction were determined on a GeneQuantII spectrophotometer (Amersdam Biosciences, Piscataway, NJ). Nearly full-length community 16S rRNA gene sequences (>1300 bp) were PCR-amplified using the eubacterial primer and pair 1492r 27f (forward, (reverse, 5'5'- AGAGTTTGATCCTGGCTCAG-3') GGTTACCTTGTTACGACTT-3') (Lane, 1991). PCR is a method that amplifies a specific DNA sequence in vitro by repeated cycles of synthesis using specific primers and DNA polymerase. Amplification was performed with a Perkin Elmer 9700 thermal cycler and AmpliTaq Gold (Applied Biosystems, Branchnurg, NJ) under the following conditions: denaturation at 94oC for 1 min, primer annealing at 42oC for 1 min, chain extension at 72oC for 1.5 min, repeated for 35 cycles. A control tube containing sterile water instead of DNA was used as a negative control; Escherichia coli DNA was used as a positive control. 16S rRNA Gene Clone Library Construction Amplified PCR products were purified with the GeneClean II Kit (Bio101, Inc., Vista, CA), following manufacturer recommendations. Purified PCR products were cloned using the TOPO TA Cloning kit with E. coli TOP10F' cells, according to manufacturer instructions (Invitrogen, Carlsbad, CA). Molecular cloning involves the isolation and incorporation of a fragment of DNA (i.e., PCR product) into a cloning vector (a plasmid), where it is transformed into and perpetuated by a 38 population of host cells descended from a single cell. A collection of clones from one sample, each containing a replicated PCR product, comprises a clone library. Bacterial 16S rDNA clone libraries were constructed from eleven microbial mat morphotype samples in the Department of Integrative Biology, Brigham Young University, Provo, Utah. Plasmids containing nearly-full length inserts were extracted using a standard alkaline lysis miniprep method (Ausubel et al., 1990). Clone plasmids were digested simultaneously using EcoRI and RsaI (1U each) according to manufacturer's instructions (New England Biolabs) for restriction fragment length polymorphism (RFLP) analysis. RFLP patterns were visualized on 2% agarose gels stained with ethidium bromide and run in TBE (Tris-borateEDTA)-buffer (refer to Appendix A for RFLP clone library results). Clones representing unique patterns from each library were selected for sequencing (Appendix A; Table A-1). Sequencing of 16S rRNA Genes and Phylogenetic Analysis Sequence inserts from plasmid minipreps for each clone to be analyzed were PCR-amplified using the plasmid-specific primer pair M13(-20) (5'GTAAAACGACGGCCAGT-3') and M13(-24) (5'-AACAGCTATGACCATG3'). PCR products were purified using Sephadex columns and sequenced using an ABI Big-Dye Ready Reaction kit (Perkin Elmer) with the primers 27f and 1492r in conjunction with the internal primers and 704f 907r (forward, (reverse, 5'5'- GTAGCGGTGAAATGCGTAGA-3') CCGTCAATTCCTTTRAGTTT-3'). Automated DNA sequencing was done on an ABI Prism 377X sequencer (Perkin Elmer) at Brigham Young University. 39 DNA sequences were submitted to the CHECK-CHIMERA program of the Ribosomal Data Base Project (RDP) II at Michigan State University (http://rdp.cme.msu.edu/html/) (Maidak et al., 2001) to screen for and to eliminate chimeric sequences. Clone sequences were subjected to BLAST searches within the GenBank database (http://www.ncbi.nlm.nih.gov/) to determine 16S rDNA sequence similarities to culturable and not yet cultured organisms (Appendix A; Table A-1). Phylogenetic analyses were conducted by Megan L. Porter, Department of Microbiology and Molecular Biology, Brigham Young University, and detailed information can be found in Engel et al. (accepted). Briefly, nucleotide sequences were initially aligned using Clustal X (Thompson et al., 1997) and then manually adjusted based on conserved primary and secondary gene structure. Analyses were done after removal of segments that could not be unambiguously aligned corresponding to E. coli 16s rRNA secondary structure helices 9 and 10 (bp 181226; all alignments), helix 17 (bp 452-481; all but the Betaproteobacteria alignment), helices 25 and 26 (bp 822-860; Gammaproteobacteria and Bacteroidetes/Chlorobi-Acidobacterium alignments), helix 30 (bp 1028-1032; Betaproteobacteria and Deltaproteobacteria alignments), and helix 33 (bp 9951045; "Epsilonproteobacteria" and Bacteroidetes/Chlorobi-Acidobacterium alignments). All base pair positions correspond to E. coli numbering (Brimacombe et al., 1988). The minimum evolution criteria in the program PAUP* (Swofford, 2002), maximum likelihood criteria using a genetic algorithim (MLga) in MetaPIGA (Lemmon and Milimkovitch, 2002), and Bayesian inference coupled 40 with Markov chain Monte Carlo techniques (BMCMC) in MrBayes version 3.0b4 (Ronquist and Huelsenbeck, 2003) were used for phylogenetic analyses. As an indication of nodal support, bootstrap analyses were performed for minimum evolution (500 replicates) criteria using full heuristic searches. For BMCMC and MLga analyses, posterior probabilities were used for nodal support (Lemmon and Milimkovitch, 2002; Ronquist and Huelsenbeck, 2003). Statistical Analysis and Sequence Population Diversity To determine if the number of clones in each of the clone libraries was representative of microbial diversity, rarefaction curves were produced using the approximation algorithm aRarefactWin (Analytic Rarefaction, version 1.3, S. Holland, http://www.uga.edu/~strata/software/). The calculations for the program are based on Hurlbert (1971) and Heck et al. (1975). Rarefaction curves having 95% confidence levels were constructed by comparing the number of clones in each 16S rRNA gene library to the number of phylotypes from a particular library. Estimates of clone library species richness and species dominance/evenness indices (combined to represent heterogeneity; e.g., Rousseau and Van Hecke, 1999; Ricotta, 2003) were calculated based on the number of phylotypes identified from RFLP and taxonomic affiliations from GenBank BLAST searches (Hughes et al., 2001; Martin, 2002; Hill et al., 2003). The nonparametric methods Abundancebased Coverage Estimator (ACE) and Chao1, and the Shannon-Wiener biodiversity function expressed as the Shannon Index (H') were computed for each library using EstimateS (version 6.0b1, R.K. Colwell, http://viceroy.eeb.uconn.edu/estimates). Although there are problems with the interpretation of H' (refer to Hill et al., 2003, 41 for a complete discussion), it is the negative sum of each phylotype proportional abundance multiplied by the log of its proportional abundance, and is a measure of the difficulty in predicting the identity of the next individual sampled. The H' value is positively correlated to richness and evenness, but because it gives more weight to rare than to common phylotypes, it is more sensitive to absolute, but not relative, changes in abundance. The Shannon Evenness (E) and the Simpson's Dominance index (D) were also calculated based on equations presented in Hill et al. (2003). E gives the ratio of H' to the maximum possible value of H' that could be obtained theoretically with the observed number of phylotypes; E is sensitive to changes in evenness of rare phylotypes such that an increase in abundance of a rare phylotype will increase E more than the equivalent reduction in abundance of a dominant phylotype (Hill et al., 2003). D strongly weights dominant phylotypes, which provides the probability that two clones chosen at random will be from the same phylotype. D is useful expression in this RFLP study because the dominant organisms are similar in all libraries (Hill et al., 2003). A sequence similarity matrix was constructed for the closely related clone sequences from the "Epsilonproteobacteria" using corrected distances based on the model selected by Modeltest 3.06 (Posada and Crandall, 1998). Nucleotide Sequence Accession Numbers Nucleotide sequence data reported in this study are available in the GenBank database under the accession numbers AY208806 to AY208817 for LKC2-labeled clones, and AY510166 to AY510267 for LKC3-labeled clones. 42 RESULTS Spring and Stream Geochemistry The cave springs are all calcium-bicarbonate-sulfate water type and the geochemistry of the springs did not vary significantly from sample period to sample period (Table 2-1). Although the cave forms from sulfuric acid dissolution of limestone (Egemeier, 1981), the spring and stream pH was buffered to circumneutral by ongoing carbonate dissolution. Incoming spring water had an average concentration of total dissolved sulfide (CTS=) of ~38 mol L-1 and non-detectable dissolved oxygen (Table 2-1). Incoming CTS= was speciated as ~60% HS-:40% H2S (pK 7.04) based on the pH of the cave waters. The concentration of dissolved oxygen and CTS= changed downstream in all transects, such that at the end of the microbial mats CTS= decreased to non detectable and the concentrations of dissolved oxygen exceeded 40 mol L-1 (e.g., from the Upper Spring transect from 190 m to 203 m; Figure 2-1). The concentration of DOC in all the spring waters was extremely low, at <80 mol L-1 including methane. Morphologic Description of the Microbial Mats Four major mat morphotypes were distinguished by color and visual inspection in the cave when illuminated with white light. All three spring orifice pools contained long white filament bundles suspended in the water column. The Lower Spring (248 m) had the densest concentration of orifice bundles (Figure 22A), although the Upper Spring had the longest bundles at more than a meter in length. The entire microbial mat below the Lower Spring orifice pool was 2 to 5 cm 43 thick and less than 1 m in length; the mat was yellowish-white in appearance (Figure 2-2B). At the Upper Spring (190 m), long filament bundles in the orifice pool (Figure 2-2C, 2-2D) transitioned to shorter bundles that coalesced on the edges of the outflow channel (195 to 198 m), corresponding to a decrease in CTS= and an increase in dissolved oxygen concentrations (Figure 2-1). Very thin, short (1 cm in length) whitish-gray filaments covered stream sediments in flowing water at the bottom of the outflow channel (195 m). Approximately 6 m downstream from the Upper Spring orifice, the gray filaments thickened and were stratigraphically below long white filaments and thin white webs that occasionally had a bumpy or knobby texture (Figure 2-2E). Oxygen microelectrode profiles at 203 m showed oxygen tension abruptly decreased ~3 mm below the mat-water interface and anaerobic conditions (pO2 < 10 Pa) persisted within the 5 cm-thick mat interior, indicating that the mats are geochemically stratified. Gray filaments within the mat at 203 m (~2 cm below the white mat surface) were sampled to test for differences in the microbial community structure. For both the Upper and Lower Springs, dense white filamentous mats, with small (1-3 cm diameter) discontinuous yellow patches and feathery bundles (i.e., short, thick, and branching filaments), dominated the lower reach of the outflow channels (Figure 2-2F). Filament bundles near the orifice of the Fissure Spring (118 m) (Figure 2-2G) were also associated with web-like structures (125 m) (Figure 2-2H). SEM examination of several mat samples revealed complex organic and inorganic structures as part of the overall mat structure. White filamentous material revealed a tight filament network (Figure 2-3A) with branching and non-branching 44 filaments averaging 1 m wide (Figure 2-3B through 2-3D). Some thin filaments (~0.5 m wide) were twisted and associated with long rods (Figure 2-3E and 2-3F). In comparison, gray sediment from the Upper Spring orifice (Figure 2-4) had few filaments and Fe-S framboids and crystallites were common (Figure 2-4A through 2-4D), indicating a reducing microenvironment within the sediments. Cell clusters were also observed (Figure 2-4E and 2-4F). C:N Ratios, Sulfur Content, and Biomass Estimates The N contents varied by mat morphotype (Table 2-2), and white filaments and white webs had the highest N content compared to gray filaments or gray sediment. Generally, the lower the C:N ratio, the higher the quality of the mat as a food source for the ecosystem (Fagerbakke et al., 1996). The mean C:N ratios for white filament morphotypes was 5.0 (1 = 0.8), suggesting a high quality food source. The C:N ratios of gray filaments were significantly higher and more variable than white mat morphotypes, with a mean of 15.0 (1 = 10.5). The C:N ratios were highest for gray filaments and sediment from spring orifice sites, suggesting that this biomass consisted of lower quality organic matter, while the C:N ratios for gray filaments at the end of the microbial mats approached those of the white mat morphotypes (Table 2-2). The sulfur content of the white filaments was higher than the gray filaments (Table 2-2), presumably reflecting intracellular sulfur (as elemental S0) rather than organosulfur compounds. Typically, in the absence of stored sulfur the highest bacterial cell sulfur content ranges up to 1% (w/w) (Fagerbakke et al., 1996). 45 However, the white filament sulfur content had a mean of 30.0% (1 = 11.2%), and the white webs had consistently the highest sulfur content. The gray filaments and sediment had significantly lower sulfur contents, with a mean of 1.9% (1 = 0.6%), consistent with predicted values for bacterial biomass (Fagerbakke et al., 1996). The sulfur content of white filaments was generally the same at the extreme upstream and downstream samples of the Upper Spring transect, but decreased by up to 10% in the middle stream reach (Table 2-2). The biomass of the microbial mat samples was generally high (~1010 cells ml-1), with less than an order of magnitude difference between white and gray filament morphotypes (Table 2-2). Biomass values may underestimate the actual biomass because cell conversion factors are for rod-shaped cells, and FISH suggests that the mats are dominated by filamentous morphotypes (Engel et al., 2003; refer to Chapter 3). Carbon Isotope Systematics The 13C value for the limestone bedrock was +0.95, and the DIC reservoir along the Upper Spring transect had an average 13C value of -7.5 (n = 7, 1 = 0.1), and DIC from the Fissure Spring orifice water had a slightly higher 13C value of -7.2. Microbial mat morphotypes had 13C values ranging from -23 to -41 (mean -34.1, 1 = 4.1) (Figure 2-5). The low 13C values reflect the large discrimination against 13 C exhibited by autotrophs (e.g., ~25 relative to total DIC for sulfur-oxidizing bacteria; Ruby et al., 1987). 46 Microbial mat morphotypes showed systematic variations in their carbon isotope compositions at most locations (Figure 2-5). At all three spring locations, gray filaments consistently had among the highest 13C values, whereas all coexisting white filaments had lower 13C values. More specifically, near the distal portions of the Upper Spring mats (203 m), white feathery bundles and yellow patches (Figure 2-2F) had some of the lowest 13C values, whereas the white webs and gray filaments had the highest 13C values (Figure 2-5). In contrast, however, the feathery bundles from the proximal region of the Upper Spring mats (196 m) had among the highest 13C values. Moving downstream, the 13C value of the white filaments in both Upper Spring and Fissure Spring decreased (Figure 2-5). Sulfur Isotope Systematics Allochthonous sulfide (CTS=) at the Upper Spring orifice had a 34S value of ~-22.5, and decreased downstream by approximately 1.6 (Figure 2-6). Dissolved sulfate had a 34S value of +13 and was invariable. The white filamentous morphotypes, containing high concentrations of sulfur (Table 2-2), had sulfur isotope values up to 2 lower than the allochthonous sulfide (Figure 2-6). Clone Library Coverage, Species Richness, and Diversity Eleven bacterial 16S rDNA clone libraries from four different microbial mat morphotypes were constructed and over 1000 clones were screened using RFLP (Appendix A). Although the RFLP method does not provide an absolute quantity of microbial groups within a community, it does provide an estimate of the proportional abundances of microbial groups in a given sample by assuming an 47 RFLP pattern of an abundant `species' will be encountered more often in the clone library (N bel et al., 1999; Morris et al., 2002). Nearly-full length 16S rRNA genes (>1300 bp) were sequences in both directions from selected clones. Sequences from the same RFLP pattern that had 98% sequence similarity were grouped as a phylotype, and this classification scheme was used to estimate community diversity (Table 2-3). This level of sequence similarity takes into account micro-variations in genetic sequences due to PCR and cloning biases and variations in 16S rRNA gene copies (N bel et al., 1999; Speksnijder et al., 2001). Approximately 2% of the clone 16S rRNA gene sequences were chimera and removed from further analyses. Of the phylotypes identified, 44% had sequences that were 95% identical to GenBank sequences, corresponding to genus-level relationships (Stackebrandt and Goebel, 1994), and 30% of the sequences were 98% identical to GenBank sequences, corresponding to species-level relationships (Stackebrandt and Goebel, 1994). The remaining phylotype sequences had 90% sequence similarity to GenBank sequences (Table 2-3; Appendix A; Table A-1). Rarefaction analysis was done to determine if the libraries had saturated coverage based on the number of clones obtained per library. The rarefaction curves indicated different patterns of diversity for different morphotype clone libraries (Figure 2-7). In the non-filament clone libraries (203g, 203w, 203y, and 248y), diversity was not fully covered compared to the saturation plateau reached for most of the white filament libraries (124f, 127f, 190f, 198f) (Figure 2-7). The non-filament libraries had higher phylotype richness overall and higher nonparametric estimates (ACE and Chao1; Table 2-4). As there was an overall 48 increase in the rate of phylotype accumulation in these unsaturated curves, major diversity within these libraries may not be well represented, although some of these libraries (e.g., 203y) do have high dominance (D) values (Table 2-4). Species heterogeneity among the clone libraries was generally low and many of the white filament libraries showed overwhelming dominance by one of two phylotypes. Species richness was higher for the non-filament morphotypes, with the white webs from 203 m and the yellow patches from 203 m and 248 m showing the most diverse taxonomic representation among the eleven bacterial clone libraries (Table 2-3), even though observed species richness was lower than expected based on ACE and Chao1 values (Table 2-4). In comparison, although species richness of the white filament libraries varied, ranging from one to ten observed phylotypes, ACE and Chao1 estimates for the white filament libraries indicated that the observed phylotype numbers were close to the calculated values because of near-complete clone coverage for most of those libraries (Table 2-4). The diversity (H')/dominance (D) indices changed for the white filament clone libraries downstream for both the Upper and Lower Spring transects, such that the H' values increased and D values correspondingly decreased (Table 2-4). Phylogenetic Analysis of 16S rRNA Gene Clone Libraries The 16S rDNA clones were affiliated with several bacterial phyla (Table 23; Figures 2-8 through 2-12). The majority of the sequences identified from the clone libraries belonged to the Proteobacteria taxonomic division, specifically the "Epsilonproteobacteria," (68%) Gammaproteobacteria (12.2%), Betaproteobacteria (11.7%), and Deltaproteobacteria (0.8%) classes, as well as other 49 bacterial divisions, including the Acidobacterium (5.6%) and Bacteroides/Chlorobi (1.7%). The "Epsilonproteobacteria" class The highest number of clones from all the libraries (68%) was assigned to the "Epsilonproteobacteria" (Table 2-3; Figure 2-8). Epsilonproteobacterial sequences from 17 phylotypes clustered into six groups based on phylogenetic position (Figure 2-8) and the sequence similarity matrix (Table 2-5), which suggests that genetic microdiversity was high (Fuhrman and Campbell, 1998). At least one epsilonproteobacterial phylotype was found in all clone libraries regardless of morphotype type or location (Table 2-3). The epsilonproteobacterial groups identified from Lower Kane Cave have few closely related sequences from the public sequence databases, suggesting that the diversity of these groups, and the "Epsilonproteobacteria" in general, is only now being realized. The most abundant epsilonproteobacterial groups from all three springs formed two distinct clades, LKC group I and group II. No other epsilonproteobacterial phylotype was detected in a clone library if either LKC group I or II was absent. The closest relatives to LKC groups I and II were the two environmental clones, sipK119 and sipK94, respectively (98-99% similar in nucleotide sequence), from microbial mats with a string-of-pearls morphology in sulfidic marsh springs at the Sippenauer Moor, Regensburg, Germany (Rudolph et al., 2001; Moissl et al., 2002). Clone sequences from LKC group I were also closely related (97-99% similar in nucleotide sequence) to environmental clones obtained from a petroleum-contaminated, sulfidic groundwater storage cavity in 50 Japan (Watanabe et al., 2000; Watanabe et al., 2002) and two clones from microbial mats from the sulfidic Cesspool Cave, Virginia (Engel et al., 2001) (Figure 2-8). The closest cultured relative for LKC groups I clones is Sulfuricurvum kujiense; this organism is a slightly curved rod isolated as a chemolithoautotrophic sulfur-oxidizer, capable of growth on thiosulfate, elemental sulfur, and hydrogen sulfide, and able to use molecular oxygen, nitrate, or ferric iron as electron acceptors (Kodama and Watanabe, 2003). LKC group II clones were more distantly related (90-94% similar in nucleotide sequence) to miscellaneous marine, hydrothermal vent field and epibiont clones (e.g., Longnecker and Reysenbach, 2001; Lopez-Garcia et al., 2002) and clones from a sulfidic cave microbial mat in Parker Cave, Kentucky (Angert et al., 1998) (Figure 2-8). The phylogenetic affinities (Figure 2-8) and the sequence similarity matrix (Table 2-5) demonstrate that group I and group II are distinct from each other at more than the genus-level (85-87% similar in nucleotide sequence). LKC group III did not form a distinct phylogenetic cluster and was defined by several phylotypes from five libraries, supported by the range of sequence similarities among the sequences and moderate boot-strap node values (Table 2-5; Figure 2-8). No LKC group III clones were found at the Lower Spring. The relatives to the epsilonproteobacterial LKC groups III and IV were similar to LKC group I clones (Table 2-5). LKC group IV, comprised of clones only from the Upper Spring, clustered closely with S. kujiense (Kodama and Watanabe, 2003), and groundwater and cave environmental clones (Figure 2-8). Based on the similarity matrix, LKC groups III 51 and IV had low sequence similarity within the groups, although each library was distinct from all the other groups (Table 2-5). LKC group V also had a wide range of sequence similarity (Table 2-5), indicating additional diversity within this group that could not be resolved by RFLP. LKC group V was most closely related to LKC group II clones (Table 2-5). Seventeen clones from four morphologically diverse libraries, but mostly white filament morphotypes, belonged to the novel sequence cluster LKC group VI, which was genetically distinct from all the other epsilonproteobacterial groups (Table 2-5). The closest relatives to LKC group VI clones were environmental clones from subsurface acid mine drainage and groundwater (95-96% similar in nucleotide sequence). The Gammaproteobacteria class Twelve percent of all the clones belonged to the Gammaproteobacteria (Figure 2-9). Eighty-one clones formed one phylotype, closely related to the environmental clone sipK4 from sulfidic marsh springs (99-100% similar in nucleotide sequence) (Rudolph et al., 2001), which is also closely related to Thiothrix unzii. The relative abundance of this phylotype generally increased downstream from the orifice to the end of the mats for all three spring transects (Table 2-3). Cultured representatives from Thiothrix oxidize a variety of reduced sulfur compounds and several Thiothrix species have been identified from other sulfidic caves, including Parker Cave (Angert et al., 1998), underwater caves and karst springs in Florida (Brigmon et al., 2003), and Cesspool Cave (Engel et al., 2001). T. unzii, an aerobic sulfur-oxidizing bacterium that utilizes sulfide and 52 thiosulfate as sole sulfur sources and stores sulfur intracellularly (Howarth et al., 1999), and environmental clones closely related to T. unzii are also commonly associated with activated sludge and wastewater treatment plants (e.g., Wagner et al., 1994; Howarth et al., 1999), as well as sulfidic springs where it is the dominant group (Rudolph et al., 2001; Moissl et al., 2002). Clone library 203g was dominated by clones belonging to the Enterobacteriaceae, specifically the Pantoea and Serratia genera (Figure 2-9). The libraries 203g and 248y had six clones closely related to Serratia marcescens (99% similar in nucleotide sequence). Nine sequences from the 124f and 203w libraries were distantly related to Beggiatoa sequences (90% similar in nucleotide sequence), with one relative being the isolate Beggiatoa sp. MS-81-1c from a salt marsh, described as a narrow and non-vacuolate filament (Ahmad et al., unpublished GenBank submission) (Table 23; Figure 2-9). The weak sequence similarity to known Beggiatoa sequences and low bootstrap values between the clone group and Beggiatoa sequences (Figure 29), however, indicate that Lower Kane Cave clones may belong to a different, unclassified bacterial group within the Gammaproteobacteria. Beggiatoa-like filaments have been described from a marine cave in Italy using microscopy (Airoldi et al., 1997) and from microbial mats in Parker Cave, Kentucky (Thompson and Olson, 1988), although detailed phylogenetic investigations from Parker Cave did not support the presence of Beggiatoa (Angert et al., 1998). 53 The Betaproteobacteria class Nearly twelve percent of the clones were affiliated with the Betaprotoebacteria, and were most closely related to Thiobacillus spp. (Figure 210). Three phylotypes were identified from two libraries. The closest relatives were the environmental clone 44a-B2-21 from acid mine drainage (Labrenz and Banfield, unpublished GenBank submission) and Thiobacillus aquaesulis (94-95% sequence similarity). Many Thiobacillus spp. oxidize reduced sulfur compounds (Kelly and Harrison, 1989), and T. aquaesulis is not an obligate sulfur-oxidizing chemolithoautotroph, but can grow as a facultative heterotroph on nutrient broth and yeast extract (McDonald et al., 1996). Thiobacilli have been described from caves (Vlasceanu et al., 1997; Angert et al., 1998; Hose et al., 2000; Vlasceanu et al., 2000; Engel et al., 2001), but environmental clones from those studies were not closely related to the Lower Kane Cave group. The Deltaproteobacteria class Less than 1% of the clones were closely related (96-97% similar in nucleotide sequence) to Desulfocapsa thiozymogenes, the environmental clone sipK94 from the string-of-pearls mats in Germany (Moissl et al., 2002), and the environmental clones SRB348 and SRB282 identified from the chemocline of the meromictic Lake Cadagno, Switzerland (Tonolla et al., 2000) (Table 2-3; Figure 211). D. thiozymogenes disproportionates thiosulfate, sulfite, or elemental sulfur to sulfate and sulfide (Janssen et al., 1996). 54 The Acidobacterium division One phylotype representing 5.6% of all the clones obtained from this study was closely related (96-97% similar in nucleotide sequence) to uncultured environmental clones within the Acidobacterium division. Library 203w was dominated by this clone group, and rare clones from this phylotype were found in five additional libraries (Table 2-3). The closest relative was clone SJA-36 identified from an anaerobic bioreactor with trichlorobenzene contamination (von Wintzingerode et al., 1999) (Figure 2-12). The Lower Kane Cave phylotype also expands the Acidobacteria-group 7 described by Liles et al. (2003), which consists of only a few environmental clones from soil, as well as the Acidobacteriasubgroup-b described by Schabereiter-Gurtner et al. (2002) from La Garma Cave, Spain. Clone LKC3_156.13 had 92% sequence similarity to clone SJA-36, but also clustered as an unclassified taxonomic group within the Bacteroidetes phylum by phylogenetic analysis (Figure 2-12). Cultivated members of the Acidobacteria division are heterotrophs, although Acidobacteria have been found in many geochemically different environments, including neutral and acidic soils, marine and freshwater sediments, hot springs, deep-sea vents, and engineered systems (Barns et al., 1999; Schabereiter-Gurtner et al., 2002; L pez-Garc a et al., 2003; Schabereiter-Gurtner et al., 2003). The Bacteroidetes/Chlorobi division Seven phylotypes, each represented by rare `singleton' or `doubleton' clones, belonged to the Bacteroidetes/Chlorobi (BC) taxonomic group (Table 2-3; Figure 2-12). Three phylotypes (BC groups I-III) were closely related to 55 environmental clones within the Bacteroides class, including clones from lakes and contaminated groundwater. Four phylotypes (BC groups IV-VII) were related to environmental clones within the Sphingobacteria class (including the genus Cytophaga) obtained from a wide habitat range, including deep-sea hydrothermal vent metazoans, gas hydrate sediment, soil, and contaminated groundwater. DISCUSSION Terrestrial subsurface environments are often inaccessible for study, limiting our understanding of ecosystem structure and dynamics, elemental cycling, and potential biogeochemical impacts to earth and atmospheric processes. The main research goals of this part of the study were to identify the bacterial groups characteristic of the cave microbial mats, to gain an understanding of how geochemistry may control microbial community diversity within the aphotic environment, and to elucidate potential ecosystem functioning and the impact of sulfur cycling and chemolithoautotrophy on the ecosystem. The results of this work suggest that similar microbial communities and concomitant microbially mediated biogeochemical cycles may be more widely dispersed in sulfidic groundwater habitats than previously recognized. "Epsilonproteobacteria" Diversity and Ecophysiology The majority of 16S rRNA gene sequences from the clone libraries were affiliated with the "Epsilonproteobacteria," and specifically with two epsilonproteobacterial groups defined as LKC Group I and II (Figure 2-8). Additionally, there was a high level of genetic microdiversity within the 56 "Epsilonproteobacteria" identified in this study based on the range of sequence similarities for the obtained epsilonproteobacterial clones (Table 2-5). Numerous 16S rDNA-based molecular surveys suggest that "Epsilonproteobacteria" successfully colonize a variety of geochemical settings, including caves (Angert et al., 1998; Engel et al., 2001; Barton et al., 2002), deep aquifers (Pedersen et al., 1997), terrestrial sulfidic springs and groundwater (Watanabe et al., 2000; Rudolph et al., 2001; Moissl et al., 2002; Watanabe et al., 2002; Elshahed et al., 2003; Kodama and Watanabe, 2003), oil fields (Voordouw et al., 1996), deep marine sediments and ocean water (Fenchel and Glud, 1998; Li et al., 1999a; Li et al., 1999b; Todorov et al., 2000; Madrid et al., 2001), hydrothermal vent sites (Moyer et al., 1995; Muyzer et al., 1995; Polz and Cavanaugh, 1995; Brinkhoff et al., 1999; Reysenbach et al., 2000; Corre et al., 2001; Longnecker and Reysenbach, 2001; Takai et al., 2003), in association with deep-sea animal life at vent sites (Haddad et al., 1995; Cary et al., 1997; Naganuma et al., 1997; Alain et al., 2002a; Lopez-Garcia et al., 2002; L pez-Garc a et al., 2003), and in engineered systems including sewage sludge and contaminated waste (Engberg et al., 2000; On, 2001). Based on the geochemical diversity of these study sites and the lack of culture-based information from most phylogenetic groups within the "Epsilonproteobacteria", especially those groups identified from terrestrial settings, it is challenging to predict what the ecophysiological roles of "Epsilonproteobacteria" in Lower Kane Cave might be (Gray and Head, 2001). In many environments "Epsilonproteobacteria" are implicated in sulfur cycling, and specifically in oxidizing reduced sulfur compounds; this is supported 57 by several culture-based studies (Finster et al., 1997; Stolz et al., 1999; Gevertz et al., 2000; Campbell et al., 2001; Nemati et al., 2001; Alain et al., 2002b; Miroshnichenko et al., 2002; Kodama and Watanabe, 2003; Takai et al., 2003). Additionally, in nearly all of these culture-based studies, the epsilonproteobacterial strains grew chemolithoautotrophically. Therefore, reasonable metabolic hypotheses can cautiously be made for the "Epsilonproteobacteria" from Lower Kane Cave based on cave biogeochemistry. It is possible that the organisms represented by LKC groups I, III, and IV clones may have metabolic similarities to S. kujiense, which grows under microaerophilic to anaerobic conditions. However, alternative electron acceptors such as nitrate and ferric iron are exceptionally low in the cave waters (Table 2-1). Closely related phylogenetic groups, however, do not necessarily indicate similar ecophysiological characteristics (Fuhrman and Campbell, 1998), as Takai et al. (2003) showed that the observed phylogenetic distribution of epsilonproteobacterial cultures isolated from deep-sea vents did not correlate with substrate or electron acceptor preferences, oxygen requirements, or geographic location. These deep-sea vent cultures were non filamentous and affiliated with five distinct epsilonproteobacterial taxonomic groups, yet had high metabolic diversity which included sulfur and thiosulfate oxidation using nitrate or molecular oxygen (at 1% or 10%) or H2-dependent sulfur-reduction (Takai et al., 2003). Clearly, the high concentrations of dissolved sulfide discharging from the springs provide a rich energy source for sulfur-oxidizing bacteria. Although it is unlikely that abiotic autoxidation (i.e., chemical oxidation) and volatilization cause 58 sulfide loss exclusively, there was an observed decrease in dissolved sulfide concentrations downstream (Figure 2-1). Abiotic sulfide autoxidation is extremely slow in disaerobic water at pH ~7.4 (the autoxidation half-life was calculated at >800 hours; H2S:HS- pK 7.04) and sulfide volatilization from the water to the atmosphere accounts for <8% of the sulfide loss in the stream based on gas flux experiments (see Chapters 5 and 6). With no other mechanism for CTS= loss, there would be, for example, significantly higher sulfide concentrations at the end of the Upper Spring microbial mat, as well as at the cave entrance 150 m further downstream. However, a very rapid decrease in CTS= is observed (Figure 2-1). Microbial catalysis under microaerophilic conditions causes rapid sulfide consumption; therefore, most of the epsilonproteobacterial groups identified must consume dissolved sulfide as sulfur-oxidizers (see Chapters 5 and 6). Another line of evidence to suggest that "Epsilonproteobacteria" in the microbial mats oxidize sulfur compounds is from the measured 34S values (Figure 2-6). The decrease in 34S values for CTS= from the cave spring to the end of the mats, by up to 2, indicates that allochthonous 34S values are being offset by biogenic sulfide generated by sulfate-reducing bacteria (e.g., Desulfocapsa thiozymogenes) within the mats (Canfield, 2001b). H2S produced by sulfatereducing bacteria would have more negative 34S values isotopes than the incoming sulfide, as the 34S discrimination expressed by sulfate reduction is variable and can be as high as 45 (Canfield, 2001a; 2001b). In comparison, the processes of sulfide volatilization (Van Everdingen et al., 1985; Fry et al., 1986) and abiotic autoxidation (Fry et al., 1988) would result in a 59 34 S enrichment in the residual dissolved sulfide. Because there is a high sulfur concentration in the microbial biomass, presumably as intracellular sulfur, the 34S values for microbial biomass are interpreted as a direct measure of the sulfide source utilized by the sulfuroxidizers. Current thought is that sulfur-oxidizing bacteria exhibit negligible (+/1) sulfur isotope fractionation during both the transformation of sulfide to elemental sulfur and elemental sulfur to sulfate (Toran and Harris, 1989). Specifically, if the bulk of the microbial biomass that oxidized reduced sulfur compounds utilized only allochthonous sulfide, then the 34S values for the mats would be nearly the same as the spring water sulfide (Kaplan and Rittenberg, 1964). The general 34 S depletion in the microbial biomass relative to the source (i.e., dissolved sulfide) suggests that sulfur-oxidizing bacteria utilize a mixture of both allochthonous and autochthonous sulfide (Toran and Harris, 1989; Canfield, 2001a). Geochemical Controls on Community Structure and Ecosystem Function Studies from aquatic environments suggest that shifts in community structure could result from changes in nutrient availability, salinity, light penetration, turbidity, oxygen content, sulfide, or pH (e.g., Skirnisdottir et al., 2000; N bel et al., 2001). At present, however, there have not been any investigations that describe the controls on changing community structure from a freshwater aphotic habitat. Specifically, light penetration, turbidity, and salinity are not critical physicochemical conditions to influence cave microbial communities, and changes in the pH of the cave waters are not important because of the buffering of pH to circum-neutral conditions by dissolving carbonate rock. Instead, I propose 60 that 1) variations in dissolved hydrogen sulfide concentrations, 2) increasing dissolved oxygen concentrations downstream, 3) colonization of the springs and outflow channels by "Epsilonproteobacteria", and 4) changes in organic carbon production and storage as a result of chemolithoautotrophy by epsilonproteobacterial groups are the most critical parameters affecting microbial community structure within the microbial mats. The relative abundances of epsilonproteobacterial and other taxonomic groups shifted moving downstream with changing dissolved sulfide and oxygen concentrations (Table 2-3). In general, the abundance of both epsilonproteobacterial LKC groups I and II decreased from the orifice pools downstream, and the highest abundance of epsilonproteobacterial LKC group I was from samples where the concentration of dissolved oxygen was very low at both the Fissure and Upper Springs. Clone libraries from the three spring orifices, which originated from areas continuously replenished by sulfidic spring water, were dominated by one epsilonproteobacterial group, whereas downstream libraries had higher bacterial diversity (Table 2-3). For example, at the Lower Spring, all clones screened by RFLP belonged to the epsilonproteobacterial LKC group II, whereas 1 m downstream in the microbial mat nine other bacterial groups were identified. At the three springs, there was also an increase in the abundance of Thiothrix- and/or Thiobacillus-like clones downstream, which is in accordance with the characterized metabolism and sulfide and oxygen preferences for these groups (e.g., Howarth et al., 1999). Specifically, Thiothrix spp. tolerate or require higher dissolved oxygen concentrations compared to what is known about the oxygen requirements for some 61 cultured "Epsilonproteobacteria". At the Upper Spring, LKC group III was most abundant in downstream libraries (203f) where the dissolved oxygen concentration was higher, suggesting that while this group may be involved with sulfur cycling, this group may prefer higher habitat oxygen content. The dominance of Acidobacterium in the 203w clone library, from the surface of the mat where dissolved oxygen is highest, suggests that this group prefers higher oxygen and lower sulfide concentrations. Sulfur storage in the microbial mats from the three springs, as indicated by the sulfur content of the mats, also changed downstream (Table 2-2), which corresponds to the types of microbial groups present in the mats. The highest sulfur content was from white filament bundles collected from the orifices and from the mat termini; lower sulfur contents were measured from the filament libraries from the middle of the stream transects. There is no indication from cultures that "Epsilonproteobacteria" store sulfur intracellularly like Thiothrix spp. (Howarth et al., 1999), possibly as a mechanism to attenuate changes in substrate availability in the environment (Chesson et al., 2002). The marine epsilonproteobacterial strain "Candidatus Arcobacter sulfidicus" forms extracellular sulfur filaments (Taylor et al., 1999; Wirsen et al., 2002) and cultures of nitrate-reducing sulfur-oxidizing "Epsilonproteobacteria" form sulfur as the metabolic end-product when nitrate is limiting or absent (Gevertz et al., 2000; Nemati et al., 2001). Some neutrophilic Thiobacillus spp. are also known to precipitate sulfur outside the cell (Kelly and Harrison, 1989). Therefore, the high sulfur content in white filaments from the spring orifice samples, dominated by "Epsilonproteobacteria" could be due to 62 extracellular sulfur or sulfur accumulation due to nitrate-reduction. Higher sulfur content in downstream samples could also be due to incomplete sulfide oxidation to elemental sulfur by Thiothrix spp. The exceptionally low sulfur content in the 203y sample (8.6%) compared to other morphotypes may be because the thiobacilli oxidize sulfur within the mat morphotype due to the diminished dissolved sulfide concentration in the stream water. Therefore, the distribution of sulfur content in the microbial mats may explain how several bacterial groups with similar metabolic requirements (e.g., presence of reduced sulfur compounds) coexist, as there appears to be distinct differences in spatial and temporal resource use by the microbial populations in the mat morphotypes moving downstream (Chesson et al., 2002). Based on experiments from deep-sea vent sites where "Epsilonproteobacteria" are the first to colonize virgin surfaces, L pez-Garc a et al. (2003) suggest that epsilonproteobacterial groups initially and rapidly diversify metabolically within a habitat (natural or artificial), and thereby create microniches (such as anoxic regions) where other bacteria will subsequently colonize. High diversity among the specialized "Epsilonproteobacteria" would essentially maximize ecosystem functionality of other microbial groups and make the entire system more productive because of high growth rates, significantly higher biomass, and quick adaptations to specific geochemical conditions of the habitat. Additionally, Chesson et al. (2002) describe the tendency for the most productive species to also be the most dominant in a habitat, and thereby push others species to comparatively lower densities. These ecological caveats may explain why the 63 microbial mats in Lower Kane Cave have high diversity within the "Epsilonproteobacteria", but lower bacterial diversity overall. The bacterial community composition of the 203g clone library is one of the most telling examples of the controls geochemistry has on community structure, and perhaps as an ecological consequence of "Epsilonproteobacteria" creating anoxic regions within the mat. The interior of the mat was dominated by clones with 99% nucleotide similarity to Pantoea agglomerans and environmental clones. Pantoea spp. are ubiquitous in natural and engineered environments, and are saprophytic, facultative anaerobes with diverse metabolic capabilities, including dissimilatory metal reduction. Recently, Northup et al. (2003) report a single Pantoea 16S rDNA clone from corrosion residues in Lechuguilla Cave, New Mexico, a cave that formed over several million years from sulfuric acid dissolution. This clone, however, is not closely related to those from Lower Kane Cave. There were also clones with 99% sequence similarity to Serratia marcescens, which is common to soil, water, and plants. S. marcescens is saprophylic and an opportunistic pathogen known to cause a variety of infections and diseases (Su et al., 2003). Although rare clones closely related to D. thiozymogenes were also identified from some samples (Table 2-3), preliminary culture investigation of gray filaments and other mat samples suggest that sulfate-reducing bacteria are present (see Chapter 4). The presence of fermenting and sulfate-reducing bacteria in the deepest, most anaerobic portion of the mat is consistent with reducing conditions measured from oxygen microelectrodes, the presence of reduced mineral phases (Figure 2-4), 64 and the low C:N ratios for the gray sediment and filaments (Table 2-2). The ratios suggest enhanced processing of biological material due to heterotrophy, and possibly fermentation. While molecular methods allow for the characterization of organisms that are difficult, if not impossible to cultivate (e.g., Head and Gray, 1998), unfortunately molecular methods can create significant biases and underestimations of particular microbial groups, especially if abundances are <107 cells per volume (von Wintzingerode et al., 1997; Speksnijder et al., 2001). Therefore, because this part of the study focused on the white filament morphotypes, it is likely that the diversity of anaerobes is underrepresented with respect to total genetic diversity, and combined culture- and molecular-based approaches are necessary. Chemolithoautotrophy in the Subsurface Most caves are traditionally energy- and nutrient-limited, commonly fed by surface streams in which photosynthetically-derived organic matter and sediments, as well as microorganisms, are washed into the subsurface and deposited (Poulson and Lavoie, 2000; Simon et al., 2003). Previous studies have shown that most microorganisms in caves are not chemolithoautotrophs, but instead are translocated soil heterotrophs, chemoorganotrophs, or fecal coliform bacteria from contaminated surface water (Poulson and Lavoie, 2000). Mikell et al. (1996) estimate that 75% of microbial communities in caves are heterotrophs. While it would be difficult to assess the metabolic pathways for all the microbial groups identified from the clone libraries using culture-based methods, or even to speculate about the metabolism for some groups with no cultured relatives, I used stable carbon isotope systematics 65 to interpret the source of carbon to the microbial mats, as well as to determine how carbon is cycled among different metabolic guilds. I propose that primary productivity was linked to the sulfur cycle through chemolithoautotrophy, as the geochemistry of the cave waters is consistent with reduced sulfur compounds being important components of the energy budget of the microbial ecosystem and because the most abundant microbial groups are associated with sulfur metabolism. While the Bighorn River may have had a role in inoculating the cave with microorganisms during past flood stages (refer to Figure 1-10), I suggest that the microbial communities in Lower Kane Cave are endemic and virtually unaffected by surface hydrologic conditions, on the basis of these findings: 1) the discharging cave springs contribute little to no allochthonous DOC or particulate organic carbon to the microbial community (Table 2-1); 2) bacterial groups common to most soil environments, as evidence of translocated soil microbes, were not identified based on 16S rDNA phylogenies; and 3) the filamentous microbial biomass in Lower Kane Cave reaches at least 1010 cells ml-1 (Table 2-2), significantly higher than 102 to 104 cells ml-1 commonly found in aquatic cave systems (Brown et al., 1994). Furthermore, the overall carbon isotope compositions of the microbial biomass reflect significant isotopic discrimination against 13 C relative to the inorganic carbon source (Figure 2-5), with 77% of the microbial mat samples having 13C values 30, well below that of terrestrial biomass (Coplen et al., 2002), demonstrating that inorganic carbon is predominately fixed through chemolithoautotrophy. Porter (1999; unpublished data) verified chemolithoautotrophic productivity from the white filamentous microbial mats at 66 the Lower Spring by H14CO3-assimilation, which suggested that there was more than six times higher autotrophic productivity than heterotrophic productivity, as tested by 14C-leucine-incorporation. The results of the 16S rRNA gene phylogenies also suggest that only a very minor component of the community is potentially heterotrophic. Chemolithoautotrophy in a cave ecosystem is important because it serves as the base for the cave food web, increasing both food quality and quantity (Kinkle and Kane, 2000; Poulson and Lavoie, 2000). Movile Cave, Romania, was the first documented chemolithoautotrophically-based cave and groundwater ecosystem (Sarbu et al., 1996), and subsequently chemolithoautotrophy has been found in several different sulfidic caves systems around the world, including marine caves from Cape Palinuro, Italy (Airoldi et al., 1997), the Frasassi Caves, Italy (Vlasceanu et al., 1997; Sarbu et al., 2000), Cueva de Villa Luz, Mexico (Hose et al., 2000), Cesspool Cave (Engel et al., 2001), and the flooded Nullarbor caves, Australia (Holmes et al., 2001). The bulk of the white filament microbial biomass in Lower Kane Cave has low C:N ratios averaging 5.0, compared to a C:N ratio of 5.7 for microbial mats from Movile Cave (Kinkle and Kane, 2000). The C:N ratios for Lower Kane Cave mats match previously reported ratios for bacterial cells (C:N = 3 to 5; Paul and Clark, 1996), but also to periphyton in surface streams (C:N = 4 to 8; Gregory, 1983) and bacteria from a marine hydrothermal vent site (C:N = 3.8 to 9.4; Gugliandolo and Maugeri, 1998). These C:N ratios are consistent with an insignificant influx and processing of allochthonous carbon, and instead suggest that carbon is provided in situ through autotrophy. The especially low C:N ratios 67 for the white filaments from Lower Kane Cave suggest a high quality food that could be used by heterotrophic microorganisms and higher trophic levels (McMahon, 1975). Incidentally, there is a large population of snails (Physa spelunca) that graze on the microbial mats in Lower Kane Cave. In contrast, the high C:N ratios in the gray filaments indicate carbon storage and accumulation of processed biomass, but a reduction in nitrogen availability. The mechanisms for inorganic carbon fixation were not evident based on carbon isotope analyses, as there are several different pathways for inorganic carbon fixation, and not all fixation pathways and their isotopic effects are known. Microorganisms that fix CO2 by the Calvin reductive pentose phosphate pathway (Calvin-Benson-Bassham cycle), the predominant and most important CO2-fixation pathway for photosynthetic and chemosynthetic bacteria, have isotopic values that fall into two categories based on the form of CO2-fixing enzyme, ribulose- 1,5bisphosphate carboxylase/oxygenase (RubisCO) (Robinson and Cavanaugh, 1995). The fixation pathway using the reductive citric acid (TCA) cycle imparts a smaller (~-10) carbon isotope fractionation (Preu et al., 1989; Taylor et al., 1999; Wirsen et al., 2002). Nearly all of the mat samples from Lower Kane Cave had 13C values that fit into the RubisCO form I group, ranging between -27 to -35 (the `-30 group') (Robinson and Cavanaugh, 1995). Physicochemical conditions, such as velocity, water depth, temperature, pH, and CO2 concentrations, can affect the effective isotope discrimination of autotrophs, which would result in tremendously different discrimination values (Preu et al., 1989; Simenstad et al., 68 1993; France, 1995; France and Cattaneo, 1998). However, the Upper Spring transect stream water maintains constant chemistry and turbulent flow. Nutrient Spiraling There were systematic differences in the carbon isotope composition among the mat morphotypes at any location, however, which suggests that there may be distinct carbon isotope effects imparted by specific populations due to autotrophic production versus heterotrophic consumption. Changing abundances of bacterial populations downstream could account for this observation if the downstream populations express larger 13 C discrimination. An alternative explanation for the downstream carbon (and sulfur) isotope trends may be that mat stratification, caused by redox conditions, creates an environment for nutrient spiraling, a process first described from surface stream ecosystems in which nutrients are cycled between multiple components of the ecosystem while being transported further downstream in each subsequent cycle (Newbold et al., 1981; Newbold et al., 1982; Newbold, 1982; Allan, 1995). Nutrient spiraling has neither been described from subsurface ecosystems, nor from chemosynthetic systems. Variations in the 13C composition among the different microbial mat morphotypes (white filaments versus gray filaments) in downstream transects suggest carbon cycling between chemolithoautotrophs and heterotrophs. The physical model of the nutrient spiral is based on the energy transfer between redox (metabolic) environments, and without the metabolic complexity and juxtaposition of aerobic and anaerobic populations, nutrient spiraling would not occur. The autotrophically-fixed carbon, which may be respired as CO2 with a low 13C value, 69 is transported downstream and preferentially reassimilated by autotrophs in the mat boundary layer; the proportion and intensity of recycling of the fixed carbon derived from respiration should increase downstream (Newbold, 1982). Downstream, filamentous sulfur-oxidizing bacteria form a thick mat that restricts the downward flux of oxygen into the mat interior, and the occupation of the exterior of the microbial mats by sulfur-oxidizers results in consumption of most of the available oxygen. This creates an anaerobic mat interior where compounds are transported by diffusion only, maintaining a habitat for obligate anaerobic populations. The anaerobic consortium, consisting of predominately sulfatereducing and fermenting bacteria (refer to Chapter 4), generates biogenic sulfide that diffuses outward for consumption by the sulfur-oxidizers. Classically, the components of a nutrient spiral are transported by stream advection, and DIC, sulfate, and allochthonous sulfide move through the system in this manner. However, the effect of the anaerobic system bounded by the oxidizing system is a partial retention of both organic carbon and biogenic sulfide relative to stream advection, which promotes spiraling of sulfur between redox environments. CONCLUSIONS Molecular techniques and carbon and sulfur isotope ratio analyses were combined to examine the dynamics of microbial community structure and nutrient cycling in an aphotic sulfidic cave system. Microbial mat bacterial diversity was low overall, with most communities being dominated by several evolutionary lineages within the "Epsilonproteobacteria". Certain bacterial groups were found only in one microbial mat morphotype, and most bacterial groups were rarely found 70 or were completely absent in other morphotypes. The concentrations of dissolved oxygen and dissolved sulfide controlled the distribution of sulfur-oxidizers with differing requirements for oxygen, such that those preferring higher oxygen conditions were found at the end of the microbial mats where dissolved oxygen was highest. The "Epsilonproteobacteria" play a significant role in providing chemolithoautotrophic energy to the otherwise nutrient-poor cave habitat. Additionally, these filamentous sulfur-oxidizing bacteria initially colonize the cave springs, forming a dense mat that provides habitat for other bacterial groups with heterotrophic or chemoorganotrophic metabolism (Horner-Devine et al., 2003), and thereby increase mat species richness. The physical model of carbon and sulfur spiraling is based on the energy transfer between redox environments within the mats, and without aerobic and anaerobic metabolic complexity, nutrient spiraling would not occur. Spiraling carbon and sulfur between aerobe and anaerobe, autotroph and heterotroph, significantly extends the mat community. 71 Table 2-1: Geochemical parameters from representative Lower Kane Cave spring and stream water samples from August 2001, reported in mmol L-1, unless otherwise noted. T ( C) o Site S mol L mol L mg C L 0.66 0.25 0.009 0.012 1.69 0.94 3.43 39.7 <0.2 -1 -1 -1 pH Cond DO a S2- b NPOCc Na+ K+ NH4+ Ca2+ Mg2+ HCO3- Cl- NO3- SO42- Si Fissure 22 580 0.12 0.001 1.19 0.17 Spring 7.3 (118 m) 21.3 577 <0.2 35.3 0 0.26 0.009 0.025 1.75 0.96 3.46 0.14 0.001 1.15 0.17 Upper Spring 7.39 (189 m) 22 587 40 5.6 0.2 0.25 0.008 0.014 1.74 0.92 3.48 0.14 0.001 1.22 0.17 72 22.1 575 <0.2 39.4 0.13 0.25 0.009 0.025 Stream Channel 7.43 (205 m) 1.66 0.91 3.42 0.13 0.002 1.18 0.17 Lower Spring 7.22 (248 m) a Dissolved oxygen, measured by the rhodazine D colorimetric method (CHEMetrics). Dissolved sulfide (as total dissolved sulfide, including H2S and HS-), measured by the methylene blue colorimetric method (CHEMetrics). c Nonpurgable organic carbon, including methane. b Table 2-2: Biomass estimates and elemental analyses for microbial mat morphotypes. Site locations refer to distance (in meters) from the back of the cave. Site (m) 118 120 125 128 189 192 198 203 204 248 248 120 128 189 192 192.5 196.5 198 201 201 203 248 201 203 248 204 203 Mat morphotype Gray sediment Gray filaments & sediment Gray sediment Gray filaments & sediment Gray sediment Gray filaments Gray filaments Gray filaments Gray filaments Gray filaments Gray filaments White filaments White filaments & webs White filaments White filaments White filaments White filaments White filaments White feathers White filaments White filaments White filaments White webs White webs White filaments & feathers White feathers Yellow patches Biomass (1010cells ml-1 ) 2.9 2.6 1.8 7.6 0.73 1.8 4.7 C:N 28.8 28.0 17.1 9.5 23.6 13.8 7.9 6.8 6.0 35.1 6.7 5.4 5.1 3.6 4.4 3.6 5.3 5.2 4.2 3.5 4.2 4.9 4.4 4.7 6.6 4.2 4.7 %N 0.3 0.3 0.4 1.2 0.2 0.2 0.6 2.3 3.9 0.2 5.5 0.7 4.2 4.1 4.0 6.1 2.4 2.4 7.3 4.6 5.5 5.8 2.3 4.1 5.6 6.1 8.1 %S 0.3 0.5 1.5 1.0 1.7 2.1 1.3 2.0 2.0 1.8 1.5 24.4 17.7 38.1 51.2 41.6 16.1 26.7 16.1 26.6 50.0 32.7 38.5 35.4 27.0 37.3 8.6 1.8 1.8 2.9 2.0 1.4 73 Table 2-3: Distribution of bacterial clones as they appeared in the microbial mat clones libraries. Representative clone sequences and phylotypes Closest relative a %a Fissure Spg 19 b 124f c 22 127f 1 1 1 1 1 1 1 1 2 2 46 3 1 57 190f 270 195f 190 198f 127 203f 156 203w Lake clone TLM10/dgge01 Digestor clone vadinHA54 Groundwater clone ECP-C1 Groundwater clone WCHA101 Gas hydrate clone Hyd.B2.1 Gas hydrate clone Hyd-B2-1 Groundwater clone SJA-36 Groundwater clone SJA-36 97 92 97 92 94 91 90 96 7 Upper Spring 102 203y 125 203g Phylogenetic affiliation a Library location and number clones in library Lower Spg 199 248f 198 248y 3 Bacteroidetes/Chlorobi Group I Group II Group III Group IV Group V Group VI Group VII Unclassified LKC3_198.43 LKC3_156.56 LKC3_ 270.15 LKC3_102B.33 LKC2_127.25 LKC3 _19.50 LKC3_102B.59 LKC3_156.13 LKC3_156.1 Acidobacterium 74 66 4 80 3 5 3 12 4 41 4 LKC3_22.5 (2) d LKC3_190.31 LKC3_127.1 (7) LKC2_270.19 (3) LKC3_127.28 (3) LKC3_156.15 Sulfidic spring clone sipK119 Sulfidic spring clone sipK94 Sulfidic spring clone sipK119 Groundwater clone 1028 Sulfidic spring clone sipK94 Acid mine clone 44a-B1-1 99 90 99 99 94 95 96 116 111 127 98 98 96 96 95 96 77 28 7 8 4 LKC3_22.33 LKC3_19B.17 LKC3_125.3 LKC3_125.46 LKC3_ 102B.25 LKC3_198.35 (2) LKC3_190.37 (3) Desulfocapsa thiozymogenes Thiobacillus clone 44a-B2-21 Thiobacillus aquaesulis Sulfidic spring clone sipK4 Beggiatoa MS-81-1c strain Pantoea agglomerans Serratia marcescens 47 54 6 1 2 4 81 2 29 30 3 1 7 1 1 1 76 10 17 11 14 5 2 2 1 6 18 6 1 70 6 117 87 81 1 74 1 79 26 76 91 6 43 1 Proteobacteria "Epsilonproteobacteria" Group I Group II Group III Group IV Group V Group VI Gammaproteobacteria Thiothrix unzii Beggiatoa spp. Pantoea spp. Serratia spp. Betaproteobacteria Group I Group II Deltaproteobacteria a Total clones Affiliation based on taxonomic classifications from BLAST searches and percent sequence similarity to closest relative. Refer to Figures 2-8 through 2-12. b Clone library reference number. c Meter location along the cave stream; letter corresponds to morphotype: f, white filaments; w, white webs; y, yellowish-white mat; g, gray filaments. d Number in parentheses represents number of phylotypes for each group if more than one, with phylotype defined as 98% sequence similarity. Table 2-4: Bacterial clone library coverage and ecological indices. Library (m) Mat typea No. clones Number phylotypes observed ACEb,c Chao1c ShannonWiener (H')c Evenness (E)d Simpson's Index (D)d 124 f 116 9 10.55 11.0 1.18 0.49 0.49 127 f 111 4 4.0 4.0 0.88 0.63 0.47 190 f 127 10 10.33 10.05 1.36 0.39 0.65 195 f 117 10 12.0 10.66 1.24 0.54 0.38 198 f 87 3 2.0 2.0 0.28 0.40 0.87 203 f 81 10 14.66 11.62 1.53 0.73 0.45 203 w 74 9 20.84 21.5 1.27 0.52 0.42 203 y 79 7 13.24 10.5 0.55 0.26 0.79 203 g 26 4 8.04 6 0.84 0.37 0.54 248 f 76 1 1.0 1.0 0 0 1.0 248 y 91 11 15.48 14.5 1.69 0.66 0.27 a Letter corresponds to morphotype: f, white filaments; w, white webs; y, yellowish-white mat; g, gray filaments. b Abundance-based coverage estimator. c Calculated by EstimateS, ver. 6.01b (http://viceroy.eeb.uconn.edu/estimates). d H', E, and D calculated from equations provided in Hill et al. (2003). Table 2-5: Percentage of 16S rDNA sequence similarity for Lower Kane Cave clones belonging to the "Epsilonproteobacteria." "Epsilonproteobacteria" Group I (n = 16) 99.1-100.0 Group I 85.0-87.4 Group II 94.6-98.6 Group III 92.9-98.2 Group IV 89.3-94.0 Group V 81.9-83.2 Group VI Group II Group III Group IV Group V Group VI (n = 12) (n = 10) (n = 3) (n = 3) (n = 5) 99.4-100.0 85.0-91.5 85.0-90.6 93.4-95.7 83.8-85.4 91.0-99.8 88.7-97.9 84.6-92.8 82.1-85.2 95.0-97.9 84.5-90.1 83.5-81.0 97.1-99.5 84.5-85.1 99.6-99.9 75 30 A: FISSURE SPRING microbial mats 70 60 Dissolved Oxygen mol L 50 40 25 Dissolved Sulfide mol L -1 20 15 30 10 20 CTS= O2 -1 5 10 0 0 117 122 127 132 Distance (m) 137 142 147 30 B: UPPER SPRING microbial mats 70 60 Dissolved Oxygen mol L 50 40 25 Dissolved Sulfide mol L -1 20 15 30 10 20 CTS= O2 -1 5 10 0 0 188 193 198 203 Distance (m) 208 213 218 Figure 2-1: Total dissolved sulfide (CTS=) ( ) and dissolved oxygen ( ) versus distance profiles from spring orifice through stream transects for the (A) Fissure Spring and (B) Upper Spring. Distance was measured from the back of the cave to the entrance. 76 Figure 2-2: Sampling sites, springs, and microbial mats in Lower Kane Cave, Wyoming. Black arrows in all photographs point in the direction of flow. (A) Lower Spring orifice (248 m) occupied by emergent sulfidic groundwater (flowing over the lip at the lower left) and white filament bundles. A thick white filamentous mat forms at the edge of the orifice. Orifice walls are made of limestone, while gypsum occurs around the edge of the orifice pool (upper right). (B) End of thick microbial mat below Lower Spring (248 m). The mat is composed of white filaments and dense gel-like yellow masses. Black spots on rocks at the edge of the mat (upper left) are snails (Physa spelunca, 2 mm long). Gas bubbles of carbon dioxide, methane, and hydrogen sulfide gases form in this portion of the mat. (C) Upper Spring Pool (190 m) area, looking upstream, with gray sediment on the orifice pool bottom and white filaments suspended in the water column. Water depth at the deepest part of the pool is ~ 2 m, although average water depth is 30 cm. (D) Upper Spring filament bundles suspended in the water column (190 m). (E) Knobby white webs on the surface of the mat within the Upper Spring channel (203 m). Thin white filaments are suspended in the water column above the webs. (F) Yellowish-white patches within white filament area at 203 m from the Upper Spring channel. Chert fragments are the stream bed substrate (lower center and lower left). Gas bubbles have also been observed at this locale. (G) Fissure Spring orifice (118 m). The orifice pool area consists of limestone cobbles and gray sediment, mostly clay. (H) Thin white filament bundles and white webs in the Fissure Spring outflow channel (125 m). Gas bubbles are also present. 77 A B 50 cm C D 0.5 m 10 cm E F 5 cm 1 cm G H 20 cm 10 cm 78 A B C D E F Figure 2-3: Scanning electron photomicrographs of white filamentous biomass from the 203 m sampling location in Lower Kane Cave, Wyoming. A 30 kV accelerating voltage was used. (A) and (B) Filament networks. (C) and (D) Branching and non branching filaments. (E) Twisted filaments and rods. (F) Filaments with intracellular sulfur. 79 A B C D E F Figure 2-4: Scanning electron photomicrographs of gray sediment from the Upper Spring orifice (189-190 m). (A-C) Organic material and Fe-S framboids; (D) Close-up of individual Fe-S crystallites; (E) Biological material and cluster of cells (outlines in box) for photomicrograph (F) Cluster of cocci. Accelerating voltage for all images was 30 kV. 80 -25.00 A: Fissure Spring -30.00 C 13 -35.00 -40.00 gray filament web -45.00 118 -25.00 120 122 Distance (m) 124 126 128 130 B: Upper Spring -30.00 C 13 -35.00 gray -40.00 filament feather web yellow -45.00 188 190 192 194 196 198 200 202 204 206 Distance (m) Figure 2-5: Carbon isotope composition of microbial mat morphotypes from (A) the Fissure Spring and (B) the Upper Spring. 81 -21 -22 34S (CDT) -23 -24 Dissolved sulfide Microbial mat -25 190 195 Distance (m) 200 205 Figure 2-6: 34S values of dissolved sulfide () and bulk samples of white filamentous microbial mats () dominated by "Epsilonproteobacteria" versus distance along the Upper Spring flowpath. 82 12 10 number of phylotypes 8 6 4 2 124f 127f 190f 195f 198f 203f 203w 203y 203g 248y 0 0 10 20 30 40 50 60 70 80 90 100 110 120 130 number of clones Figure 2-7: Rarefaction curves of the diversity in ten of the eleven 16S rRNA gene sequence bacterial clone libraries based on phylotypes identified from restriction fragment length polymorphism (RFLP) patterns. Library 248f was excluded because only one RFLP phylotype was identified. 83 Figure 2-8: 16S rRNA gene-based phylogenetic tree showing the phylogenetic position of clones from Lower Kane Cave within the "Epsilonproteobacteria". Clones are labeled in bold with corresponding sample and clone numbers. Reference sequences (with GenBank accession numbers) were chosen to represent the diversity of the "Epsilonproteobacteria". The tree was rooted with Proteobacteria representatives and other bacterial divisions. The tree is a representative topology from 188 trees of the same score inferred from minimum evolution analysis, with the differences among the minimum evolution trees due only to changes in the relative position of sequences within clades of Lower Kane Cave clones. The phylogenetic affiliations of the clones were confirmed by comparison with different reconstruction methods. Numbers along tree branches refer to support values for each node, corresponding to minimum evolution bootstrap proportions, MLga and BMCMC posterior probabilities (see text for details). 84 -/-/81 100/100/100 LKC3 102.21 [AY510214] LKC3 57.4 [AY510210] LKC3 270.57 [AY510215] LKC3 159.12 [AY510213] LKC3 156.14 [AY510208] LKC3 127.29 [AY510216] 85/88/100 LKC3 125.31 [AY510207] 98/94/80 LKC3 57C.13 [AY510212] LKC3 199.1 [AY510205] 86/-/LKC3 198.26 [AY510211] 85/88/100 LKC2 127 53 [AY208813] Deep-sea hydrothermal field strain EM9I37-1 [AB091299] Alvinella pompejana epibiont [L35521] LKC3 127.36 [AY510203] LKC3 127.28 [AY510204] 100/100/99 LKC3 198B.17 [AY510202] 60/100/- 85/100/100 Parker Cave clone SRang51 [AF047630] Activated sludge clone rA10 [AF047626] -/62/Hydrocarbon seep sediment clone GCA014 [AF154101] 77/-/99 Hydrothermal vent clone 49MY [AB091293] -/81/100 -/-/64 Marine sediment clone NKB9 [AB013261] Marine sediment clone JTB315 [AB015258] 78/-/65 94/100/100 Marine sediment clone a2b004 [AF420345] Deep-sea hydrothermal field clone 42BKT [AB091292] Deep-sea hydrothermal field strain E9S37-1 [AB091300] 77/100/71 73/-/56 Sulfidic spring clone ZB50 [AY327163] Hydrothermal vent clone VC2_1 Bac1 [AF068783] Riftia pachyptila's tube clone R103-B22 [AF449234] Estuarine sediment clone 2BP-7 [AF121887] Benzene-mineralizing consortium clone SB-17 [AF029044] 67/75/100 Rimicaris exoculata ectosymbiont [U29081] Parker Cave clone SrangJ [AF047633] Sulfidic spring clone ZB43 [AY327156] Parker Cave clone SRang1.27 [AF047626] 96/88/100 68/100/100 Sulfidic groundwater clone 1065 [AB030598] Candidatus `Arcobacter' sulfidicus [AY035822] 91/100/100 Marine sediment clone NB1-k [AB013832] 96/100/100 Arcobacter butzlerii [L14626] 67/100/100 Oilfield groundwater strain FWKOB [AF144693] Geospirillium sp. [Y18254] 56/-/90/-/69 100/100/100 Petroleum-contaminated groundwater clone 1014 [AB030587] Sulfurospirillum deleyianum [Y13671] -/69/Campylobacter jejuni [AF393203] 99/100/100 71/-/Helicobacter winghamensis [AF363063] 87/100/100 Flexispira rappini [AF034135] Helicobacter pylori isolate MC123 [U01328] 99/100/100 Wolinella succinogenes [AF273252] LKC3 156.15 [AY510218] 65/-/94/100/100 LKC3 102B.55 [AY510219] 100/100/100 LKC3 156.74 [AY510217] LKC3 156.38 [AY510221] 100/100/100 LKC3 102B.15 [AY510220] Acid mine drainage clone 44a-B1-40 [AY082468] 83/100/100 Acid mine drainage clone 44a-B1-1 [AY082456] Petroleum-contaminated groundwater clone 1070 [AB030590] 100/100/100 Caminibacter hydrogeniphilus [AJ309655] 100/100/100 Nautilia lithotrophica [AJ404370] Desulfocapsa thiozymogenes [X95181] Desulfovibrio fairfieldensis [U42221] -/88/100 Hydrogenophaga pseudoflava [AF078770] 92/100/100 100/100/100 Leptothrix.discophora [L33975] Thiobacillus 44a.B2.21 [AY082471] 95/100/81/-/79 Thiothrix unzii [L79961] Beggiatoa sp. [AF110276] 91/-/Escherichia coli K12 [NC_000913] Cytophaga .sp [AB015525] Bacteroides sp. [AB021162] 100/100/100 Thermotoga.subterranea [U22664] Thermus aquaticus [L09663] LKC Group II LKC Group V Miscellaneous strains & clones (marine & freshwater) Arcobacter Geospirillum Sulfurospirillum Campylobacter Helicobacter/ Flexispira Wolinella LKC Group VI Caminibacter Nautilia 0.01 substitutions/site 85 Outgroups "Epsilonproteobacteria" LKC3 22.17 [AY510189] LKC3 57B.57 [AY510193] Sulfidic spring clone sipK119 [AJ307940] LKC3 22.54 [AY510180] LKC3 57B.49 [AY510194] LKC3 57B.17 [AY510188] LKC3 22.53 [AY510181] LKC3 57C.15 [AY510190] LKC3 57C.33 [AY510195] LKC2 270.64 [AY208817] LKC2 57.8 [AY208807] LKC3 57B.2 [AY510196] LKC3 22.5 [AY510183] LKC3 198.20 [AY510185] LKC3 57.20 [AY510184] LKC3 127B.2 [AY510182] LKC3 19.39 [AY510191] 99/89/LKC3 22.81 [AY510192] LKC3 198.15 [AY510186] Petroleum-contaminated groundwater clone 1043 [AB030601] Uncultured groundwater clone FTL212 [AF529098] LKC3 22.72 [AY510167] LKC3 57B.41 [AY510197] Petroleum-contaminated groundwater clone 1049 [AB030606] 64/-/Petroleum-contaminated groundwater clone 1011 [AB030607] Sulfuricurvum kujiense [AB080643] 99/89/61/-/- LKC3 57B.22 [AY510200] Cesspool Cave clone group CC-4 [AF207530] LKC3 57C.10 [AY510199] LKC2 270 19 [AY208816] Cesspool Cave clone group CC-9 [AF207534] Groundwater clone RA9C8 [AF407391] LKC3 127.40 [AY510174] LKC3 127.14 [AY510175] LKC2 127.32 [AY208810] LKC3 127.6 [AY510169] LKC3 127.46 [AY510170] LKC3 127.23 [AY510173] LKC3 127B.27 [AY510171] LKC3 127.39 [AY510172] LKC3 57B.54 [AY510168] LKC3 127B.26 [AY510177] LKC3 270.5 [AY510187] 64/-/LKC3 127.43 [AY510176] Petroleum-contaminated groundwater clone 1023 [AB030610] 56/-/Petroleum-contaminated groundwater clone KB2C [AB07495] LKC2 270.16 AY208815] 68/-/LKC3 270.58 [AY510178] 52/-/LKC3 127.1 [AY510179] LKC3 270.13 [AY510198] LKC3 57B.56 [AY510201] /-/100 Thiomicrospira sp. [U46506] 97/100/100 99/100/100 Thiomicrospira denitrificans [L40808] Pele's Vent clone PVB 55 [U15105] Sulfurimonas autotrophica [AB088432] 90/100/100 72/100/100 Thiovulum sp. [M92323] Meromictic lake sediment clone PENDANT-10 [AF142923] 100/100/100 Sulfidic spring clone sipK94 [AJ307941] 53/-/LKC3 190 31 [AY510209] 50/-/- LKC3 127.4 [AY510206] LKC Group I LKC Group IV LKC Group III Pig gastrointestinal gut clone p-2172-s959-3 [AF371852] Pantoea agglomerans [AF199029] Folsomia candida gut clone Pantoea sp. isolate 8 [AJ002811] Pantoea group LKC3 125.2 [AY510236] 94/100/100 LKC3 125.3 [AY510237] 95/55/84 LKC3 125.60 [AY510238] 100/100/95 Serratia marcescens [AJ296308] 100/96/100 Serratia group LKC3 125.59 [AY510235] LKC3 125.46 [AY510234] 100/100/100 Escherichia coli K12 [NC_000913] Aeromonas sobria [X60412] 100/100/100 Pseudomonas pseudoalcaligenes [Z76675] Pseudomonas putida [AY332610] 50/-/Hydrothermal vent sulfur-oxidizing bacterium NDII1.2 [AF181991] 100/100/ Achromatium oxaliferum [L79967] 100 -/-/67 Achromatium minus [AJ010596] 100/100/100 Dechloromarinus chlorophilus [AF170359] -/87/Codakia costata [L25712] Thiorhodospira sibirica [AJ006530] 100/100/100 Ectothiorhodospira Bogoria Red [AF084511] Nullarbor Caves aquatic formations clone wb1 P19 [AF317768] LKC3 127.2 [AY510228] Thiothrix unzii [L79961] 57/-/Sulfidic spring clone sipK4 [AJ307933] LKC3 270.18 [AY510222] LKC3 22.33 [AY510233] LKC3 19.29 [AY510231] LKC3 22.89 [AY510223] Thiothrix unzii group LKC3 22.97 [AY510224] LKC3 127.33 [AY510230] LKC3 159.7 [AY510226] LKC3 127.26 [AY510229] 60/-/LKC3 19B.29 [AY510232] 100/62/100 LKC2 127.27 [AY208809] 91/74/77 LKC3 22.73 [AY510225] 98/-/100 51/-/- LKC3 22 3 [AY510227] 95/ Parker Cave clone SRang1.40 [AF047621] 100/ Thiothrix fructosivorans [L79963] 100/100/100 100 Thiothrix eikelboomii strain COM-A [AB042542] LKC3 156.55 [AY510239] 100/100/100 93/100/100 82/-/88 LKC3 156.46 [AY510166] Salt marsh Beggiatoa isolate MS-81-1c (non-vacuolate) [AF110276] Beggiatoa Marine Beggiatoa `Bay of Concepcion' isolate (40 m filament) [AF035956] group LKC3 19B.45 [AY510241] -/100/99 100/100/100 LKC3 156.41 [AY510242] 74/-/79 LKC3 19B.17 [AY510240] 100/100/100 Leptothrix.discophora [L33975] Hydrogenophaga pseudoflava [AF078770] 77/100/100 Thiomicrospira denitrificans [L40808] 100/100/100 100/100/100 Campylobacter sp. [L14632] 63/-/76 Helicobacter winghamensis [AF363063] Desulfovibrio fairfieldensis [U42221] 71/100/100 Desulfocapsa thiozymogenes [X95181] 100/100/100 Cytophaga sp. [AB015525] Bacteroides sp. [AB021162] Thermus aquaticus [L09663] Thermotoga.subterranea [U22664] 0.05 substitutions/site Figure 2-9: 16S rRNA gene-based phylogenetic tree showing the phylogenetic position of Gammaproteobacteria bacterial clones from Lower Kane Cave. Clones are labeled in bold with corresponding sample and clone numbers. Reference sequences (including GenBank accession numbers) were chosen from the RDP to represent the diversity of each division. Each tree was rooted with different members of the Proteobacteria and other bacterial divisions. Tree topology was inferred from the results of minimum evolution (ME) analysis, and the phylogenetic affiliations of the clones were confirmed by comparison with different reconstruction methods. This tree is a representative topology from 14 trees. Numbers along tree branches refer to support values for each node corresponding to ME bootstrap proportions, MLga and BMCMC posterior probabilities. 86 Outgroups Gammaproteobacteria LKC3 102B.25 [AY510243] Lower Kane Cave LKC3 102B.27 [AY510244] Thiobacillus LKC3 102B.28 [AY510245] Group I 68/51/100 LKC3 198.8 [AY510250] LKC3 198.31 [AY510247] LKC3 198.44 [AY510248] 92/73/100 Lower Kane Cave Thiobacillus 99/ LKC3 198.35 [AY510246] Thiobacillus 100/ group 100 LKC3 198.29 [AY510251] Group II LKC3 198B.29 [AY510249] 100/ 100/ Thiobacillus aquaesulis [U58019] 100 Contaminated groundwater clone RB7C6 [AF407385] Thiobacillus denitrificans [ AJ243144] 99/100/100 -/-/59 99/100/Thiobacillus thioparus [M79426] 60/ 96/ Acid mine drainage clone 44a-B2-2 [AY082471] Yellowstone hot spring clone OPB37 [AF026985] 100/100/100 Azoarcus denitrificans [L33689] 52/ Trichlorobenzene consortium clone SJA.21 [AJ009455] -/ 84 Thiomonas sp. Ynys1 [AF387302] 81/96/100 77/100/100 Hydrogenophaga pseudoflava [AF078770] 88/100/100 Leptothrix.discophora [L33975] -/79/98 Beggiatoa sp. [AF110276] -/-/71 Thiothrix unzii [L79961] 77/96/93 Escherichia coli K12 [NC_000913] Desulfovibrio fairfieldensis [U42221] 99/98/100 51/ Desulfocapsa thiozymogenes [X95181] 79/ 97 Helicobacter winghamensis [AF363063] 100/100/100 Thiomicrospira denitrificans [L40808] 88/100/100 Campylobacter sp. [L14632] -/-/88 Cytophaga sp. [AB015525] 100/100/100 Bacteroides sp. [AB021162] Thermotoga.subterranea [U22664] Thermus aquaticus [L09663] 0.05 substitutions/site 100/100/100 Figure 2-10: 16S rRNA gene-based phylogenetic trees showing the phylogenetic position of Betaproteobacteria bacterial clones from Lower Kane Cave. Clones are labeled in bold with corresponding sample and clone numbers. Reference sequences (including GenBank accession numbers) were chosen from the RDP to represent the diversity of each division. Each tree was rooted with different members of the Proteobacteria and other bacterial divisions. Tree topology was inferred from the results of minimum evolution (ME) analysis, and the phylogenetic affiliations of the clones were confirmed by comparison with different reconstruction methods. This tree is a representative topology of 2 trees of the same score inferred from ME analyses, with the differences among the ME trees from the same search due only to changes in the relative position of sequences within clades of the LKC clones. Numbers along tree branches refer to support values for each node corresponding to ME bootstrap proportions, MLga and BMCMC posterior probabilities. 87 Outgroups Betaproteobacteria -/84/79 Desulfocapsa thiozymogenes [X95181] Lake clone SRB-348 [AJ389628] LKC3 190.63 [AY510263] 84/100/Sulfidic spring clone sipK108 [AJ307944] 83/100/100 Lake clone SRB-282 [AJ389626] Desulfocapsa isolate Cad626 [AJ511275] 100/98/100 LKC3 190.28 [AY510266] LKC3 190.37 [AY510265] 63/-/LKC3 190.75 [AY510267] -/94/ 100 LKC3 102.22 [AY510264] 100/95/100 Contaminated groundwater clone WCHB1-67 [AF050536] Vent sediment clone C1 B030 [AF420366] 82/53/100 73/-/62 Desulfocapsa sulfoexigens [Y13672] Geobacter sulfurreducens [U13928] -/64/85 96/100/87 Antarctica sediment Desulfobacula clone SB4-98 [AY177803] 56/-/Antarctica sediment Desulfuromonadales clone LH5-30 [AY177804] -/-/81 Desulfovibrio fairfieldensis [U42221] 90/76/88 Beggiatoa sp. [AF110276] 77/94/69 Thiothrix unzii [L79961] Escherichia coli K12 [NC_000913] 99/99/100 62/77/Acid mine drainage clone 44a-B2-2 [AY082471] 100/100/100 Hydrogenophaga pseudoflava [AF078770] 100/100/100 Leptothrix.discophora [L33975] 100/100/100 84/100/100 Thiomicrospira denitrificans [L40808] Campylobacter sp. [L14632] 100/100/100 Helicobacter winghamensis [AF363063] Bacteroides sp. [AB021162] 100/100/100 Cytophaga sp [AB015525] Thermotoga subterranea [U22664] Thermus aquaticus [L09663] Desulfocapsa 0.05 substitutions/site Figure 2-11: 16S rRNA gene-based phylogenetic trees showing the phylogenetic position of Deltaproteobacteria bacterial clones from Lower Kane Cave. Clones are labeled in bold with corresponding sample and clone numbers. Reference sequences (including GenBank accession numbers) were chosen from the RDP to represent the diversity of each division. Each tree was rooted with different members of the Proteobacteria and other bacterial divisions. Tree topology was inferred from the results of minimum evolution (ME) analysis, and the phylogenetic affiliations of the clones were confirmed by comparison with different reconstruction methods. The ME analysis of the Deltaproteobacteria alignments resulted in a single tree. Numbers along tree branches refer to support values for each node corresponding to ME bootstrap proportions, MLga and BMCMC posterior probabilities. 88 Outgroups Deltaproteobacteria Acid mine drainage clone 44a-B1-14 [AY082459] Dechlorinating aquifer clone SHA-13 [AJ306737] BC 100/100/100 LKC3 198.43 [AY510261] Group 100/100/LKC3 198.17 [AY510262] I Arctic lake clone TLM10/TLMdgge01 [AF534434] 99/100/Tuber borchii symbiont clone b_17BO [AF070444] Acid mine drainage clone 253c [AY082449] 100/100/100 Bacteroides acidofaciens [AB021162] 92/100/ Chlorobenzene-removing clone IA-16 [AJ488070] 100 BC Anaerobic digestor clone vadinHA54 [U81722] 64/-/Gas-contaminated aquifer clone ECP-C1 [AF529225] Group II & III LKC3 156 56 [AY510259] 84/ 100/100/100 98/ LKC3 270.15 [AY510260] 100 Basalt pipe smoker clone Ko710 [AF550591] 59/83/LKC3 102b.33 [AY510256] 62/100/100 93/ Anaerobic digestor clone vadinHA17 [U81712] BC 100/ Deep-sea sediment clone BD1-16 [AB015525] 100 71/100/98 Group Contaminated aquifer clone WCHB1 53 [AF050539] IV Contaminated aquifer clone WCHA1.01 [AF050541] 98/100/100 LKC2 127.25 [AY208811] 98/100/100 LKC3 19.50 [AY510258] BC Paralvinella palmiformis clone P.palm-A10 [AJ441240] 53/100/ 98 Gas hydrate clone Hyd-B2-1 [AJ535256] Group 93/-/65/-/LKC3 102B.59 [AY510257] V & VI 100/100/100 78/100/100 Vent worm clone R76-B13 [AF449261] Swamp sediment clone FSA6 [AY193038] LKC3 156.13 [AY510255] Unclassified Campylobacter sp. [L14632] 92/100/100 Thiomicrospira denitrificans [L40808] 100/100/100 Helicobacter winghamensis [AF363063] -/64/98 53/-/Desulfovibrio fairfieldensis [U42221] 64/-/56 Desulfocapsa thiozymogenes [X95181] Acid mine drainage clone 44a-B2-2 [AY082471] Proteobacteria 100/100/100 Hydrogenophaga pseudoflava [AF078770] 92/100/100 70/ Leptothrix discophora [L33975] 100/ 99/100/100 100 Beggiatoa.sp. [AF110276] Thiothrix unzii [L79961] 97/89/100 74/100/51 Escherichia coli K12 [NC_000913] Anaerobic trichlorobenzene clone SJA.36 [AJ009461] LKC3 156.1 [AY510252] 100/100/100 LKC2 127.11 [AY208806] LKC3 156.4 [AY510253] 60/100/95 Acidobacterium LKC3 156.19 [AY510254] Uranium mine waste clone KCM-C-25 [AJ581628] 51/-/90/100/ Soil clone 32-20 [Z95713] 100 Soil clone iii1.88 [Z95729] Thermotoga.subterranea [U22664] Outgroups Thermus aquaticus [L09663] 0.05 substitutions/site 100/100/100 Figure 2-12: 16S rRNA gene-based phylogenetic trees showing the phylogenetic position of Bacteroidetes/Chlorobi and Acidobacterium bacterial clones from Lower Kane Cave. Clones are labeled in bold with corresponding sample and clone numbers. Reference sequences (including GenBank accession numbers) were chosen from the RDP to represent the diversity of each division. Each tree was rooted with different members of the Proteobacteria and other bacterial divisions. Tree topology was inferred from the results of minimum evolution (ME) analysis, and the ME analysis of the Bacteroidetes/Chlorobi-Acidobacterium alignments resulted in a single tree. Numbers along tree branches refer to support values for each node corresponding to ME bootstrap proportions, MLga and BMCMC posterior probabilities. 89 Sphingobacteria Bacteriodetes/Chlorobi group Bacteriodes Chapter 3: Prevalence of Novel "Epsilonproteobacteria" from Filamentous Microbial Mats in Sulfidic Caves and Springs ABSTRACT1 A molecular phylogenetic approach and fluorescence in situ hybridization (FISH) were used to survey white filamentous microbial mat populations in Lower Kane Cave, Wyoming. FISH probes were designed from thirty-two 16S ribosomal DNA (rDNA) gene sequences obtained from Lower Kane Cave clones belonging to two distinct clusters within the "Epsilonproteobacteria," designated LKC group I and LKC group II. FISH proved to be an efficient tool for identifying and quantifying the previously uncharacterized, filamentous "Epsilonproteobacteria" from the cave microbial mats. Bacterial group quantification from six different Lower Kane Cave mat samples indicated that filamentous LKC group II populations dominated most of the bacterial communities (70% of the total bacteria). The microbial mats from Lower Kane Cave represent the first nonmarine natural system demonstrably Moreover, to driven by the that activity these of novel "Epsilonproteobacteria". demonstrate "Epsilonproteobacteria" are broadly distributed throughout sulfidic terrestrial habitats, occupying a range of physicochemical conditions, microbial mats from three additional sulfidic caves and nine surface-discharging mesothermal and thermal sulfidic springs were examined using lineage-specific A portion of this chapter originated from the publication A.S. Engel, N. Lee, M.L. Porter, L.A. Stern, P.C. Bennett, M. Wagner, 2003, Filamentous "Epsilonproteobacteria" dominate microbial mats from sulfidic springs: Applied and Environmental Microbiology, vol. 69(9), p. 5503-5511. 1 90 epsilonproteobacterial PCR primers based on the LKC sequences. Microbial mats from four geographically-distinct sulfidic caves in the USA and Italy, and nine surface-discharging springs were examined. Of the sites surveyed, positive amplification of 16S rDNA was obtained for both LKC groups I and II from two caves and five springs, ranging in water temperature from 8oC to 55oC. Several sites only had one group present. This work expands the evolutionary history and biogeographic diversity of the "Epsilonproteobacteria" and demonstrates the importance of this class to widespread biogeochemical cycling in terrestrial sulfidic habitats. INTRODUCTION The "Epsilonproteobacteria" class is, by and large, the most poorly characterized division within the Proteobacteria (Garrity and Holt, 2001; On, 2001). At present, the "Epsilonproteobacteria" consists of two genetically distinct orders, the "Campylobacterales", and the Nautiliales (Miroshnichenko et al., 2004). The family Campylobacteraceae comprises the genera Campylobacter, Arcobacter, Sulfurospirillum, and Thiovulum, the family "Helicobacteraceae" is formed by the genera Helicobacter and Wolinella, and the family Nautiliaceae consists of the genera Nautilia and Caminibacter (Vandamme and De Ley, 1991; Miroshnichenko et al., 2004). While several genera within this group have been well characterized, such as Helicobacter and Campylobacter because of pathogenicity (On, 2001), most lineages have only been described from environmental 16S rRNA-based phylogenetic studies. Moreover, of all the genera described, Thiovulum and Sulfurospirillum are not associated with metazoans. 91 Many as yet uncultured phylogenetic groups have been characterized from environmental sequences retrieved from marine settings, including seawater and muds (Todorov et al., 2000; Madrid et al., 2001), deep-sea hydrothermal vent sites (Reysenbach et al., 2000; Corre et al., 2001; Longnecker et al., 2001; Takai et al., 2003), and associated with vent metazoans (Moyer et al., 1995; Reysenbach et al., 2000; Corre et al., 2001; Longnecker and Reysenbach, 2001; L pez-Garc a et al., 2003; Takai et al., 2003; Alain et al., 2004). Recent culture-based investigations expanded the metabolic diversity of evolutionary groups from marine habitats (Takai et al., 2003; Alain et al., 2004), and several studies have resulted in new genera and species descriptions (Alain et al., 2002; Miroshnichenko et al., 2002; Alain et al., 2004; Miroshnichenko et al., 2004). In many investigations, "Epsilonproteobacteria" are characterized as mesophiles and occupy environments with low oxygen tensions, but many strains that have been isolated can also grow in the absence of oxygen (e.g., Luijten et al., 2003), and have been implicated in sulfur cycling, and especially oxidation (e.g., Finster et al., 1997; Gevertz et al., 2000; Nemati et al., 2001; On, 2001; Takai et al., 2003). Comparatively, epsilonproteobacterial groups identified from terrestrial environments are only now being recognized (e.g., Engel et al., 2003), as little is known about the diversity, ecophysiology, and biogeochemical importance of terrestrial sulfur-based habitats. Environmental sequences of "Epsilonproteobacteria" have been retrieved from microbial mats at surface-discharging sulfidic springs (Rudolph et al., 2001; Barton et al., 2002; Moissl et al., 2002; Elshahed et al., 2003; Rzonca and Schulze-Makuch, 2003), groundwater associated with 92 hydrocarbons (Gevertz et al., 2000; Watanabe et al., 2000; Watanabe et al., 2002; Kodama and Watanabe, 2003), and caves with sulfidic springs and streams (Angert et al., 1998; Engel et al., 2001); Engel et al. 2003; Engel et al., in review). Kodama et al (2003) and Gevertz et al. (2000) isolated strains of rod-shaped cells from petroleum-contaminated groundwater and oil field production waters, respectively. Relatively little is known about "Epsilonproteobacteria" from non-marine settings because the terrestrial subsurface, including groundwater, is often inaccessible for study. However, the subsurface is reachable through caves and springs. Although springs vary geochemically and hydrogeologically, some of the most common types are associated with sulfidic water, and sulfidic caves and springs could be exceptional habitats for "Epsilonproteobacteria". The main purpose of this chapter was to examine terrestrial sulfidic habitats for "Epsilonproteobacteria", based on initial 16S rRNA gene characterization of the filamentous microbial mats from Lower Kane Cave, Wyoming (Chapter 2) and previous work from other sulfidic caves (Angert et al., 1998; Engel et al., 2001; Barton et al., 2002). To identify and reliably quantify "Epsilonproteobacteria", I designed, evaluated, and applied two new 16S rRNA-targeted oligonucleotide probes specific for the detection of novel "Epsilonproteobacteria" using fluorescence in situ hybridization (FISH). With the complementary oligonucleotide sequences to target DNA, I also designed PCR primers to amplify lineage-specific epsilonproteobacterial DNA from microbial mats in other sulfidic caves and surface-discharging springs. The results of this work expand the geographic diversity of "Epsilonproteobacteria" to many different sulfidic springs and caves 93 that have been previously overlooked. The "Epsilonproteobacteria" play an important role in the widespread biogeochemical cycling of carbon and sulfur nutrients, and in subterranean geological processes. MATERIALS AND METHODS Sample Acquisition from Lower Kane Cave, Wyoming Two major and two minor anaerobic, sulfidic springs discharge into Lower Kane Cave along a fracture zone (Figure 1-2; see Chapter 1). Samples for DNA extraction were obtained from microbial mats, and 16S rDNA clone libraries were constructed from a subset of six samples from the cave: Fissure Spring orifice white filament bundles (sample 21), Upper Spring orifice white filament bundles (samples 57 and 58), Upper Spring orifice white filament bundles (sample 114), Upper Spring thin white filaments (sample 190), Lower Spring orifice white filament bundles (sample 199), and Lower Spring yellowish-white mat (sample 198). Mat samples used for FISH were collected from the Fissure Spring orifice, Lower Spring orifice, the white filamentous mat from the Lower Spring, Upper Spring orifice, and three white filamentous mat samples from the Upper Spring stream channel (white mat 1, 2, and 3). Sample Acquisition from Other Sulfidic Caves and Springs White filamentous microbial mats from four sulfidic cave springs (12 to 22oC), four mesothermal (10 to 33oC) sulfidic surface-discharging springs, and eight thermal sulfidic surface-discharging springs (45 to 65oC) were sampled from the United States and Italy (Table 3-1). The broad geography and geochemical conditions were of interest in order to survey for "Epsilonproteobacteria" related to 94 those from Lower Kane Cave using epsilonproteobacterial-specific PCR primer sets. Refer to Appendix B for descriptions of the field sites. Spring pH and sulfide content were not significantly different (Table 3-1). There were several surface springs with phototrophic microbial biomass associated with the white filamentous mat morphotypes (Appendix B, Figures B-1 through B-10). All of the caves that were sampled formed in limestone or travertine, and almost all of the surface springs discharged from carbonates, either limestone or travertine (Table 3-1). The Lazio springs in Italy issued from volcanics (e.g., rhyolite), although the spring waters were also actively depositing travertine, and Palmetto Spring, Texas, discharged from sandstone, but the spring orifice was in an artificial concrete basin. DNA Extraction Approximately 0.2 to 0.5 ml of microbial mat from caves and springs were aseptically collected in the cave and transferred into 0.5 to 1 ml DNA extraction buffer containing 10 mM Tris-HCl (pH 8), 100 mM EDTA, and 2% sodium dodecyl sulfate (SDS). Total community DNA was extracted using an extraction protocol similar to the commercially available Purgene DNA extraction kits (Gentra Systems, Minneapolis, Minnesota), with the following modifications: 9 l Proteinase K (20 mg/ml) was added to each DNA extraction buffer prior to digestion; a freeze-thaw (3 times at 80oC to 65oC) series was used; samples were incubated at 55oC overnight to digest cellular material; RNase was added to the digests and incubated at 37oC for up to 1 hr; and nucleic acids were precipitated in isopropanol overnight at 20oC. 95 PCR Amplification For Lower Kane Cave samples, nearly full-length 16S rDNA gene sequences were PCR-amplified using the primer pairs 27f (forward, 5'AGAGTTTGATCCTGGCTCAG-3') and 1492r (reverse, 5'-GGTTACCTT GTTACGACTT-3') for the domain Bacteria, according to the protocol described by Lane et al. (1991). Amplification was performed with a Perkin Elmer 9700 thermal cycler by Megan L. Porter at Brigham Young University, Department of Integrative Biology (Provo, Utah). The following conditions were used: denaturation at 95oC for 1 min, primer annealing at 42oC for 1 min, chain extension at 72oC for 1.5 min. Fifty PCR cycles were used. A control tube containing sterile water instead of DNA was used as a negative control. For other cave and spring samples, 16S rDNA gene sequences were PCRamplified using lineage-specific epsilonproteobacterial PCR primers designed during FISH probe design from the PROBE-DESIGN tools of the ARB software package (http://www.arb-home.de) (Ludwig and Strunk, 1996). For epsilonproteobacterial LKC group I, the primer pairs `eps59f' (forward, 5'AGTCGAACGATGAGAGGA-3') and 1492r for the domain Bacteria were used. For epsilonproteobacterial LKC group II, the primer pairs `eps174f' (forward, 5'CCCCATACTCCTTCTCAT-3') and 1492r. Amplification was performed in the Jackson School of Geosciences at the University of Texas at Austin with a MJ Research thermal cycler under the following conditions: denaturation at 95oC for 1 min, primer annealing at 45oC for eps59f and 47oC for eps174f for 1 min, chain extension at 72oC for 1.5 min. A control tube containing sterile water instead of 96 DNA was used as a negative control. Thirty cycles were done. PCR bands were visualized by electrophoresis on a 1% agarose gel. Cloning, Sequencing, and Phylogenetic Analysis of 16S rRNA Genes Amplified PCR products for Lower Kane Cave samples were purified with the GeneClean II Kit (Bio101, Inc., Vista, California), as recommended by the manufacturer. Purified PCR products were cloned using the TOPO TA Cloning Kit (Invitrogen, San Diego, California), following manufacturer's instructions. The 16S rDNA clones obtained from Lower Kane Cave were lysed in 50 l buffer (10mM Tris-HCl; 0.1 mM EDTA, pH 8.0) for 10 min at 96oC. Clones were randomly selected for sequencing, and sequence inserts were then PCR-amplified from lysed cells using plasmid-specific primer pairs M13(-20) (5'- GTAAAACGACGGCCAGT-3') and M13(-24) (5'-AACAGCTATGACCATG-3') and the following PCR conditions: denaturation at 94oC for 1 min, primer annealing at 55oC for 1 min, chain extension at 72oC for 3 min, for 35 cycles. PCR products were purified using Sephadex columns and sequenced using an ABI BigDye Ready Reaction kit (Perkin Elmer) using the primers 27f and 1492r in conjunction with internal primers 907r (reverse, 5'-CCGTCAATTC CTTTRAGTTT-3') and 704f (forward, 5'-GTAGCGGTGAAATGCGTAGA-3'). Automated DNA sequencing was done on an ABI Prism 377XL sequencer (Perkin Elmer) at Brigham Young University. The DNA sequences were submitted to the CHECK-CHIMERA program of the Ribosomal Data Base Project (RDP) II at Michigan State University (http://rdp.cme.msu.edu/html/) (Maidak et al., 2001). Clone sequences were 97 subjected to BLAST searches within the GenBank database (http://www.ncbi.nlm.nih.gov/) to determine 16S rRNA gene sequence similarities to culturable and not yet cultured organisms. The retrieved nucleotide sequences were first aligned using Clustal X (Thompson et al., 1997) and then manually adjusted based on conserved gene primary and secondary configuration. Phylogenetic analyses were done by Megan L. Porter at Brigham Young University, using maximum likelihood, minimum evolution, and maximum parsimony criteria in PAUP* (Swofford, 2000) and Bayesian inference in MrBayes (Huelsenbeck and Crandall, 1997). For minimum evolution, Bayesian inference, and maximum likelihood searches, a model of evolution was chosen based on likelihood ratio tests (Huelsenbeck and Crandall, 1997), as implemented in Modeltest 3.06 (Posada and Crandall, 1998). As an indication of nodal support, bootstrap analyses were performed for maximum likelihood (100 replicates), minimum evolution (500 replicates), and maximum parsimony (1000 replicates). For Bayesian inference analyses, posterior probabilities were used for nodal support. All trees were rooted using Desulfocapsa thiozymogenes (X95181) and Hydrogenophaga pseudoflava (AF078770) as outgroups. 16S rRNA Oligonucleotide Probes Oligonucleotide probes specific for two epsilonproteobacterial clone groups from Lower Kane Cave (LKC group I and group II) were designed using the PROBE-DESIGN tools from ARB and probe designations according to Alm et al. (Alm et al., 1996). Probe LKC59 (S-*-eProt-0059-a-A-18) is specific for LKC 98 group I clones, and probe LKC1006 (S-*-eProt-1006-a-A-18) targets LKC group II clones. Probe specificity was verified using the RDP PROBE-MATCH function (Maidak et al., 2001) and the PROBE-MATCH tool of the ARB software package, and a 16S rRNA data set including all publicly available sequences from "Epsilonproteobacteria" (at the time of probe design, 238 epsilonproteobacterial GenBank sequences were available for comparison). Sequences and optimal hybridization conditions for probes LKC59, LKC1006, and other probes used, are listed in Table 3-2, including probe EPS710, designed to target environmental clones within the "Epsilonproteobacteria" (Watanabe et al., 2000) and Sulfuricurvum kujiense (Kodama and Watanabe, 2003). More information about the applied probes can be found at probeBase (http://www.microbial- ecology.net/probeBase) (Loy et al., 2003). Fluorescence In Situ Hybridization (FISH), Microscopy, and Quantification For FISH, microbial mat samples were collected and shipped on dry ice to Dr. Michael Wagner and Dr. Natuschka Lee in the Department of Microbial Ecology, Technische Universit t in Munich, Germany. Samples were preserved in two ways (with respect to the general requirements for successful FISH of gramnegative and gram-positive bacteria) within 48 hr of collection: (i) with 4% (wt/vol) paraformaldehyde for 3 hr before final wash with saline phosphate buffer as described by Manz et al. (1992), and (ii) with ice-cold ethanol according to Roller et al. (1994). I preformed all hybridizations and examined samples using a LSM510 confocal laser scanning microscopy (CLSM) (Zeiss, Oberkochen, Germany) in 99 Germany. Prepared cells were attached to Teflon-coated nonfluorescence slides and air-dried overnight before dehydrating by sequential washes in 50, 80, and 100% (vol/vol) ethanol for 3 min each. The probes were synthesized and directly labeled with the monofunctional, hydrophilic, sulfoindocyanine dyes indocarbocyanine (Cy3) and indodicarbocyanine (Cy5), or with FluosPrime (5,6-carboxyfluoresceinN-hydroxysuccinimide ester), purchased from Hybaid-Interactiva (Ulm, Germany). Hybridization and washes were performed as described by Manz et al. (1992). Briefly, the hybridization solution contained 5 M NaCl, 1 M Tris/HCl (pH 8), 10% [wt/vol] SDS, and formamide (as given in Table 3-2 for each probe). Cy3 and Cy5labelled probes were applied at 30 ng l-1 each, while FluosPrime probes were applied at 50 ng l-1. Washing buffer consisted of 5 M NaCl, 1 M Tris/HCl (pH 8), 10% SDS, and 0.5 M EDTA (pH 8) and was preheated to 48oC. The wash buffer salt concentration was adjusted to the hybridization buffer formamide concentration according to Manz et al. (1992). Optimal hybridization stringency for probes EPS710, LKC59, and LKC1006 was determined by increasing the formamide concentration of the hybridization buffer in increments of 5 or 10% while maintaining a constant hybridization temperature of 46oC. Because of a lack of suitable reference cells, the optimal hybridization stringency for the probes was defined by the highest stringency allowing unambiguous visual detection of probe target cells in fixed samples of the white filamentous microbial mats from two different sampling locations (`Upper Spg white mat 1' and `Upper Spg white mat 3'). 100 To determine the percentage of all cells detected with the bacterial probe set, EUB338I-III mix-hybridized mat samples were additionally stained with 10 l of a 10,000-fold dilute SYBR Green I (FMS Bioproducts, Rockland, Maine) working solution in the dark for 10 min at room temperature. Slides were then washed briefly with double-distilled H2O and air-dried. Before examination, samples were covered with the antifading agent Citifluor AF1 (Chemical Laboratory, Caterbury, England). A CLSM equipped with a UV laser (351 and 364 nm), an Ar ion laser (450 to 514 nm), and two HeNe lasers (543 and 633 nm) was used to visualize FISH results. All images were recorded with a Plan-Apochromat 63x (1.4; oil immersion) objective. Image processing was performed with the LSM510 software (version 1.6). Quantification of probe-detected cells was achieved using Carl Zeiss Vision KS400 software in conjunction with the RAM (Relative Area Measurement) macro (Schmid et al., 2000). Nucleotide Sequence Accession Numbers The 16S rRNA gene sequences determined in this study, labeled with the prefix "LKC1", were submitted to GenBank, with accession numbers from AY191466 to AY191497. RESULTS Phylogenetic Analysis of 16S rRNA Clone Sequences from Lower Kane Cave From six mat samples, 44 clones were randomly selected for sequencing in order to conduct a broad survey of the microorganisms present in Lower Kane Cave mat communities. Nearly full-length 16S rRNA gene fragments from the 101 clones were amplified and sequenced. None of the sequences were identified as chimera from RDP analysis. All clone sequences belonged to the Proteobacteria division; 85% of the clones were affiliated with the "Epsilonproteobacteria", 11% of the clones belonged to the Betaproteobacteria, and 4% of the clones clustered within the Gammaproteobacteria, and specifically Thiothrix spp. The Lower Kane Cave epsilonproteobacterial clones clustered into two different clades, referred to as LKC group I and LKC group II, with high bootstrap values supporting their phylogenetic position (Figure 3-1), similar to results described in Chapter 2. The closest relatives to both LKC group I and group II were two environmental clones, sipK119 and sipK94, obtained from microbial aggregates with a string-of-pearls-like morphology in sulfidic springs at the Sippenauer Moor, Regensburg, Germany (Rudolph et al., 2001; Moissl et al., 2002). Group I clones, identified from the Fissure and Upper Spring orifice samples, clustered closely (98-99% similar in nucleotide sequence) with clone sipK119 and Cesspool Cave clone group CC-4 (Engel et al., 2001). The closest cultured representative of LKC group I sequences was Sulfuricurvum kujiense (Kodama and Watanabe, 2003). LKC group II clones, obtained from Upper and Lower Spring orifice and white mat samples, were closely related to the sipK94 clone (99% similar in nucleotide sequence), and more distantly to various marine and hydrothermal vent clones (91-94% similarity), as well as to Parker Cave clones (92-94% identical) (Angert et al., 1998). 102 Fluorescence In Situ Hybridization of Lower Kane Cave Microbial Mats FISH probes were used to identify and to quantify specific microorganisms in the filamentous microbial mats from Lower Kane Cave (Appendix B). Thirtytwo clone sequences, thirteen from LKC group I and nineteen from LKC group II (Figure 3-1), were used to design rRNA-specific oligonucleotide probes. Probes LKC59 and LKC1006 targeted clone sequences from group I and group II, respectively, based on the PROBE-MATCH function in the ARB software (Table 3-3). Although LKC group I is closely related to other environmental clones from groundwater and caves (Figure 3-1), probe LKC59 has at least one mismatch with these and all other rRNA gene sequences in the database. Probe LKC1006 also did not target any other sequences in the database, including clone sipK94 which has 99% 16S rRNA gene sequence similarity to LKC group II sequence (Figure 3-1). Another probe for LKC group II was LKC174, targeting base position 174; however, the LKC174 probe did not work for FISH (no hybridization signal was observed), possibly because the 16S rRNA target region was inaccessible; probe LKC174 was disregarded for further FISH analyses. The target sequence was used instead as a PCR primer for LKC group II. Three of the LKC group II clones possess a single mismatch within the target site of probe LKC1006 and might not be detectable by this probe (Table 3-3). These mismatches indicate either actual genetic microheterogeneity or originate from PCR and/or sequencing artifacts. Optimal hybridization stringency was determined for the newly designed probes, as well as for the previously published probe EPS710 (Watanabe et al., 2000); (Kodama and Watanabe, 2003). Because no cultured strains possessing the 103 target sites for probes LKC59 and LKC1006 are available, optimal hybridization stringency was inferred by visual comparison of filament fluorescence from cave samples (Figure 3-2; Appendix B). For all three probes, mat filaments showed bright fluorescence if hybridization buffers with up to 30% formamide were used (Figure 3-2); under more stringent conditions (higher formamide concentrations), signal intensity decreased. In the six samples analyzed, between 68% and 88% of the cells stainable with a general nucleic acid dye (SYBR green I) could be detected with a bacterial probe set (Table 3-2; Appendix B). Of the ten different group- and genus-specific probes applied, positive hybridization signals were observed with the probes BET42a, GAM42a, G123T, and ARCH915. Long filamentous cells hybridized strongly with probes GAM42a and G123T, indicating the presence of Gammaproteobacteria, and in particular Thiothrix spp., which also hybridized to EUB338I-IIImix (Figure 3-3A and 3-3B). Probe BET42a labeled small rods specifically, but weakly, in all samples (Figure 3-3C), indicating the presence of rare Betaproteobacteria. The FISH results for Betaproteobacteria and Gammaproteobacteria are similar to the results obtained from the 16S rRNA clone libraries (Chapter 2), in that these bacterial groups occur in the mats but are not dominant. In addition to the filamentous Gammaproteobacteria, the other filaments (Figure 3-3D) hybridized with EUB338I-IIImix, are likely "Epsilonproteobacteria". Interestingly, filaments that hybridized with GAM42a and G123T also hybridized with ARCH915. The PROBE-MATCH function in ARB suggested that the probe ARCH915 targeted some Thiothrix sequences, 104 particularly T. unzii, and therefore may not be a suitable probe when Thiothrix is present. No further analyses with the ARCH915 probe were done (Appendix B). The three probes targeting subgroups within the "Epsilonproteobacteria" were used in different combinations to survey probe overlap. All probes exclusively hybridized to filamentous microbes, and conferred very bright signals to their target cells, indicating high rRNA contents (Amann et al., 1995). Filaments detected by probe LKC59 had an average diameter of 1 m and appeared kinked or twisted, while straighter, longer, and slightly thicker filaments were detected by LKC1006 (Figure 3-4). Twisted filaments were observed with scanning electron microscopy (Figure 2-3E). Simultaneous hybridization with the epsilonproteobacterial probes LKC59 and EPS710 showed that they stained the same filamentous cells (Figure 3-4, row I), and as expected, neither probe EPS710 nor probe LKC59 overlapped with probe LKC1006 (Figure 3-4, rows II and III). In the study by Watanabe et al. (2000), probe EPS710 hybridized only to short rods during FISH. The different morphologies that hybridize to the EPS710 probe may indicate the potential for more group diversity than previously identified, and that the probe may confer genus-level or higher taxonomic specificity. Epsilonproteobacterial filaments belonging to LKC group II dominated five of the six microbial mats examined and made up to 70% of the biovolume of those cells detectable by FISH with a bacterial probe set (Table 3-4). Only in Lower Spring white mats, which were dominated by Thiothrix spp., epsilonproteobacterial filaments occurred at relatively low numbers (4% of the bacterial biovolume). In contrast, epsilonproteobacterial filaments of LKC group I were below the detection 105 limit in three of the six samples investigated, and made up less than 10% of the biovolume in the other three samples (Table 3-4). Distribution of "Epsilonproteobacteria" in Other Caves and Springs The primer sequences for eprot59f (group I) and eprot174f (group II) were analyzed in GenBank for lineage-specificity (Table 3-5). Primer eprot59f showed a perfect match for the clone sipK119, the closest relative to LKC group I sequences. Other sequences, including members of the Actinobacteria, had one or more mismatches at the end of the primer. Primer eprot174f showed a perfect match for the epsilonproteobacterial clone T6-ph07-987 from deep-sea experimental chamber associated with Alvinella (Alain et al., 2004), but not for clone sipK119 (99% sequence similarity). Previous 16S rRNA-based studies of several sulfidic springs have revealed the presence of "Epsilonproteobacteria" (Engel et al., 2001; Barton et al., 2002; Elshahed et al., 2003; Rzonca and Schulze-Makuch, 2003), and microbial mat samples from several of these sites (e.g., Table 3-1; Lower Kane Cave, Cesspool Cave, Glenwood Springs, and Soda Dam Spring) were used to test the utility of the new primers. Certainly, to verify that the phylogenetic groups of interest were amplified completely, the resulting PCR products should be cloned and sequenced. Additionally, a positive result does not always indicate that the organisms of interest are present if primers are not optimally applied (e.g., by using annealing temperatures that are not stringent). However, positive amplification using lineagespecific primers is a first step to circumvent the need to sequence many products randomly; if PCR is not successful following multiple approaches, no further 106 analyses are likely needed. Moreover, quantification is not possible by using this PCR-based method, so to determine if the groups are numerically important, additional studies using FISH are needed. FISH will also reveal the morphology of the microbial groups, which is important to demonstrate that the epsilonproteobacterial groups that hybridize with the LKC probes are filamentous in other habitats. Positive PCR amplification with the lineage-specific primers was achieved for many white filamentous microbial mat samples; the general bacterial primer set was also used to verify results. Both epsilonproteobacterial primer sets had positive PCR amplification to Lower Kane Cave samples, as well as from Hellspont Cave and PBS Spring, sites downstream from Lower Kane Cave along the Bighorn River (Table 3-6; Figure 1-3). Based on phylogenetic relationships, the epsilonproteobacterial clone groups identified from Cesspool Cave were closely related to LKC group I (Engel et al., 2001), and PCR amplification with the eps59f/1492r primer set showed positive amplification, but not with the LKC group II primers (Table 3-6). "Epsilonproteobacteria" were also described from 16S rDNA investigations from the Drinking Spring at Glenwood Springs, Colorado (Barton et al., 2002), and Soda Dam spring, New Mexico (Rzonca and SchulzeMakuch, 2003); successful PCR amplification was obtained using both primer sets for Glenwood Springs, and only the eps59f/1492r set for Soda Dam spring (Table 3-6). PCR amplification with the eps59f/1492r primers was successful for mat samples from all the sulfidic caves and several springs (Table 3-6). The group II 107 primers successfully amplified all samples except those from Cesspool Cave and several springs (Table 3-6). DISCUSSION Defining the composition of microbial communities can aid in our understanding of biogeochemical cycling that occurs in remote and difficult-tocharacterize habitats. Particularly for the terrestrial subsurface, little is known about microbial community structures because of the limited number of investigations done on such systems. The application here of the full-cycle rRNA approach to characterize microbial mats and "Epsilonproteobacteria" from cave and spring habitats, with Lower Kane Cave as the feature site, enabled assessment of the biogeochemical significance of these previously unknown "Epsilonproteobacteria" groups in a variety of environments. The majority of 16S rRNA gene clones from Lower Kane Cave microbial mats from this investigation were assigned to two evolutionary lineages within the "Epsilonproteobacteria", designated LKC group I and II, neither of which have closely related cultured representatives. Several 16S rDNA sequences retrieved from groundwater at an underground petroleum storage cavity (Watanabe et al., 2000; Watanabe et al., 2002) and S. kujiense (Kodama and Watanabe, 2003), an anaerobic sulfur oxidizer recently isolated from this habitat, cluster with LKC group I (Figure 3-1). Two clone groups (CC-4 and CC-9) from Cesspool Cave (Engel et al., 2001) are also closely related to LKC group I (Figure 3-1). In contrast, LKC group II forms a monophyletic grouping with the 16S rRNA gene sequence of an uncultured filamentous epibiont associated with the vent annelid 108 Alvinella pompejana (Campbell et al., 2001), and clone groups from phylogenetic studies of Parker Cave, another sulfidic cave system (Angert et al., 1998), are found in sister clades (Figure 3-1). Quantitative FISH with two newly developed 16S rRNA-targeted oligonucleotide probes specific for LKC group I and II revealed that both groups in Lower Kane Cave are filamentous bacteria. Filamentous "Epsilonproteobacteria" have not previously been identified, as most "Epsilonproteobacteria" are demonstrably rod-shaped, helical, curved, or ovoid in cell morphology (Miroshnichenko et al., 2004). Bright FISH signals observed for both groups also suggest that the microbes were physiologically active when the mats were collected (Amann et al., 1995). LKC group I filaments were detected in low abundances in three samples, and were not detected in other samples. In contrast, LKC group II filaments dominated five of six samples by biovolume, and represented 50% to 70% of the community bacterial biovolume (Table 3-4). The FISH results do complement the 16S rDNA clone libraries, as described in Chapter 2, but disparate results also point to the importance of using more than one molecular method to characterize microbial communities. Specifically, the quantification results with FISH for LKC groups I and II (Table 34) are slightly different than the phylotype results from restriction fragment length polymorphism (RFLP) that suggest both LKC groups I and II have nearly the same population sizes in some of the microbial mat samples (Chapter 2; Table 2-3), although in any one filamentous mat sample either one of the groups could 109 dominate. For example, LKC group I clones were prevalent by RFLP in samples from the Fissure Spring, but not LKC group II as indicated by FISH (Table 3-4). Results from FISH did not target high microdiversity within the "Epsilonproteobacteria" that was identified from RFLP (Chapter 2; Table 2-3 and Table 2-5; Figure 2-8). The FISH probes designed in this part of the study were intended for LKC groups I and II only, but probe LKC59 (LKC group I) could also target three of 17 clone sequences from LKC group III (Figure 2-8). Probe LKC1006 targeted only LKC group II clone sequences. Conversely, probe EPS710, designed by Watanabe et al. (2000) for a group of "Epsilonproteobacteria" from groundwater, targeted LKC group I, III, and IV clone sequences, indicating that the EPS710 probe could broadly hybridize to a large group of "Epsilonproteobacteria" in which most of the environmental clones are from groundwater and caves, much more broadly than the LKC probes or primers. Sequences for the LKC groups V and VI are not targeted by any 16S rRNA probe currently known, indicating additional diversity within the "Epsilonproteobacteria". Since these groups were not numerically important by RFLP, probes or primers were not constructed, although future work to describe complete epsilonproteobacterial diversity will require targeting these groups. While high in situ abundance of free-living and eukaryote-associated "Epsilonproteobacteria" have been described from many marine environments, including hydrothermal vent and field communities and marine sediments, there has been no evidence that members of the "Epsilonproteobacteria" are also numerically important in terrestrial systems. The only exceptions currently known 110 are from two engineered systems and caves in which "Epsilonproteobacteria" exist in significant numbers. In the petroleum-contaminated groundwater associated with a storage cavity in Japan, between 12% and 24% of the total prokaryote cells could be identified via FISH as epsilonproteobacterial curved rods (Watanabe et al., 2000; Watanabe et al., 2002). Relatively low abundances of Arcobacter (4%) were identified with FISH from activated sludge in a wastewater treatment plant (Snaider et al., 1997). Epsilonproteobacterial abundance estimates from 16S rRNA clone libraries in Parker and Cesspool Caves, at 73% and 47% "Epsilonproteobacteria", respectively, are similar to the FISH biovolume estimates for LKC groups I and II (Angert et al., 1998; Engel et al., 2001). Although the mats from the sulfidic springs in Sippenauer Moor harbor "Epsilonproteobacteria" closely related to LKC group I and II, FISH analysis revealed that these mats are dominated not by "Epsilonproteobacteria", but by Thiothrix spp. and novel Archaea (Rudolph et al., 2001; Moissl et al., 2002). Consequently, the mats from Lower Kane Cave represent the first non-marine natural system demonstrably driven by the activity of "Epsilonproteobacteria". "Epsilonproteobacteria" obviously are successful at colonizing a range of marine habitats, and one of the goals of this study was to determine if "Epsilonproteobacteria" potentially are as widespread in terrestrial sulfidic environments as they are in marine settings. Results suggest that terrestrial "Epsilonproteobacteria" can occupy a variety of physicochemical conditions, and specifically a range of thermal conditions (Table 3-1; Appendix B). Most of the sampling locations were associated with discharge from limestone or travertine 111 (Table 3-1), suggesting that the "Epsilonproteobacteria" may prefer to colonize habitats where acidity generated from their metabolism (e.g., sulfur oxidation) is potentially buffered by dissolving limestone. The prevalence of "Epsilonproteobacteria" in terrestrial environments may be even more widespread than results of this study indicate, and the utility of the epsilonproteobacterial-specific probe and primer sets is significant. There are fewer terrestrial epsilonproteobacterial 16S rDNA gene sequences available in the public databases compared to marine-originating sequences, indicating a lack of information regarding the presence of "Epsilonproteobacteria" in terrestrial settings. The clone sequences previously retrieved from Cesspool Cave (Engel et al., 2001) and Sippenaeuer Moor (Rudolph et al., 2001) provided the first indications that at least LKC group I was more widely distributed in terrestrial sulfidic spring and cave habitats than previous research suggested. It is possible that other sites are occupied by members of the "Epsilonproteobacteria" that are not targeted by the eps59f or eps174f PCR primers. For instance, epsilonproteobacterial clone groups that are phylogenetically distinct from LKC clone groups have been reported from the sulfidic Parker Cave, Kentucky (Angert et al., 1998), and Zodletone Spring, Oklahoma (Elshahed et al., 2003) (Figure 2-8), indicating additional diversity not targeted by the lineage-specific primers applied in this study. Because of the specificity of the epsilonproteobacterial primers designed in this study, however, it is highly probable that the epsilonproteobacterial groups in the systems I examined are closely related in nucleotide sequence to the Lower 112 Kane Cave groups, and may not be genetically distinct enough to suggest different microbial species. A low degree of genetic divergence within these groups would indicate that modern geographic isolation may not be a driving factor in the speciation of the LKC epsilonproteobacterial groups, in contrast to what has been demonstrated for some cyanobacteria from hot springs around the world (Papke et al., 2003). Obtaining the full-length 16S rRNA gene sequences, as well as other gene sequences, are required in the future to examine the significance of geographic isolation and genetic evolution for "Epsilonproteobacteria". At the onset, however, this study expands the geographic diversity of "Epsilonproteobacteria" to a variety of terrestrial photic and aphotic sulfidic habitats, suggesting that these groups may be more important ecologically and geologically than previously considered. 113 Table 3-1: Geographic and physicochemical information for sampling sites. Refer to Appendix B for complete site descriptions. Location pH 7.3 7.1 7.4 7.2 7.4 6-7 7.2 NM 6.9 6.4 NM NM NM NM 6.5 6.57.5 Limestone Limestone Sandstone Limestone Limestone Limestone Limestone Limestone Volcanics, travertine Limestone Limestone Travertine Limestone Limestone Rock type Big Horn County, Wyoming Big Horn County, Wyoming Allegheny County, Virginia Trigg County, Kentucky Genga, Italy Schoharie County, New York Big Horn County, Wyoming Gonzales County, Texas Hot Springs County, Wyoming Garfield County, Colorado Washington County, Utah Utah County, Utah Sandoval County, New Mexico Viterbo, Italy 45 46-65 48 45-55 50-54 1.6 NM NM 10 13 17 53-57 NM 5 NM 4.5 Literature sources Cave, Spring, or Stream Lower Kane Cave springs Hellspont Cave spring Cesspool Cave spring Big Sulphur Cave stream water Frasassi Cave resurgent spring Temp o C 21 22 12 15 13 H2Sa ppm 6-8 12 14-21 NM 6-24 White Sulphur Springs PBS Spring Palmetto Spring Thermopolis Hot springs 114 Glenwood Hot Springs Pah Tempe Mineral Hot Springs Fifth Water Hot Spring (aka Diamond Fork) Soda Dam Spring Engel et al., 2003 This study Engel et al., 2001 This study Sarbu et al., 2000; Vlasceanu et al., 2000 This study This study This study Breckenridge and Hinckely, 1978 Glendon, 1989 This study This study Le Zitelle Springs Goff and Grigsby, 1982; Rzonca and Schulze-Makuch., 2003 This study a NM: not measured Table 3-2: Probe sequences used to screen cave microbial populations. Refer to Appendix B for screening results. Target group Probe sequence (5' 3') FAb References Probe 115 EUB338 EUB338-II EUB338-III NonEUB ARCH344 ALF968 BET42a GAM42a HGC69a LGC345A LGC354B LGC354C CF319a G123Tc EPS710 16S (59) 16S (1006) 30 30 Target Sitea GCT GCC TCC CGT AGG AGT 16S (338) GCA GCC ACC CGT AGG TGT 16S (338) GCT GCC ACC CGT AGG TGT 16S (338) ACT CCT ACG GGA GGC AGC 16S (338) TCG CGC CTG CTG CIC CCC GT 16S (344) GGT AAG GTT CTG CGC GTT 16S (968) GCC TTC CCA CTT CGT TT 23S (1027) GCC TTC CCA CAT CGT TT 23S (1027) TAT AGT TAC CAC CGC CGT 23S (1901) TGG AAG ATT CCC TAC TGC 16S (354) CGG AAG ATT CCC TAC TGC CGG CGT CGC TGC GTC AGG TGG TCC GTG TCT CAG TAC 16S (319) CCT TCC GAT CTC TAT GCA 16S (697) CAG TAT CAT CCC AGC AGA 16S (710) 0-40 0-40 0-40 0 0 20 35 35 25 20 20 20 35 40 30d Daims et al., 1999 Daims et al., 1999 Daims et al., 1999 Wallner et al., 1993 Roller et al., 1994 Neef et al., 1998 Manz et al., 1992 Manz et al., 1992 Roller et al., 1994 Meier et al., 1999 Meier et al., 1999 Meier et al., 1999 Manz et al., 1996 Kanagawa et al., 2000 Watanabe et al., 2000 This study This study LKC59 LKC1006 a Eubacteria Planctomycetes Verrucomicrobia (and others) Negative control Archaea Alphaproteobacteria Betaproteobacteria Gammaproteobacteria Actinobacteria Firmicutes (with two other probes) Same as LGC344A Same as LGC344A Some members of "Flavobacteria" Thiothrix "Epsilonproteobacteria" Thiovulum groundwater subgroup Epsilonproteobacterial Group 1 from TCC TCT CAT CGT TCG ACT Lower Kane Cave Epsilonproteobacterial Group 2 from CTC CAA TGT TTC CAT CGG Lower Kane Cave E. coli rRNA position (Brosius et al., 1981). Formamide percentage (vol/vol) in the FISH hybridization buffer. c Used in conjunction with a competitor probe G123T-C (5'-CCTTCCGATCTCTACGCA-3') (Kanagawa et al., 2000). d This formamide concentration differs from the one suggested in the original publication (Watanabe et al., 2000) because a different hybridization temperature was used. b Table 3-3: Difference alignment of the target regions of the 16S rRNA for LKCspecific probes. Probe and target LKC59 probe sequence (5'-3') Target sequence Target sequence a TCCTCTCATCGTTCGACT AGUCGAACGAUGAGAGGA ------------------------------U----------------U---CTCCAATGTTTCCATCGG CCGAUGGAAACAUUGGAG --------------------G--------------U------------------------U-------- LKC Group I clonesb Uncultured Cesspool Cave clone CC-4 [AF207530] Uncultured groundwater clone 1023 [AB030610] Most LKC Group II clonesc LKC1.114_5 [AY191480] LKC1.199_5 [AY191494] LKC1.199_6 [AY191495] a -, identical to the probe sequence. b Based on nine LKC group I clones with 16S rRNA gene sequences at this position. c Based on fifteen LKC group II clones. LKC1006 probe sequence (5'-3') Target sequence 116 Table 3-4: Quantification of epsilonproteobacterial filaments of LKC Group I and II with specific FISH probes.a Relative Biovolume, % (SE) Fissure Spring Upper Spring White mat 1 White mat 2 Orifice b Group Lower Spring White mat 3 Orifice White mat 117 88 (2) <8c (2) <1 70 (2) 64 (5) 67 (4) <10c (1) 87 (3) 77 (3) 80 (3) <1 57 (2) 74 (1) 2 (1) 50 (1) 68 (4) <1 4 (1) EUB338I-III mix / SYBR Green I ratio LKC Group I / EUB338I-III mix LKC Group II / EUB338I-III mix a Relative biovolumes are given as percentages, and the number in parentheses is the standard error (SE). White mat 1 and white mat 2 were separated by 4 m, and white mat 2 and white mat 3 were separated by 5 m. c This value is most likely overestimated because of relatively weak filament signal intensity and high background. b Table 3-5: Difference alignment of the LKC-specific PCR primers. Primer and Sequences eprot59 primer sequence (5'-3') LKC Group I clones Uncultured spring clone sipK119 [AJ307940] Uncultured Actinobacteria clone ARK10173 [AF468440] and 2 others Uncultured clone T2772c [AF019024] Sequence Matcha AGTCGAACGATGAGAGGA ---------------------------------------------------G -----------------G CCCCATACTCCTTCTCAT --------------------------------------------------CC ----------------TC eprot174 primer sequence (5'-3') LKC Group II clones Uncultured "Epsilonproteobacteria" clone T6-ph07-987 [UEP576005] Uncultured "Epsilonproteobacteria" clone BD7-9 [AB015584] and 2 other deep-sea clones [AB015582 and AF468745] a -, identical to primer sequence. Table 3-6: Geographic and physicochemical information for additional sampling sites. Refer to Appendix B for PCR and electrophoresis gel details. Location Bacterial primers eprot59f eprot174f 8f/1492r a Lower Kane Cave b b Hellspont Cave Cesspool Cave Big Sulphur Cave PBS Spring Frasassi Caves Spring White Sulphur Springs Palmetto Spring Thermopolis Hot Springs Glenwood Hot springs b b Pah Tempe Hot Springs -b Diamond Fork Spring -Soda Dam Spring Le Zitelle Spring a , positive PCR amplification; , negative PCR amplification; --, not attempted. b Determined by M.L. Porter, Brigham Young University (refer to Appendix B). 118 Figure 3-1: 16S rRNA gene-based phylogenetic tree showing the positions of 32 clones from Lower Kane Cave (designated `LKC1' and labeled in bold with corresponding sample and clone numbers) within the "Epsilonproteobacteria". Reference sequences (with GenBank accession numbers) were chosen from the RDP to represent the diversity of "Epsilonproteobacteria" members. The topology of the tree was inferred from results of the maximum likelihood analysis and the phylogenetic affiliations of the LKC clones were confirmed by comparison with different reconstruction methods. Specificity of the "Epsilonproteobacteria" FISH probes applied in this study is shown. The tree was rooted with the sequences of Desulfocapsa thiozymogenes and Hydrogenophaga pseudoflava, shown as an arrow `to outgroups.' Numbers at branch intersections refer to bootstrap values for each node from maximum likelihood / maximum parsimony / minimum evolution / and Bayesian inference posterior probabilities (values below 50% or where only one method supports a node are not shown). 119 Parker Cave clone SrangJ [AF047633] Parker Cave clone SRang1.27 [AF047626] 62 / 61 / 64 / 100 Activated sludge clone rA10 [AB021361] Marine sediment clone NKB9 [AB013261] 60 / 85 / - / 90 Marine sediment clone SB-17 [AF029044] Hydrothermal vent clone VC2.1Bac1 [AF068783] Estuarine sediment clone 2BP-7 [AF121887] Rifta pachyptila's tube clone R103-B22 [AF449234] Marine sediment clone a2b004 [AF420345] LKC1.57_5 [AY191473] 68 / 61 / 59 / 91 LKC1.198_2 [AY191489] LKC1.57_1 [AY191469] LKC1.199_3 [AY191492] LKC1.199_1 [AY191490] LKC1.57_9 [AY191474] LKC1.199_2 [AY191491] Lower Kane LKC1.190_2 [AY191487] LKC1.199_6 [AY191495] Cave Group II LKC1.199_5 [AY191494] LKC1.57_25 [AY191476] FISH LKC1.57_13 [AY191475] 51 / - / - / 69 LKC1.190_1 [AY191486] Probe LKC1006 100 / 97 / 97 / 100 LKC1.199_9 [AY191497] LKC1.190_3 [AY191488] 91 / 98 / 99 / 100 LKC1.199_4 [AY191493] LKC1.199_8 [AY191496] LKC1.114_5 [AY191480] LKC1.57_3 [AY191471] Sulfide spring clone sipK94 [AJ307941] Alvinella pompejana epibiont [L35521] Rimicaris exoculata ectosymbiont [U29081] Sulfurospirillum barnesii [U41564] 85 / 72 / 72 / 100 Campylobacter jejuni [AF393203] LKC1.114_6 [AY191481] LKC1.57_4 [AY191472] LKC1.21_5 [AY191468] LKC1.114_7 [AY191482] Lower Kane LKC1.114_8 [AY191483] Cave Group I LKC1.114_12 [AY191485] LKC1.58_1 [AY191477] 81 / 91 / - / 74 LKC1.21_2 [AY191467] 81 / 65 / 89 / 100 LKC1.58_3 [AY191478] LKC1.57_2 [AY191470] FISH LKC1.114_2 [AY191479] Probe LKC59 LKC1.21_1 [AY191466] LKC1.114_11 [AY191484] Sulfide spring clone sipK119 [AJ307940] Cesspool Cave clone CC-4 [AF207530] 86 / 71 / 72 / 99 Groundwater clone 1023 [AB030610] 90 / 77 / - / 100 Deep Groundwater clone JN5bf [Z69270] 63 / 93 / 99 / 95 Sulfuricurvum kujiense strain YK-1 [AB053951] Groundwater clone KB2 [AB074955] 100 / 100 / 100 / 100 Shallow Groundwater clone FTL212 [AF529098] Shallow Groundwater clone RA9C8 Cesspool Cave clone CC-9 [AF207534] Thiovulum sp. [M92323] 53 / - / - / 82 Thiomicrospira denitrificans [L40808] 100 / 99 / 100 / 100 Pele's Vent clone PVB 12 [U15104] Flexispira rappini [AF034135] 100 / 100 / 100 / 100 Helicobacter pullorum [L36144] Acid mine drainage clone 44a-B1-1 [AY082456] 100 / 94 / 51 / 100 FISH Probe EPS710 To outgroups 0.01 substitutions/site 120 A B 10% Formamide 20% Formamide 25% Formamide 30% Formamide 40% Formamide Figure 3-2: A series of fluorescence images for probe LKC1006 with varying formamide concentrations to optimize hybridization stringency (optimal formamide is 30% for this probe). Columns: A, LKC1006 (labeled with Cy3, colored in red); B, EUB338I-III mix (labeled in Cy5, colored in light blue). When the two probes overlap, the fluorescence color is pink. Scale bar is 20 m. 121 A B C D Figure 3-3: (A) Overlapping fluorescence in situ hybridization probes; green, EUB338I-III mix; light blue, GAM42a for Gammaproteobacteria (e.g., Thiothrix spp.); red and arrows, BET42a for Betaproteobacteria (e.g., Thiobacillus spp.). (B) EUB338I-III probes mix; (C) GAM42a probe only; (D) BET42a probe only. Scale bar is 20 m. 122 A B C D I. LKC59 EPS710 II. LKC1006 EPS710 III. LKC1006 LKC59 Figure 3-4: Fluorescence in situ hybridization of Lower Kane Cave microbial mat samples with probes EUB338I-III mix, newly designed probes LKC59 and LKC1006. Rows: I, Fissure Spring orifice filaments; II, Upper Spring white mat 1; III, Upper Spring white mat 2. Columns: A, EUB338I-III mix (labeled with FluosPrime, colored in green); B and C, epsilonproteobacterial LKC group probe or EPS710 probe (labeled with Cy3 and colored in red, or labeled in CY5 and colored in light blue); D, overlap of columns B and C. If Cy3- and Cy5-labeled probes overlap, the filmaents appear pink. Scale bar is 20 m. c 123 c Chapter 4: Diversity of Anaerobic Microorganisms in Cave Microbial Mats: Using a Culture-based Approach to Understand Carbon and Sulfur Cycling ABSTRACT Hydrogen sulfide-rich groundwater discharges from springs into Lower Kane Cave and Hellspont Cave, Wyoming, where filamentous sulfur-oxidizing bacteria form thick microbial mats, and through chemolithoautotrophy, these bacteria serve as the energetic base of the cave ecosystems. Spring waters entering the caves, as well as the interior of the microbial mats, are devoid of oxygen, and a culture-based approach was used to characterize and to enumerate anaerobic metabolic guilds, including sulfate- and sulfur-reducing bacteria, fermenting bacteria, iron-reducing bacteria, sulfur-oxidizing nitrate- reducing bacteria, and methanogens. Anaerobic microbes were more abundant in mat samples, with up to 106 cells ml-1, compared to water or sediment samples. Sulfate-reducers and fermenters represented the dominant culturable groups from the mats, while sulfurreducers, iron-reducers, and methanogens were in low abundance (<103 cells ml-1). Eight fermentation pathways were identified from Lower Kane Cave enrichments, suggesting that fermenters play a significant role in degrading the chemolithoautotrophically produced organic carbon. This work expands the diversity of some anaerobic metabolic guilds to microbial mats in sulfidic caves, and demonstrates that anaerobic processes are as vital to the cave ecosystem as chemolithoautotrophy. 124 INTRODUCTION Ecosystems are arranged structurally and functionally such that there is a producer component and a consumer component, and the primary goal in ecology is to understand the spatial arrangements of these organisms (Eiler et al., 2003; Horner-Devine et al., 2003). While autotrophs generate organic carbon, predominately carbohydrates (e.g., Hayes, 2001), consumers decompose the carbon, releasing and recycling new compounds back into the ecosystem (Wardle, 2002). While characterizing ecosystem primary productivity is essential, understanding the diversity of microbial consumers and the variety of carbon breakdown pathways are also crucial aspects for differentiating total ecosystem functioning. A significant finding from previous molecular-based investigations of the microbial mats in Lower Kane Cave, as presented in Chapter 2, is that sulfuroxidizing bacterial communities do not occur in isolation, but have the potential to form a complex mat structure with diverse metabolic guilds (Engel et al., 2003; Engel et al., accepted). While sulfate-reducing bacteria (SRB) have long been implicated in the degradation of organic contaminants in many different habitats (e.g., Widdel and Bak, 1992; Stoessell et al., 1993; Voordouw et al., 1996; Ulrich et al., 1998; Minz et al., 1999a; Minz et al., 1999b; Rios-Hernandez et al., 2003), colonization of sulfidic caves by SRB, or even sulfur-reducing bacteria (S0RB), has not been previously recognized. Other microorganisms responsible for fermentation or methanogenesis have been characterized from total community 16S rRNA gene 125 sequences retrieved from subaqueous cave mats (Angert et al., 1998; Engel et al., 2001), but most groups have not been intensely studied in detail. Molecular methods allow characterization of a microbial community that may be difficult, if not impossible, to cultivate (Head et al., 1998). Unfortunately, molecular techniques can create significant biases and underestimations of particular microbial groups, especially if certain organisms have extremely high dominance, or conversely abundances 107 cells per volume (von Wintzingerode et al., 1997; Speksnijder et al., 2001). Although Leff et al. (1995) suggest that only 1% of environmental bacteria can be cultured, and that standard culturing methods do often introduce a selective bias toward microorganisms able to grow quickly and to utilize substrates provided in the medium ("r-strategists") more efficiently than other organisms in the community (McDougald et al., 1998), culturing does allow quantification of metabolically active organisms (Palleroni, 1997). Based on previous geochemical and molecular investigations (Chapter 2), I anticipated that redox stratification of the mats would result in a range of metabolically active anaerobic microorganisms in the microbial mats, and that most would be concentrated in their anaerobic interior. Molecular methods may have overlooked less abundant populations of anaerobic microorganisms in the microbial mats, and this chapter describes a culture-based approach to characterize anaerobic microbes in both Lower Kane Cave and Hellspont Cave, Wyoming (Figure 1-1). I used the most probable number (MPN) method for enumeration and enrichment to examine a variety of anaerobes from mat samples, including many different groups 126 of fermenting bacteria, sulfur-oxidizing nitrate-reducing bacteria, iron-reducing bacteria, three different types of sulfate-reducing bacteria, sulfur-reducing bacteria, and three methanogenic groups. This work demonstrates that anaerobic microbial processes that cycle carbon and sulfur are as vital to the cave ecosystem as chemolithoautotrophy. MATERIALS AND METHODS Sampling Strategy and Protocol Several iterations of the enrichment culturing and biomass estimate techniques were done from 2000 to 2003. Microbial mat samples from Lower Kane Cave and Hellspont Cave were collected aseptically, placed into 15-ml sterile Falcon tubes, and shaken vigorously to disrupt the mat structure prior to inoculation into enrichment media. Shaking proceeded for 1 to 5 min, depending on the type of mat being sampled (i.e., longer times for white filaments, shorter durations for gray sediment). No dispersion amendments were added to the mat samples (e.g., Tween20) because some microbes can utilize these substrates (Holdman and Moore, 1972). Aliquots of disrupted mat material, ranging from 0.1 ml to 0.5 ml depending on the dilution series, were inoculated by syringe into pre-reduced and sterile (PRAS prepared; Holdman and Moore, 1972) medium in the first bottle in a dilution series, per media type. Serial dilutions were performed in triplicate per sampling site, per enrichment medium; for a 10-fold dilution series, there would be a 30 total bottles in three separate dilution series. For Lower Kane Cave samples, 127 all of the first bottle inoculations were done in the cave. At Hellspont Cave, because of sampling restrictions and hazardous sulfide levels, samples were brought out of the cave and manipulated. Samples were removed from the cave within 1-3 hr of inoculation, and bottles were sonicated for 2 min to promote disaggregation and homogenization prior to material transfer. All serial dilutions were initiated within 3-7 hr of collection. Several bottles of sterile media (no inoculations) were used as controls. All bottles were incubated in the dark at room temperature (~21-23 oC). Anaerobic Biomass Estimates from Enrichment Cultures Microbes were enumerated for each metabolic group using the MPN method (Hurely and Roscoe, 1983). A 10-fold serial dilution series was used for cultivable fementers, and SRB, and a 5-fold serial dilution series was used for S0RB, methanogens, and iron-reducers. Upon returning to the laboratory, inoculated bottles were transferred to a Coy anaerobic chamber with N2:H2 mixed gas to maintain anaerobic conditions. The computer program MPN Calculator (Build 2.0; M. Curiale, http://members.ync.net/mcuriale/mpn/index.html) was used to calculate MPN biomass estimates from bottles having positive growth, using 95% confidence intervals (Appendix C). Fermenting Bacteria Fermentative bacteria were initially enumerated using 25% Schaedler's Broth (Difco Laboratories, Detroit, MI) dispensed into 25 ml serum bottles. The medium was prepared per manufacturer instructions. Growth, as visible turbidity, 128 was scored positive after two days. Strain isolates were obtained non-selectively on solid 25% Schaedler's Broth and screened for a variety of fermenting abilities and possible H2S production using triple-sugar iron (TSI) broth (Difco). TSI agar is traditionally used to screen for the physiological activities of some Enterobacteriacea groups (Hajna, 1945). Cells were grown on TSI agar slants in an anaerobic chamber, and growth was screened within 18-24 hr. A combination of cell growth, acid production, gas generation, and medium blackening caused by H2S production in the butt of the tube and/or in the slant describes a specific pathway (although it should be noted that a particular combination of attributes may not be specific to just one group of organisms; (Hajna, 1945). Following TSI screening, strains were maintained on a modified low-nutrient (MLN) broth of (w/v) 1% sucrose, 0.5% peptone, 0.3 % yeast extract, 0.2% glucose, and 0.2% NaCl. Sulfate-reducing Bacteria Sulfate-reducing bacteria (SRB) are divided into two broad physiological subgroups: Group I SRB use lactate, pyruvate, ethanol, or other fatty acids as carbon sources, and Group II SRB oxidize acetate (Widdel and Bak, 1992). Certain species from both SRB groups are capable of growing chemolithoautotrophically with hydrogen as the electron donor, sulfate as the electron acceptor, and CO2 as the sole carbon source (Widdel and Bak, 1992). Carbon-utilizing SRB were cultured from media designed for this work from modified Baar's and Postgate's C media (MacFarlane and Gibson, 1991). Three components were mixed: 400 ml of 129 Component I with 2 g MgSO4 7H2O, 2 g NaSO4, 0.5 g NH4Cl, 0.06 g CaCl2 2H2O; 200 ml of Component II with 0.5 g K2HPO4; and 400 ml Component III having the carbon source(s) and 0.1% yeast extract (Fisher Scientific). Components I and II were pre-autoclaved (121 oC, 20 min), before mixing with boiled Component III. Non-acetate oxidizers (Group I SRB) were provided with a mixture of 20 mM 60% Na-lactate and 20 mM Na-formate. Group II SRB were provided with 20 mM acetate. SRB Groups I and II capable of autotrophic growth were cultured using only Components I and II. Per liter, 10 ml Wolfe's trace element solution was added to each media mixture. Media were PRAS-prepared, with the exception that to reduce the potential for blackening of the media from abiotic iron reduction by cysteine or evolved microbial H2S during growth, 0.01% L-cysteine (Fisher) was added as a PRAS reducing agent. Media were dispensed anaerobically into 10 ml serum bottles, crimp-sealed with butyl rubber caps and aluminum seals, and autoclaved to 121oC (20 min). Following inoculation, Groups I and II SRB bottles were pressurized with N2 and CO2, whereas the autotrophic bottles were pressurized with a 70:30 mixture of H2:CO2 to 140 kPa. Instead of scoring positive growth from visual medium blackening due to the formation of iron-sulfides, from cell and iron-sulfide turbidity, or by using leadacetate paper (e.g., Bekins et al., 1999), bottles were scored for positive growth by measuring evolved headspace H2S with gas chromatography (GC) (refer to Chapter 5 for GC details). This is a much more sensitive and accurate method for 130 determining sulfate-reducing activity. SRB growth was found to be very rapid and growth was scored by GC after 7 days. Sulfur-reducing Bacteria A sulfur-reducing bacteria (S0RB) medium was designed for this study, and consisted of the following components: Component I, 0.5g NH4Cl in 400 ml, and Component II, 0.5 g K2HPO4 in 200 ml; Component III, 20 mM Na-formate, 2 mM Na-acetate, and 1 g Difco yeast extract in 400 ml. Components I and II were autoclaved separately (121oC, 10 min) and mixed with pre-boiled Component III and PRAS preparation. No cysteine was added to the medium. Elemental sulfur flowers were washed with distilled H2O, and crushed with a sterile mortar and pestle under water to hydrate surfaces. Approximately 5 ml sulfur slurry were dispensed anaerobically into 25 ml serum bottles prior to the addition of 10 ml media. Bottles were individually purged with a 90:10 mixture of N2:H2, crimpsealed with butyl rubber caps and aluminum seals, and autoclaved at 112 oC (S0 melts at 121oC) for 20 min. Evolved gases were measured using GC after 2 weeks. Uninoculated bottles were also incubated to ensure no contamination occurred due to the lower autoclaving temperature. Iron-reducing Bacteria Iron-reducing medium was previously described by Lovely and Phillips (1986) and Bekins et al. (1999). The medium was dispensed in 25 ml serum bottles and autoclaved for 15 min. After inoculation, bottles were pressurized with a 70:30 131 mixture of H2:CO2 to 140 kPa. Positive growth was scored after 6 weeks by the bipyridine method (Bekins et al., 1999). Methanogens A variety of methanogens were enriched for using PRAS-prepared dilute mineral salts media previously described in Bekins et al. (1999). Briefly, mineral salts media were amended with 20 mM Na-acetate for methanogens that utilized acetate, and in separate medium 20 mM 60% Na-formate was amended for formate-utilizing methanogens. Hydrogen-oxidizing methanogens were enumerated on only mineral salts medium pressurized with a 70:30 mixture of H2:CO2 to 140 kPa. The use of acetate, formate, or hydrogen distinguishes among different groups of methanogens based on nutrition (Garcia et al., 2000); methanogens that produce methane from methyl-compounds (methylotrophs) were not enumerated. After inoculation, bottles were incubated a minimum of 6 weeks, and scored positive from GC analysis of evolved headspace methane. Denitrifying Bacteria Nemati et al. (2001) suggest that if sulfur-oxidizers use nitrate as an alterative electron acceptor at low O2 tensions, and if sufficient nitrate is not available, then elemental sulfur will accumulate in culture as an indication of positive growth, 5HS- + 2NO3 + 7H+ 5S0 + N2 + 6H2O (4-1) Using this scheme to test for the presence of nitrate-reducing sulfur-oxidizing bacteria from the microbial mats, the following solutions were combined: Solution 132 A, 2.0 g KH2PO4, 2.0 g KNO3, 1.0 g NH4Cl, 0.8 g MgSO4 7H2O in 940 ml dH2O; Solution B, Na2S2O3 5H2O in 40 ml dH2O; Solution C, 1.0 g NaHCO3 in 20 ml dH2O; Solution D, 2.0 g FeSO4 7H2O in 1.0 ml 0.1N H2SO4. Solutions A, B, and D were autoclaved separately at 121 oC (15 min). Solution C was 0.22 m-filtersterilized. The four solutions were combined anaerobically, 2.0 ml sterile Wolfe's trace element solution was added, and medium was dispensed into 250 ml Erlenmeyer flasks in an anaerobic chamber. Flasks were sealed with rubber stoppers to limit oxygen diffusion following inoculation, and flasks were shaken for 2 weeks at ~200 rpm. Medium color was noted (yellow representing sulfur, S0, accumulation) and cell growth was visualized using phase-contrast microscopy. RESULTS Diversity and Biomass of Anaerobic Enrichment Cultures Several attempts were made to estimate biomass from the Lower Kane Cave and Hellspont Cave microbial mats (Table 4-1; Appendix C). In most of the mat samples analyzed, sulfate-reducers and/or fermenters represented the highest MPN estimates, with up to 106 cells ml-1. However, compared to the overall biomass estimates from the microbial mats of approximately ~1010 cells ml-1 (Table 2-2), the MPN estimates of anaerobic microorganisms were up to four orders of magnitude less. There was little correlation in cell count or metabolic guild to the type of mat morphotype cultured (e.g., between white versus gray morphotypes). For instance, iron-reducers grew from gray sediment in the Upper Spring, but were also present in the white webs and stream water (Figure 4-1; Appendix C). 133 Moreover, there were nearly equal abundances of S0RB in white and gray filament types, although less S0RB in the stream and orifice water. Excluding the higher abundances for methanogenic and iron-reducing groups in the Hellspont Cave sample (Figure 4-2), there were no statistical differences in the abundances of fermenters or SRB between Hellspont and Kane cave samples. Sampling was focused at the Lower Kane Cave Upper Spring microbial mats (Figure 4-1). SRB abundance increased from the orifice downstream through the microbial mats, but SRB were less abundant in the cave stream at 215 m. Both SRB Groups I and II were present in nearly all the samples, including mat and water, with less than 1- to 1000 fold differences in cell abundance overall (Figure 4-1; Appendix C). In some samples, fermenting bacterial biomass was almost equivalent to or surpassed SRB (Figure 4-1). Chemolithoautotrophic SRB were not detected in all samples, and generally had up to four orders of magnitude less cells than SRB Groups I or II. S0RB were detected in most samples, with relatively low biomass overall with up to 102 cells ml-1. There were a few iron-reducers enumerated (up to 103 cells ml-1) in microbial mat samples, but none were detected in spring or stream water. Methanogens were rarely enumerated, with <14 cells per 100 ml-1 from the Upper Spring gray sediment and stream channel white filament samples, consistent with low detectable CH4 in the water. (Figure 4-1); hydrogenotrophic methanogens most abundant in Hellspont Cave (Figure 4-2). Four of eight enrichment cultures for nitrate-reducing sulfur-oxidizing bacteria, 134 although not enumerated, had positive growth, and cells from the enrichments were single-celled rods (~ 1-2 m long) (Table 4-1). Fermentation Diversity Of the 28 fermenting bacterial strains isolated from 13 mat samples, most of the strains were classified as `A/A, G', representing acid production in the tube butt and slant (A/A) and gas production (G) (Table 4-2). There were three variations in gas production; gas was produced throughout the agar, only in the butt, or only in the slant (Table 4-2). Eight strains produced H2S, as well as other gases and acidity. Overall, there were eight distinct types of growth identified. Three strains did not change medium pH or generate gas, although they did grow. DISCUSSION Diversity and Ecology of Anaerobic Microorganisms Anaerobic microorganisms are common in marine and freshwater habitats, from anoxic sediments and bottom waters to rice paddies (e.g., Bak and Pfenning, 1991; Widdel and Bak, 1992; Wind et al., 1999; Scholten et al., 2002), and even in oxic regions of microbial mats (Minz et al., 1999a; Minz et al., 1999b). Of the anaerobic microbial guilds, especially SRB and S0RB, many have not been studied in detail from groundwater and springs from karst terrains. Although one group of SRB and several types of fermenters were identified from 16S rDNA sequences retrieved from the microbial mats (Chapter 2), culture-based methods indicate that anaerobic microbial diversity is greater than previously determined, as is subsequent ecosystem functional diversity. 135 There are several reasons why the results of this culture-based study must be considered `estimates' of microbial diversity. First, culturing techniques can distort the original population abundances and community structure, as there is no one medium that can suit all the organisms (e.g., McDougald et al., 1998). Secondly, enrichment techniques can narrow community structure by providing selective media that eliminate large groups of physiologically distinct bacteria. A rich nutrient source may allow a rare, lesser abundant metabolic group to compete ecologically, and thereby artificially offset community structure. Moreover, assessments of ecosystem function can be distorted if organisms utilize multiple electron acceptors in enrichments that are not available in the natural habitat. The apparent dominance of SRB among the anaerobic metabolic groups enumerated here from Lower Kane and Hellspont Cave microbial mats was not surprising, as dissolved sulfate concentrations in the cave waters are high. Interestingly, the abundance of SRB in white and gray mat morphotypes was not significantly different, although there were more SRB in both groups in the more distal mat samples compared to mats proximal to the orifice. These results suggest that either microhabitats with low oxygen tension exist in more aerobic portions of the mat (white morphotypes), or that the SRB tolerate more oxygenated conditions (e.g., Minz et al., 1999a). The estimated abundances of Group I and II SRB were similar; in most samples these groups had equal biomass values (Figure 4-1; Appendix C). Although SRB can use a variety of electron donors, the most common low136 molecular weight organic compounds are lactate and formate (Group I SRB) and acetate (Group I SRB) (Widdel and Bak, 1992). There is some evidence that Group I SRB associate closely with Group II SRB, as some formatotrophs do not completely oxidize lactate to CO2, but instead oxidize it to acetate that is then completely oxidized to CO2 by a Group II SRB (Ehrlich, 1996). This may explain why the abundance of these groups overlaps in some samples, suggesting that carbon is actively cycled between the groups. Fermenting bacteria generate low molecular weight compounds, including amino acids, carbohydrates, and organic acids and alcohols, as intermediaries from the breakdown of complex organic matter (Ehrlich, 1995). TSI agar was used to test for mixed-acid fermentation from sugar breakdown, which yields either lactic, acetic, and succinic acids, formic acid (or CO2 and H2), ethanol, or 2,3-butanediol (Stainer et al. 1986). Other fermentation pathways common to anaerobic microorganisms, such as propionate, butyrate, or succinate fermentation, or fumarate reduction, could not be examined using TSI agar. Therefore, although it is apparent that a range of fermentation processes are evident based on culturing, actually assigning a specific pathway to an organism or genera is only a speculative process. A combination of culture- and molecular-based approaches will be essential to characterize the fermenting bacteria in the future. Eight fermentation pathways were detected from culture, indicating there is a range in metabolic preferences and capabilities for the microbes from the cave microbial mats (Table 4-2). The most common fermentation pathway identified 137 involved acid production from mixed-sugar metabolism to organic acids, indicated by a reduction in medium pH below 4.5. CO2 production was also common, and bacteria associated with sugar metabolism and gas production include Escherichia, Proteus, and most species of Salmonella, Aeromonas (Stainer et al., 1986). CO2 production is an indication of formate cleavage by the enzyme formic hydrogenase, and formic acid (the end fermentation product) is replaced by CO2 and H2. Four of the 28 strains generated acidity but not CO2 gas, a pathway characteristic of organisms that do not have formic hydrogenase, such as some species of Shigella. Of the 28 strains, eight produced H2S, which is characteristic of species from Citrobacter, Salmonella, and Proteus. Sulfide production results from breakdown of the sulfur-containing amino acid cysteine, or thiosulfate disproportionation. However, Kolmos and Schmidt (1987) caution that some H2S-producing Enterobacteriaceae were not detected using TSI agar. For three strains, medium pH was not lowered and no gas was produced, which could indicate one of four possibilities: 1) no fermentation; 2) fermentation occurred but did not reduce pH because large amounts of 2,3-butanediol or ethanol were produced, which would also not generate CO2; 3) only lactose fermentation was occurring, but strains lacking certain enzymes for efficient breakdown may not grow fast enough to illicit a medium pH reaction; or 4) since colony growth was obvious for these strains, it is also possible that peptone catabolism was occurring, in which there neither would be acid nor gas production. Butanediol and ethanol production are common to Serratia, and species from several other genera 138 including Erwinia, produce butanediol (Stainer et al. 1986); Serratia spp. was identified in a 16S rDNA clone library from a gray filament sample (Chapter 2; Table 2-3), suggesting that Serratia may have been at least one of the strains isolated. Lactose fermentation is characteristic of Escherichia and Enterobacter, but absent in Shingella, Salmonella, and Proteus (Stainer et al. 1986). While not abundant, iron-reducers were enumerated from several mat samples. The concentration of Fe3+ is low in the cave (Table 2-1), and could be one reason why iron-reducers are not abundant. The presence of Fe-S framboids in the gray mat interior (Figure 2-4), however, may indicate microbial Fe3+ reduction. Iron-reducers can also use humic substances (Lovley and Phillips, 1986), and acetate is likely the major electron donor. Some iron-reducers ferment glucose, or can use monoaromatic compounds and fatty acids (Lovely, 2001). Competition with SRB, which represents a much larger biomass, for carbon substrates may be another reason why iron-reducers are not prevalent. Enrichment cultures for nitrate-reducing sulfide-oxidizing bacteria were successful for half of the mat samples examined. However, although some sulfuroxidizing organisms could reduce nitrate in the laboratory, this does not mean that sulfur oxidation through nitrate reduction also occurs in the cave microbial mats. Using the enrichment scheme from Nemati et al. (2001) for Thiobacillus denitrificans and Thiomicrospira spp., incomplete oxidation of sulfide to sulfate occurs if nitrate is not sufficient; the result is an accumulation of elemental sulfur. Brunet and Garcia-Gil (1996) suggest that if sulfide concentrations are <50 mol 139 L-1, as they are in Lower Kane Cave, nitrate is reduced to N2. Conversely, at higher sulfide concentrations incomplete nitrate reduction to nitrite, NO, and N2O occurs, which can inhibit sulfate reduction and methanogenesis (Scholten et al., 2002). As the concentration of sulfide is below the reported threshold, nitrate is likely reduced completely to N2, if reduction is occurring at any significant rate. More often than not, however, nitrate is a limiting nutrient in freshwater habitats, as it is in Lower Kane Cave (Table 2-1), making sulfide-dependent nitrate reduction either nonexistent or indicating that nitrate is kept at an exceedingly low concentration because is so tightly cycled between production and consumption. Methanogens are ubiquitous in most anaerobic environments with decomposing organic material (Garcia et al., 2000). Conversely, methanotrophy, a metabolic pathway not investigated in this study, is an important metabolic pathway in Movile Cave, Romania (Hutchinson et al., 2003), where the concentration of methane in the cave water is greater than that measured at Lower Kane Cave (Sarbu et al., 1996). It is well known that competition between methanogens and SRB is significant, and if the concentration of dissolved sulfate is high, this has been shown to inhibit methanogenesis (e.g., Garcia et al., 2000). Therefore, the low concentration of methane in Lower Kane Cave, lack of methanogens, and high sulfate concentration supporting high SRB biomass, suggest that methanogenesis is not a prominent physiology in the microbial mats. 140 Implications for Carbon and Sulfur Cycling There have been many ecological studies (albeit from surface environments such as forests and soils) that indicate habitat diversity is an important determination for organism diversity, and therefore the organisms that affect niche diversity also affect the biological diversity of other organisms in that habitat (Naeem, 2002; Wardle, 2002). The microbial mats are grossly structured with white filamentous mat morphotypes of sulfur-oxidizing "Epsilonproteobacteria"and Thiothrix-dominated communities in contact with flowing stream water, covering a microbial consortium within a gray mat interior. Niche development by the sulfur-oxidizers provides habitat for anaerobic microorganisms within the mat interior, while conversely the benthic orifice and stream sediments are depleted in anaerobic microbes by the absence of a mat structure. Clearly, organic carbon quality and quantity are important factors in influencing the types of physiological processes that will occur in an ecosystem (Eiler et al., 2003; Horner-Devine et al., 2003). Horner-Devine et al. (2003), based on work in shallow phototrophic ponds, also suggest that primary productivity influences the composition and richness of bacterial communities. Although chemolithoautotrophically-based cave ecosystems have been previously described with respect to metazoans (Sarbu et al., 1996; Airoldi et al., 1997; Hose et al., 2000; Sarbu et al., 2000; Vlasceanu et al., 2000; Engel et al., 2001; Garman, 2002), the diversity and dependence of anaerobic microbial guilds from chemosynthetic primary productivity have not been considered. Based on 16S rDNA sequences 141 retrieved from the microbial mats (refer to Chapter 2), bacterial diversity increased downstream, including both the "Epsilonproteobacteria" and the other bacterial groups. In caves, one of the consequences of chemolithoautotrophy is that the quality and quantity of organic carbon is greater than most aphotic ecosystems typically have (Sarbu et al., 1996; Kinkle and Kane, 2000; Poulson and Lavoie, 2000; Simon et al., 2003). The bulk of the white filamentous microbial biomass in Lower Kane Cave has low C:N ratios (refer to Chapter 2; Table 2-2), indicative of an extremely high quality food source (McMahon, 1975), but also consistent with an insignificant influx and processing of allochthonous carbon. In Lower Kane Cave, the trend of microbial biomass stable carbon isotope values demonstrates nutrient spiraling (Chapter 2), which suggests that carbon is cycled through an active microbial detrital loop and possibly that a significant portion of autotrophic biomass is cycled heterotrophically (Allan, 1995; Porter, 1999). A percentage of heterotrophic carbon recycling can be based on the raw ratio of heterotrophic productivity to autotrophic productivity, and this ratio is doubled to assume 50% growth efficiency (Kirchman et al., 1993; Engel et al., 2001). Porter (1999) determined the rate of chemolithoautotrophic primary productivity in Lower Kane Cave, by H14CO3 assimilation, to be 96.5 6.0 mg C grams dry weight (gdw-1) hr-1 for the white channel mats. By comparison, the heterotrophic productivity by 14 C-leucine incorporation was only 14.8 5.7 mg C gdw-1 hr-1 (Porter, 1999). Therefore, ~ 30% of the autotrophic productivity in Lower Kane Cave is processed through 142 heterotrophy. Comparatively, bacterioplankton utilization of phytoplankton production in a variety of open ocean systems ranges from 10-40% (Kirchman et al., 1993, and references therein). While allochthonous sulfide most likely originates from dissimilatory sulfate reduction deeper in the Bighorn Basin, as determined on the basis of sulfur isotope values (see Chapter 2; Carmody et al., 1998), SRB within the cave also generate autochthonous sulfide (refer to Chapter 5). Sulfur isotope values support that there is a component of autochthonous sulfide with lower 34S values, compared to allochthonous sulfide, that is incorporated by sulfur-oxidizing bacterial biomass (Figure 2-6; Chapter 2 for discussion). While fermenting bacteria in mixed anaerobic communities are key to carbon cycling, the contribution of fermenting bacteria to sulfur cycling has not been well established. Results from this study indicate that some of the fermenting bacteria have the ability to generate H2S from reduced inorganic and organic sulfur compounds, thereby also affecting sulfur cycling inside the cave. This led to the hypothesis that fermenting bacteria, in addition to SRB and S0RB, can contribute to autochthonous H2S gas production, as well as other reduced sulfur gases, in the microbial mats (Chapter 5). In conclusion, although resolving the detailed structural and functional aspects of the anaerobic microbial community are not possible based only on the culture-based assessments used in this study, results do indicate 1) higher abundances of culturable anaerobes in the mats than sediment or water; 2) little difference in the abundance of SRB groups in white or gray mat morphotypes, suggesting that the conditions for SRB anaerobic growth are favorable throughout 143 the subaqueous cave environment; 3) many different fermentation pathways, indicating a variety of ways the chemolithoautotrophically produced organic carbon can be processed in the microbial mats; 4) methanogenesis is not a significant carbon cycling pathway in the anaerobic microbial mats; and 5) a large component of autotrophically produced carbon is cycled through a detrital microbial loop. This study expands the diversity and importance of anaerobic microorganisms in cave microbial mats and demonstrates the importance of these organisms to the cave ecosystem, despite low biomass. 144 Table 4-1: Culture groups, sampling sites, and microbial mat morphotypes in Lower Kane and Hellspont Caves, Wyoming. 2001 Sampling Sites (meters and morphotype)b 189 w c Groupa 189 g 193 g 195 g 195 f 198 g 198 f 201 g 201 f 203 g 203 f 203 k 203 y 203 fe 215 r 216 w HC 118 g 248 w 248 g 248 f FB ASRB LFSRB HSRB IRB AM LFM HM 2002 Sampling Sites 145 2003 Sampling Sites FB ASRB LFSRB HSRB IRB MM HM S0RB SONR Type of physiologic group: FB, fermenting bacteria; ASRB, acetate-utilizing sulfate-reducing bacteria (SRB); LFSRB, lactate-/formateutilizing SRB; HSRB, autotrophic SRB; IRB, iron-reducing bacteria; AM, acetate-utilizing methanogens; LFM, lactate-/formate-utilizing methanogens; HM, autotrophic methanogens; MM, mixed acetate- and formate-utilizing methanogens; S0RB, sulfur-reducing bacteria; SONR, sulfur-oxidizing nitrate-reducing bacteria b Number represents meter location along cave stream from back of the cave. HC: Hellspont Cave spring sediment. Letter represents mat morphotype: w, water; g, gray filaments or sediment; f, white filaments; fe, white feathers; k, knobby-web; y, yellow patches; r, red mats c Box symbols: , positive growth; , negative growth; no symbol means the enrichment was not attempted. a Table 4-2: Results from isolation and screening of fermenting bacterial strains using TSI agar. Triple Sugar Iron (TSI) Agar Growth Strain Colony Description General Shape 1-1-2 1-3-2 2-2-2 2-3-2 3-1-2 3-2-2 4-2-2 4-3-2 5-3-2 6-3-2 7-2-2AA 7-22AB1 7-22AB2 8-2-2 10-3-2 2ferm4-1-5A 2ferm4-1-5B 2ferm7-1-3A 2ferm1-1-1A Ferm-2-1-5 Ferm-4-1-5A Ferm7-1-5A Ferm8-1-5A Ferm10-1-5A Ferm11-1-5-A Ferm13-1-5A Ferm15-1-5A Ferm19-1-5-A a b a Growth Acid Slant Butt + + + + + + + + + + + + + + + + + + + + + + + + Gas Slant + + + + + + + + + + + + Butt + + + + + + + + + + + + + + + + + + + H2 S + + + + + + + + - Symbolb (slant/butt, gas, black) A/A, G A/A A/A, G, B A/A, G, B A/A, G A/A, G, B A/A, G A/A A/A A/A, G, B K/A, G NC/NC NC/NC A, K A/A, G A/A, G A/A, G A/A, G K/A, G NC/NC A/A, G, B A/A, G, B K/A, G A/A K/A, G, B A/A, G A/A, G, B A/A, G Yellow, shiny colony White, smooth colony edges, smooth White, spreading colony edges White, shiny colonies White, slimy and shiny colonies, smooth edges White, slimy and puffy colonies, swarming White, shiny colony, smooth, swarming White, tiny, smooth Yellow, small colonies, swarming White, slimy, small Brown, flat and spreading colonies White, irregular spreading colony edges White, smooth edges, grows into medium Pink, tiny colonies, smooth edges White, bumpy colonies, shiny White, shiny colonies White, shiny colonies White, smooth colony edges White, slimy colonies White, flat and small colonies White, small and shiny White, shiny colonies Pink, shiny colonies White White, swarming White, smooth edges Yellow, shiny and slimy colonies White 0.7 m rods Dumb-bell rods Rod Rods Rods ~1 m rods Rods Rods Rods Rods Rods ~1 m fat rods Rods Rods Rods ND ND ND ND ND ND ND ND ND ND ND ND ND + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + + ND, not determined. Symbols and interpretation: K, alkalinity, medium turns red; A, acid production, medium turns yellow; NC, no medium color change; G, gas production in medium; B, black precipitate in medium due to H2S gas. 146 Figure 4-1: Most-probable number estimates for various anaerobic bacteria from Lower Kane Cave water, sediment, and microbial mat samples. Number at left represents site location, in meters, from the back of the cave forward (the spring orifice is the lowest number). SRB, sulfate-reducing bacteria (groups I and II, and autotrophic); S0RB, sulfur-reducing bacteria; IRB, ironreducing bacteria. Error bars are 95% confidence intervals calculated by MPN program (Hurley and Roscoe, 1983). 147 cells.ml-1 0.1 1 10 100 1000 104 105 106 107 108 189 orifice water Fermenting bacteria Group II SRB Group I SRB 189 gray sediment Autotrophic SRB S0RB 193 gray filaments IRB Methanogens detected 195 white filaments 195 gray filaments 198 white filaments 198 gray filaments 201 white filaments 201 gray filaments 203 white webs 203 filaments 203 gray filaments 215 stream water 148 1 10 100 cells.ml-1 1000 104 105 106 Acetoclastic methanogens Formatotrophic methanogens Hydrogenotrophic methanogens Fermenting bacteria Group II Sulfate-reducing bacteria Group I Sulfate-reducing bacteria Autotrophic Sulfate-reducers Iron-reducing bacteria Figure 4-2: Most-probable number estimates for various anaerobic bacteria from Hellspont Cave orifice sediment. Error bars are 95% confidence intervals. 149 Chapter 5: Production and Consumption of Hydrogen Sulfide and Volatile Organosulfur Compounds in a Sulfidic Cave System ABSTRACT1 Hydrogen sulfide gas (H2S(g)) and volatile organosulfur compounds (VOSC) were examined in filamentous microbial mats formed in anaerobic, sulfidic water in Lower Kane Cave, Wyoming. Total dissolved sulfide (CTS=) decreases from upstream to downstream through the microbial mats, while H2S(g) at the air-water interface directly over the microbial mats averages 30 ppmv, corresponding to an average H2S(g) flux of 44 mol m-2 min-1. The measured H2S(g) volatilization accounts for less than 8% of the CTS= flux into the cave, and abiotic sulfide autoxidation accounts for <0.014% CTS= loss, suggesting that the primary CTS= loss mechanism is caused by oxidation of CTS= by subaqueous sulfuroxidizing bacteria. Incubations of cave water and microbial mat samples demonstrate that H2S(g) and VOSC are of biogenic origin, and are cycled by aerobic and anaerobic mat communities. Anaerobic laboratory enrichment cultures of fermenting, sulfate-, and sulfur-reducing bacteria from the cave microbial mats were screened for sulfur gas production. Enrichments of sulfur-reducing bacteria generated only H2S(g), but sulfate-reducing and fermenting bacteria produced VOSC, in order of abundance: H2S(g), methanethiol, dimethyl sulfide, and carbonyl sulfide. Twenty-nine percent of fermenting bacteria strains generated H2S(g) with A portion of this chapter originated from the paper A.S. Engel, L.A. Stern, and P.C. Bennett, 2004, Microbial contributions to cave formation: new insights into sulfuric acid speleogenesis, Geology, v. 32 (5) p. 369-372. 1 150 growth medium, and two of the isolated strains produced H2S(g) when thiosulfate and cysteine were present in the growth medium, but not with sulfate; methanethiol was produced when methionine was present. In anaerobic enrichment cultures and natural mat incubations, generation of methanethiol and dimethyl sulfide was always preceded by the generation of H2S(g). Inhibition of microbial sulfate reduction by addition of molybdate in the incubations resulted in diminished to no VOSC production. There was no correlation between VOSC concentration and methane gas production, indicating that methanogenesis is not an important sulfur gas consumption mechanism in this system. Natural mat samples, dominated by active sulfur-oxidizing bacteria, degraded methanethiol and dimethyl sulfide aerobically when supplemented with VOSC. Active cycling of VOSC by anaerobes and aerobes from a freshwater, subterranean environment has not been previously demonstrated. As photochemical degradation of these gases is not possible, sulfurbased microbial mat communities in the subsurface represent a new terrestrial source for sulfur gases and the impact of subterranean microorganisms to the global sulfur cycle should be considered. INTRODUCTION Bacterial processes are largely responsible for recycling inorganic and organic sulfur-containing compounds within the sulfur cycle, and volatile components are released into the atmosphere (Taylor, 1991; Kelly et al., 1994). Hydrogen sulfide gas (H2S(g)) and volatile organosulfur compounds (VOSC), including carbonyl sulfide (COS), methanethiol (MT), dimethyl sulfide (DMS), and 151 dimethyl disulfide (DMDS), contribute to the global sulfur cycle, and consequently these sulfur gases have been intensively studied with respect to their contribution to acid precipitation and global warming (Legrand et al., 1991; Bates et al., 1992; Bodenbender et al., 1999; Watts, 2000). Understanding natural abiotic and biogenic emission is vital to evaluate the magnitude of anthropogenic perturbations to the global sulfur cycle. Marine and volcanic gas emissions make up a significant fraction of the global sulfur flux; while terrestrial biogenic emissions may be insignificant at the global scale, they are likely more important to local cycles (e.g., Andreae and Andreae, 1988; Bates et al., 1992; Watts, 2000). Attempts to estimate natural terrestrial emissions from surface environments have had variable results, showing disparate emission patterns spatially and temporally. Most studies of natural H2S(g) and VOSC production and consumption rates have investigated marine environments (Zinder et al., 1977; Visscher et al., 1991; Bodenbender et al., 1999; Fritz and Bachofen, 2000; Kristensen et al., 2000; Visscher et al., 2003), freshwater limnic systems (Stoner et al., 1994; Fritz and Bachofen, 2000), hot springs (Stoner et al., 1994), soil (Conrad, 1996; Yang et al., 1998), and flooded sediments and wetlands (Lomans et al., 1997; Lomans et al., 1999a; Lomans et al., 1999b; DeLaune et al., 2002). However, virtually nothing is known about the production of reduced sulfur gases from terrestrial, aphotic environments (caves) or sulfidic groundwater systems, although H2S(g) has been detected in several caves (Galdenzi and Menichetti, 1995; Sarbu et al., 1996; Angert et al., 1998; Hose et al., 2000). H2S(g) and VOSC have been identified rarely in caves, including from Lower Kane Cave, Wyoming (Egemeier, 1981), and 152 Cueva de Villa Luz, Mexico (Boston et al., 2001). VOSC in the subsurface do not photodegrade, and consequently cycling of reduced sulfur gases in the aphotic unsaturated zone may be a significant source of sulfur to the global budget. This is particularly important geologic environments from which gases can diffuse into the atmosphere, such as fractured bedrock or karst. Sulfur-based microbial communities can utilize the ubiquitous reduced sulfur compounds in anaerobic, sulfidic groundwater, and sulfur cycling may be more widely dispersed in the terrestrial subsurface than currently recognized. This study describes the production and consumption of H2S(g) and VOSC in Lower Kane Cave by sulfur-based microbial communities. While there have been investigations of VOSC utilization by some microbial groups, including Thiobacillus spp. (Kanagawa and Kelly, 1986) and Hyphomicrobium (DeBont et al., 1981), these organisms have mostly been studied in engineered systems (e.g., sewage sludge) or in photic microbial mats (e.g., Visscher et al., 1991). "Epsilonproteobacteria", the dominant sulfur-oxidizing bacteria in the Lower Kane Cave microbial mats (refer to Chapter 2 and 3; Engel et al., 2003), have not been previously linked to VOSC consumption or utilization. Based on gas chromatography (GC) methods designed specifically for the cave environment, I report H2S(g) and MT gas flux rates from the subterranean sulfidic springs and microbial mats, as well as detailed regarding microbially mediate sulfur gas transformations from native microbial mat samples and laboratory enrichment cultures. 153 MATERIALS AND METHODS Cave Gas Sampling and Flux Measurements A description of Lower Kane Cave, Wyoming, and its geological and hydrological setting are presented in Chapter 1 (Figure 1-1 and 1-2). Cave atmosphere components (CO2, CH4) and sulfur gases (H2S, COS, SO2, CS2, MT, DMS) were determined in the cave by direct-inject GC on a SRI 310 (SRI Instruments, Torrance, CA) using either a Restek XL sulfur column and TCD/FID detectors, or a 60m 0.53mm MXT-1 column with FID/FPD detectors, respectively. In the cave, the MXT-1 column resulted in better gas separation with an isothermal run. The GC was placed in an area of the cave previously determined to have extremely low to negligible sulfur gases, away from the cave stream (Figure 5-1A). A gas-powered Honda generator was placed at the cave entrance and 150 m of heavy-duty-outdoor extension cord was extended through the cave to the GC. H2S(g) flux at the water-mat boundary layer was measured using a 16-cm2 flux chamber suspended over the water and partially submerged to approximately 2 to 10 mm into the cave water (Figure 5-1B). Cave air was pumped through a reactive copper intake tube connected to the chamber to remove ambient VOSC (Figure 51C), and the chamber gas was withdrawn and pumped through Teflon tubing to a 1 ml Teflon sample loop for injection into the GC (Figure 5-1D). The entire gas flow path from the flux chamber to the GC was Teflon due to the reactivity of sulfur gases. The chamber gas outflow was monitored continuously for CO2 (Comfort 154 Chek 100/200 CO2 analyzer, Bacharach, Inc., Pittsburgh, PA) and flow rate using a mass flow meter. Replicate flux measurements were determined with variable pumping rates to achieve a representative flux rate. Individual gas samples were also collected throughout the cave in cleaned and non-reactive plastic syringes, and samples were analyzed immediately by GC. Dissolved gases in the stream were measured from sample bottles by headspace GC and by the bubble-strip method (Chapelle et al., 1997). Enumeration of Anaerobic Enrichment Cultures Microbes from the cave water and microbial mats were enriched using the most probable number (MPN) method in pre-reduced anaerobic media specific to certain physiologic groups, including fermenting bacteria, chemolithoautotrophic, acetate-, and lactate-/formate-utilizing sulfate-reducing bacteria (SRB), sulfurreducing bacteria (S0RB), methanogens, and iron-reducers. Gas evolution (H2S, COS, MT, DMS, CH4, CO2) was measured by GC in the enrichment headspace (Chapter 4). Isolation of Fermenting Bacteria and Metabolism of Sulfur Compounds Some strains of fermenting bacteria (isolates obtained as described in Chapter 4) were tested for utilization of different sulfur substrates, including thiosulfate, sulfate, cysteine, and methionine. 100 l of cell suspension was harvested from strain isolates, washed with filter-sterilized tap water, and inoculated into fresh serum bottles containing 10 ml MLN media (Chapter 4) supplemented with one of the following: 2.5 mM NaS2O3; 4 mM MgSO4 7H2O + 9 mM NaSO4; 0.01% L-cysteine (Fisher); or 0.01% L-methionine (FisherBiotech). 155 MLN broth with no addition of sulfur compounds was a live-control. In each sulfur substrate series, a killed cell suspension was used as a sterile control (121oC, 20 min). All bottles were pressurized with N2 to ensure anaerobic conditions. One bottle was used to measure microbial growth as optical density (OD) using a Perkin Elmer Lambda 6 UV/VIS spectrophotometer at a wavelength of 588 nm (traditionally, 600 nm is used to measure cell absorbance, but there was interference at 600 nm by the medium). pH was measured by electrode and headspace gas was quantified by GC. Growth rate for each strain was calculated using the modified Monod expression OD(t) = ODo e t (5-1), where OD(t) is the optical density at time t, ODo is the optical density at the start, and is the specific growth rate. Doubling times (td) were calculated using = ln 2 / td Gas Cycling by Native Microbial Mat Communities H2S and VOSC production from homogenized mat samples In separate cave-incubated and laboratory-incubated gas production experiments, approximately 100 ml of aseptically collected and homogenized microbial mat from the end of the Upper Spring and Lower Spring, and 150 ml of 0.22- m-filtered (cold-sterilized) spring water, were dispensed into sterile 500 mL black Teflon chambers equipped with gas-sampling and electrode ports. Microcosms were amended with 10 ml of a gypsum-saturated, cold-sterilized solution to provide dissolved sulfate. For the cave incubations, a control chamber (5-2) 156 containing 250 ml filter-sterilized spring water and 10 ml gypsum-saturated solution was used. The laboratory incubations had two controls: one sterile control with 250 ml filtered spring water and 10 ml gypsum-saturated solution, and another killed control of autoclaved (121 oC, 30 min) microbial mat. Microcosms were incubated in the cave or at room temperature, and pH in the live chambers was monitored continuously using an Accumet portable pH/mV meter with a doublejunction pH electrode (Fisher Scientific). For the laboratory incubations, sterilized pieces of Iceland Spar calcite (Wards Scientific), totaling 1 g for closed-chamber microcosms and 0.1 g for serum bottles, were added prior to mat inoculation to maintain circum-neutral pH and to provide an inorganic carbon source to autotrophic populations. Emitted sulfur gases and headspace CO2 from caveincubated chambers were measured in the cave by GC and using the portable Comfort Chek CO2 monitor, respectively, and O2 was measured by GC using a CTR-1 column. For the laboratory chambers, sulfur gases, methane and hydrocarbons, and CO2 were separated by GC with FPD, FID, and TCD, respectively. Inhibition of H2S and VOSC production from homogenized mat samples Aerobic and anaerobic gas production and consumption were measured in sterile 60-ml glass serum bottles inoculated with 10 ml homogenized microbial mat material, 20 ml cold-sterilized spring water, and 2 ml gypsum-saturated, filtered solution. Some bottles were supplemented with 4mM Na-molybdate to inhibit microbial sulfate reduction, while others were supplemented with 25 mM 2bromoethanesulfonic acid (BES) to inhibit methanogenesis (Oremland and Capone, 157 1988; Lomans et al., 1997; Scholten et al., 2000), or with ~30 l HgCl-saturated solution for a killed control. Abiotic controls were prepared from bottles of 30 ml cold-sterilized spring water and 2 ml gypsum-saturated solution, and of sterilized mat (121 oC; 20 min). Bottles were sealed with butyl rubber stoppers and aluminum seals, and were incubated in the dark at room temperature (~21-23 oC). VOSC consumption by homogenized mat samples Cold-sterilized spring water was purged with a mixed-sulfur gas standard (71.0 ppmv COS, 75 ppmv MT, 68.5 ppmv DMS) for 2 hr to examine VOSC degradation. Purged water was cold-sterilized into glass serum bottles and 10 ml homogenized aliquots of microbial mat were added. Some bottles were amended with 25 mM BES or HgCl2 for a killed control. One bottle with mat was sterilized by autoclaving (121 oC; 20 min). Immediately following sealing and gentle shaking, headspace samples were taken to determine starting gas concentrations. Samples were incubated in the dark at room temperature. RESULTS Hydrogen Sulfide Dynamics in the Cave Field description Cave spring waters had pH values ranging from 7.1 to 7.3 and a nearly constant temperature of 21.5 oC (Table 2-1). Total dissolved sulfide (CTS=) at all orifice discharge pools averaged 30 mol L-1, but decreased rapidly from the discharge point downstream through the microbial mats (refer to Chapter 2; Figure 2-1). The total flux of CTS= at the Upper Spring was calculated from the incoming concentration and the stream discharge determined by salt dilution tracing. 158 Discharge CTS= flux, determined from the 191-m concentration of dissolved sulfide, was ~8700 mol min-1 (Table 5-1). CTS= was not detected downstream from the mat terminus at the Lower and Upper Spring (2 and 20 m flow path distance from the orifice, respectively), although CTS= (~20 mol L-1) was measured 25 m from the Fissure Spring where the mats ended (Figure 2-1A). At the Upper Spring orifice, water had no detectable dissolved O2 (Table 51) and H2S(g) was not detectable directly over this anaerobic spring water (Figure 52; Appendix D). Along the outflow channel, CTS= was approximately constant as dissolved O2 increased. From the orifice and along the stream channel, the water transitioned from anaerobic to disaerobic (O2 = 0.1 to 5.0 mol L-1), with 21 mol L-1 CTS=. The mats abruptly terminated at 205 m, and here dissolved O2 exceeded 45 mol L-1 while CTS= decreased to <6 mol L-1 (Figure 2-1B). The approximately constant, and subsequently steep and concave-down CTS= vs. distance profile (Figure 5-3A) is not the expected shallow, concave-up first-order loss profile characteristic of abiotic first-order loss mechanisms (Millero et al., 1987; Brezonik, 1994). A first-order loss curve was estimated from the initial CTS= at the spring and the distance at half-CTS= [t1/2 = ln2/k; Brezonik, 1994] and an estimated stream velocity of 0.5 m s-1 along the proximal 6 m channel from the orifice. The observed t1/2 was ~0.5 min (k = 1.5) (Figure 5-3A). Stream velocity decreased to 0.2 m s-1 at the mat terminus as the stream channel widened, and another first-order loss curve was estimated from the CTS= at 203 m, near the end of the mat, with an observed t1/2 ~ 0.2 min (k = 3.0) (Figure 5-3A). 159 Field flux experiments and theoretical volatilization Although H2S(g) flux varied along the outflow channel at different sampling times, the highest H2S(g) flux was consistently quantified from directly over the microbial mats from the 193 to 194-m locations where dissolved CTS= was relatively lower than CTS= at the spring orifice (Table 5-1; Figure 5-2). MT was measured above the detection limit (<1 ppmv) from a flux chamber at 191 m, but upon repeating the experiment, MT was not detected again. The average concentration of H2S(g) at the air-water interface for the entire stream length was 22 ppmv; this steady state concentration resulted in an average H2S(g) flux of 44 mol m-2 min-1 (Table 5-1). The measured H2S(g) flux was compared to a theoretical volatilization rate calculated from the 2-film model of Liss and Slater (1974) (Table 5-1), using the average water depth of 10 cm and an average channel width of 1 m. It was assumed that the shallow stream channel results in a completely mixed aqueous system and a purely fluid film-controlled volatilization rate. Volatilization half-life was determined from the first-order liquid phase mass transfer coefficient, KL = (H ' RT ) k k (H ' RT ) k + k g l g (5-3) l where kg is the gas phase exchange coefficient, kl is the liquid phase exchange coefficient, H' is the dimensionless Henry's constant, T is temperature in Kelvin, and R is the gas constant; kg and kl are estimated from the ratio of the mass of water vapor and CO2, respectively, to the mass of H2S (Liss and Slater, 1974). Dissolved H2S was speciated from CTS= for pH (H2S:HS- pK = 7.04; Stumm and Morgan, 160 1996). The theoretical volatilization rate yielded an estimated flux of 23 mol m-2 min-1, with a first-order volatilization half-life (t 1/2) of 13 min (Table 5-1). This compares very closely to the actual volatilization t1/2 of ~6 min based on the mean measured H2S(g) flux. Over the entire length of the Upper Spring stream channel, however, the total H2S(g) volatilization accounted for less than 8% of the total influx CTS= (Figure 5-3B), suggesting that the bulk of subaqueous CTS= is lost by other mechanisms, not by volatilization. Abiotic autoxidation rates The rates of abiotic autoxidation were estimated to evaluate their contribution to CTS= loss. The rate of sulfide autoxidation in water is a complex function of temperature, salinity, reactant concentration, dissolved metal concentration, pH, and the presence of ferric hydroxides (Millero, 2001; Millero et al., 1987). At low sulfide concentrations and low temperatures, the sulfide loss rate is first-order with respect to sulfide concentration and first-order with respect to oxygen, and second-order overall for the disappearance of sulfide (Millero et al., 1987): d [H 2 S]T dt = k 2 [C T S = ] [O 2 ] (5-4) where [O2] is the concentration of dissolved oxygen, [CTS=] is the total concentration of all sulfide species, and k2 is the second-order rate constant. At pH values near the pK of H2S, k2 consists of two components, k0 and k1, corresponding to the oxidation of H2S and HS-, respectively. Therefore, the second-order rate of sulfide oxidation, in water at 25oC at pH <6, is dominated by k0 of ~11 L mol-1 hr-1, 161 with an activation energy of 43.5 kJ mol-1. For pH >8 the rate is dominated by HSoxidation, with a second-order rate constant of ~48 L mol-1 hr-1 at 25oC, with an activation energy of 53.5 kJ mol-1 in freshwater (Millero et al., 1987). Using a pH of 7.3 at 22oC, a second-order rate constant of 40 L mol-1 hr-1 was estimated. Using these rate constants, the calculated CTS= autoxidation under the conditions in the disaerobic cave waters (20 mol L-1 CTS= and a constant O2 of 20 mol L-1; conditions at 201 m) is extremely slow. The autoxidation rate of dissolved sulfide was estimated from the kinetic data determined at 25.0 oC for low ionic strength aqueous systems using a constant [O2] to yield a pseudo-first order rate constant and half-life (Zhang and Millero, 1983; Millero et al., 1987). For the analysis, the point along the stream where [O2] = CTS= was chosen, because for kinetic evaluation, choosing the point where starting concentration [A]0 = [B]0 for a second-order overall reaction, greatly simplifies the evaluation. For a closed system under these conditions, the half-life is (Brezonik, 1994): t1 / 2 = 1 k 2 [C T S = ]0 (5-5) and was estimated to be 1250 hr at the 201 meter-location along the stream channel. Alternatively, the concentration of dissolved oxygen can be fixed so that the second-order reaction can be evaluated as a pseudo first-order reaction, where the rate is independent of the variable component (H2S) and dependent only on the fixed concentration of dissolved oxygen: t1 / 2 = ln 2 k 2 [O 2 ] 0 (5-6) 162 and was estimated at 866 hr at 201 m. Upstream, where the concentration of oxygen was lower, the rate was much slower, while the rate increased slightly downstream with increasing oxygen, and the half-life decreased by a factor of ~3 at 215 m (Table 5-1). Therefore, CTS= autoxidation rate represented ~0.014% of the potential abiotic loss in this oxygen-limited system with low concentrations of dissolved metals (Figure 5-3B). Microbial Mat Sulfur Gas Production and Consumption Experiments Enrichment cultures of anaerobic microorganisms Microbial mat sampling was focused at the Upper Spring mats. Sulfur gas, and specifically H2S(g), production was used to detect SRB and S0RB growth in MPN enrichment cultures, and sulfur gases were also measured from the headspaces of fermenting bacteria enrichment bottles (Table 5-2). SRB cell abundance based on MPN increased downstream, from the orifice water and orifice sediment samples moving through the microbial mats. Both lactate/formate (Group I)- and acetate (Group II)-utilizing SRB were present in nearly all the microbial mat samples, with 1- to 1000-fold differences in cell abundance overall (refer to Chapter 4; Figure 4-1). In some samples, fermenting bacterial biomass was almost equivalent to or surpassed SRB abundances (Figure 4-1). S0RB were detected in most samples, with relatively low biomass overall (102 cells ml-1). Methanogens were rarely detected by MPN, with <14 cells 100 ml-1 (Figure 4-1). While H2S(g) was generated in SRB, S0RB, and some fermenting enrichment bottles, MT was detected in cultures of SRB Groups I and II and some fermenter enrichments (Table 5-2). COS was only generated from Group I SRB and fermenter enrichments from 163 the Upper Spring orifice samples (Table 5-2). DMS was produced in the headspace of rare fermenter and SRB Group II enrichment cultures (Table 5-2), Sulfur gas production from fermenting bacteria Twenty-nine percent of the fermenting bacterial strains produced H2S(g) when provided with thiosulfate or sulfur-containing amino acids. Two of these strains (LKC-232 and LKC-322) were used to measure rates of H2S(g) production given various inorganic and organic sulfur sources, and to test for possible VOSC formation from natural precursors (Figure 5-4). Both strains produced H2S(g) from thiosulfate (Figure 5-4A and 5-4B); MT was only detected in bottles supplemented with methionine (Figure 5-4C). Strain LKC-232 reached exponential growth earlier when supplemented with thiosulfate than LKC-322, and had a faster doubling time at 0.19 hr-1compared to 0.43 hr-1 for LKC-322 (Figure 5-4D and 5-4E). The addition of thiosulfate strongly stimulated H2S(g) formation, compared to the small H2S(g) concentrations measured in bottles supplemented with SO42-, cysteine, and methionine at levels approximately that of the control, suggesting those H2S(g) concentration profiles represent background sulfur assimilation rates. Native mat incubations H2S(g) was present in the headspace initially, but decreased to nondetectable after 57 hr (Figure 5-5A; M-series). After a lag phase (~15 hr), the concentration of H2S(g) in live mat samples increased rapidly, while MT was not generated in the live samples until after 200 hr (Figure 5-5B). COS and DMS were not detected in any bottles (Figure 5-5C). Decreases in oxygen concentration in the live mat incubations corresponded to increases in H2S(g), but not in the sterile water 164 or killed controls (Figure 5-5D). The observed increase in headspace oxygen in the live sample incubations, with a corresponding rapid decrease in H2S(g), may be related to oxygen leaking into the chambers during the course of the experiment and subsequent sulfide oxidation. Homogenized mat incubations and inhibition experiments Two different types of microbial mats were collected for three separate incubation experiments (Figure 5-6 through 5-8). In the A- and B-series, the concentration of headspace H2S(g), MT, COS, and DMS were measured (Figures 56 and 5-7). H2S(g) concentrations in the A- and B-series increased rapidly within the first day (6-15 hr) (Figures 5-6A and 5-7A), but in the C-series, H2S(g) production occurred from 16-79 hr (Figure 5-8A). Headspace MT in the live mat incubations increased after 50 hr (e.g., Figure 5-6B), faster than observed from the M-series. MT was not detected from the C-series. While H2S(g) concentration varied over time, the concentration of MT steadily increased in the live incubations (Figures 56B and 5-7B). COS was not measured from filtered spring water, suggesting that COS was produced biogenically in the mat environment (Figures 5-6C and 5-7C). Inhibition of sulfate reduction and methanogenesis allowed for an examination of the possible roles of these anaerobic groups to sulfur gas cycling. Limited H2S(g) evolved in molybdate-amended samples in any incubation series, resulting from either 1) unsuccessful inhibition of sulfate reduction, or 2) sulfur assimilation and H2S(g) production by fermenting bacteria at low O2 levels (e.g., Figure 5-6A). For the B-series, significant H2S(g) was only produced in the BESamended bottle, but not in the molybdate-amended bottle (Figure 5-7A). MT 165 headspace evolution was the same for BES-amended samples, but not the molybdate-amended samples, suggesting that MT production may be from SRB activity. In the A- and B-series, however, MT was detected in the molybdateamended bottles, although at lower concentrations than the unamended incubations or BES-amended incubations. This indicates that other metabolic groups, such as the fermenting bacteria (e.g., Figure 5-4D and 5-4E), may also be responsible for MT production under low O2 conditions. In the B-series (Figure 5-7C), COS increased in the unamended live mat incubations under low oxygen conditions, above the concentration in the filtered water (Figure 5-9C, 5-9F), but not in the BES-amended sample, indicating that COS production may be linked to methanogenic activity. Conversely in the A-series, COS increased in the BESamended sample, suggesting association with sulfate reduction (Figure 5-6C). DMS was detected earlier in the BES-amended incubation than in an unamended sample in the A-series, but at the same rate in the B-series (Figures 5-6D and 5-7D). Rapid increases in H2S(g) concentrations corresponded to an increase in headspace CO2 in the A- and B-series (Figures 5-6E and 5-7E), but to a more subdued increase in the C-series (Figure 5-8E). CO2 did not increase in the headspaces of the sterile water and killed controls in any of the incubation series, indicating the CO2 production was a results of microbial activity. The rapid increase in headspace CO2 in the A- and B-series, coinciding with decreases in O2, may be related to aerobic CO2 utilization by aerobic chemolithoautotrophs (e.g., sulfur-oxidizers), and that CO2 consumption slows when O2 becomes low. This is 166 supported by the rates of H2S(g) increase corresponding to decreases in headspace O2 to <15% (Figure 5-6F, 5-7F, 5-8F). VOSC consumption by homogenized mats No H2S(g) was detected from the spring water following sparging with VOSC (Figure 5-9); however, H2S(g) was measured in the autoclaved microbial mat control, possibly because of VOSC degradation. The concentrations of both MT and DMS in the filtered water were nearly equal to the concentrations of the standard gases used for sparging. COS was extremely low following sparging, suggesting that the gas concentration in the water had not equilibrated and should have been sparged longer. Initially, headspace concentrations of MT and DMS were lower in the live microbial mat incubations compared to those from the headspace of the sterile water sample, suggesting aerobic microbial consumption of both gases, as the samples still had oxygen present (Figure 5-9F). MT increased above the concentration in the sterile sample after 16 hr (Figure 5-9B), corresponding to a rapid increase in headspace CO2 (Figure 5-9E) and decreases in O2 (Figure 5-9F). DISCUSSION The processes affecting the consumption and production of sulfur gases in aphotic spring systems and associated microbial mats were studied by gas flux measurements and by incubations of native and homogenized microbial mats. Changes in CTS= along the Upper Spring stream channel result from the combined effects of abiotic volatilization, autoxidation, and microbial processes, including biological production and consumption (microbial consumption includes both 167 catalyzed sulfide oxidation, as well as sulfur utilization for biosynthesis). VOSC production from incubations of the cave microbial mats, but low amounts measured from the habitat, suggest that the microbes in Lower Kane Cave, as well as other aphotic subsurface environments, play a role in VOSC flux to the atmosphere. At the Upper Spring the observed CTS= loss at the orifice is much faster than can be accounted for from the combined effects of abiotic autoxidation and volatilization loss mechanisms (Figure 5-3B). Based on the estimated autoxidation, and actual and theoretical volatilization rates alone, CTS= at the cave entrance should be only slightly lower than the measured Upper Spring CTS=. In this anaerobic to disaerobic cave system, autoxidation t1/2 is considerably slower (t 1/2 = 866 to 1250 hr) than measurements from air-saturated natural waters with low salinity (Avrahami and Golding, 1969; Chen and Morris, 1972) and several deep waters from anoxic marine basins, such as Framvaren Fjord (t 1/2 = 19-20 min) and the Chesapeake Bay (t1/2 ~ 8.3 min), where the autoxidation rates are enhanced as a result of reaction with dissolved constituents (e.g., metals) in the water and mixing with surface waters (Millero, 2001). Therefore, the observed rapid decrease in CTS= near the mat terminus requires subaqueous microbial sulfide consumption. Microbial consumption of CTS= occurs under disaerobic to aerobic conditions in the cave stream (Figure 5-3A). Using the full-cycle 16S rRNA approach to describe the microbial communities in the Lower Kane Cave mats (refer to Chapters 2 and 3), mat biovolume is dominated by "Epsilonproteobacteria" (~70% of the bacterial biovolume), with minor assemblages of known sulfur-oxidizing bacterial groups belonging to the genera 168 Thiothrix and Thiobacillus (McDonald et al., 1996; Cha et al., 1999). Although cultures of the "Epsilonproteobacteria" from Lower Kane Cave have not been successfully established to date, closely-related taxonomic groups consume dissolved sulfide in culture (Fenchel and Glud, 1998; Gevertz et al., 2000; Campbell et al., 2001; Nemati et al., 2001; Kodama and Watanabe, 2003), and even at oxygen tensions as low as 1% (Takai et al., 2003). Qualitative assessment of microbial sulfide oxidation was done from the mat incubation experiments. In the M-, A-, and B-series incubations, there were low to non-detectable concentrations of headspace H2S(g) when O2 levels were >15%. Headspace H2S(g) increased when O2 decreased, suggesting that microbial sulfide consumption occurs over a narrow range of O2 levels for aerobic to microaerophilic sulfide consumption. Microbial consumption could also be occurring at lower O2 tensions, but consumption rates may not be as rapid nor as competitive compared to microbial H2S(g) production by SRB, S0RB, and fermenting bacteria at low O2. The observed CTS= versus distance profile showing nearly constant CTS= through the microbial mats (Figure 5-3A) and enhanced H2S(g) flux directly over the microbial mats in the stream channel (Table 5-1) indicate that the mats in the cave stream are sites for in situ H2S(g) production by SRB and S0RB, and possibly, to a lesser extent, from sulfur assimilation or growth on dissolved thiosulfate by fermenting bacteria (Figure 5-4). MPN biomass estimates demonstrate that anaerobic metabolic guilds are ubiquitous in the microbial mats, and that SRB are the most abundant anaerobic metabolic guilds (refer to Chapter 4; Figure 4-1). The sulfur isotope values of the dissolved sulfide in Lower Kane Cave, averaging 169 22.5, support autochthonous microbial sulfate reduction. The 34S values for dissolved sulfide in the spring waters indicate that dissimilatory sulfate reduction is the source of sulfide to the cave (refer to Chapter 2; Figure 2-6) (Canfield, 2001a). However, the decrease in 34S values at the end of the Upper Spring microbial mats suggests that sulfide, with a lighter 34S value, is being generated within the stream; in situ sulfate reduction would generate a decrease in the sulfide 34S composition. MT, DMS, and COS generated in incubations were biogenic in origin because VOSC were not emitted from filtered spring water or killed microbial mat samples. The prevalence of MT production from cultures (Table 5-2), bulk mat incubations (Figure 5-5), and from the small flux from the cave stream suggest that MT is the most abundant VOSC in the cave. These results are in agreement with studies from other anoxic freshwater and marine settings where MT is also the dominant VOSC (Kiene, 1988; Kiene et al., 1990; Lomans et al., 1997; Bodenbender et al., 1999; Lomans et al., 1999a; Fritz and Bachofen, 2000). Lomans et al. (1997) suggest that MT and DMS form from the methylation of H2S(g) due to the degradation of sulfur-containing amino acids and methoxylated compounds by SRB during degradation of organic material in anoxic sediments. Some microbes can demethiolate methionine to MT (Taylor, 1991; Dias and Weimer, 1998); (Seiflein and Lawrence, 2001). Taylor (1991) describes that various enzymes are responsible for VOSC production from successive methylation of precursor sulfur gases. If these processes were occurring, a decrease in H2S(g) would be expected. However, decreasing H2S(g) could also be from microbial sulfide consumption. Only when aerobic or microaerophilic consumption was 170 lessened could the effects of methylation be resolved. For the M-series and B-series (Figures 5-5 and 5-7), decreases in H2S(g) under anaerobic conditions could be correlated to MT increases. Therefore, MT production by the microbes from Lower Kane Cave may be due to both amino acid breakdown and sulfide methylation. Disparate results for COS from the incubations of homogenized mat samples may be due to differences in the microbial populations within the sample, such that the Upper Spring sample (A-series) may have a greater abundance of methanogens compared to the Lower Spring sample (B-series). Previous research supports that COS is primarily derived from bacterial activity in marine environments, but COS can also form chemically in soil stimulated by thiocyanate (Conrad, 1996), from photochemical oxidation of CS2 in the atmosphere (Bodenbender et al., 1999; Watts, 2000), or by autoclaving or -irradiating soils (Lehmann and Conrad, 1996). COS evolved in the headspace of two live mat incubations and BES-amended samples (Figures 5-6C and 5-7C), suggesting that COS may be produced by SRB or fermenters (Table 5-2). However, because the COS concentrations were relatively low for all of the incubations, additional work is required to verify microbial COS cycling. VOSC consumption by microorganisms may be a mechanism by which VOSC concentrations are kept low in the cave. MT, and DMS to a lesser extent, were degraded under aerobic conditions (Figure 5-9B), which may have been due to microbial consumption. Previous work with sulfur-oxidizing bacteria, including Thiobacillus thioparus strain 5 and Thiobacillus novellas strain SRM, indicates that these organisms grow using DMS, MT, DMDS (Visscher et al., 1991). Some 171 sulfur-oxidizing bacteria (e.g., Smith and Kelly, 1988; Jordan et al., 1995; Conrad, 1996) and other metabolically diverse bacteria (e.g., Smith et al., 1991; Ensign, 1995; Seefeldt et al., 1995) can also grow using COS. Headspace DMS remained relatively low during the incubations, except for the BES-amended bottles (e.g., Figure 5-6D) which may suggest that methanogens are important for DMS cycling. Several researchers suggest that methanogens are vital to VOSC cycling (Kiene et al., 1986; Rajagopal and Daniels, 1986; Lomans et al., 1999a; Lomans et al., 1999b). Specifically, Lomans et al. (1999a) demonstrate that high concentrations of VOSC result in methane production through methanogenesis in marine and brackish settings. Anaerobic heterotrophs, such as proteolytic (fermenting) clostridia, can also generate methane from methionine and MT (Rimbault et al., 1988). However, in the S-series incubations, aerobic consumption of DMS was evident (Figure 5-9D), and because the BES-amended sample had the same concentration profile as the unamended samples, DMS may also be consumed by SRB or aerobic bacteria, but not by methanogens. Moreover, rare methanogens were cultured from some of the Lower Kane Cave microbial mats (Chapter 4), and low levels of methane were measured from the cave spring water (Table 2-1). During the A- and B-series, however, methane levels did not increase above the background, which is expected if methanogenesis or proteolytic sulfur amino acid breakdown processes were occurring (Figures 5-6G and 5-7G). In conclusion, microorganisms living in sulfur-rich, subterranean habitats contribute to sulfur gas cycling, and the terrestrial subsurface represents both a source and sink for H2S(g) and VOSC. This research demonstrates for the first time 172 that H2S and VOSC are cycled by microbial communities in an aphotic freshwater habitat. Photochemical degradation processes are absent in the cave, so the microbial contributions to gas cycling can be studied in detail. Even if microbial production is balanced by consumption, sulfur gas cycling by microorganisms may be important in subsurface ecosystems by providing additional sulfur substrates for the microbial communities. The results from Lower Kane Cave are consistent with those from previous studies from marine and freshwater habitats with SRB (e.g., Stoner et al., 1994; Lomans et al., 1997; Visscher et al., 2003). One notable exception of this work is that methanogenesis does not appear to be a significant process by which VOSC are degraded in Lower Kane Cave. Less than 10% of the world's karst is associated with sulfidic waters (Palmer, 1991), and yet caves like Lower Kane Cave can serve as a proxy for larger sulfidic aquifers that may be inaccessible for study. As groundwater in non-karst areas can often have high concentrations of dissolved sulfide, especially in regions near hydrocarbon reservoirs (Ulrich et al., 1998; Nemati et al., 2001; Kodama and Watanabe, 2002; Elshahed et al., 2003), the contribution of subterranean sulfur gas cycling is potentially significant globally. 173 Table 5-1: Aqueous geochemical data, observed CTS=, and theoretical and measured H2S(g) volatilization flux. CTS= and flux measurements were collected at different times over a three day time period, August 2003. 2-film Theoretical Volatilization Model Measured Volatilization Mass Flux Loss by Volatilization Actual [H2S] ppmv ml H2S min-1 Mean ol m-2 min-1 9.13 48.5 15.62 40.58 Vapor Flux H2S(g) flux mol m-2 min-1 Theoretical Observed Parameters Site min ml min-1 O2 CTS = pH Flux CTS= KH t 1/2 mol L-1 0.00 ppmv mol L-1 26.67 7.36 7.36 7.36 8727 83.4 13.07 30.98 8727 83.4 13.07 30.98 mol min-1 9600 Mean H2S(g) Flux mol m-2 min-1 Discharge from chamber t 1/2 min CTS= mol L-1 26.67 26.58 Theoretical Flux mol m-2 min-1 34.08 30.98 CTS= mol L-1 26.67 26.59 0.00 26.24 0.00 26.2 11.7 174 7.38 7964 73.9 13.07 27.43 26.68 7.42 7745 67.6 13.07 25.09 24.39 7.42 7.42 7.42 7527 6764 7309 24.39 21.91 23.68 41.43 26.08 87.52 26.42 2.34 21.52 84.48 62.43 71.45 4.38 25.82 26.33 2.66 20.91 (m) 190 191 191 191 191 191 191 191 191 191 191 191 191 193 193 193 193 193 193 193 193 194 194 194 194 194 194 195 196 197 86 53 80 84 57 84 84 56 52 49 82 82 54 84 84 25 27 59 85 103 57 84 84 53 87 87 4.5 38.46 30.38 27.87 9.3 7.2 6.9 26.5 5.4 26.8 19.7 19.72 47 41.7 42.4 51.73 46.3 47.2 46.9 42.1 46.43 37.56 35.31 41.2 32.6 32.4 0.00038 0.00204 0.00243 0.00234 0.00053 0.00061 0.00058 0.00148 0.00028 0.00131 0.00161 0.0016 0.0025 0.0035 0.00356 0.00129 0.00125 0.00278 0.00399 0.00433 0.00264 0.0031 0.0030 0.00218 0.00284 0.00282 10.58 55.74 66.46 64.02 14.50 16.54 15.85 40.58 7.68 35.91 44.17 44.22 69.40 95.78 97.39 35.36 34.18 76.15 109.01 118.58 72.37 86.27 81.11 59.71 77.56 77.08 4.21 25.57 25.32 25.17 26.25 26.17 26.08 2.55 2.19 7.5 20.91 18.79 20.3 Table 5-1: Continued. 2-film Theoretical Volatilization Model Measured Volatilization Mass Flux Actual [H2S] ppmv ml H2S min-1 37.91 24.88 46.61 44.44 7.03 24.73 24.59 40.61 24.44 25.75 25.67 25.59 25.83 0.00121 0.00156 0.00113 0.00228 0.00129 0.0018 0.00174 33.09 42.72 30.82 62.41 35.25 50.53 47.55 Vapor Flux Observed Parameters Loss by Volatilization Theoretical Meter min ml min-1 O2 CTS= pH KH Mean mol m-2 min-1 Flux CTS= t 1/2 t1/2 Discharge from chamber mol L-1 14.69 ppmv mol L-1 22.42 0.67 0.62 0.64 7.42 7.43 7.43 7.36 62.8 58.1 66.8 13.07 13.07 13.07 23.31 21.57 24.78 40 63 27 84 44 84 84 30.25 24.8 41.74 27.17 29.3 22 20.7 mol min-1 8073 7309 6764 6982 Mean H2S(g) Flux mol m-2 min-1 H2S(g) flux mol m-2 min-1 min Theoretical Flux mol m-2 min-1 26.16 23.22 CTS= mol L-1 25.02 CTS= mol L-1 25.92 19.38 33.44 54.8 50.6 50.6 13.07 13.07 13.07 20.34 18.79 18.79 25.13 26.72 20.76 175 0.44 0.34 0.41 7.4 7.4 7.4 4800 3709 4473 43.2 33.4 40.3 13.07 13.07 13.07 16.04 12.39 14.95 32.3 30.0 13.07 13.07 11.99 11.13 14.46 11.56 6.61 56 84 13.1 10.25 7.4 7.44 7.43 7.43 7.43 4473 3818 3491 2073 1200 40.31 10.09 20.3 0.51 0.54 0.54 7.34 7.34 7.34 7.43 7.43 6873 7309 5564 5891 5891 34.85 7.27 40 60 85 42 42 80 80 38 57 85 83 43 43 31 25.4 19.9 27.1 25.7 18.3 17.7 35 34.4 29 27.9 7.41 7.7 0.0012 0.0015 0.00169 0.00114 0.00108 0.00146 0.00142 0.00133 0.00196 0.00246 0.00231 0.00031 0.00033 0.00073 0.00086 33.91 41.67 46.25 31.12 29.52 40.03 38.72 36.37 53.62 67.40 63.32 8.71 9.05 20.06 23.54 55.18 4.14 23.95 23.81 21.80 7.25 23.41 25.03 24.63 25.38 25.27 m 199 200 200 200 200 200 200 200 201 202 203 203 203 203 203 203 203 205.5 205.5 205.5 205.5 205.5 205.5 206 207 207 210 215 47.50 12.42 0.35 0.32 5.76 3.33 Table 5-2: Reduced sulfur gases produced from all enrichment cultures, listed per microbial mat site in the Lower Kane Cave. H2S + SoRB COS MT DMS - Site (m) and Samplea 176 189 orifice water 215 orifice water 248 orifice water 189 gray orifice sediment 189 gray orifice sediment 189 gray orifice sediment 193 gray filaments 198 gray filaments 201 gray filaments 203 gray filaments 203 deep gray filaments 248 gray filaments 248 gray orifice sediment 198 white filaments 201 white filaments 203 white filaments 203 white filaments 248 white filaments 203 white webs 203 white feathers 249 feathers 203 yellow patches 249 yellow patches + + + + + + + + + + + + + + - a H2S -b + + + + + + + + + + + + + + + + + + + + + + Group I SRB COS MT + + + + + + + + + + + + + + + + + + + DMS H2S + + + + + + + + + + + + + + + + + + + + + + DMS + + - Group II SRB COS MT + + + + + + + + + + + + + + + + + + + + + + + H2S + + + + + + + + + + + + + + + + + + + + Fermenters COS MT + + + + + + + + + + + + + + + + + + + DMS + + - b Repeated sampling sites represent replicate bottles scored for serial dilutions from different sampling trips. - gas not produced; + gas detected; no symbol represents a sample was not analyzed. A C B D 177 Figure 5-1: Gas chromatograph (GC) setup in Lower Kane Cave. (A) Passage below Upper Spring, with flux chamber in foreground and GC to the left (arrow). (B) Close-up of Teflon 16-cm2 flux chamber (arrow). (C) Flux chambers over microbial mats and cave stream at 197 m. A copper tube was used to scrub incoming H2S(g). (D) GC in Lower Kane Cave; flux chamber running in the background (arrow). 70 microbial mats 70 H2S gas CTS= 60 50 mol L CTS= 40 30 20 10 0 190 60 50 40 30 20 10 0 215 ppmv H2S 195 200 205 210 Distance (meters) Figure 5-2: Total dissolved sulfide concentration (CTS=) and average H2S gas concentrations measured along the Upper Spring flowpath in Lower Kane Cave, starting downstream from the spring orifice (189 m). Gas was measured by gas chromatography directly in the cave from individual gas samples collected from the air-water boundary layer. 178 A 30 25 20 microbial mats t1/2 = 0.46 min, velocity = 0.5 m/s sulfate reduction Dissolved Sulfide Calculated Sulfide at 190 m Calculated Sulfide at 203 m CTS= ( mol L-1) sulfur oxidation t1/2 = 0.23 min, velocity = 0.2 m/s 15 10 spring orifice 5 0 sulfur oxidation 189 194 199 B 30 25 CTS= ( mol L-1) 204 209 Distance (meters) 214 219 60 50 40 microbial mats 20 15 10 spring orifice Loss by Autoxidation Loss by Actual Volatilization Loss by Theoretical Volatilization Dissolved Sulfide Dissolved Oxygen 30 20 10 0 5 0 189 194 199 204 209 Distance (meters) 214 219 Figure 5-3: Total dissolved sulfide (CTS=) loss from the Upper Spring orifice through the microbial mats downstream. (A) Observed concave-down CTS= vs. distance profile and two expected concave-up first-order loss profiles estimated from the initial CTS= at the spring orifice (190) and CTS= at 203 m. Microbial processes proposed responsible for offsetting the CTS= are labeled. (B) Observed CTS= and dissolved oxygen vs. distance profiles compared to volatilization and autoxidation loss profiles. Actual volatilization based on H2S(g) flux measurements and theoretical volatilization loss curve estimated from 2-film Liss and Slater (1974) model. 179 Dissolved oxygen ( mol L-1) 160 140 120 A Strain LKC-232 160 140 120 B Strain LKC-322 H2S ppmv 100 80 60 40 20 0 0 30 25 5 10 time (hr) H2S ppmv 15 20 25 100 80 60 40 20 0 0 5 10 time (hr) 15 20 25 C 20 15 10 5 0 233 Methionine 1 233 Methionine 2 322 Methionine 1 322 Methionine 2 0 50 100 150 200 250 300 350 Fermenter broth + thiosulfate 1 Fermenter broth + thiosulfate 2 Fermenter broth + sulfate 1 Fermenter broth + sulfate 2 Fermenter broth only 1 Fermenter broth only 2 Fermenter broth + cysteine 1 Fermenter broth + cysteine 2 Fermenter broth + methionine 1 Fermenter broth + methionine 2 1 0.8 0.6 CH3SH ppmv 1 0.8 0.6 D Strain LKC-232 E Strain LKC-322 abs 0.4 0.2 0 0 5 10 abs 0.4 0.2 0 15 20 25 0 time (hr) 5 10 15 20 25 time (hr) Figure 5-4: Time course series for two fermenting bacterial strains. (A and B) H2S production was tested for using different inorganic and organic sulfur substrates. (C) Methanethiol production on methionine. (D and E) Growth curves measured by spectrophotometry. 180 1000 A 3 2.5 D H2S (ppmv) CO2 (%) Ar+ O2 (%) 100 2 1 .5 1 10 0.5 0 1 100 20 B 15 10 5 0 8 Methanethiol (ppmv) E 10 1 10 F 7.5 C pH 7 COS & DMS (ppmv) 6.5 5 6 1 10 102 103 Minutes Filtered spring water Live microbial mat Live microbial mat Live microbial mat Killed microbial mat 104 105 1 1 10 102 103 Minutes 104 105 Figure 5-5: M-series time courses of gases and pH from Teflon reactor incubations prepared from natural microbial mat samples (Lower Kane Cave, 248 m mat sample). pH was monitored in reactor M2 and M3. 181 10000 A A-series incubations Filtered spring water Live microbial mat Live microbial mat Live microbial mat Molybdate-amended mat BES-amended mat Killed microbial mat 15 H2S ppmv 1000 100 10 1 1000 E Methanethiol ppmv 100 CO2 (%) B 10 5 0 10 20 1 10 C Ar + O2 (%) 15 10 5 0 F COS ppmv 100 5 G 1 100 CH4 ppmv 10 1 1 10 102 103 104 105 D Minutes 10 1 1 10 102 103 Minutes 104 105 Figure 5-6: Time courses of gas production for A-series incubations prepared with microbial mat samples from Lower Kane Cave, 203 m. Some samples were amended with Namolybdate ( ) and others with BES ( ). Solid lines in A, B, C represent addition of BES. DMS ppmv 182 10000 A B-series incubations Filtered spring water Live microbial mat Live microbial mat Live microbial mat Molybdate-amended mat BES-amended mat Killed microbial mat 15 1000 H2S ppmv 100 10 1 E Methanethiol ppmv 10 CO2 (%) Ar + O2 (%) CH4 ppmv 100 B 10 5 0 20 15 10 5 0 F 1 10 C COS ppmv 20 5 G 10 1 10 0 D 1 10 102 Minutes 103 104 105 5 1 1 10 102 103 Minutes 104 Figure 5-7: Time courses of gas production for Bseries incubations prepared with microbial mat samples from Lower Kane Cave, 248 m. Some samples were amended with Namolybdate ( ) and 105 others with BES ( ). DMS ppmv 183 1000 A C-series incubations Filtered spring water Live microbial mat Live microbial mat Live microbial mat Molybdate-amended mat BES-amended mat Killed microbial mat 15 H2S ppmv 100 10 1 10 Methanethiol ppmv 5 CO2 (%) B E 10 5 0 1 3 5 7 Days 9 11 13 20 8 COS ppmv 6 4 2 0 10 8 DMS ppmv 6 4 2 0 1 C Ar + O2 (%) 0 10 15 10 5 0 F 1 3 5 7 Days 9 11 13 D Figure 5-8: Time courses of gas production for Cseries incubations prepared with microbial mat samples from Lower Kane Cave, 193 m. Some samples were amended with Namolybdate ( ) and others with BES ( ). 10 103 102 Minutes 104 105 184 10000 1000 A H2S (ppmv) 100 Filtered spring water Live microbial mat Live microbial mat Live microbial mat BES-amended mat Killed microbial mat 20 10 1 1000 Methanethiol (ppmv) B E 16 100 CO2 (%) 12 8 4 0 10 1 10 20 COS (ppmv) 5 Ar+ O2 (%) C 15 F 10 5 0 1 10 1 100 102 103 Minutes 104 105 D DMS (ppmv) 10 Figure 5-9: VOSC sparged spring water incubation S-series. Microbial mat samples originated from 248 m, Lower Spring. 1 1 10 102 103 Minutes 104 105 185 Chapter 6: Microbial Contributions to Cave Formation: New Insights into Sulfuric Acid Speleogenesis ABSTRACT1 The sulfuric acid speleogenesis model was introduced in the early 1970s from observations of Lower Kane Cave, Wyoming, and was proposed as a cave enlargement process resulting primarily from H2S autoxidation to sulfuric acid and replacement of carbonate by gypsum on subaerially exposed cave-wall surfaces. This sulfuric acid speleogenesis type-locality was reexamined using uniquely applied geochemical and microbiological methods. Little H2S escapes into the cave atmosphere, or is lost by abiotic autoxidation, and instead the primary H2S loss mechanism is from consumption of reduced sulfur compounds by subaqueous sulfur-oxidizing bacteria. Filamentous "Epsilonproteobacteria" and Gammaproteobacteria, characterized by fluorescence in situ hybridization, colonize carbonate surfaces and generate sulfuric acid as a metabolic byproduct. The bacteria focus carbonate dissolution by locally depressing pH. These exceptional findings show that sulfuric acid speleogenesis occurs in the shallow subaqueous cave environment, and potentially at much greater phreatic depths in carbonate aquifers, thereby offering new insights into the microbial roles in subsurface karstification. A portion of this chapter was published in the paper "Microbial contributions to cave formation: new insights into sulfuric acid speleogenesis" by A.S. Engel, L.A. Stern, and P.C. Bennett, 2004, Geology, vol. 32 (5) p. 369-372. 1 186 INTRODUCTION Karst landscapes form where soluble carbonate rocks dissolve by chemical solution, resulting in distinct geomorphic features including caves and conduit drainage systems (White, 1988; Ford and Williams, 1989). Karst comprises a significant percentage of the continental earth's surface, and karst formations are important resources for water, hydrocarbon energy, and tourism (see reviews by White, 1988; Ford and Williams, 1989; Palmer, 1991; Daoxian, 1998). The classic model for cave and karst development (speleogenesis) is one of carbonate rock dissolution by carbonic acid, generally at the water table or within the epikarst. Palmer (Palmer, 1991) estimates that <10% of karst globally has developed from hypogenic processes involving CO2- or H2S-rich fluids. Examples of extensive hypogenic karst include the caves of the Black Hills, South Dakota (Palmer and Palmer, 1989), the caves of the Guadalupe Mountains, New Mexico and Texas, including Carlsbad Cavern (Hill, 1996; Polyak et al., 1998), caves in Central Italy (Galdenzi and Menichetti, 1995), and the karst of the Caucasus Mountains (Shelley, 1956) and Turkmenistan (Maltsev, 1993). Many hypogenic caves are polygenic in origin (Hill, 1995; 1996), reflecting processes whereby mixing of meteoric water with waters enriched in CO2 or H2S has played a role in formational processes (e.g., Ford and Williams, 1989). Because of the complexity of speleogenesis processes, as well as the fact that many of these systems are no longer forming today, many of the mechanisms governing cave formation must be inferred. 187 Historically, several researchers have proposed that carbonates could dissolve by acids other than carbonic acid, including acids derived from inorganic or bacterial oxidation of sulfides or organic matter (Kaye, 1957; Howard, 1964). For many years, however, carbonate dissolution by sulfuric acid was considered insignificant, and more recently Lowe and Gunn (1995) suggest that sulfuric acid may be important for all nascent subsurface carbonate porosity generation. Palmer (1991; 1995) speculates that sulfuric acid dissolution, as a major cave formation process, is of more importance for the evolution of carbonate petroleum reservoirs than it is for the origin of caves. Indeed, sulfuric acid dissolution has been proposed for the karstification of significant hydrocarbon reservoirs, including the Lisburne field in Prudhoe Bay, Alaska (Jameson, 1994; Hill, 1995), and Mississippi Valleytype deposits (Hill, 1990). Recently, several active caves with hydrogen sulfiderich groundwater, which discharges into the cave passages as springs, have been recognized (Hubbard et al., 1990; Galdenzi and Menichetti, 1995; Sarbu et al., 1996; Hose et al., 2000). The process of sulfuric acid speleogenesis from the `replacement-solution' process was proposed by Egemeier (Egemeier, 1981; Hill, 1990; Jagnow et al., 2000) from work in Lower Kane Cave, Wyoming. Dissolved hydrogen sulfide in the groundwater volatilizes into the cave atmosphere, where hydrogen sulfide oxidizes on moist, sub-aerial cave-wall surfaces to sulfuric acid; H2S + 2O2 H2SO4 (5-1) The acid reacts with and replaces limestone with gypsum (CaSO4 2H2O), CaCO3 + H2SO4 + H2O CaSO4 2H2O + CO2 188 (5-2) which eventually dissolves into groundwater undersaturated with respect to gypsum. The net result is the removal of mass and an increase in void volume. Hydrogen sulfide is a rich energy source for microorganisms, and sulfidic cave systems are often colonized by thick microbial mats (e.g., Angert et al., 1998; Hose et al., 2000; Engel et al., 2001). However, the potential role of sulfuric acidgenerating microorganisms to cave formation has only been alluded to (Symk and Drzal, 1964; Hubbard et al., 1990; Hill, 1996; Angert et al., 1998; Hose et al., 2000; Vlasceanu et al., 2000; Engel et al., 2001). The present investigation reexamines the Lower Kane Cave sulfuric acid speleogenesis process by considering the presence of active microbial communities in subaqueous microbial mats, and in particular sulfur-oxidizing bacteria. Because only a small percentage of hydrogen sulfide volatilizes into the cave air, most of the dissolved sulfide is consumed within the subaqueous environment by microbial oxidation (refer to Chapter 5). This exceptional finding led to the hypothesis that speleogenesis within Lower Kane Cave occurs in the subaqueous environment, where sulfur-oxidizing bacteria directly contribute to carbonate dissolution. MATERIALS AND METHODS Aqueous Geochemistry The focus of this chapter is on the Upper Spring transect and the 17 m of mat-filled outflow channel (Figure 1-2), although similar results were found at the other spring-stream complexes in the cave. Stream and gas chemistries were characterized seasonally over three years by standard field-based techniques, as well as by methods developed specifically for the cave environment. Unstable 189 aqueous parameters (pH, EH, O2, temperature) were determined in situ by electrode methods every meter along the spring outflow channel. Low-level dissolved oxygen and total dissolved sulfide (CTS=) were measured in the field by colorimetric analysis on a field spectrophotometer using Rhodazine D and Methylene Blue complex (CHEMetrics, VA), respectively. Dissolved, moderatelevel oxygen was measured in the field also by colorimetry using the IndigoCarmine method (CHEMetrics). Other dissolved constituents were determined as previously described in Chapter 2. Stream water dissolved solute speciation and activity, equilibrium gas partial pressure, and saturation state with respect to mineral phases were calculated from the in situ temperature, dissolved solute concentration, pH, and ionic strength using the geochemical speciation model PHREEQC (Parkhurst and Appelo, 1999). Atmosphere Gases As a result of the unstable and fugitive nature of some sulfur gases, gas measurements and H2S flux were performed in the cave by direct-inject field gas chromatography (refer to Chapter 5). Calcite Field Chambers and Microcosms Sterile and non-sterile field chambers (in situ microcosms) containing chips (0.5 to 1 cm3) of Iceland Spar calcite (Wards Scientific) and native Madison Limestone (mineralogy confirmed by x-ray diffraction) were deployed to test whether microorganisms or the bulk stream water chemistry controlled carbonate dissolution. Microcosms were constructed from 2.5 cm wide by 5 cm long PVCpipes with screw-caps on either end. Sterile microcosms had 0.1 m-PVDF 190 hydrophilic filters on the end (similar to `peepers'), while non-sterile microcosms had 0.5 mm polyethylene mesh on either end to allow for fluid flow, and also for microbial colonization of the chips. Paired sterile and non-sterile microcosms were placed throughout the cave in the stream and within microbial mats, and remained in the cave to react for 2 weeks to nine months (Figure 6-1). Additionally, a technique similar to the buried-slide technique (Parkinson et al., 1971) was used: polished chips (with the largest surface areas being 1-2 cm2) of limestone and pieces of Iceland Spar were surrounded by a 0.5 mm-mesh sack. At each microcosm site, a mesh sack was also deployed (which contained glass slides for structural support). Electron Microscopy When field chambers and mesh sacks were retrieved, the rock and mineral chips were processed separately and chips were preserved for microscopy, including conventional scanning electron microscopy (CSEM), environmental SEM (ESEM), and florescence in situ hybridization (FISH). Chips examined by ESEM were not preserved, but instead were frozen to -20oC. Unpreserved chips were examined using a Philips XL30 ESEM, with energy dispersive x-ray analysis system, and gaseous secondary and backscatter electron detectors. Chamber conditions were varied from 10%-95% relative humidity using a peltier cooling stage and variable water pressure (0.9 to 6.4 torr, with corresponding accelerating voltages from 4 to 20 kV). For CSEM, chips were fixed using a chemical criticalpoint drying method modified from Nation (1983). Chips were fixed in 2.5% gluteraldehyde, followed by dehydration in a series of ethanol washes to preserve 191 biological material on the surfaces. Air-dried samples were mounted on aluminum stubs, sputter-coated with gold for 30 sec, and examined using a JEOL JSM-T330A SEM with a 30 kV accelerating voltage. Fluorescence In Situ Hybridization Because filamentous sulfur-oxidizing bacteria are difficult to isolate or to grow in cultures, as well as to enumerate by standard culturing methodology, culture-independent phylogenetic characterization and quantification of microbial communities comprising the filamentous mats, including the construction of 16S rRNA gene sequence clone libraries (Chapter 2) and fluorescence in situ hybridization (FISH) using 16S rRNA-specific probes (Chapter 3; Engel et al., 2003), was done for Lower Kane Cave microbial mats. FISH probes were designed to target specific and dominant epsilonproteobacterial groups within the mats (Amann et al., 1995). For fluorescence in situ hybridization (FISH), chips were fixed in two ways within 12 hr of collection: (i) with 4% paraformaldehyde for 3 hr, as described by Manz et al. (1996), and (ii) with 50% ice-cold ethanol according to Roller et al. (1994). Fixed chips were air-dried and dehydrated by sequential washes in 50, 80, and 100% ethanol for 3 min prior to hybridization. Previously synthesized oligonucleotide probes (LKC1006, GAM42a, and EUB338I-IIImix; Chapter 3) were applied to the chips using hybridization and washing buffers. Refer to Chapter 3 for details. Probe LKC1006 (5'-CTCCAATGTTTCCATCGG-3') was designed to target one epsilonproteobacterial group (LKC Group II) from the microbial mats; this group was the most abundant by biovolume using FISH (Table 3-4). Probe 192 GAM42a (5'-GCCTTCCCACATCGTTT-3') targeted Gammaproteobacteria, including Thiothrix spp. (Manz et al., 1996). Probe EUB338I-IIImix hybridized with Eubacteria, with equal concentrations for Eubacteria, of EUB338I EUB338II (5'(5'- GCTGCCTCCCGTAGGAGT-3') GCAGCCACCCGTAGGTGT-3') specific for Plantomycetes, and EUB338III (5'GCTGCCACCCGTAGGTGT-3') specific for Verrucomicrobia and other bacterial groups (Daims et al., 1999). A Leica TCS 4D confocal laser scanning microscope (CLSM) at the Institute for Cellular and Molecular Biology Core Facility, University of Texas at Austin, was used for three-channel simultaneous monitoring of fluorescence results. The CLSM was equipped with a Kr/Ar mixed gas laser and a UV laser, with DIC optics in an inverted microscope. The lasers supply the excitation wavelengths at 488, 568, and 647 nm. Examination was done using the 100x oil immersion objective. Image processing was done using software provided with the Leica instrument, with manual adjustment of contrast and brightness using Adobe Photoshop. RESULTS AND DISCUSSION Hydrogen Sulfide Transport and Reaction Based on Egemeier's original work (Egemeier, 1973, 1981), it was hypothesized that subaerial replacement of limestone by gypsum was a significant mechanism for cave enlargement in Lower Kane Cave because most of the H2S would quickly escape into the cave atmosphere. Volatilization was tested by measuring the dissolved and atmospheric gases, and by determining the actual flux of H2S(g) from the cave water to the atmosphere (Chapter 5), and subsequently the 193 available H2S(g) that could oxidize on the moist cave-wall surfaces (refer to Chapter 7 for additional information). At the study spring (Upper Spring), anaerobic water has 34 mol L-1 total dissolved sulfide (CTS=) at 20.5 C and pH 7.2 (40% H2S:60% HS-; pK= 7.04), (Table 6-1). The total flux of CTS= at the Upper Spring, calculated from the stream velocity and the incoming concentration of sulfide, was ~8700 mol min-1, with an average H2S(g) flux of 44 mol m-2 min-1 from the stream and microbial mats (Table 5-1 and 5-3). Therefore, over the entire length of the microbial mat at the Upper Spring, the transfer H2S(g) to the cave atmosphere represents less than 8% of the total discharged CTS=, suggesting that the bulk of CTS= is lost by other mechanisms in the subaqueous environment. One of those loss mechanisms is abiotic autoxidation, but it was found to be very slow in the disaerobic proximal stream water (Zhang and Millero, 1983; Millero et al., 1987), representing only ~0.014% of the potential abiotic loss. Therefore, the CTS= loss rate was much faster than could be accounted for by volatilization and autoxidation. The distinctly concave-down concentration vs. distance profile (Figure 5-3) is unlike the characteristic concave-up first-order loss profile expected for both abiotic loss mechanisms. At the mat terminus, however, the increase in dissolved O2 concentration corresponds to rapid biotic consumption of CTS= (Figure 5-3). Near the Upper Spring mat terminus, both allochthonous and autochthonous (see Chapter 5) sulfide is consumed by subaqueous biotic consumption, evidenced by the extremely rapid decrease in CTS=. The high 194 background [SO42 ] in the stream water (Table 6-1), however, makes it impossible to measure small changes in oxidation byproducts. Subaqueous Microbial Carbonate Dissolution The dominant mechanism for CTS= loss is subaqueous microbial oxidation, and most sulfur-oxidizing bacteria oxidize H2S completely to sulfate with a substantial energy yield: H2S + 2O2 SO42 + 2H+ ( 798 kJ/reaction) (6-3) Others initially form elemental sulfur (S0) as an intermediate that is stored intracellularly and further oxidized during periods of limiting sulfide: HS + O2 + H+ S0 + H2O + 1 O2 S0 + H2O SO42 + 2H+ ( 209 kJ/reaction) ( 587 kJ/reaction) (6-4) (6-5). As a result of two of these three energetic oxidation reactions (Equations 6-3 and 65), acidity is generated in the form of sulfuric acid, attacking the geologic matrix supporting the community and modifying their ecological surroundings. Although some sulfur-oxidizers are acidophiles (Johnson, 1998), most are neutrophilic. Colonization of carbonate surfaces, therefore, buffers excess acidity and maintains pH homeostasis (Engel et al., 2001). The dominant microorganisms in Lower Kane Cave are phylogenetically grouped within the "Epsilonproteobacteria", with lesser abundant sulfur-oxidizing communities of Thiothrix spp. (Gammaproteobacteria) and Thiobacillus spp. (Betaproteobacteria) (Chapter 2; Table 2-3). Although many "Epsilonproteobacteria", including those identified from Lower Kane Cave, have not been obtained in pure culture, many cultured epsilonproteobacterial groups 195 oxidize reduced sulfur compounds using molecular oxygen (under microaerophilic conditions) or alternative electron acceptors (such as nitrate or metals) (e.g., Campbell et al., 2001; Madrid et al., 2001; Lopez-Garcia et al., 2002; Takai et al., 2003). On the basis of phylogenetic affiliation, habitat geochemistry, and sulfur isotope data (Chapter 2), I hypothesize that the "Epsilonproteobacteria" in Lower Kane Cave are also sulfur-oxidizers. Most cultured "Epsilonproteobacteria" do not store sulfur intracellularly, in contrast to Gammaproteobacteria, specifically Thiothrix spp. (Larkin, 1989; Nielsen et al., 2000). Deeply corroded native carbonate fragments (pebble- to cobble-size limestone clasts) were observed in the cave stream, with dissolution effects only on surfaces exposed to stream water and the filamentous microbial mats (Figure 62A). Gypsum replacement of these clasts is obvious above the water surface (Figure 6-2B). Examination of these rock surfaces by CSEM and ESEM revealed a complex reacting environment with dissolving carbonate surfaces, secondary gypsum, and a thick cover of predominately filamentous bacteria and exopolysaccharide material (Figure 6-3). The observed carbonate dissolution and gypsum precipitation associated with a surface biofilm suggests that sulfuric acid speleogenesis occurs within a mineral surface microenvironment maintained by the microbial community, rather than being caused by changes in bulk aqueous geochemistry. The water exiting the Upper Spring pool had a calculated equilibrium partial pressure of CO2 (pCO2) of 10-1.95 atm, was only slightly undersaturated with respect to calcite (SIcal ~-0.2), but was significantly undersaturated with respect to 196 gypsum (SIgyp ~ -1.5) (Table 6-1). Within a few meters from the orifice, the cave waters became supersaturated with respect to calcite (SIcal =+0.06), and at the end of the microbial mat SIcal = +0.2 due to the volatilization of CO2 and the contribution of alkalinity by dissimilatory sulfate reduction. The bulk waters were undersaturated with respect to gypsum (SIgyp = -1.54 to -1.62) (Table 6-1). Although the waters were in equilibrium or supersaturated with respect to calcite, SEM examination of the chamber calcite revealed etching localized where microbial filaments or biofilms were attached to the mineral surfaces (Figure 6-4A through 6-4E; Appendix E). Sterile calcite chips exposed to the stream water show no microbial colonization and no apparent dissolution (Figure 6-4F). Two filament types were observed on nonsterile calcite chips with ESEM, one containing sulfur globules and another without (Figure 6-5A). Dissolution trenches directly under filaments without sulfur (white arrows) were deeper than trenches associated with sulfur-containing filaments (black arrows) (Figure 6-5B). Differences in dissolution intensity at the microbial filament may correspond to the predominance of each of the two sulfide oxidation mechanisms (Equation 6-3 vs. Equation 6-4); greater localized acidity may be generated under the filaments that do not store sulfur 6-3). (Equation FISH probes were used on the experimental chip surfaces to determine the identities of the filaments colonizing the surfaces. By using CLSM, the three FISH probes applied to rock and mineral surfaces showed positive hybridization signals to predominantly filamentous organisms (Figure 6-6). The topographic signal range was ~25 m from the native limestone and 5 m on the Iceland Spar surfaces. 197 However, these topographic variations in the surfaces, presumably because of dissolution of the carbonate during the experiment, made it difficult to obtain one focal plane with the CLSM. Exceptionally bright hybridization signals for each of the probes indicated high rRNA content, suggesting metabolically active populations when the samples were retrieved (Amann et al., 1995). Nearly all observed filaments simultaneously hybridized with the EUB338I IIImix and LKC1006 probes on limestone surfaces (Figure 6-6A and 6-6B), and fewer filaments hybridized with the GAM42a and EUB338I IIImix probes (Figure 6-6A and 6-6C). The FISH results genetically identify the two filamentous sulfuroxidizing bacterial groups that colonize subaqueous carbonate surfaces, consistent with the ESEM observations of filaments with and without intracellular sulfur (Figure 6-5A). A New Model for Microbial Sulfuric Acid Speleogenesis The rapid loss of sulfide from the stream, carbonate dissolution associated with microbial filaments, and the dominance of "Epsilonproteobacteria" on the experimental limestone surfaces support the hypothesis that these organisms have a direct role in sulfuric acid speleogenesis by oxidizing sulfide to sulfuric acid. The classic sulfuric acid speleogenesis model relied in part on H2S(g) volatilization from the cave stream to the cave atmosphere for replacement-solution processes to proceed (Egemeier, 1981; Palmer, 1991), yet negligible volatilization and abiotic autoxidation of CTS= in the cave stream was measured in Lower Kane Cave (Chapter 5). Instead, CTS= is consumed by subaqueous sulfur-oxidizing bacteria and cave enlargement occurs via microbially-enhanced dissolution of the cave 198 floor. In the cave stream, the system is very close to equilibrium, and the calcite dissolution rate is a function of [Ca2+], pCO2, and solution pH. The rate has a nonlinear dependence on the degree of undersaturation () that can be described as the difference between the actual and equilibrium saturation pH (pH = 0.5 log ) (e.g., (Morse and Berner, 1972; Berner and Morse, 1974). At conditions of small (pH < 0.15), such as found in the bulk stream water receiving diffuse proton input from abiotic sulfide oxidation, calcite dissolution is very slow with little effect from small changes in pH. In contrast, where bacterial filaments are in contact with carbonate, excess acidity is focused at the reacting surface (Equation 3) and local increases. Although the stream water is near calcite equilibrium, the [Ca2+] increases along the stream region where microbial growth is greatest, whereas [SO42-] increases only slightly (Table 6-1). These observations show that sulfuroxidizing bacteria colonize subaqueous carbonates, localize dissolution by generating acidity, and therefore are integral to sulfuric acid speleogenesis. CONCLUSIONS While a microbial role to cave formation has been alluded to for sulfidic caves (Hubbard et al., 1990; Hill, 1996; Angert et al., 1998; Hose et al., 2000; Vlasceanu et al., 2000; Engel et al., 2001), previous explanations for sulfuric acid speleogenesis assumed abiotic chemical and hydrologic controls. These controls operate predominantly near a shallow groundwater table because of oxygen requirements for abiotic processes (e.g., Palmer and Palmer, 2000) or subaerially after H2S volatilization on the basis of extensive gypsum in these cave systems 199 (e.g., Egemeier, 1981; Hill, 1990; Galdenzi and Menichetti, 1995; Hill, 2000). This work reexamined Lower Kane Cave and confirmed that some H2S(g) does indeed volatilize into the cave atmosphere, and consequently subaerial speleogenesis does occur. However, the long-term rate is unknown. The H2S(g) volatilization flux today cannot explain modern speleogenesis processes, but the volatilization loss into the cave atmosphere represents the maximum potential proton transfer to the cave walls, which affects subaerial cave-wall speleogenesis processes (Chapter 7). Almost all CTS= in Lower Kane Cave is consumed by subaqueous sulfuroxidizing bacteria. These bacteria drive sulfuric acid speleogenesis by their attachment to carbonate surfaces and generation of sulfuric acid, which focuses local carbonate undersaturation and dissolution. As the sulfur-oxidizing "Epsilonproteobacteria" can be metabolically active under low oxygen tensions, they can catalyze sulfide oxidation in environments where autoxidation is kinetically limited. Therefore, microbial catalysis extends the phreatic depths to which porosity and conduit enlargement could occur in carbonate systems, including oil-field reservoirs and aquifers. One such setting for future investigations, and a location to test the microbial dissolution model, is within the Edwards Aquifer, Central Texas, where it has been proposed that a portion of the karstic aquifer has formed from sulfuric acid speleogenesis (Grubbs, 1991). The metabolic and geologic consequences of an active microbial ecosystem fundamentally change the model for sulfuric acid speleogenesis in the deep subsurface, which may shed light on carbonate porosity development in large aquifers and petroleum reservoirs. 200 Table 6-1: Aqueous geochemical data, total dissolved sulfide (CTS=), and saturation indices (SI) for calcite (SIcal) and gypsum (SIgyp) from Lower Kane Cave Upper Spring stream water. Ca2+ O2 mol L mol L Log Atm mmol L mmol L mmol L mmol L mmol L Meter pH Mg2+ Na+ Major Constituents HCO3SO42CTS= Saturation Index pCO2 SIcal SIgyp (m) 1.583 1.637 0.98 0.25 3.38 1.11 0.94 0.25 3.39 1.13 -2.08 -2.14 -0.06 0.01 -1.62 -1.62 201 1.762 1.765 1.691 1.890 1.914 0.96 0.27 3.45 1.00 0.99 0.97 0.33 0.25 0.31 3.23 3.28 3.41 1.08 1.11 1.14 1.18 0.99 0.26 3.38 1.12 190 191 193 194 195 196 197 199 200 201 202 203 206 207 210 215 7.36 7.36 7.38 7.42 7.42 7.42 7.42 7.42 7.36 7.34 7.34 7.43 7.40 7.43 7.43 7.43 0.00 0.00 2.34 2.66 2.55 2.19 7.50 14.69 14.69 19.38 33.44 40.31 47.50 47.81 -2.08 -2.17 -2.13 -2.15 -2.14 -0.02 0.03 -0.01 0.08 0.09 -1.59 -1.60 -1.61 -1.56 -1.54 26.67 24.24 21.52 20.91 20.91 18.79 20.30 22.42 19.49 19.09 20.30 16.82 12.42 10.15 5.76 3.33 20 cm AS Engel 5 cm AS Engel C D 10 cm 10 cm AS Engel E F 10 cm 5 cm Figure 6-1: Examples of field chambers and buried slides used in Lower Kane Cave. (A) Fissure Spring orifice (hole at center right) at 118 m; (B) Microcosms at 127 m with sparse filaments; (C) Arrows point to microcosms covered by microbial mats at 203 m; (D) Exposed and buried microcosms at 249 m (arrows); (E) Arrows point to partially buried microcosms (white caps are showing) in Lower Spring orifice pool, 189 m; (F) Microcosms and buried slide mesh pouch in stream channel at 215 m. 202 AS Engel AS Engel AS Engel A B A limestone filaments B limestone filaments Secondary gypsum limestone limestone above water below water Figure 6-2: Examples of deeply corroded limestone cobbles in Fissure Spring stream channel. (A) Microbial filaments and webs discontinuously cover limestone clasts. (B) When limestone is exposed above the water surface, secondary gypsum replaces the limestone (lower right). Scale bars are 10 cm. 203 AS Engel AS Engel A g f g f f g g g f B g f 204 f Figure 6-3: Environmental scanning electron photomicrographs showing surface textures of native limestone. (A) Biofilm and filaments (f) coating surface and gypsum (g) crystals on native limestone (0.9 torr, 12 kV). Scale bar represents 10 m. (B) Biofilm showing deeply etched limestone surface filled with gypsum (6.4 torr, 20 kV). Scale bar represents 50 m. A B C D E F 10 m Figure 6-4: Scanning electron photomicrographs of calcite chip surfaces from in situ microcosm in microbial mats at 203 m. (A-E) Preserved chip examined with CSEM with 30 kV accelerating voltage. All strands are microbial filaments; calcite surface is etched. (F) Unpreserved chip examined with ESEM at 5 kV and 1.6 torr. Filaments and surface dissolution were not visible. 205 A B 206 Figure 6-5: Environmental scanning electron photomicrographs of calcite chip surface from field microcosm. (A): Two filament morphologies, one with intracellular sulfur globules (black arrows) and another filament without (white arrows) (1.0 torr, 6.0 kV). Scale bar represents 5 m. (B): Calcite chip surface with two filament morphologies showing deep trenches directly under filaments without intracellular sulfur (white arrows) in contrast to shallow trenches under filaments with sulfur (black arrows) (1.6 torr, 4.0 kV). Scale bar represents 20 m. A B C 207 Figure 6-6: Fluorescence in situ hybridization (FISH) of filaments attached to polished chip of native limestone from mesh-covered buried slides with probes "Epsilonproteobacteria" LKC1006 (red), EUB338I IIImix (green) specific for all Eubacteria, and GAM42a (light blue), specific for Gammaproteobacteria, including Thiothrix spp. Scale bar in all images represents 20 m. Chapter 7: Geochemistry and Interfacial Phenomena of Acidic Condensation Droplets on Cave-wall Surfaces: Implications for Authigenic Quartz Precipitation and Sulfuric Acid Speleogenesis ABSTRACT Volatilization of hydrogen sulfide in Lower Kane Cave, Wyoming, and sulfide oxidation to sulfuric acid on moist subaerial cave-wall surfaces causes carbonate rock dissolution and replacement by gypsum during sulfuric acid speleogenesis. A slight temperature gradient from the cave springs to the rock walls and high relative humidity contribute to condensation. Condensate forms on cavewall surfaces, including gypsum and organic-rich reddish-brown crusts (13C = 36.3 ; n = 11, 1 = 0.84). The brown crusts discontinuously cover the gypsum throughout the cave and are composed principally of carbon, oxygen, and silicon. Average droplet pH is 1.7 (n = 40), ranging from pH 1.25 on brown crusts to 2.92 on gypsum. Droplets on crusts had pH values below the critical HSO4-:SO42- pK as a result of crust hydrophobicity and acid-producing bacteria; droplets with a pH < 2 were undersaturated with respect to gypsum, while droplets with pH > 2 were in equilibrium with gypsum, as a result of buffering by the bisulfate-sulfate weak acid/base pair (pK = 1.92) combined with the gypsum-sulfate. Microbial cells and filaments, although evident from nucleic acid staining, are metabolically inactive based on application of 16S rRNA-specific oligonucleotide probes. Low pH droplets were supersaturated with respect to quartz (SIquartz = +0.55, n = 4), and euhedral quartz microcrystals were found in the crusts. The cave-wall environment enhances silica mobilization from acid dissolution of the limestone and liberation 208 of insoluble residues (clays and feldspars). Quartz precipitation may be accelerated by bacterial cells and organic material acting as nucleation points for crystal growth, especially for the very small crystals. Armoring of the cave walls by gypsum and organic biofilms fundamentally affects how the cave enlarges during sulfuric acid speleogenesis. Microbial colonization of the low pH, moist gypsum habitat forms an organic film that eventually becomes impermeable through time. Condensation droplets become separated from the underlying gypsum, thereby precluding diffusion of sulfuric acid through the gypsum to the limestone, limiting or shutting off sulfuric acid dissolution completely. Subaerial sulfuric acid speleogenesis, as the replacement of the limestone by gypsum, will commence only when fresh limestone is exposed. INTRODUCTION Tiny authigenic quartz crystals have been found in gypsum deposits from ancient caves formed by sulfuric acid speleogenesis, including several caves in Italy (Forti, 1994), Turkmenistan (Maltsev et al., 1997), and Carlsbad Cavern, Lechuguilla Cave, and others in the Guadalupe Mountains, New Mexico (Polyak and Provencio, 2001; Polyak and G ven, 2004). In Carlsbad Cavern, micrometerto deci-micrometer quartz crystals are found in unconsolidated powders in association with gypsum and carbonate mineralization (Polyak and Provencio, 2001; Polyak and G ven, 2004). These cave systems did not form under hydrothermal processes, and therefore quartz precipitation in the caves occurred at low, earth-surface temperatures. Moreover, as most of these cave systems are no 209 longer active, there has been little explanation for how the quartz crystals originated. Quartz precipitation at low temperatures is a perplexing chemical and kinetic occurrence (e.g., Chafetz and Zhang, 1998). Although quartz is one of the most abundant minerals on the Earth, it is kinetically unreactive at low temperatures and it is unlikely to precipitate (or dissolve) at any significant rate without a catalyst (Rimstidt and Barnes, 1980; Rimstidt, 1997). Quartz precipitation from solution is hindered by the presence of humic acids, Al, Fe, and Ca, and removal of these impurities is necessary for precipitation (Thiry and Millot, 1987). There is evidence that microorganisms, acting as potential nucleation sites, catalyze silica precipitation at low pH, by cell wall sorption of H4SiO4 ions (Fortin and Beveridge, 1997). In Lower Kane Cave, Wyoming, a small system actively forming from sulfuric acid speleogenesis (Egemeier, 1981), quartz microcrystals are found in brown organic-rich crusts associated with gypsum cave-wall mineralization and acidic condensation droplets. Sulfuric acid speleogenesis in Lower Kane Cave is principally by subaqueous hydrogen sulfide oxidation to sulfuric acid by microbial catalysis. A small percentage of hydrogen sulfide does volatilize into the cave atmosphere (refer to Chapters 5 and 6), and it is subsequently oxidized on moist cave-wall surfaces. The sulfuric acid on exposed cave-wall surfaces creates an aggressive solution that rapidly corrodes the carbonate host rock and replaces it with gypsum, the replacement-solution process of Egemeier (1981): CaCO3 + H2SO4 + H2O CaSO4 2H2O + CO2 210 (7-1) To date, sulfuric acid speleogenesis has been linked to the development of <10% of the carbonate karst worldwide (Palmer, 1991), including the formation of some of the world's largest caves such as Carlsbad Cavern, New Mexico (Hill, 1996), and the Frasassi Caves, Italy (Galdenzi and Menichetti, 1995). In some cave systems that are actively forming from sulfuric acid speleogenesis, microbial communities colonize subaqueous and subaerial habitats, including acidic cave-wall surfaces (Hose et al., 2000; Vlasceanu et al., 2000; Engel et al., 2001). Microbial life at pH <4 in natural and artificial environments has been characterized, as studies of life in extreme environments has surged recently (e.g., Johnson, 1998; Bond et al., 2000). Warm (22.6 oC) incoming sulfidic water and cooler (~21 oC) cave walls enhance condensation on subaerial cave-wall surfaces in Lower Kane Cave. I hypothesized that the chemistry of condensation on cave walls influences limestone dissolution, and gypsum and authigenic quartz precipitation reactions. Subaerial condensation is significant not just for cave formation mechanisms and speleothem alteration or precipitation (Dublyansky and Dublyansky, 1998), but also for microbial communities that may colonize the cave-walls. Condensation has been noted in many different caves (e.g., Eraso, 1969; Dublyansky and Dublyansky, 1998), and several studies of caves forming by carbonic acid dissolution describe aggressive condensate chemistries (Gergedeva, 1970; Pasquini, 1973; Dublyansky and Dublyansky, 1998), demonstrating that condensed waters are responsible for carving cave features and even wide-scale speleogenesis. Understanding cave-wall condensate properties is important because the nature of condensate and the 211 surfaces upon which they form affect the dissolving or precipitating ability of the solution, as well as its ability to entrain gas(es). The role of condensation in sulfuric acid speleogenesis and cave-wall modification has not been previously investigated. Moreover, there has not been demonstrable evidence of quartz precipitation from a naturally acidic, microbiallyactive environment. Therefore, the goals of this study were to characterize the physical and chemical nature of the subaerial cave-wall surfaces from Lower Kane Cave in order to speculate about the formation of quartz in other caves formed from sulfuric acid speleogenesis. The quartz may also serve as a biomarker for an environment in these ancient caves where microorganisms once lived. MATERIALS AND METHODS Study Site Several caves in the Bighorn Basin, Wyoming, formed from sulfuric acid speleogenesis, including Lower Kane Cave (Egemeier, 1981). Lower Kane Cave air temperatures average ~21 oC with a relative humidity of 99-100%. The spring waters average 22.6 oC, and have a pH of ~7.3. Incoming total dissolved sulfide is ~34 mol L-1, and volatilization of H2S(g) is <8% of the total dissolved sulfide flux (refer to Chapter 5). Crust and Gypsum Characterization Moist and dry brown crusts and gypsum deposits were collected aseptically throughout Lower Kane Cave. Crusts were digested in warm HCl and HNO3 for 4 hr and the acid solution was analyzed by inductively coupled plasma mass 212 spectroscopy (ICP-MS) for major cations and high pressure liquid chromatography (HPLC) for anions. Crusts were fixed using a chemical critical-point drying method modified from Nation (1983); samples were washed overnight with 25% gluteraldehyde, and then dehydrated with a series of ethanol washes. Air-dried, preserved samples were mounted on aluminum stubs, sputter-coated with gold, and examined using a JEOL JSM-T330A scanning electron microscope (SEM) in conventional-mode (CSEM) at an accelerating voltage of 30 kV with electron dispersive analysis (EDS) system. A Philips XL30 SEM operated in environmental mode (ESEM) with EDS was used for visualizing hydrated crusts and EDS elemental mapping without gold-coating. The brown crusts were HCl-acidified to ensure removal of carbonate mineral phases for carbon isotope ratio analysis. Most carbon isotope measurements were made by elemental analyzer interfaced with a continuous flow FinniganMAT Delta Plus mass spectrometer, but some measurements were also made by sealed tube combustion, vacuum purification, and dual-inlet VG Prism II mass spectrometer (refer to Chapter 2). Carbon isotope values are expressed in delta () notation with respect to the international standard V-PDB. Brown crusts were prepared with the stain 4',6-diamidino-2-phenylindole (DAPI) to visualize nucleic acids within the crusts. Some crust samples were also preserved in two ways with respect to the general requirements for successful fluorescence in situ hybridization (FISH) of various gram-negative and grampositive bacteria: (i) with 4% (wt/vol) paraformaldehyde for 3 hr before final wash with saline phosphate buffer as described by Manz et al. (1992), and (ii) with one213 time 96% ice-cold ethanol according to Roller et al. (1994). Preserved crusts were attached to a thin agarose layer on Teflon-coated nonfluorescence slides and airdried before dehydrating by sequential ethanol washes (refer to Chapter 3 for detailed FISH methodology). Hybridizations with multiple 16S rRNA-specific oligonucleotide probes to target specific taxa were preformed as described by Manz et al. (1992) (Table 7-1); following hybridization, DAPI was applied to the crusts for cross-referencing cells. DAPI and FISH results were visualized on an Olympus BX41 phase-contrast microscope with a reflected fluorescence system, and a 100x oil-immersion objective. Droplet Collection and Characterization Condensation droplets were drawn into a sterile, plastic, and non-reactive syringe, whereby ~1 ml of droplet solution over a 5-10 cm2 area of the same substrate was collected and homogenized. Droplets from brown crusts and gypsum were collected separately. Condensate solutions were filtered to remove gypsum crystals. Solution chemistry was determined by HPLC and ICP-MS, and pH was measured from an aliquot with a IQ150 pH/mV/temperature meter with a stainless steel micro pH electrode (IQ Scientific Instruments, Inc., San Diego, CA) calibrated to pH 4, 2, and 1.8. Dissolved solute speciation and activity and saturation state with respect to mineral phases were calculated from in situ temperature, dissolved solute concentration, pH, and droplet ionic strength using the geochemical speciation model PHREEQC (Parkhurst and Appelo, 1999). pH of individual droplets in the cave was measured using the IQ150 pH microelectrode. 214 Surface free energy affects the saturation state of a solution with respect to mineral stability. Condensation droplet morphology was measured in the cave and interfacial tension was determined from digital images by measuring the contact angles droplets on brown crust (n = 21) and on gypsum (n = 3; droplets on gypsum were rare). In order to reduce complications between advancing and receding edge contact angles, because droplets did not form on completely flat surfaces and the inclination of the solid can deform the droplet (Extrand and Kumagai, 1995), only droplets that appeared to be free-hanging on nearly horizontal cave walls (ceilings) were measured. Interfacial tension was determined from a modified Young's relationship, LV cos = SV - SL (7-2), such that the surface forces are of a liquid (L) drop on a surface (S) in contact with air (V), and a definite contact angle, , between the liquid and solid phase. If the contact angle can be estimated, then an equation-of-state approach that relates S and LV can be derived. Kwok et al. (1998) suggest that, for a contact angle- based surface analysis, the measure of contact angles of a single liquid drop would suffice for determination of the surface tension of the underlying solid, cos = -1 + 2 (S / LV) (7-3) Once the contact angle and S are known, SL can be calculated (Kwok et al., 2000). Using Equation 7-3 assumes a smooth, homogeneous, and incompressible solid, all aspects of natural systems that are difficult to quantify (e.g., solids are not completely smooth surfaces; adsorbed gas can reduce the solid-liquid interfacial 215 tension and smooth effective roughness at the solid surface). These calculations were used to estimate surface hydrophobicity, which would also serve as a measure for the potential reactions that could be occurring between the droplet and the underlying mineral or crust surface(s). Quartz Separation and Cathodoluminescence Microscopy Gypsum deposits and brown crusts were dissolved in dilute HCl to collect the insoluble residue phase. Gypsum was further dissolved by gentle shaking in distilled water with NaCl to complex free Ca2+. Bedrock from several units within the Madison Limestone was digested with HCl and EDTA separately. Insoluble residues from the various digestions were centrifuged and the supernatants were removed by pipet. Residues from gypsum and the brown crusts were visually inspected by stereoscopic microscopy, and individual euhedral crystals were separated from quartz clusters. Residues from the limestone were analyzed by x-ray diffraction and quartz grains were separated. Quartz crystals from the brown crusts, ~30 m long (the lower limit for manual manipulation using a stereo-microscope), were mounted individually in epoxy and polished flat to obtain cross-sections of the crystals at random orientations. Clusters were also mounted in epoxy in several orientations and polished. Crystal sections were imaged by cathodoluminescence (CL) microscopy using the ESEM with a Philips PanaCL detector and RGB (red/green/blue) filters. Digital images were processed in Adobe Photoshop for RGB color merging, and brightness and contrast alterations. 216 RESULTS Cave-wall Crusts and Condensate Morphology The walls of the cave are nearly all replaced by gypsum (Figure 7-1A), formed as a result of active sulfuric acid speleogenesis; limestone is rarely exposed. Discontinuous patches of reddish-brown crusts cover the gypsum (Figure 7-1B). Acid digestion of the brown crusts revealed they were composed of 56% carbon by dry weight. ESEM with elemental mapping of crusts supported this, demonstrating dominance by carbon (Figure 7-2A), with rare sulfur and isolated areas of silicon (Figure 7-2B). Calcium was not detected in the brown crusts using ESEM, although there was 14% calcium detected by dry weight. Condensation droplets, averaging 2-3 mm in diameter, were observed on brown crusts, gypsum, and rarely on freshly exposed limestone. The highest abundance of droplets was on brown crusts in areas of the cave directly over flowing stream water (Figure 7-3). Where there was no flowing stream water, condensation was not observed on the walls, and the brown crusts appeared dehydrated (i.e., cracked and pulling away from the gypsum underneath). Contact angle estimates for condensation droplets on brown crusts were >90o, with an average angle of 121.6o (n = 21; Table 7-2), and 20% of these droplets were noticeably cloudy. In contrast, the average contact angle of droplets on gypsum was 71o (n = 3) (Table 7-2). The surface tension (S) values for the brown crust were estimated using Equation 7-3, assuming that the LV was pure water at 72.8 mJ m-2 (although the ionic strength and the high concentration of sulfate-ions, speciated as sulfate and 217 bisulfate, would slightly decrease interfacial tension of the liquid (Zhou et al., 1998). For the crusts with contact angles >90o, the mean interfacial tension was 17.3 mJ m-2, while the average surface tension for droplets with contact angles <90o was higher at 47.5 mJ m-2 (Table 7-2). The corresponding interfacial tension between the solid and water was 55.3 mJ m-2 for droplets on brown crust, while droplets on gypsum had less SL values, averaging 25.1 mJ m-2 (Table 7-2). The small contact angles and tension estimates indicate that the brown crusts were more hydrophobic surfaces than the gypsum. Condensate Geochemistry Condensation droplets on brown crusts had a mean pH of 1.7 (n = 40), whereas gypsum droplets had pH values ~2-3. Higher pH droplets (~pH 4, n = 20) were measured from exposed limestone surfaces near the cave entrance. Condensation droplets were collected from fifteen locations for geochemical analysis. Table 7-3 summarizes overall droplet geochemistry. Droplets with the lowest pH had the highest [SO42-] and ionic strengths. Sulfate (as CTSO42-) was speciated using dissociation constants for sulfatebisulfate-sulfuric acid (Stumm and Morgan, 1996). There was up to 85 mM HSO4for the lowest pH droplet (Figure 7-4). The concentrations of metals increased with decreasing condensate pH, including Si (Figure 7-5A). Total Al3+ (measured as CTAl) was complexed as AlSO4+ and Al(SO4)2-, at 0.047 and 0.011 mM, respectively (Figure 7-5B). Equilibrium calculations of condensate solutions showed that most droplets at pH >2 were in equilibrium with gypsum, while the droplets at pH <2 were undersaturated with respect to gypsum (Tables 7-3 and 7-4; 218 Figure 7-4). The droplets from gypsum that were not in equilibrium with gypsum have varying concentrations of Ca2+ in solution, and especially low concentrations. (Figure 7-4). Droplets on crusts and gypsum were undersaturated with respect to calcite (Table 7-4), but most of the droplets on brown crusts were supersaturated with respect to quartz (Table 7-4; Figure 7-5B). Quartz Quartz crystals observed from the brown crusts ranged in size from <1 m to 10 m along the longest axis (Figure 7-6; Appendix F). Some crystals had organic debris covering them (Figure 7-6C), or were imbedded within a fibrous matrix (Figure 7-6E). The surfaces of the quartz crystals showed no evidence of abrasion or pitting common to detrital quartz. Crystal size ranged from <0.5 m to >19 m, and the median crystal length of crystals observed by ESEM was 1.28 m (n = 495). All sizes fell within a statistically significant log-normal distribution (with four degrees of freedom) (Figure 7-7). Quartz crystals were also found within gypsum, in large complex clusters with odd morphologies; individual crystals could not be discerned (similar to Figure 7-6D). No euhedral quartz crystals were found in limestone, although chert fragments were identified and x-ray results showed a `quartz' peak that most likely coincided with chert interference. Fifteen quartz crystals or clusters ~30 m or larger were examined using CL. It was not possible to examine thin sections of the averaged-size quartz crystals (1.28 m), and the larger crystal size distribution was not measured using ESEM. Therefore, the significance of this size range is not known. The smallest crystal 219 analyzed by CL (~10 m in length) had only dark brown to black luminescence color, indicating an authigenic phase (e.g., Pagel et al., 2000; Ramseyer and Mullis, 2000). In contrast, larger crystals showed authigenic overgrowths on a quartz core with higher luminescence color (Figure 7-8). High luminescence color is characteristic of detrital quartz grains (light blue) (Ramseyer and Mullis, 2000). Depending on the sectioning of an individual crystal, the overgrowth rim width varied, but an average thickness was <5 m. In Figure 7-8, the detrital grain is fractured and two stages of authigenic overgrowth precipitation events were observed, one brown layer and another purplish-black layer. Brown Crust Microbiology Eleven brown crust samples were analyzed for the carbon isotope composition in order to determine if there was a signature of biological activity. The brown crusts had carbon isotope values of -36.3 (n = 11; 1 = 0.84). The limestone had a carbon isotope composition of +0.95. The carbon isotope values for the crusts reflect significant discrimination against chemolithoautotrophic fractionation (Preu et al., 1989). DAPI inspection of the crusts indicated a variety of microbial cell morphologies throughout the brown crusts had, including thin filaments ~1-2 m wide and larger fungal hyphae with filament widths averaging 10 m, and small rods and cocci (Figure 7-9). However, FISH with 16S rRNA-specific oligonucleotide probes was not successful. After multiple attempts, no fluorescence signals were observed with any of the probes hybridized to the brown crusts. 220 13 C, typically exhibited by DISCUSSION Sulfuric Acid Speleogenesis and the Role of Cave-wall Surfaces Lower Kane Cave formation mechanisms, including mineral alteration or precipitation reactions, are affected by the properties of the solids and liquids on cave-wall surfaces and microorganisms using the cave-wall surfaces as habitat. The cave walls are coated with gypsum, formed from the dissolution of the host limestone by sulfuric acid produced via sulfide oxidation (either biotic or abiotic): H2S + 2O2 HSO4- + H+ (the dominant species at pH ~2) 2CaSO4 2H2O + CO2 (7-4) (7-5) HSO4- + H+ + CaCO3 + H2O If the condensate solution is in contact with the underlying gypsum, the solution will approach pH 2, but typically will not go below this pH due to the buffering by the sulfate-bisulfate weak acid/base pair: HSO4H+ + SO42(pKa = 1.98) (7-6) and also from the gypsum-sulfate, H+ + HSO4- + CaSO4 2H2O 2HSO4- + Ca2+ + 2H2O (7-7) In contrast, droplets on brown crusts have pH values below the critical bisulfatesulfate pKa, possibly as a result of sulfuric acid-producing bacteria, as well as crust hydrophobicity that physically separates the condensate from the gypsum and allows the pH to pass the bisulfate buffer point (Figure. 7-4). The S values calculated for the brown crusts are as high as hydrophobic hydrocarbon or plastic surfaces (Stumm and Morgan, 1996). Changes in surface tension resulting from the effects of solution pH and ionic strength have been previously investigated (Zhou et al., 1998). According to 221 Butkus and Grasso (1998), solution pH has two distinct effects on the interfacial energy between surfaces. pH indirectly affects hydration forces by changing surface charge, which also affects the magnitude of electrolyte adsorption. Additionally, solution pH changes surface functional group characteristics (Holmes-Farley et al., 1985). When the pH of water droplets decreased (as in the example provided by Holmes-Farley et al., 1985, from droplets on polyethylene carboxylic acid surfaces), there was an increase in the hydrophobicity of the surface and water contact angles increased due to the protonation of surface groups. This is further supported by the work of H rd and Johanson (1977) which demonstrated that the conjugate base of strong acids, and not the proton, may be responsible for decreases in surface tension, and that an increase in surface tension caused by the conjugate acid of a strong base is more pronounced than the decrease in surface tension caused by the conjugate base of a strong acid. In Lower Kane Cave, the droplets on brown crust had a lower pH and higher contact angles than those on gypsum, resulting in the crusts themselves being were more hydrophobic than the gypsum. Ionic strength will also influence contact angle, and consequently surface tension. The experiments by Butkus and Grasso (1998) demonstrate that an increase in ionic strength results in an increase in contact angle, approximately 2-5o for a 10-fold increase in ionic strength. The highest ionic strength of the droplets with the lowest pH also had the lower surface tension value (Tables 7-2 and 7-3). Speleogenetic Quartz Formation At pH ~2 or less, total silica (CTSi) was dominated by H4SiO4. As pH decreased, CTSi increased, as did the SI with respect to quartz (Tables 7-3 and 7-4), 222 indicating that the observation of quartz crystals in the brown crusts is matched by the saturation state of the solutions on the surfaces. The log-normal distribution in quartz crystal sizes suggests that the crystals have not grown uniformly on the cave walls. These differences may be due to variable adsorption of silica from the bulk condensate solution, depending on the surrounding physical surface chemistry of the gypsum or organic crusts, as well as the presence of microorganisms. Parks (1990) describes that if a solution is supersaturated with respect to quartz, quartz is expected to precipitate, but unless particles of quartz are already present, precipitation begins with nucleation of very small particles (<5 nm) (Iler, 1979). At this size, quartz is very soluble (Parks, 1990) and amorphous silica will precipitate, not quartz. If the rate of dissolution of amorphous silica is larger than the rates of nucleation and growth of quartz, then amorphous silica will precipitate. Conversely, precipitation in the presence of seed quartz crystals larger than 5 nm should allow quartz to grow directly. Quartz overgrowths on large detrital quartz grains suggest that the latter is true (Figure 7-8). As the authigenic, euhedral crystals are also quartz, this suggests that nucleation of small particles may have also occurred (Figure 7-6). Sources of Silica Several hypothetical sources of dissolved silica are evaluated to examine what processes may have contributed to high silica saturation states in the condensate solutions. Sources of silica from meteoric water, dissolving silicates, and weathering of insoluble clays are discussed. 223 Influx of quartz-saturated meteoric water Surface waters are often supersaturated with respect to quartz because the reaction kinetics of quartz precipitation are slow compared to the dissolution rate of, for example, feldspars at temperatures <80 C (Rimstidt and Barnes, 1980; Rimstidt, 1997). However, Egemeier (1981) proposes that there is little infiltration of meteoric water into Lower Kane Cave because of the depth of the cave in Little Sheep Mountain and the low precipitation rates in the northern Bighorn Basin (<20 cm yr-1). Additionally, abundant cave gypsum would most likely not be preserved if meteoric water, typically undersaturated with respect to gypsum, percolated into the cave. Meteoric influx has caused gypsum to dissolve in the caves of the Guadalupe Mountains (Polyak and Provencio, 2001), as well as the Frasassi system, Italy (Galdenzi and Menichetti, 1995). If quartz-saturated meteoric water did flow along fractures into the cave, quartz would most likely be precipitating at the contact between limestone and gypsum, which was not observed. Therefore, the source of silica in the condensation droplets on the cave walls most primarily results from condensation of water vapor, and not a source outside into the cave. Silicates dissolution Dissolution of feldspars by hydrolysis (e.g., KAlSi3O8), 2KAlSi3O8 + 2H+ + 9H2O Al2Si2O5(OH)4 + 4H4SiO4 + 2K+ (7-8) or precipitation of secondary clay minerals such as kaolinite or illite on the cavewall surfaces, could be occurring. KAlSi3O8 + Al2Si2O5(OH)4 KAl3Si3O10(OH)2 + 2SiO2 + H2O (7-9) There is limited evidence of primary feldspar within the limestone or of significant 224 diagenetic clay precipitation within the gypsum deposits, as has been observed in Carlsbad Cavern (Polyak and Provencio, 2001). Clay minerals are most likely not forming because concentrations of accessory cations in condensate solutions are not high enough to reach mineral saturation, and SI values for various clays are extremely low. Therefore, the high silica concentrations in the cave condensate do not result from feldspar dissolution and precipitation of clays. Weathering of insoluble (residual) clays Dissolution of insoluble residues from the limestone during sulfuric acid speleogenesis could be occurring. Previous descriptions of the Madison Limestone suggest that the most dominant insoluble residues in the limestone units are clays, such as kaolinite and illite (Plummer et al., 1990). Clay solubility increases with decreasing pH, and as a result, silica could be released into solution (Nagy, 1995): Al2Si2O5(OH)4 + 6H+ 2Al3+ + 2H2SiO4 + H2O (7-10) Aggressive condensate containing sulfuric acid would enhance clay mineral weathering. Organic acids and ligands may also increase clay dissolution by forming complexes with Al on the clay surface, weakening the Al-O-Si bonds (Nagy, 1995). Because rates of silica dissolution vary with changes in exposed surface area of a silicate mineral, and with changes in the volume of water reacting with the dissolving clays, the high surface area of dissolving clays would provide more silica to solution with constant reacting water volumes. For the Lower Kane Cave condensate solutions, liberation of Al during clay dissolution could inhibit the activity of silica (Iler, 1979). At pH values <4, Al exists as Al3+, and in the lowest pH condensate solution, free Al3+ is almost completely complexed with sulfate 225 (Figure 7-5B). Therefore, of the possible silica sources, clay weathering during sulfuric acid dissolution of the host carbonates is the most likely. Significance of Microorganisms to Cave Formation and Quartz Precipitation Authigenic quartz formation at earth-surface temperatures is extremely slow, and not typically considered an important process. Kinetic barriers still must be overcome to precipitate quartz in the cave. Because metals (e.g., Ca and Al) that would otherwise poison quartz precipitation are complexed with sulfate, it is hypothesized that organic matter, and specifically microorganisms that once were metabolically active on the cave walls and formed organic biofilms, may have acted as catalysts for quartz precipitation. Any microorganisms living on the cave-walls are most likely acidophilic bacteria. DAPI staining suggests that microbes are present in the wall crusts (Figure 7-9), but FISH might suggest that they are not metabolically active. As the FISH probes target rRNA, perhaps the lack of positive hybridization indicates the metabolic status (or lack thereof) of the organisms in the low-pH environment when the samples were collected and preserved (Amann et al., 1990; Amann et al., 1995). Previous investigations of 16S rRNA gene sequences retrieved from cavewall biofilms in other active sulfidic caves demonstrated the occurrence of known sulfur-oxidizing bacteria, including Thiobacillus and Sulfobacillus (Vlasceanu et al., 2000), and Thiobacillus spp. and Acidomicrobium (Hose et al., 2000). However, the metabolic viability of these organisms was not established. Acidic biofilms also have been reported from mines, and cultures of Leptospirillum, Acidomicrobium, 226 Ferromicrobium acidophilus, and the Archaean Thermoplasmales have been obtained (Bond et al., 2000). The occurrence of quartz within the organic crusts may not be coincidental. Acidophilic sulfur-oxidizing bacteria generate sulfuric acid, which is important for maintaining an acidic habitat, but cells may also function as nucleation sites for quartz precipitation. Fortin and Beveridge (1997) suggest that sorption of silicate ions (speciated as H4SiO4) onto acidophiles (e.g., Thiobacillus spp.) serves as a nucleation surface, possibly by templating, for the precipitation of amorphous silica at pH ~2 - 3. Metal-bridging of multivalent cations on bacterial cells also has been shown to promote aluminosilicate precipitation by enhancing silicate anion binding (Urrutia and Beveridge, 1993). Currently, however, there is no known relationship between microbes and quartz precipitation at any temperature range, Microbes that colonization the subaerial cave-wall surfaces play a role in speleogenesis by forming a thick organic biofilm on the gypsum. These biofilms have high surface tensions relative to the gypsum, which eventually creates an impermeable surface layer on the cave walls. This hydrophobic surface precludes diffusion of sulfuric acid through to the gypsum and, consequently, to the underlying limestone. CONCLUSIONS Low-temperature euhedral quartz crystals found in caves formed from sulfuric acid speleogenesis indicate potentially microbially active acidic environments during periods of cave formation. While it is possible that the speleogenetic quartz precipitated in Lower Kane Cave prior to the cave formation, 227 it is more likely, based on condensate geochemistry and the association of the quartz with gypsum and brown crust, that the quartz formed since cave inception. The relationship between euhedral quartz and the cave-wall surfaces colonized by microorganisms (active or not) suggests that the rate of quartz precipitation might be enhanced by biologically active surfaces or microbially-produced ligands. This finding would be a fundamental discovery for both geomicrobiology and geochemistry. Identification of quartz, as a geologically stable signature of acidsulfur bacterial metabolism, could then be used to identify such integrated bio- and geo- processes in ancient cave systems, or other more diverse terrestrial or extraterrestrial systems, modern or ancient. 228 Table 7-1: Oligonucleotide probes used to screen cave-wall biofilms using fluorescence in situ hybridization. Target group Probe sequence (5' 3') GCT GCC TCC CGT AGG AGT GCA GCC ACC CGT AGG TGT GCT GCC ACC CGT AGG TGT ACT CCT ACG GGA GGC AGC TCG CGC CTG CTG CIC CCC GT GGT AAG GTT CTG CGC GTT GCC TTC CCA CTT CGT TT GCC TTC CCA CAT CGT TT TAT AGT TAC CAC CGC CGT TGG AAG ATT CCC TAC TGC 16S (319) 35 Target Sitea 16S (338) 16S (338) 16S (338) 16S (338) 16S (344) 16S (968) 23S (1027) 23S (1027) 23S (1901) 16S (354) FAb 0-40 0-40 0-40 0 0 20 35 35 25 20 Reference Daims et al., 1999 Daims et al., 1999 Daims et al., 1999 Wallner et al., 1993 Raskin et al., 1994 Neef et al., 1998 Manz et al., 1992 Manz et al., 1992 Roller et al., 1994 Meier et al., 1999 Manz et al., 1996 Probe EUB338 EUB338-II EUB338-III NonEUB ARCH344 ALF968 BET42a GAM42a HGC69a LGC345mix 229 Eubacteria Planctomycetes Verrucomicrobia (and others) Negative control Archaea Alphaproteobacteria Betaproteobacteria Gammaproteobacteria Actinobacteria Firmicutes (together with two other probes) CF319a Some members of TGG TCC GTG TCT CAG TAC "Flavobacteria" a E. coli rRNA position (Brosius et al., 1981). b Formamide percentage (vol/vol) in the FISH hybridization buffer Table 7-2: Contact angles of condensation droplets on brown crust and gypsum measured from digital images, with corresponding surface tension (S) and interfacial tension (SL) calculations between the solid and liquid droplets. Contact Anglea Brown Crust 101.5 102.5 105 115 115 115.5 116.5 117 120 120 121.5 123 124 127.5 130 130 131 131.5 132.5 133.5 141 Gypsum 62 74 80 S (mJ m-2) 29.06 28.44 26.9 20.96 20.96 20.67 20.1 19.82 18.15 18.15 17.33 16.53 16 14.2 12.97 12.97 12.48 12.24 11.78 11.31 8.09 53.34 46.3 42.72 SL (mJ m-2) 43.53 44.15 45.69 51.64 51.64 51.92 52.58 52.78 54.55 54.45 55.37 56.07 56.6 58.4 59.64 59.64 60.11 60.48 60.83 61.28 64.51 19.16 26.29 30.11 To avoid complications with receding versus advancing contact angles, contact angles were measured from droplets with equal (within 0.5o) angles on both sides. a 230 Table 7-3: Major geochemical constituents for condensation droplets. Cave air temperature was 21oC. Analyses are reported in mg L-1. 231 pH Substratea SO42- Na K Ca Mg Fe Mn Sr Zn 1.17 BC 3679 22.4 3.9 338 81.2 0.40 0.46 1.17 0.14 1.24 BC 1297 7.6 8.2 400 259 1.06 0.38 1.46 0.11 1.24 BC 9491 2.4 0.72 309.1 71.8 1.12 0.14 0.57 0.13 1.45 BC 6221 25.3 1.13 427.3 110.3 0.38 0.50 1.40 0.04 1.5 BC 4339 2.7 1.18 319.0 65.1 2.56 0.11 1.62 0.06 2.01 G 2281 2.1 0.24 572.1 37.0 0.67 0.04 0.78 0.07 2.15 G 1955 21.2 2.3 545 141 0.15 0.05 1.06 0.03 2.35 G 3297 4.0 0.0 517 19.7 0.10 0.08 1.05 0.05 2.45 G 1835 1.8 0.22 536.5 20.2 0.60 0.03 0.24 --c 2.55 G 1760 6.1 0.33 63.1 24.0 0.01 -0.74 -2.72 G 919 1.6 0.0 470 11.6 2.02 0.07 0.36 0.03 2.92 G 1493 2.1 0.10 578.3 5.1 0.08 0.01 0.18 0.01 a Substrate from which droplets were collected: BC, brown crust; G, gypsum. b Ionic strength. c not determined. Al Si ISb 0.47 14.5 0.06 1.20 15.2 0.05 1.85 5.77 0.14 1.67 15.84 0.10 5.54 17.62 0.08 1.71 6.66 0.05 0.45 6.8 0.05 0.18 8.3 0.04 2.83 5.00 0.05 0.10 4.26 0.03 0.19 1.6 0.03 0.10 2.13 0.04 Table 7-4: Calculated mineral saturation indices (SI)a for condensation droplets. Modeled using the web-based PHREEQC (ver. 2) computer software (Parkhurst et al., 1999). Droplet pH 1.17 1.2 1.24 1.45 1.55 2.01 2.15 2.35 2.45 2.55 2.72 2.92 Calcite SIb -4.33 -7.71 -2.79 -7.08 -7.05 -5.72 -4.15 -4.15 -4.97 --d -3.36 -4.41 Gypsum SIc -0.48 -0.27 -0.49 -0.11 -0.28 -0.04 -0.21 -0.28 -0.04 --0.41 -0.03 Quartz SIc 0.59 0.04 0.61 0.48 0.52 0.09 0.26 0.35 -0.03 --0.37 -0.4 a SI = (log IAP/Ksp), where IAP is ion activity product and Ksp is the equilibrium constant b Modeled as pre-batch reaction with a CO2 phase c Modeled as post-batch reaction d Not modeled due to charge balance problems. 232 A B Figure 7-1: (A) Gypsum coatings ("paste") and condensation droplets from the cave ceiling. Scale bar = 2 mm. (B) Clear and mucous-like droplets on brown cave-wall crust. White area to the upper left of the center droplet is gypsum. Scale bar = 2 mm. 233 A B Figure 7-2: ESEM photomicrographs of brown crust combined with elemental mapping. (A) Map of carbon (red). (B) Map of sulfur (yellow) and silicon (blue). Scale bar = 10 m. 234 235 Figure 7-3: Condensation droplets on brown crusts (hydrated and dehydrated) on gypsumcovered cave-wall surface in Lower Kane Cave, Wyoming. Droplets are roughly 2 mm wide. 0.5 90 Gypsum Saturation Index (log IAP/Ksp) 0.4 0.3 0.2 0.1 SI SIgypsum HSO4HSO4Ca2+ Ca 80 70 60 50 mmol L-1 HSO4- and Ca2+ 0 40 -0.1 -0.2 -0.3 -0.4 -0.5 1.0 1.5 2.0 2.5 3.0 30 20 10 0 pH Figure 7-4: Condensation droplet chemistry, with HSO4-, speciated from CTSO4, CTCa, and Saturation Index (SI) with respect gypsum versus pH of the droplets. [HSO4-] increases with decreasing pH. Droplets near pH 2 are at the the pKa of the weak acid-base pair HSO4-:SO42- (pKa = 1.98) and are in equilibrium with respect to gypsum. Deviations from equilibrium are due to the low concentration of CTCa for that droplet. 236 0.7 0.6 0.5 Si -1 A R 2 = 0.8885 0.4 0.3 0.2 0.1 0.0 1 1.5 2 pH 2.5 3 mmol L 0.25 B 0.2 mmol L -1 A l species 0.15 0.1 0.05 0 1 1.5 2 pH 2.5 CTAl AlSO4+ Al(SO4)2Al3+ 3 Figure 7-5: Si and Al concentrations in condensation droplets. (A) The [Si] from the condensation droplets strongly correlates to decreasing pH. (B) Al and Al-SO4 complexes modeled by PHREEQC; free Al (Al3+) has negligible concentrations in the droplets, as a result of Al complexation with sulfate. There is no correlation with Al-species and pH. 237 A B 10 m 6 m C D E F Figure 7-6: Scanning electron photomicrographs of authigenic quartz crystals from brown crusts on cave-wall surfaces. 238 140 120 100 number 80 60 40 20 0 0 1 2 3 4 5 6. 5 7. 5 8. 5 9. 5 Median = 1.28 microns n = 495 crystal size (c-axis) microns 10 .5 11 .5 12 .5 13 .5 14 .5 15 .5 16 .5 17 .5 18 .5 Figure 7-7: Quartz crystal lengths measured using ESEM. 239 Figure 7-8: Cathodoluminescence image of polished quartz crystal imaged by ESEM. The crystal is approximately 50 mm wide. The core of the crystal is a fractured detrital quartz grain, although quartz was rarely found in the limestone as an insoluble phase. Two stages of authigenic overgrowths occurred on this grain (dark purple and dark orange-red). The overgrowths are not evenly distributed around the grain, possibly due to polishing artifact or to authigenic growth in the brown crust. 240 A B C D Figure 7-9: Fluorescence photomicrographs of brown crust stained with DAPI. Bright blue areas are those that have nucleic acids and bright yellow areas (D) are those with elemental sulfur. Scale bar = 10 m. 241 Chapter 8: Conclusions The subsurface is a major habitat for diverse microbial communities, as reactive rock surfaces and mineral-rich groundwater provide a variety of energy sources for microorganisms. Caves, although typically <50 m below the earth's surface, offer a valuable opportunity to examine biologic-geologic interactions under subsurface conditions. In Lower Kane Cave, Wyoming, groundwater bearing dissolved hydrogen sulfide, but negligible allochthonous carbon, discharges as springs into the cave passage. Both subaqueous and subaerial microbial communities inhabit this cave system, and the microbial ecology and geochemistry of both of these habitats were investigated. The bulk of the subaqueous microbial mats has high sulfur content and expresses chemolithoautotrophic metabolism. 16S rDNA sequences retrieved from these mats indicate low overall taxonomic diversity, and the mats are dominated by several novel evolutionary lineages within the class "Epsilonproteobacteria". Lower Kane Cave represents the first non-marine natural system demonstrably driven by the metabolic activity of "Epsilonproteobacteria". The epsilonproteobacterial groups identified from Lower Kane Cave are not endemic to Lower Kane Cave, as molecular evidence suggests that these epsilonproteobacterial groups occupy a variety of terrestrial sulfidic habitats, both under photic and aphotic conditions. These results expand the geographic and ecological diversity of "Epsilonproteobacteria", suggesting that geographic isolation may not be a driving factor in speciation based on minimal genetic divergence among these groups. 242 Ecologically, the "Epsilonproteobacteria" are chemolithoautotrophic sulfur-oxidizing bacteria. As they diversify genetically and metabolically, they create habitats within the microbial mats for other bacterial groups. The biomass of the diverse anaerobic microbial groups, within the interior of the microbial mats, is substantially less than the total biomass of the microbial mat. Stream geochemistry and the spatial relationship of aerobic and anaerobic metabolic guilds allow for tight carbon and sulfur cycling constrained by redox boundaries within the mat environment. A significant component of autotrophically produced carbon is cycled through a detrital microbial loop, demonstrating the ecological importance of these anaerobes. The physical model of carbon and sulfur nutrient spiraling is based on the energy transfer between redox environments, and without aerobic and anaerobic metabolic complexity, nutrient spiraling would not occur. Specifically, chemoautotrophically-fixed carbon produced by the sulfur-oxidizers is degraded by the fermenting bacteria, as well as transported downstream. Fermenters produce a variety of breakdown products that are consumed by different sulfate-reducing bacterial groups, methanogens, and other microbial groups such as iron-reducers or denitrifiers. In turn, the sulfate-, sulfur- and fermenting bacteria generate autochthonous hydrogen sulfide and other volatile organosulfur gases that are consumed primarily by sulfur-oxidizers. Active VOSC cycling by microbial communities from an aphotic habitat has not been previously demonstrated. Sulfur gases produced in terrestrial subsurface habitats, such as sulfidic groundwater, can be important potential sources to the global sulfur cycle. 243 Almost all of the dissolved sulfide coming into the cave is consumed by subaqueous sulfur-oxidizing bacteria within the microbial mats, including the "Epsilonproteobacteria", but also Gammaproteobacteria (e.g., Thiothrix spp.) and Betaproteobacteria (e.g., Thiobacillus spp.). These bacteria drive subaqueous sulfuric acid speleogenesis by attachment to carbonate surfaces and generation of sulfuric acid, which focuses local carbonate undersaturation and dissolution. As the "Epsilonproteobacteria" can be metabolically active under low oxygen tensions, these organisms can potentially catalyze sulfide oxidation where autoxidation would be kinetically limited, such as in deep anoxic aquifers. Prior to this work, sulfuric acid speleogenesis was considered a subaerial process (e.g., Egemeier, 1981; Hill, 1990; Galdenzi et al., 1995; Hill, 2000) or a process limited to shallow groundwater environments because of oxygen requirements for abiotic autoxidation mechanisms (e.g., Palmer, 2000). However, chemolithoautotrophy and microbial sulfur oxidation under microaerophilic conditions extend the phreatic depths to which porosity and conduit enlargement can occur in carbonate systems. Groundwater in karst and non-karst areas can often have high concentrations of dissolved sulfide, especially in regions near hydrocarbon reservoirs (Ulrich et al., 1998; Nemati et al., 2001; Kodama et al., 2002; Elshahed et al., 2003), making the contribution of sulfur cycling and microbially enhanced carbonate dissolution more significant globally. As "Epsilonproteobacteria" are the major microbial groups so far identified in many sulfidic habitats associated with carbonate caves and springs, these organisms may not only be responsible for sulfuric acid speleogenesis in Lower Kane Cave, but may also be important for carbonate porosity development 244 globally. The metabolic and geologic consequences of an active microbial ecosystem in the subsurface fundamentally change the model for sulfuric acid speleogenesis. 245 APPENDIX A Scanning Electron Photomicrographs and 16S rRNA Gene Sequence Clone Libraries: Chapter 2 Supplement Appendix A Table of Contents............................................................................ 246 Scanning Electron Photomicrographs of Upper Spring white mat, 203 m ........ 247 Scanning Electron Photomicrographs of Upper Spring white mat, bottom, 203 m ........................................................................................... 248 Scanning Electron Photomicrographs of Upper Spring gray orifice Sediment, 189 m...................................................................................... 249 Scanning Electron Photomicrographs of Upper Spring orifice red mats ............ 250 Scanning Electron Photomicrographs of Upper Spring orifice orange film ....... 251 Scanning Electron Photomicrographs of 215 m red mats ................................... 252 Scanning Electron Photomicrographs of 72 m orange mats ............................... 253 Clone Library 19 (124f) ...................................................................................... 254 Clone Library 19B (124f).................................................................................... 255 Clone Library 22 (127f) ...................................................................................... 256 Clone Library 57 and 57C (190f) ........................................................................ 258 Clone Library 57B (190f).................................................................................... 259 Clone Library 270 (195f) .................................................................................... 260 Clone Library 270B (195f).................................................................................. 261 Clone Library 190 (198f) .................................................................................... 262 Clone Library 127 (203f) .................................................................................... 263 Clone Library 127B (203f).................................................................................. 264 Clone Library 159 (203f) .................................................................................... 265 Clone Library 156 (203w)................................................................................... 266 Clone Library 102 (203y).................................................................................... 267 Clone Library 102B (203y) ................................................................................. 268 Clone Library 125 (203g).................................................................................... 269 Clone Library 199 (248 f) ................................................................................... 270 Clone Library 198 (248y).................................................................................... 271 Clone Library 198B (248y) ................................................................................. 272 Table A-1 Clone sequences and closest relatives................................................ 273 246 File SEM5201usm_b.doc Date May 2, 2001 Sample upper spring white mat bottom jelly mat Sample # LKB-O1M OO3b (jelly mat) Description brown-golden to darkbrw mat under white mats at Site 3 Duration in place Date emplaced Date retrieved March 2001 Method of preservation gluteraldehyde, frozen in N2, Cracked, freeze-dried overnight Coating material gold Time of coating 2x 15 sec Accelerating voltage 30 kv Are bugs present? No. types present Radius of bugs Etching? EDAX File names Singles? cocci Length Secondary minerals? Groups? rods branchy Comments: Pictures from two sample stubs White mat bottom jelly brown mat slimy with some sediment (evidence of fine-grained stuff (50-100 um) stuff inside of mat). Lots of filaments on the mineral substrates and it looks like the filaments are ordered structurally on the substrates in a lattice structure. Really strange thick filaments are oriented in one direction, with smaller ones in between them (1 um wide)- may be for nutrient acquisition ("closest- packing" arrangement) Need to look for more of this Saw spiky mineral (calcite?)- within the mats maybe dissolving maybe precipitating.... Other strange mineral forms maybe sulfur globules REALLY AWESOME spiral-guys... but very very very small (100-200 um wide) but long... free-floating in filament matrix but also adhered to substrates (last pic) No. photomicrographs 20 File names M2_upb2_*.tif 247 File SEM5201usm.doc Date 5-2-01 Sample Upper Spring White Mat top layer Sample # LKB-01M-003a Description white filamentous mat from U Spg Duration in place Date emplaced Date retrieved March 2001 Method of preservation gluteraldehyde, N2frozen, fractured w/ exacto, froze whole bottle in N2, Then freeze-dried overnight (~ 15 hrs) Coating material gold Time of coating 2x 15 sec Accelerating voltage 30 KV Are bugs present? No. types present Radius of bugs Etching? EDAX File names Singles? cocci Length Secondary minerals? Groups? rods branchy Comments: White mat preserved in gluteraldehyde in field collected from 203 m (same spot as always Site 3) Mat is very slimy and difficult to pull apart had to cut sections to look at in freeze-dry and look at Lots of filaments very dense mat size of filaments is generally the same (between 3 and 1 m). Larger filaments look a little rough on the outside, but smaller (around 1 um wide) are very smooth See some minerals like the spiky calcite and other rhombs within filament matrix Sulfur globules 248 File SEM21301framdoc Date 2-13-01 Sample Upper Spring Gray sediment top layer Sample # LKC-01M-004 Duration in place Date emplaced Date retrieved March 2001 Method of preservation gluteraldehyde, N2frozen, fractured w/ exacto, froze whole bottle in N2, Then freeze-dried overnight (~ 15 hrs) Coating material gold Time of coating 2x 15 sec Accelerating voltage 30 KV Are bugs present? Etching? EDAX Singles? rods Groups? branchy No. types present 3 cocci Comments: Gray sediment mat preserved in gluteraldehyde in field collected from 189 m (pool bottom) Description gray sediment from U Spg orifice area Sediment is very flocky Lots of black specs Fe-S stuff Not many biological thingssome filaments and cocci "sacks" lots of framboids and crystallites 350.00 300.00 Fe 250.00 200.00 15 0 . 0 0 10 0 . 0 0 50.00 0.00 1 10 1 201 301 401 501 601 701 801 901 10 0 1 Secondary minerals? File names attached peaks Fe and S (small peak) 249 File SEM62601_up_red.doc Date 6-26-01 Sample up spg 189 m, blood red mat next to black Sample # lkb-01m-004r Description blood red, thin biofilm/mat near black Duration in place Date emplaced Date retrieved 3-01 trip Method of preservation glut, freeze-fract, freeze-dry Coating material gold Time of coating 30 sec Accelerating voltage 30 kv Are bugs present? No. types present Radius of bugs variable Etching? Singles? cocci Length rods Groups? branchy variable Secondary minerals? Comments: Upper Spring orifice 189 m (Figure 16A). Most impt images were the first ones (esp the third in the 1st row), showing that a framboid is associated with limestone (Ca, and shows extensive dissolution). Perhaps the framboids ARE coming from the limestone.... Need to look at hostrock in SEM to see b/c thin sections show Fe present.... Some of the framboid crystals are "coated" compared to others, maybe they are being replaced? Rest of the sample showed thick glycocaylx with lots and lots of cells (mostly cocci <1mm), but also with some rods 1-2 mm long. The biofilm is generally thin. Some evidence for filamentous cells, but could be sheaths what is making the blood red color? Saw lots of small framboids also encased in biofilm (looked for density/composition variation in TV with contrast turned way up.... Very dark objects were framboids!). 250 ile SEM62601org.doc Date 6-26-01 Sample 189 m , up spg, orange mat next to black Sample # lkb-01m-004o Description rusty org mat in Upper Spg pool, near blk Duration in place Date emplaced Date retrieved 3-01 trip Method of preservation 2.5% glut, freeze-fracture, Freeze-dry Coating material gold Time of coating 30 sec Accelerating voltage 30 KV Are bugs present? No. types present Radius of bugs Etching? EDAX File names Singles? cocci Length Secondary minerals? Groups? rods branchy Comments: Upper Spring orange film next to gray and red. Really bad charging problems and the SEM was jumping all over the placedrifting and jumping during picturecapture. Sample was really really porous. For the most part, it looked like the samples from the red mats at Site 1, with sheaths. Didn't see any Gallionella-like stuff. There was a thick biofilm on solid particles (sediment?), with clumps of cocci, less then 1 m across. Some indication of spirochetes. No. photomicrographs File names 626site4org1 thu 9.tif Red stuff on right (whole picture ~0.75 m wide). 251 File SEM62601red Date 6-26-01 Sample 215 m red mats discharing into stream Sample # lkb-01m-001 Description red mats at 215 m, sampled from top 2 cm Duration in place Date emplaced Date retrieved 3-01 trip Method of preservation 2.5% glut, freeze-fracture, Freeze-dry for at least 18 hours Coating material gold Time of coating 30 sec Accelerating voltage 30 KV Are bugs present? No. types present Radius of bugs ~ 1 m Etching? EDAX Singles? cocci rods Groups? branchy Comments: 215 m red mats (Figure 1-8), thick goopy stuff freeze-dried sample was very soft and porous, little bit of charging problem, but not too bad over all. Lots and lots of sheaths (Leptothrix) this sample didn't have as much of the bumpy stuff all over it like before (maybe too much gold before?) clumps of cocci and rods in between sheaths. Sheaths have broken ends, but couldn't find growing tips. Sheaths approx 1 m across, and more than 20 m long, avg about 10 m, but this is probably a function of breaking them up. Thin filaments, about 100 nm across, twisted around each other and sheaths (see image below!) perhaps Gallionella...very nice twisty segments, no more than about 10 m long, but could have been broken Length see comments Secondary minerals? Mostly Fe just general , mostly Fe 252 File SEM2201p Date 2-2-01 Sample pumpkin guts - ~72 m Sample # Description red mats @ 72 m- very dense Duration in place Date emplaced Date retrieved 9-00 trip Method of preservation tube-glut-ethanol Coating material gold Time of coating 30 sec Accelerating voltage 30 Kv Are bugs present? Sheaths Radius of bugs varies Etching? EDAX Length varies Secondary minerals? File names pumpEDS.xls Singles? Groups? rods spiral No. types present ?? cocci Comments: Pumpkin guts from stream channel at 72 m charge a little, have to reduce spot size and brightness- concentrate on small areas, then move because of charge need to coat better Seems to be just a sheath matrix with balls (or cells) all around. Really cool stufforiented? Sheaths - look at mats under Epi or confocal to see (but might not stain...?) EDS of ball material has Fe Groups/clumps of stuff mineral mattermixed with Sheaths... don't know what it is? Spiral sheaths maybe Gallionella but don't know because diameter is pretty small Straight sheaths Leptothrix-like No. photomicrographs 7 253 1 2 3 4 5 6 7 8 9 10 11 1213 1415 161718 1920 21222324 25 26272829 30 31 3233343536373839404142434445 2 X 2X 2 2 2 2 2 1 5 X X 2 X 2 X 2 4 2 2 6 5 XX 5 4 45 6 5 2X X 2 2 2 2 2 1 2 5 2 7 LKC19_1-15 61 62 63 64 65 66 67 68 69 70 71 72 LKC19_16-30 LKC19_31-45 73 74 7576 Restriction fragment length polymorphism results 254 2 2 2 1X 2 X 2 2 2 2 X 4647 48 49 50 51 52 53 54 55 56 57 58 59 60 5 2 X 2 13 2 17 2 2 12 2 2 X X 2 X X LKC19_46-60 LKC19_61-72 LKC19_ 73.76 Clone Library 19 (124 m filaments) Pattern Number 1 3 2 36 4 3 5 7 6 2 7 1 12 1 13 1 17 1 X 19 Total 74 1 2 3 4 5 6 7 8 9 10 11 12 13 1415 16 17 19 20 222324 25 26 2728 2930 31 32 33 34 35 36 37 38 39 40 4142 43 44 45 46 47 2 2 2 2 2 2 X 5 XX 2 2 2 2 5 2 17 6 X 4 2 4 2 2 X 2 5 5 2 X 2 X 2 2 2 2 2 2 X X 2 X 17 23 2 LKC19B_1-15 LKC19B_16,17,19,20,22-32 63 646566 6768697071 LKC19B_33-47 Restriction fragment length polymorphism results 255 2 2 2 5 2 2 X 2 2 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 1 2 2 2 2 X2 2 X 2 2 2 2 2 2 LKC19B_48-62 LKC19B_63-71 Clone Library 19B (124 m filaments) Pattern Number 1 1 2 44 4 2 5 5 6 1 17 2 23 1 X 13 Total 69 1 2 3 4 5 6 7 8 9 10 11 121314 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 2 2 5 5 2 4 5 X 2 5 5 5 2 2 5 2 2 X 2 5 2 2 5 2 2 2 5 5 5 5 2 5 5 5 5 2 5 2 2 2 2 2 2 2 5 LKC22_1-15 LKC22_16.30 616263 6465 66 67 6869 707172 73 7475 LKC22_31.45 76 77 78 79 80 81 82 83 84 85 86 87 88 Restriction fragment length polymorphism results 256 5 2 2 2 5 5 5 5 2 2 2 4 5 5 2 46 47 48 4950 5152 53 54 5556 575859 60 5 2 2 4 2 2 2 5 2 2 5 2 5 2 2 5 2 5 2 2 12 12 2 2 2 2 2 5 LKC22_46.60 LKC22_61.75 LKC22_76.88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 Clone Library 22 (127 m filaments) 2 2 2 2 5 5 2 5 X 2 2 2 2 4 5 2 2 2 Pattern 2 4 5 12 X Total Number 61 4 36 2 3 106 Restriction fragment length polymorphism results 257 LKC22_104-106 LKC22_89-103 1 2 3 4 5 6 7 8 9 1011 12131415 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 2 1 1 X 2 2 2 2 2 2 2 X 2 2 2 Clone Library 57 (190 m filaments) Pattern 1 2 X Total Number 7 24 25 56 X 2 2 1 X 2 1 2 1 2 2 2 2 1 2 LKC57_1-15 46 47 48 49 50 51 52 53 54 55 56 LKC57_16-30 Clone Library 57C (not shown) (190 m filaments) XX X X X XX XX X X Restriction fragment length polymorphism results 258 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 X X X X1 X X X X X 2 2 2 2X LKC57_31-45 LKC57_46-56 Pattern 1 2 4 8 10 Total Number 16 16 2 6 2 42 1 2 3 4 5 6 7 8 9 10 11121314 15 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 16 17 1819 2021 22 23 24 25 2627 2829 30 2 2 21 2 22 2 2 2 95 2 21 2 2 2 1 2 10 8 X 4 X 1 2 5 2 2 2 2 X2 2 2 2 1 2 2 X 2 1 X 2 LKC57B_31-45 LKC57B_16-30 61 62 63 64 65 66 67 68 69 70 71 72 Restriction fragment length polymorphism results 259 2 2 2 2 X2 X2 4 5 2 2 LKC57B_1-15 Clone Library 57B (190 m filaments) 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 X X10 2 X 4 X X 4 2 X 9 2 5 12 LKC57B_46-60 LKC57B_61-72 Pattern 1 2 4 5 8 9 10 12 X Total Number 6 39 4 4 1 2 2 1 13 72 1 2 3 4 5 16 17 18 19 20 21 22 23 24 25 26 2728 29 30 6 7 8 9 10 11 12 13 14 15 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 1 10 2 1 2 1 2 1 1 2 2 1 9 1 24 5 1 2 2 4 2 5 8 2 1 2 2 1 1 1 5 1 2 1 2 1 1 2 2 1 8 1 X X X LKC270_1-15 49 50 51 52 53 54 55 56 57 58 59 60 73 74 75 LKC270_16-30 LKC270_31-45 76 77 78 79 80 8182 83 84 85 86 87 Restriction fragment length polymorphism results 260 2 2 2 10 1 2 1 2 1 4 X 1 7 2 2 46 47 48 6162 63 64 65 66 67 68 69 70 71 72 1 2 10 2 2 1 8 8 2 2 1 2 2 2 2 1 2 2 2 2 2 5 4 7 2 1 2 LKC270_46.48_61.72 Pattern 1 2 4 Number 27 38 3 Pattern 5 7 8 LKC270_49.60_73.75 LKC270_76.87 Number 4 2 4 Pattern 9 10 24 Number 1 3 1 Pattern X Total Number 4 87 Clone Library 270 (195 m filaments) 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 Clone Library 270B (195 m filaments) 1 1 1 2 1 2 1 1 1 10 1 1 1 1 X 1 2 1 2 8 1 1 X 2 1 1 1 Pattern 1 2 4 8 10 X Total Number 27 10 1 1 2 7 48 LKC270B_1-12 43 44 45 46 4748 LKC270B_13-27 Restriction fragment length polymorphism results 261 1 2 2 1 10 1 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 1 2 X 1 4 X 2 2 1 1 1 X 1 XX LKC270B_28-42 LKC270B_43-48 31 32 33 34 35 36 37 38 3940 41 42 43 44 45 Clone Library 190* (198 m filaments) Pattern 1 11 X Total 1 X 1 1 1 1 11 1 1 1 1 Number 81 6 5 92 1 1 1 1 * Additional gels not shown LKC190_31-45 76 77 7879 80 81 82 83 8455 86 87 8889 90 91 9293 94 Restriction fragment length polymorphism results 262 1 1 1 1 1 1 1 1 X 1 1 1 1 1 1 1 1 1 46 4748 49 50 51 52 53 54 55 56 5758 59 60 1 1 1 1 1 1 1 1 1 1 1 1 X 1 1 1 LKC190_46-60 LKC190_76-90 LKC190_91-94 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 Clone Library 127 (203 m filaments) 2 5 5 1 X 2 4 5 X 2 1 2 2 4 1 5 1 2 4 X 13 5 5 5 7 1 5 10 1 1 LKC127_1-15 LKC127_16-30 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Restriction fragment length polymorphism results 263 4 2 4 X 4 X 1 Pattern 1 2 4 5 7 10 13 X Total Number 13 9 10 12 2 3 1 10 60 31 32 33 3435 36 37 38 39 40 41 42 43 44 45 1 4 5 1 4 10 4 5 2 4 X 7 1 X 1 10 X 1 2 5 5 X X LKC127_31-45 LKC127_46-60 1 2 3 4 5 6 7 9 10 11 12 13 14 15 8 19 20 21 22 23 24 25 26 27 28 29 30 16 17 18 4 4 4 1 1 1 4 2 1 X 1 X 4 4 4 1 4 5 X X 4 4 4 1 2 4 4 4 X 1 Restriction fragment length polymorphism results 264 LKC127B_1-15 LKC127B_16-30 Clone Library 127B (203 m filaments) Pattern 1 2 4 5 X Total Number 8 2 14 1 5 30 1 2 3 4 5 6 7 8 9 10 11 12 1 7 X1 X 5 1 1 X 1 1 X Clone Library 159 (counted with library 127) (203 m filament) Pattern Number 1 6 5 1 7 1 X 4 Total 12 Restriction fragment length polymorphism results 265 LKC159_1.12 1 2 3 4 5 6 16 17 18 19 20 21 22 23 24 25 26 27 2829 30 7 8 9 10 11 1213 14 15 31 32 33 34 3536 37 38 394041 42 43 44 45 7 7 7 7 7 7 7 1 7 7 X 7 15 1 6 X 1 7 7 7 7 7 7 7 7 7 7 7 7 6 7 1 1 7 7 X 7 6 7 7 17 7 6 6 7 LKC156_1-15 LKC156_16-30 6162 63 64 65 66 67 68 69 70 71 72 73 74 75 LKC156_31-45 76 77 78 79 80 Restriction fragment length polymorphism results 266 7 7 7 7 7 7 7 6 6 5 7 7 X 6 11 7 7 11 7 6 46 47 4849 50 51525354 55 56 5758 59 60 Clone Library 156 (203 m white webs) 17 6 6 7 1 7 7 7 7 17 16 6 7 1 7 LKC156_46-60 LKC156_61-75 LKC156_76-80 Pattern 1 5 6 7 11 15 16 17 X Total Number 7 1 12 49 2 1 1 3 4 80 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 1 2 3 4 5 6 7 8 9 10 1112 13 14 15 29 29 29 29 29 29 29 X X X X 29 7 X X 29 X 29 7 29 1 11 X X X 29 29 29 29 29 LKC102_1-15 Clone Library 102 (203 m yellow patches) LKC102_16-30 Restriction fragment length polymorphism results 267 Pattern 1 7 11 29 X Total Number 1 2 1 26 15 45 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 X X X X X 29 29 29 29 29 29 29 29 29X LKC102_31-45 1 2 3 4 5 6 7 8 9 10 11 12 1314 15 1617 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 29 29 29 6 X X 29 29 29 29 29 29 29 29 29 29 X X X 29 X 29 29 29 29 X 29 29 X 29 X 29 27 29 X 29 29 29 29 29 X 29 X X 29 LKC102B_1-15 LKC102B_16-30 61 62 63 64 65 66 67 68 71 72 LKC102B_31-45 Clone Library 102B (203 m yellow patches) Restriction fragment length polymorphism results 268 X 29 29 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 29 X 29 29 29 29 6 29 29 7 28 29 29 29 X 29 X X 29 29 29 29 LKC102B_46-60 LKC102B_61-68, 71-72 Pattern 6 7 19 27 28 29 X Total Number 2 1 1 1 1 47 17 70 1 2 3 4 5 6 7 8 9 10 11 1213 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 3637 38 39 40 41 42 43 44 45 21 21 21 X X X X X X X 22 X X X X 21 X 21 1 12 21 21 X X 21 X X X X X X 21 21 X X X 21 X 21 X X 21 X X X LKC125_1-15r 61 62 63 64 65 66 67 68 69 70 71 72 LKC125_16-30 LKC125_31-45 Clone Library 125B (203 m gray filaments) Restriction fragment length polymorphism results 269 X 21 X 22 X 21 21 X 21 X X 21 X 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 22 X X 22 X X 22 21 X 21 X 21 X X Pattern 1 12 21 22 X Total Number 1 1 20 6 44 26 LKC102B_46-60 LKC125_61-72 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 46 47 48 49 5051 52 53 5455 56 57 58 59 60 1 1 1 1 1 1 1 1 1 1 1 1 1 X 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 LKC199_1-15 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 LKC199_16-30 LKC199_46-60 91 92 93 94 Restriction fragment length polymorphism results 270 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 X1 1 1 Clone Library 199 (248 m filaments) Pattern 1 X Total LKC199_ 91-94 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Number 77 2 79 LKC199_61-75 LKC199_76-90 30 9 10 11 12 13 14 Plas 15 16 17 181920 21 22232425 2627 28 29 30 31 32 33 34 35 36 37 38 3940 4142 4344 1 2 3 4 5 6 7 8 X X 1 5 18 231818 18 5 1 X X 1 2 X X 18 1 2 18 X 1 X 6 1 X X 18 7 18 X X 2 18 18 5 1 181018182518 LKC198_1-14 60616263 646566 6768697071 7273 74 75 76 LKC198_15.29 LKC198_30.44 45 46 4748 49505152 535455 56 5758 59 Restriction fragment length polymorphism results 271 18 5 X X 18 5 181818X 181818 1 18 1 18 X 18 1818181818 18X 2 2 5 1 LKC198_45.59 LKC198_60.74 Clone Library 198 (248 m yellow-white mat) Pattern Number 1 11 X 1 2 5 5 6 6 1 7 1 LKC198_ 18 31 75.76 23 1 25 1 X 18 Total 75 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 X X2 2 1 2 5 X X X X 23 X1 X 23 10 1 X X X X 18 1 X X 18 X18 X LKC198B_1-15 46 47 LKC198B_16-30 Restriction fragment length polymorphism results 272 2 1 23 11 7 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 18 18 X X 18 X XX X 2 1818 Clone Library 198B (248 m yellow-white mat) Pattern Number 1 5 2 5 5 1 7 1 10 1 11 1 18 8 23 3 X 22 Total 47 LKC198B_31-45 LKC198B_ 46-47 Table A-1: Clone 16S rRNA gene sequences from Lower Kane Cave libraries with phylogenetic affiliations and percent sequence similarity to closest relative. LKC clone accession numbers in Chapter 2. Library 124m filament Clonea LKC3_19.29 LKC3_19B.29 LKC3_19.39 LKC3_19.50 LKC3_19B.17 LKC3_19B.45 LKC3_22.9 LKC3_22.13 LKC3_22.5 LKC3_22.81 LKC3_22.3 LKC3_22.17 LKC3_22.33 LKC3_22.73 LKC3-22.89 LKC3_22.72 LKC3_22.6 LKC3_22.53 LKC3_22.54 LKC3_57B.17 LKC3_57B.49 LKC3_57.8 LKC3_57.20 LKC3_57C.15 LKC3_57C.33 LKC3_57B.57 LKC3_57B.2 LKC3_57.4 LKC3_57C.13 LKC3_57B.54 LKC3_57B.22 LKC3_57C.10 LKC3_57B.41 LKC3_270.5 LKC3-270.64 LKC3-270.57 LKC3_270.16 LKC3_270.58 LKC3_270.18 LKC3_270.19 LKC3_270.13 LKC3_270.15 LKC3_190.31 RFLP Pattern 5 2 13 17 2 Closest relativesb Sulfidic spring clone sipK4 [AJ307933] Sulfidic spring clone sipK119 [AJ307940] Rifta clone R76-B13 [AF449261] Beggiatoa 'Bay of Concepcion' strain [AF035956] Groundwater clone FTL212 [AF529098] Groundwater clone 1043 [AB030601] Sulfidic spring clone sipK4 [AJ307933] Percent Similarityc 99 99 91 90 98-99 127m filament 5 99 4 4 12 2 190m filament Groundwater clone 1043 [AB030601] Wolinella succinogenes [AF273252] Sulfidic spring clone sipK119 [AJ307940] Groundwater clone FTL212 [AF529098] Sulfidic spring clone sipK119 [AJ307940] 98 90 98-99 99 1 4 8 9 2 1 4 5 8 9 24 1 Sulfidic spring clone sipK94 [AJ307941] Groundwater clone FTL212 [AF529098] Sulfidic spring clone sipK119 [AJ307940] Groundwater clone FTL212 [AF529098] Sulfidic spring clone sipK119 [AJ307940] Sulfidic spring clone sipK119 [AJ307940] Sulfidic spring clone sipK94 [AJ307941] Groundwater clone 1043 [AB030601] Sulfidic spring clone sipK4 [AJ307933] Groundwater clone 1028 [AB030605] Sulfidic spring clone sipK119 [AJ307940] Bacteroides sp. ECP-C1 [AF529225] Sulfidic spring clone sipK94 [AJ307941] 98 97 99 97-98 99 98 97-98 100 96 95 94 98 195m filament 198m filament 273 Table Library 198m filament 203m filament A-1 (Continued). Clone LKC3-190.28 LKC3_190.63 LKC3_190.37 LKC3_190.75 LKC3_127.47 LKC3_127.18 LKC3_127.7 LKC3_127B.2 LKC3_127.11 LKC3_127.25 LKC3_127.4 LKC3_159.2 LKC3_159.10 LKC3_159.12 LKC3-127.29 LKC3_127.1 LKC3_127.6 LKC3_127.23 LKC3_127.32 LKC3_127.14 LKC3_127.39 LKC3_127.40 LKC3_127.43 LKC3_127.46 LKC3_127B.26 LKC3_127B.27 LKC3_127.2 LKC3_127.33 LKC3_159.7 LKC3_127.27 LKC3_127.26 LKC3_127.28 LKC3_127.36 LKC3_127.53 LKC3_156.14 LKC3_156.17 LKC3_156.1 LKC3_156.4 LKC3_156.19 LKC3_156.13 LKC3_156.15 LKC3_156.38 LKC3_156.74 LKC3_156.46 LKC3_156.55 LKC3_156.41 LKC3_156.56 RFLP Pattern 11 Closest relativesa Desulfocapsa sp. Cad626 [AJ511275] Desulfocapsa thiozymogenes [X95181] Lake clone SRB-348 [AJ389628] Groundwater clone G1070 [AB030590] Sulfidic spring clone sipK94 [AJ307941] Campylobacter sp. [L14632] Sulfidic spring clone sipK119 [AJ307940] Groundwater clone SJA-36 [AJ009461] Polychaete clone P.palmA10 [AJ441240] Sulfidic spring clone sipK94 [AJ307941] Percent Similarityb 96-97 19 19 2 7 13 1 94-95 98 99 98 90 97-98 4 Groundwater clone FTL212 [AF529098] Sulfidic spring clone sipK119 [AJ307940] Groundwater clone 1043 [AB030601] 96-98 5 Sulfidic spring clone sipK4 [AJ307933] 99-100 10 1 7 15 6 17 Sulfidic spring clone sipK94 [AJ307941] Sulfidic spring clone sipK94 [AJ307941] Groundwater clone SJA-36 [AJ009461] Groundwater clone SJA-36 [AJ009461] Acid mine clone 44a-B1-1 [AY082456] Beggiatoa sp. strain MS-81-1c [AF110276] Beggiatoa 'Bay of Concepcion' strain [AF035956] Clone vadinHA54 [U81722] 203m web 98 92 95 98 97 96 95-96 90-91 16 92 274 Table Library 203m yellow A-1 (Continued). Clone LKC3-102B.15 LKC3-102B.55 LKC3_102B.2 LKC3_102B.27L KC3 _102B.25 LKC3_102B.28 LKC3_102.21 LKC3_102.22 LKC3_102B.18 LKC3-102B.33 LKC3_102B.59 LKC3_125.2 LKC3_125.3 LKC3_125.60 LKC3_125.31 LKC3_125.46 LKC3_125.59 LKC3_199.1 LKC3_198.8 LKC3_198.29 LKC3_198.31 LKC3_198.35 LKC3_198.44 LKC3_198.17 LKC3_198.15 LKC3_198.20 LKC3_198.17 RFLP Pattern 6 29 Closest relativesa Acid mine clone 44a-B1-40 [AY082468] Thiobacillus aquaesulis [U58019] Groundwater clone FW119 [AF523954] Forest-wetland soil clone FW119 [AF523954] Sulfidic spring clone sipK94 [AJ307941] Lake clone SRB-348 [AJ389628] Acid mine clone 44a-B1-40 [AY082468] Clone WCHA1-01 [AF050541] Cytophagales clone Hyd-B2-1 [AJ535256] Pantoea agglomerans [AB004691] Uncultured clone p-2172-s959-3 [AF371852] Sulfidic spring clone sipK94 [AJ307941] Serratia marcescens strain CPO1(4)CU [AJ296308] Sulfidic spring clone sipK94 [AJ307941] Uncultured clone RB7C6 [AF407385] Leptothrix discophora (SS-1) [L33975] Acid mine clone 44a-B2-21 [AY082471] Sulfidic spring clone sipK94 [AJ307941] Groundwater clone FTL212 [AF529098] Percent Similarityb 95 93-95 203m gray filament 1 11 19 27 28 21 1 22 1 18 97 97 97 91 96 99 98 99 98 94 248m filament 248m yellowwhite 10 2 14 96 98 Lake clone TLM10/TLMdgge01 91 [AF534434] LKC3_198.26 1 Sulfidic spring clone sipK94 [AJ307941] 98 LKC3_198.43 25 Marsh clone FSA6 [AY193038] 97 a Twelve clone sequences were found to be chimera based on results from the CHECK_CHIMERA program in the RDP. b Based on BLAST search. GenBank accession numbers in brackets. c Based on alignable base pairs. 275 APPENDIX B Supplemental Information for Chapter 3 Appendix B Table of Contents ........................................................................... 276 Description of Additional Sampling Sites for Chapter 3 .................................... 277 Sulfidic Caves ......................................................................................... 277 Hellspont Cave, Big Horn County, Wyoming .................................. 277 Cesspool Cave, Allegheny County, Virginia .................................... 277 Big Sulphur Cave, Trigg County, Kentucky ..................................... 278 Surface-discharging Mesothermal Sulfidic Springs................................ 279 Frasassi Caves and resurgence, Genga, Italy .................................... 279 White Sulphur Springs, Schoharie County, New York..................... 279 Palmetto Spring, Gonzolas County, Texas........................................ 280 Surface-discharging thermal Sulfidic Springs......................................... 280 Thermopolis Hot Springs, Hot Springs County, Wyoming .............. 280 Glenwood Springs, Garfield County, Colorado ................................ 280 Pah Tempe, Washington County, Utah............................................. 281 Soda Dam Spring, Sandoval County, New Mexico .......................... 281 Lazio Volcanic Province ................................................................... 282 Figure B-1: Microbial mats in Hellspont Cave, Wyoming ................................. 283 Figure B-2: Microbial mats in Big Sulphur Cave, Kentucky.............................. 284 Figure B-3: Microbial mats at Frasassi Cave, Italy, resurgence springs............. 285 Figure B-4: Microbial mats at White Sulphur Springs, New York..................... 286 Figure B-5: Microbial mats at Palmetto Spring, Texas....................................... 287 Figure B-6: Microbial mats at Thermopolis Hot Springs, Wyoming ................. 288 Figure B-7: Microbial mats at Glenwood Springs, Colorado ............................. 289 Figure B-8: Microbial mats at Pah Tempe Hot Springs, Utah ............................ 290 Figure B-9: Microbial mats at Soda Dam, New Mexico..................................... 291 Figure B-10: Microbial mats at hot springs, Viterbo, Italy................................. 292 Figures of results from electrophoresis gels ....................................................... 293 Table B-1: Screening Lower Kane Cave microbial mats samples and FISH experimental plan............................................................................................ 298 Table B-2: Sample numbers listed in Table B-1 and corresponding location and microbial mat morphotypes ..................................................................... 303 276 DESCRIPTION OF ADDITIONAL SAMPLING SITES FOR CHAPTER 3 Sulfidic Caves Hellspont Cave, Big Horn County, Wyoming This small cave (20 m long) is located along the Bighorn River downstream from Lower Kane Cave, but across the river (Figure 1-3; Figure B-1). Hellspont Cave has one spring orifice at the back of the cave (Egemeier, 1981), and microbial mat filament bundles are suspended in the stream water column discharging from the spring (~15 cm deep). There is a thick white microbial mat at the entrance of the cave. A gypsum crust of moist white gypsum and discontinuous patches of elemental sulfur cover the walls. Mat samples from inside the cave and at the entrance were collected on several occasions in 2000 through 2002. Cesspool Cave, Allegheny County, Virginia Cesspool Cave is a small opening (<20 m of passage) developed in a travertine-marl complex of Quaternary age located along the Sweet Springs Creek (Hubbard et al., 1986, 1990; Engel et al., 2001). A sulfidic, non-thermal spring discharges into a back pool, flows out of the cave, and resurges into a surface pond. The hydrogen sulfide is thought to originate from oil-field brine solutions that flow up-dip along local faults (Hubbard et al., 1990). Some incidental sunlight reaches the back pool area due to the limited passage length and large surface entrance. The white filamentous microbial mats in the cave and resurgent spring were previously sampled (Engel et al., 2001), and two groups of "Epsilonproteobacteria" were identified from 16S rDNA gene sequences. Both of these groups were closely 277 related to the Lower Kane Cave group I (Figure 3-1), and glycerol-preserved samples and samples stored at -80oC were used to verify that the LKC group I PCR primer could amplify this group. Big Sulphur Cave, Trigg County, Kentucky The only entrance to Big Sulphur Cave, near Cadiz, Kentucky, is as a resurgence spring, approximately 5 m above the south side of the Little River. Big Sulphur Cave is large, with 15,750 ft of passage. A very small side passage, with a sulfidic stream, feeds into the main cave trunk passage within approximately 100 m of the entrance. Sulfide is only detected in this small side passage, but H2S(g) can be smelled from the entrance and in the vicinity of the side passage within the main trunk passage. The side channel is relatively small (no more than 0.5 m wide x 10.3 m high) and white filamentous microbial mats are attached to cobbles in the stream. The mats are discontinuous along the stream bed, and first occur 5-6 m from the main trunk passage (Figure B-2). Due to passage constriction, it was not possible to see a spring orifice, if there is any, or even where the sulfidic water was originating from (e.g., a spring orifice or from a fissure in the stream bed). Collembolan, flatworms, crayfish and a significantly large population of isopods, were associated with the mats. Gypsum and elemental sulfur were observed on small exposed limestone cobbles in the sulfidic stream. Mat samples were acquired in 2002. 278 Surface-discharging Mesothermal Sulfidic Springs Frasassi Caves and resurgence, Genga, Italy The Frasassi Caves, including Grotta Grande del Vento, Grotta del Fiume, and Grotta Sulfurea, have been studied extensively by Italian speleologists, and Galdenzi and Menichetti (1995) suggest that the caves formed from sulfuric acid speleogenesis. An active sulfidic stream flows through lower levels of the system, originating beyond the passages in Grotta Grande del Vento. Abundant gypsum and sulfur accumulations occur throughout the entire cave system at all cave passage elevation levels. Subaqueous white microbial mats, as discontinuous patches of filaments and webs, have developed within an anaerobic, sulfidic cave stream in two different portions of the cave (Grotta del Fiume and Grotta Sulfurea), and filamentous mats again occur at the resurgence cave spring into the Sentio River (Figure B-3). Samples from the cave microbial mats were acquired in 1998 and frozen at -80 oC prior to DNA extraction. Resurgent spring samples were collected in 2001. White Sulphur Springs, Schoharie County, New York White Sulphur Springs is part of a circa 1805-1940s resort in Sharon Springs, New York. A sulfidic spring discharges from a large pool inside an unused bathhouse behind the White Sulphur Temple (Figure B-4), and the water overflows from the floor of the bathhouse, under the walls of the building, and into a small stream outside the building. Thin white filaments covered the outflow basin from the bathhouse. Other sampling locations included two cisterns, one that was covered and another that was open to sunlight; both had white filaments and web279 like microbial mats. Elemental sulfur deposits were common on surfaces above the water level and the cistern walls. Samples were collected in 2002. Palmetto Spring, Gonzolas County, Texas Sulfidic water discharges from an artificial (concrete) basin in the parking lot of a picnic area in Palmetto State Park, Texas (Figure B-5). A thin white filamentous mat forms over the concrete, and discontinuous white filament bundles occur ~ 5 m from the concrete basin, although green phototrophic material was also present. White mat samples were collected in 2003. Surface-discharging thermal Sulfidic Springs Thermopolis Hot Springs, Hot Springs County, Wyoming Sulfidic water flows along faults in the Thermopolis anticline and emerge from the Madison Limestone, discharging into the Bighorn River within Hot Springs State Park, Thermopolis, Wyoming (Figure B-6). Water flows from one orifice at 7000 m3 day-1. Thick white microbial mats occur in the upper reaches of the spring before the water spills over travertine pools and dams. Samples were collected in 2002 and 2003. Glenwood Springs, Garfield County, Colorado The hot spring at Glenwood Springs, Colorado, is also known as Yampah Hot Springs, and feeds an extremely large swimming pool at the resort. Before reaching the pool there is a cooling pond, piping, a small cistern (referred to as the Drinking Spring), and more pipe work. Microbial mat samples were collected at the cistern (Figure B-7). 280 Pah Tempe, Washington County, and Fifth Water, Utah County, Utah Sulfidic water at Pah Tempe Mineral Hot Springs discharges along the Hurricane Fault in Quaternary basalts near the Virgin River, Hurricane, Utah, and the water flows through underground faults. The resort was closed due to groundwater pumping along the river, and hot spring flow has diminished. Microbial mats form on the artificial (concrete) pool bottoms, as well as below breaks in the pool walls where a stream developed (Figure B-8). Mats are suspended on the water surface, supported by large bubbles. Megan L. Porter and Katharina de la Cruz-Dittmar, Brigham Young University (BYU), sampled the mats from these springs in 2002. M.L. Porter extracted total environmental DNA and amplified the epsilonproteobacterial groups using specific PCR primers; universal bacterial primers were also used. Springs at Fifth Water Hot spring, also known as Diamond Fork hot springs, in Utah County, Utah, were also sampled by M.L. Porter and used in this study. Soda Dam Spring, Sandoval County, New Mexico Numerous active fumeroles, hot springs, and mudpots attest to active thermalism in the Valles Caldera, associated with the Rio Grande rift (Rzonca et al., 2003). At the Soda Dam near Jemez Pueblo, water discharges from a fault where Precambrian granite juxtaposes Paleozoic carbonates (Madera Limestone, Pennsylvanian). Waters are characterized as Na-Cl-HCO3 type with minor sulfate concentrations (Goff et al., 1982). A small outlet opposite the Travertine Dam has a small microbial mat (referred to here as Soda Dam Spring) (Figure B-9), which is mostly green from phototrophic organisms; thin patches of white filaments are 281 found closest to the orifice. Samples were collected in 2001 and 2003. Rzonca and Schulze-Makuch (2003) describe the microbiology of numerous spring waters from the region, finding evidence that an organism genetically related to the Lower Kane Cave groups were present in the water coming out at the spring, although the resulting sequence from the study has not been deposited into GenBank to verify taxonomic relatedness. Lazio Volcanic Province Hot springs in central Italy occur along the flanks of the Monte Cimino caldera near the city of Viterbo. Sulfidic waters emerge at the Bagnaccio, Bullicame, Paliano, and Le Zitelle Springs, where white and salmon-colored microbial mats and secondary travertine deposits are common (Figure B-10). Microbial mats from Le Zitelle were sampled in 2001, and were included in this study. Thick white microbial mats occur in the upper reaches of the spring before the waters spill over travertine pools and dams. 282 A B AS Engel C D SA Engel Figure B-1: (A) Entrance of Hellspont Cave, Bighorn Co., Wyoming, on the eastern side of the Bighorn River. (B) White filamentous microbial mats on bottom of stream. (C) Spring at back of cave. Full-face respirators are required because the concentration of H2S is very high. (D) White filaments and phototrophs at entrance at river level. AS Engel SA Engel entrance 283 SA Engel AS Engel A B Figure B-2: Microbial mats in Big Sulphur Cave, Trigg Co., Kentucky. (A) Very small side passage. (B) Microbial mats. (C) Closeup of mats and mud. (D) Flashlight next to mats for scale. Isopods are commonly found in the mats. AS Engel AS Engel D C AS Engel 284 A B AS Engel Figure B-3: Microbial mats at Frasassi Cave resurgence spring, Genga, Italy. (A) The spring discharges from a cave passages ~ 0.5 m high. (B) White microbial mats extend for 20 m inside the passage. AS Engel 285 C SA Engel SA Engel Figure B-4: (A) Microbial mats coating a concrete discharge pool from the Sulphur Temple bathhouse, White Sulphur Springs, Sharon Springs, Schoharie Co., New York. (B) The pipes running from the bathhouse were exposed under access panels. (C) Long filaments on concrete. SA Engel AS Engel A B 286 A B C AS Engel Figure B-5: Microbial mats associated with an artificial concrete mound at Palmetto Spring, Gonzales Co., Texas. White mats were also found ~3 m downstream in a small creek. AS Engel AS Engel 287 A B C AS Engel Figure B-6: (A) Microbial mats in stream channel discharging from one of the hot springs at Thermopolis Hot Springs, Hot Spring Co., Wyoming. (B) Floating organic matter with white filaments. (C) Thick white filaments on cobbles in stream. AS Engel AS Engel 288 A B AS Engel Figure B-7: Drinking Spring cistern, Glenwood Springs, Garfield Co., Colorado. (A) Water discharges in the upper center (arrow) from a pipe under the platform and flows into a channel ringing the cistern pool. (B) Channel around pool with white filaments. AS Engel 289 A B K. Dittmar de la Cruz C K. Dittmar de la Cruz K. Dittmar de la Cruz 290 Figure B-8: Microbial mats consisting of webs and bubbles floating on the surface of the water at Pah Tempe hot Springs, Washington Co., Utah. (A) Sampling mats. (B) Filamentous mat where wading pool wall was broken. (C) Floating mat, webs and bubbles. A B C Figure B-9: Microbial mats at Soda Dam Spring, Jemez Co., New Mexico. (A) Small discharge hole (~2 in diameter) in travertine (arrow). (B) Closeup of white filamentous area (arrow) in mostly a phototrophic mat. (C) Small discharge hole (probably from a pipe) and minor rivulet with white filaments. SA Engel AS Engel AS Engel 291 A C B SA Engel Figure B-10: Microbial mats at Zitelle mineral hot springs, near Viterbo, Italy. (A) Orifice with boiling spring. (B) Sampling white filaments on channel edge with travertine. (C) Channel below spring. SA Engel AS Engel 292 Gel B1 Both LKC groups MW DKS-02-001A PS-03-002Aa BSC-02-002 JM-01-002 SS-02-003B I II I II I II I II I II PCR conditions: 2 min hot start 47oC annealing temperature Columns: MW- molecular weight marker I - eps59f/1492r primers II - eps174f/1492r primers ND no DNA control Samples: DKS - Drinking Spring, CO PS Palmetto Spring, TX BSC - Big Sulphur Cave, KY JM - Soda Dam Spring, NM SS - White Sulphur Spring, NY LKC - Lower Kane Cave, WY Frasassi Frasassi resurgence spring, Italy FC Frasassi Cave, Italy CC - Cesspool Cave, VA LeZit Le Zitelle Spring, Italy 2000 1000 LKC-02-1 BSC-02-004 Frasassi-5A MW I II I II I II 2000 1000 MW FC-01-01 CC-00-01 LeZit-01-1A ND I II I II I II 293 Eprot59/1492r DKS-02-001A DKS-02-002 JM-01-002A JM-01-002B DKS-02-001A 8f/1492r DKS-02-002 JM-01-002A JM-01-002B SS-02-003B BSC-02-004 SOX-03-001Ca CC1 ND Gel B2 LKC group I MW CC1 PCR conditions: 2 min hot start 47oC annealing temperature 1500 Columns: MW- molecular weight marker ND no DNA control Primer sets used are labeled. SS-02-002A2 Eprot59/1492r SS-02-002B1 PS-03-002Ab SS-02-002A2 PS-03-002Aa SS-02-003B 8f/1492r SS-02-002B1 PS-03-002Aa SOX-03-001B Samples: CC - Cesspool Cave, VA DKS - Drinking Spring, CO JM - Soda Dam Spring, NM SS - White Sulphur Spring, NY PS Palmetto Spring, TX BSC - Big Sulphur Cave, KY SOX - Edwards church well, TX MW 1500 8f/1492r PS-03-002Ab BSC-02-002 MW Eprot59/1492r SOX-03-001Ca SOX-03-001Ba SOX-03-001Bb BSC-02-004 8f/1492r BSC-02-002 SOX-03-001Bb 1500 8f/1492r 8f/1492r MW ND ND 1500 294 Eprot174f/1492r Frasassi-01-5A LKC-02-01 DKS-02-001A Le Zit-01-1A Le Zit-01-1A MW FC-01-001 DKS-02-001A FC-01-001 SS-02-002Aa SS-02-002Aa ND BSC-02-004 BSC-02-004 Gel B3 LKC group II PCR conditions: 2 min hot start 47oC annealing temperature Columns: MW- molecular weight marker ND no DNA control Primer sets used are labeled. 2000 1000 8f/1492r LKC-02-01 Frasassi-01-5A PS-03-002Aa PS-03-002Ab MW 2000 1000 8f/1492r PS-03-002Aa PS-03-002Ab DKS-02-002 MW BSC-02-002 2000 1000 2000 1000 295 BSC-02-002 DKS-02-002 Samples: LKC - Lower Kane Cave, WY Frasassi Frasassi resurgence spring, Italy LeZit Le Zitelle Spring, Italy DKS- Drinking Spring, CO FC Frasassi Cave, Italy PS Palmetto Spring, TX BSC - Big Sulphur Cave, KY SS - White Sulphur Spring, NY MW ND 2000 1000 Eprot174f/1492r Eprot59f/1492r DKS-02-001A PS-03-002Aa SS-02-003B BSC-02-002 JM-01-002 Eprot174f/1492r DKS-02-001A PS-03-002Aa SS-02-003B BSC-02-002 JM-01-002 Gel B4 LKC group I and II MW ND 2 min hot start 47oC annealing temperature Columns: MW- molecular weight marker ND no DNA control Primer sets used are labeled. 2000 1000 Samples: DKS- Drinking Spring, CO SS - White Sulphur Spring, NY JM Soda Dam Spring, NM PS Palmetto Spring, TX BSC - Big Sulphur Cave, KY 296 Eprot59f/1492r PS-03-002Ab PS-03-002Aa BSC-02-002 BSC-02-004 JM-01-002A Eprot174f/1492r PS-03-002Aa PS-03-002Ab BSC-02-002 BSC-02-004 JM-01-002A Gel B5 LKC Group I and II MW ND PCR conditions: 2 min hot start 45oC annealing temperature Columns: MW- molecular weight marker ND no DNA control Primer sets used are labeled. 2000 1000 Samples: PS Palmetto Spring, TX BSC - Big Sulphur Cave, KY JM Soda Dam Spring, NM (Not the right size) 297 Table B-1: Screening Lower Kane Cave microbial mat samples and experimental FISH plan. FA% 0 0 35 35 35 35 0 40 35 20 35 22, 11, 203g, 198w 22, 11, 203g, 198w 22, 11, 203g, 198w 22, 11, 203g, 198w 22, 11, 203g, 198w 22, 11, 203g, 198w 22, 11, 203g, 198w 22, 11, 203g, 198w 22, 11, 203g, 198w 22, 11, 189w, 189g 22, 11, 203g, 198w Target General Bacteria Planctomyces Verrucomicrobium Archaea Samplesa 22, 11, 189w, 189g 22, 11, 203g, 198w Comments (13-03-02) needed to repeat with different samples (22-03-02) nothing, filaments to EUBI, tiny spots EUBII (13-03-02) needed to repeat with different samples and filaments.... (22-03-02) filaments, very weird (18-03-02) nothing (24-03-02) Beta weak to single cells, gamma to filaments (18-03-02) Beta weak, gamma strong to filaments (24-03-02) nothing (18-03-02) nothing (18-03-02) many thick filaments (24-03-02) nothing (24-03-02) nothing (18-03-02) many thick filaments Probe EUBI-FP EUBII-cy3 EUBIII-cy5 Arch915-cy3 EUBmix-cy5 Alphaproteobacteria Betaprotoebacteria Gammaproteobacteria Betaprotoebacteria Gammaproteobacteria Cytophaga Eukaryotes Thiothrix Gram+ high GC Gram+ low GC Gammaproteobacteria Thiothrix Bacteria to all cells Controls 0 298 22, 11, 203g, 198w, 15, 10, 9, 12, 1, 3, 4, 28, 29 22, 11, 203g, 198w ALF968-cy3 EUBmix-cy5 Bet42a- cy3 Gam42a- cy5 EUBmix-FP Bet42a- cy5 Gam42a cy3 EUBmix FP CF19a-cy3 EUBmix-cy5 EUK516-cy3 EUBmix-cy5 G123T-cy3 EUBmix-cy5 HGC69a-cy3 LGCmix-cy3 Gam42a cy5 G123T-cy3 EUBmix- FP EUBmix cy5 SyberGreen onEUB-cy3 EUBmix-FP (21-03-02) more cells with SyberGreen than with EUB calculated percentages of EUB biovolume to SyberGreen (sample quantification) (21-03-02) test autofluorescence, nothing Continued FA% 0 (21-03-02) nothing 22, 11 22, 11 22, 11 22, 11 22, 11 22, 11 22, 11 22, 11 Sewage sludge Sewage sludge 0 35 Sewage sludge Sewage sludge Target Controls, test autofluorescence All (maybe) Epsilonprotoebacteria LKC group I and if there are other epsilons LKC group II and if there are other epsilons LKC group II, different target site Nongreen sulfur bacteria Nongreen sulfur bacteria LKC group I and Group II LKC group I and all epsilons Bacteria 0 0 20 20 23 35 Samples 22, 11, 203g, 198w Comments (20-03-02 to 27-03-02) lots of different filaments, had to repeat with higher FA concentrations to get optimal (optimal FA 2030%) (20-03-02 to 22-03-02) lots, EPS710 optimal FA 20%, some EPS710 not hybridized to Eprot549 (25-03-02) lots, LKC1006 optimal FA 30% (20-03-02) didn't hybridize, so didn't repeat for FA optimization; use for PCR primer (21-03-02) maybe a couple of filaments (21-03-02) maybe a couple of filaments (21-03-02) LKC1006 and EPS710 to different filaments (no overlap) (21-03-02) EPS710 and Eprot549 overlap, but a couple of thin filaments with EPS710 didn't hybridize to Eprot549... hmmm (24-03-02) Used to see how all three Eubacterial groups are in sludge 0,10,20 25,30, 40,50 0,10,20 25,30, 40,50 0,10,20 30,40, 50 0-xx 299 Archaea Eukaryotes Alphaproteobacteria Table B-1: Probe Nonsense-cy3 EUBmix FP Eprot549-cy3 Eprot549-cy5 EUBmix- FP EPS710-cy3 Eprot549-cy5 EUBmix-FP LKC1006-cy3 Eprot549-cy5 EUBmix-FP LKC174-cy3 Eprot549-cy5 EUBmix-FP GNSB-941-cy3 EUBmic-cy5 CFX1223-cy3 GNSB941-cy5 EUBmix-FP LKC1006 cy3 EPS710-cy5 EUBmix-FP EPS710-cy5 Eprot549-cy3 EUBmix-FP EUBI-FP EUBII-cy3 EUBIII-cy5 Arch915-cy3 EUBmix-cy5 EUK516-cy3 EUBmix-cy5 ALF968-cy3 EUBmix-cy5 (24-03-02) Used to see how all three Eubacterial groups are in sludge (24-03-02) Used to see how all three Eubacterial groups are in sludge (24-03-02) Used to see how all three Eubacterial groups are in sludge Continued Target Betaprotoebacteria Gammaproteobacteria Betaprotoebacteria Gammaproteobacteria Cytophaga Gram+ high GC Gram+ low GC LKC group I and II LKC group II LKC group II LKC group II LKC group II LKC group II LKC group II LKC group II LKC group II LKC group II LKC group II 30 30 30 30 1 3 29 4 30 15 30 12 30 9 30 10 30 20 30 11 35 20 30 Sewage sludge Sewage sludge 10, 9, 15, 12, 20, 11 35 Sewage sludge 35 Sewage sludge FA% 35 Samples Sewage sludge Comments (24-03-02) Used to see how all three Eubacterial groups are in sludge (24-03-02) Used to see how all three Eubacterial groups are in sludge (24-03-02) See how all three Eubacterial groups in sludge (24-03-02) See how all three Eubacterial groups in sludge (24-03-02) See how all three Eubacterial groups in sludge (26-03-02) Probe screening and quantification (26-03-02) quantification; lots of filaments, very clean sample (26-03-02) quantification; thinner filaments and weaker signals, clean sample, some sulfur crystals and rosettes (26-03-02) quantification; sediment blobs, lots of samples and too many crystals (Fe-S stuff), few filaments (26-03-02) quantification; only 2 fields that had filaments (26-03-02) quantification; little sediments and a lot of filaments, very thin, hard to get good pictures because sample was too thick (26-03-02) quantification; lots of filaments, clean sample (29-03-02) quantification; lots of filaments, clean sample (29-03-02) quantification; lots of filaments, clean sample (29-03-02) quantification; lots of filaments, clean sample (29-03-02) quantification; lots of filaments, clean sample 300 Table B-1: Probe Bet42a- cy3 Gam42a- cy5 EUBmix-FP Bet42a- cy5 Gam42a cy3 EUBmix FP CF19a-cy3 EUBmix-cy5 HGC69a-cy3 LGCmix-cy3 LKC1006-cy3 EPS710-cy5 EUBmix-FP LKC1006-cy3 EUBmix-cy5 LKC1006-cy3 EUBmix-cy5 LKC1006-cy3 EUBmix-cy5 LKC1006-cy3 EUBmix-cy5 LKC1006-cy3 EUBmix-cy5 LKC1006-cy3 EUBmix-cy5 LKC1006-cy3 EUBmix-cy5 LKC1006-cy3 EUBmix-cy5 LKC1006-cy3 EUBmix-cy5 LKC1006-cy3 EUBmix-cy5 Continued Target LKC group II 28 22 11, 22 11, 22 11 11 15 20 1 3 29 10 9 30 30 12 22 LKC group II LKC group I to large epsilon group LKC group I to LKC group II to show they don't overlap LKC group I 30 LKC group I LKC group I LKC group I LKC group I LKC group I LKC group I LKC group I LKC group I LKC group I LKC group I 30 30 30 30 30 30 30 0, 10, 20 25,30,35 40, 50 30 0 30 FA% 30 Samples Comments (29-03-02) quantification; no filaments anywhere in the sample (just sediment) (26-03-02) quantification; lots and lots of sediment, impossible to get a good pictures (too much topography), lots of filaments, too (28-03-02) check that LKC59 and EPS710 overlap, they do (28-03-02) check that probes don't overlap (they don't) (29-03-02) FA optimization for LKC59 probe (30% optimal FA) (29-03-02) quantification; lots of filaments, clean sample (29-03-02) quantification; lots of filaments, lots of sediment (29-03-02) quantification; background very high, short small filaments (29-03-02) quantification; very few filaments, weak signal (29-03-02) quantification; poor signal with lots of sediment (29-03-02) quantification; lots of filaments, clean sample (29-03-02) quantification; some filaments, background high (29-03-02) quantification; too much sediment and couldn't see filaments (29-03-02) quantification; like sample 9, not a good sample because of too much sediment, topography too high (29-03-02) quantification; lots of filaments, clean sample Table B-1: Probe LKC1006-cy3 EUBmix-cy5 LKC1006-cy3 EUBmix-cy5 LKC59-cy3 EPS710-cy5 EUBmix-FP LKC59-cy3 LKC1006-cy5 EUBmix-FP LKC59-cy3 EUBmix-cy5 301 LKC59-cy3 EUBmix-cy5 LKC59-cy3 EUBmix-cy5 LKC59-cy3 EUBmix-cy5 LKC59-cy3 EUBmix-cy5 LKC59-cy3 EUBmix-cy5 LKC59-cy3 EUBmix-cy5 LKC59-cy3 EUBmix-cy5 LKC59-cy3 EUBmix-cy5 LKC59-cy3 EUBmix-cy5 LKC59-cy3 EUBmix-cy5 FA% 30 4 28 30 (29-03-02) quantification; no hybridized filaments Table B-1: Continued Probe Target LKC59-cy3 LKC group I EUBmix-cy5 LKC59-cy3 LKC group I EUBmix-cy5 a Samples numbers are listed on Table B-2. Comments (29-03-02) quantification; nothing obvious 302 Table B-2: Sample numbers listed in Table B-1 and corresponding location and microbial mat morphotype. Samples that were labeled 1 though 29 were fixed in the Munich laboratory within two days after collection, while other samples were fixed in the field immediately. 303 Sample 1 3 4 9 10 11 12 15 20 22 28 29 203g 198w 189w 189g Location in Cave 248 248 248 190 190 194 194 198 203 203 123 123 203 198 189 189 Mat type White filaments in orifice White and yellow mat near orifice Gray mat beneath sample #3 Gray orifice sediment White filaments floating in orifice water White filaments, side of channel Gray filaments in center of channel White and gray filaments in channel White surface webs on mat Gray mat beneath white Gray sediment near orifice White filaments floating in water near orifice Gray filaments beneath white, fixed later White filaments in channel White filaments floating in orifice water Gray orifice sediment APPENDIX C Most Probable Number Results Chapter 4 Supplement Appendix C Table of Contents............................................................................ 304 Locations of Sampling Sites................................................................................ 305 March 2001 and August 2001 MPN Fermenters/Heterotrophs Results.............. 306 August 2002 Fermenters and Iron-reducers MPN Results.................................. 307 August 2001 Sulfate-reducing bacteria MPN Results and August 2003 Sulfurreducing bacteria MPN Results........................................................................ 308 August 2001 and 2002 Acetate Sulfate-reducers and Methanogens MPN Results .............................................................................................................. 309 August 2001 and 2002 Lactate/formate and hydrogen Sulfate-reducers and Methanogens MPN Results.............................................................................. 310 MPN Results from 2001 and 2002 for Sulfur-oxidizing Bacteria ...................... 311 304 Locations of sampling sites meter 118 125 189 193 195 197 198 201 203 203 203 203 203 203 248 248 Cave-wall Hellspont PBS location Fissure Spring orifice Fissure Spring stream Upper Spring orifice Upper Spring stream Upper Spring stream Upper Spring stream Upper Spring stream Upper Spring stream Upper Spring stream Upper Spring stream Upper Spring stream Upper Spring stream mat type water, gray sediment, white filaments water, gray sediment, white filaments water, gray sediment, white filaments gray filaments gray filaments white filaments gray filaments, white filaments gray filaments, white filaments white webs, white filaments, gray filaments knobby web white filaments white feathers yellow patches on mat surface Upper Spring stream gray filaments, directly under white Upper Spring stream gray filaments, deeper in mat Lower Spring orifice water, gray sediment, white filaments Lower Spring orifice white filaments, gray filaments, yellow 175 m cave wall biofilm gray filaments and sediment from back of cave white filaments and mat mixutre from surface spring 305 Most Probable Number Results for Lower Kane Cave and Hellspont Cave March 2001 Fermenters meter 216 (red) 215 (water) 203 (white) 189 (black) cells ml-1 7.60E+00 4.00E+03 5.40E+03 2.60E+02 July-August 2001 Fermenters meter orifice black white stream red 248 (Lspg gray) 248 (LSpg white) Hellspont PBS Spg cell ml-1 8.20E+02 7.60E+00 5.10E+03 2.70E+03 1.10E+02 1.60E+02 1.30E+02 1.50E+04 1.30E+06 95% UL 1.60E+01 0.37 1.10E+02 5.50E+02 2.10E+01 3.10E+01 2.50E+01 2.70E+03 2.50E+05 95%LL 4.30E+00 0.15 2.30E+01 1.40E+02 5.80E+00 8.70E+00 7.10E+00 8.60E+02 7.10E+04 March 2001 Heterotrophs meter 216 215 203 189 cells ml-1 4.00E+04 1.50E+02 4.00E+03 2.80E+03 August 2001 Heterotrophs meter 189 (water) 189 (gray) 203 (white) 215 (water) 216 (red) Hellspont PBS Spring 248 (gray) 248 (white) Cave-wall biofilm cells ml-1 3.60E+07 1.20E+01 7.20E+08 2.20E+03 9.30E+00 2.20E+03 1.60E+06 7.20E+09 3.70E+00 0.024 95% UL 6.40E+07 2.40E+00 1.20E+09 4.00E+03 4.7 4.00E+03 3.10E+06 1.20E+10 1.20E+01 0.006 95%LL 2.00E+06 0.59 4.20E+08 1.20E+03 0.18 1.20E+03 8.60E+05 4.20E+09 1.20E+00 0.096 306 August 2002 Fermenters meter 118water 118g 125w 189water 189g 193g 195w 195g 198w 198g 201w 201g 203kw 203kw 203fe 203fil 203y 203g 203g 203dg 215water 248water 248g 5.90E+06 5.90E+04 5.70E+06 5.90E+04 5.90E+06 5.90E+04 5.70E+04 5.90E+04 2.40E+02 2.90E+03 5.90E+05 1.10E+07 1.10E+05 1.10E+07 1.10E+05 1.10E+07 1.10E+05 1.10E+05 1.10E+05 4.50E+02 5.40E+03 1.10E+06 2.90E+06 2.90E+04 2.90E+06 2.90E+04 2.90E+06 2.90E+04 2.90E+04 2.90E+04 1.20E+02 1.50E+03 2.90E+05 cells ml-1 1.50E-02 5.90E+04 5.90E+04 3.10E-02 5.70E+03 2.80E+02 UL 1.10E-01 1.10E+05 1.10E+05 1.20E-01 1.10E+04 5.30E+02 LL 2.10E-03 2.90E+05 2.90E+04 0.0077 2.90E+03 1.50E+02 5.90E+06 4.30E+06 1.00E+06 8.10E+06 2.90E+06 2.20E+06 August 2001 Iron-reducers meter 189 water 189 gray sediment 203 white mat 215 water 248 gray sediment 248 white mats Hellspont Cave PBS Spring cells ml-1 0.023 7.70E+01 1.60E+02 4.10E-01 1.60E+02 1.60E+02 2.80E+03 2.70E+03 UL 0.07 1.60E+01 3.10E+02 9.90E-01 3.10E+02 3.10E+02 5.50E+03 LL 0.0072 3.80E+00 8.70E+01 1.70E-01 8.70E+01 8.70E+01 1.40E+03 307 August 2001 MPN Results location U.S. orifice U.S. black U.S. white stream red L.S. black L.S. white Hellspont PBS Spring H-SRB 2.40E+00 1.00E+00 0.01 0.45 0.00 1.90E+03 1.50E+01 3.30E+04 5.70E+05 95% UL 0.96 2.30 0.10 0.14 1.40E-01 3.70E+03 3.10E+01 6.10E+04 95%LL 0.06 0.46 0.00 0.014 1.50E-02 9.40E+02 7.60E+00 1.70E+04 Ac-SRB 1.00E+01 1.90E+01 2.20E+04 5.70E+05 1.00E+01 2.90E+02 2.60E+04 5.70E+05 5.70E+05 95% UL 2.30E+01 37.00 4.00E+04 2.30E+00 5.60E+02 4.60E+04 95%LL 4.60E+00 9.40E+00 1.20E+04 0.46 1.60E+02 1.40E+04 August 2001 MPN Results location U.S. orifice U.S. black U.S. white stream red L.S. black L.S. white Hellspont PBS Spring LF-SRB 0.63 2.20E+01 1.00E+02 1.90E+02 0.46 2.20E+02 1.90E+02 5.40E+05 5.70E+05 95% UL 1.70 4.30E+01 2.30E+02 3.70E+02 1.40 430.00 3.70E+02 1.10E+05 95%LL 0.23 1.20E+01 4.60E+00 9.40E+01 0.15 100.00 9.40E+01 2.70E+04 TSRB 1.30E+01 2.20E+02 4.40E+05 5.70E+05 1.10E+00 1.40E+05 2.60E+05 5.70E+05 5.70E+05 95% UL 2.70E+01 4.30E+02 8.00E+05 2.30E+00 7.70E+05 5.00E+05 95%LL 6.00E+00 1.20E+02 2.40E+05 4.60E-01 2.20E+04 1.40E+05 August 2003 SoRB location 189 orifice water 189 gray sediment 193 gray filaments 195 white filaments 195 gray filaments 198 white filaments 198 gray filaments 201 white filaments 201 gray filaments 203 white webs 203 filaments 203 gray filaments 215 stream water cells ml-1 0 0.023 5.20E+00 1.70E+02 9.30E+01 5.20E+00 5.20E+00 1.70E+02 9.30E+01 1.70E+02 9.30E+01 1.70E+02 0 0.16 2.10E+01 3.00E+02 2.10E+01 2.10E+01 3.00E+02 3.00E+02 0.0032 1.30E+00 2.90E+01 1.30E+00 1.30E+00 2.90E+01 2.90E+01 UL LL 308 August 2002 SRB Acetate meter 118water 118g 125w 189water 189g 193g 195w 195g 198w 198g 201w 201g 203kw 203kw 203fe 203fil 203y 203g 203g 203dg 215water 248water 248g 0.015 5.70E+03 1.60E+01 6.00E+03 1.40E+01 5.90E+06 1.00E+06 3.60E+04 2.00E+03 5.90E+06 4.50E+05 5.90E+05 6.00E+06 5.90E+05 3.60E+06 1.1 1.10E+04 3.30E+01 1.20E+04 3.00E+01 1.80E+06 6.70E+04 4.00E+03 8.50E+05 0.0021 2.90E+03 8.00E+00 3.00E+03 6.60E+00 6.10E+05 1.90E+04 1.00E+03 2.40E+05 SRB cells ml1 UL 95% LL 95% 6.70E+06 1.90E+06 3.20E-01 0 5.90E+05 7.70E-01 1.30E-01 August 2001 Methanogens meter orifice black white stream red Hellspont PBS Spring 248 (Lspg gray) 248 (LSpg white) H-Met 0.00E+00 0.00E+00 1.40E-02 1.40E-02 0.00E+00 3.30E+02 2.30E+03 0.00E+00 0.00E+00 95% UL 1.40E-01 1.40E-01 1.00E-01 1.00E-01 1.40E-01 6.10E+02 4.40E+03 1.40E-01 1.40E-01 95% LL 1.50E-02 1.50E-02 0.002 0.002 1.50E-02 1.70E+02 1.20E+03 1.50E-02 1.50E-02 Ac-Met 0.00E+00 1.40E-02 0.00E+00 0.00E+00 0.00E+00 1.50E+01 2.20E+02 0.00E+00 0.00E+00 95% UL 1.40E-01 1.00E-01 1.40E-01 1.40E-01 1.40E-01 3.10E+01 4.30E+02 1.40E-01 1.40E-01 95%LL 1.50E-02 0.002 1.50E-02 1.50E-02 1.50E-02 7.60E+00 1.20E+02 1.50E-02 1.50E-02 309 August 2002 SRB Lactate/Formate meter 118water 118g 125w 189water 189g 193g 195w 195g 198w 198g 201w 201g 203kw 203kw 203fe 203fil 203y 203g 203g 203dg 215water 248water 248g 0.015 4.30E+03 2.00E+02 3.60E+02 0.05 5.90E+06 5.90E+05 3.60E+04 1.70E+03 5.90E+06 6.00E+06 5.90E+05 2.90E+05 5.90E+05 1.70E+03 1.1 8.10E+03 3.90E+02 6.70E+02 0.15 0.0021 2.20E+03 1.00E+02 1.90E+02 0.016 0 6.70E-01 6.70E-01 1.70E+01 0.05 3.40E+03 9.60E+00 2.90E+02 2.00E+01 2.40E+02 5.50E+05 3.40E+03 1.60E+05 8.30E+02 3.60E+02 5.90E+05 1.70E+01 SRB cells ml1 Hydrogen UL 95% LL 95% cells ml-1 SRB UL 95% LL 95% 1.80E+00 1.80E+00 3.40E+01 0.15 6.40E+03 2.30E+01 5.50E+02 4.00E+01 4.70E+02 6.70E+02 3.40E+01 2.50E-01 2.50E-01 8.30E+00 0.016 1.80E+03 4.00E+00 1.60E+02 1.00E+01 1.30E+02 1.90E+02 8.30E+00 6.70E+04 3.40E+03 1.90E+04 8.30E+02 6.90E+01 0 5.90E+05 1.30E+02 3.70E+01 3.20E-01 0 5.30E+01 7.70E-01 1.10E+02 1.30E-01 2.60E+01 August 2001 Methanogens meter orifice black white stream red Hellspont PBS Spring 248 (Lspg gray) 248 (LSpg white) F-Met 0.00E+00 0.00E+00 0.00E+00 0.00E+00 0.00E+00 1.90E+01 1.50E+01 0.00E+00 2.90E-02 95% UL 1.40E-01 1.40E-01 1.40E-01 1.40E-01 1.40E-01 3.70E+01 3.10E+01 1.40E-01 1.20E-01 95%LL 1.50E-02 1.50E-02 1.50E-02 1.50E-02 1.50E-02 9.40E+00 7.60E+00 1.50E-02 7.20E-03 310 2001 S-Oxidizers orifice black white stream red Hellspont March 1.10E+01 1.00E+08 0.04 1.40E+06 UL 8.10E+01 1.60E+07 0.11 2.70E+06 LL 1.60E+00 2.70E+06 0.01 7.10E+05 August 8.20E+00 0.00E+00 1.90E+03 5.60E+00 5.80E+08 5.00E+06 0.00E+00 0.00E+00 0.00E+00 0.00E+00 UL 2.00E+01 1.40E-01 3.50E+03 1.60E+01 6.40E+08 1.30E+06 1.40E-01 1.40E-01 1.40E-01 1.40E-01 LL 3.40E+00 1.50E-02 1.00E+03 5.60E+00 2.00E+08 1.90E+05 1.50E-02 1.50E-02 1.50E-02 1.50E-02 PBS Spring Cave-wall biofilm 248 (gray) 248 (white) 2002 Sulfur-oxidizers meter 118g 125w 189g 193g 195w 195g 198w 198g 201w 201g 203kw 203kw 203fe 203fil 203y 203g 203g 203dg 248g cells ml 1.40E+00 2.40E+05 2.40E+01 2.10E+02 2.00E+01 1.50E+05 2.40E+02 2.40E+04 3.30E+03 2.80E+02 2.00E+01 2.00E+01 2.90E+02 2.00E+02 2.00E+01 2.80E+02 2.40E+01 2.10E+02 2.00E+01 UL 3.00E+00 4.40E+05 4.70E+01 3.90E+02 4.00E+01 3.00E+05 4.40E+02 4.70E+04 5.90E+03 5.10E+02 4.00E+01 4.00E+01 5.50E+02 5.10E+02 4.00E+01 5.10E+02 4.70E+01 3.90E+02 4.00E+01 LL 6.60E-01 1.30E+05 1.30E+01 1.10E+02 1.00E+01 7.90E+04 1.30E+02 1.30E+04 1.90E+03 1.60E+02 1.00E+01 1.00E+01 1.60E+02 1.60E+02 1.00E+01 1.60E+02 1.30E+01 1.10E+02 1.00E+01 311 APPENDIX D Chapter 6 Supplement Appendix D Table of Contents............................................................................ 312 FPD (Sulfur Gases) Results Table ..................................................................... 313 TCD (Carbon Dioxide) Results Table................................................................. 320 FID (Methane) Results Table .............................................................................. 324 TCD-HP (Oxygen) Results Table ...................................................................... 330 312 Appendix D - Chapter 5 Gas chromatography data from Nov 2003 FPD Results INJECT SIZE 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 12/2/2003 12/4/2003 250 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/4/2003 20:44:03 2:23:06 8:09:44 16:31:05 12:30:36 12:08:54 10:21:19 18:07:16 17:09:45 H2S H2S H2S H2S H2S H2S H2S H2S H2S 0 0 0 2.641 2.616 2.633 2.6 2.633 2.625 0 0 0 42.67 1038.64 55.95 5.25 441.88 94.04 ppm ppm ppm ppm ppm ppm ppm ppm ppm COS COS COS COS COS COS COS COS COS 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 ppm ppm ppm ppm ppm ppm ppm ppm ppm CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH 0 0 0 0 0 0 0 7.475 0 0 0 0 0 0 0 0 4.75 0 1 1 1 1 1 1 1 1 1 ppm ppm ppm ppm ppm ppm ppm ppm ppm DMS DMS DMS DMS DMS DMS DMS DMS DMS 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 2:15:30 8:04:27 16:26:23 12:24:20 12:04:10 17:53:08 17:04:53 H2S H2S H2S H2S H2S H2S H2S 2.65 2.633 0 0 0 0 0 3.77 2.56 0 0 0 0 0 ppm ppm ppm ppm ppm ppm ppm COS COS COS COS COS COS COS 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 ppm ppm ppm ppm ppm ppm ppm CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 ppm ppm ppm ppm ppm ppm ppm DMS DMS DMS DMS DMS DMS DMS 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm date time gas reten area conc units gas reten area conc units gas reten area conc units gas reten area conc units gas reten area conc SAMPLE Nov03-FPD61.chr Nov03-FPD106.chr Nov03-FPD158.chr Nov03-FPD197.chr Nov03-FPD237.chr Nov03-FPD368.chr Nov03-FPD428.chr A-1 A-1 A-1 A-1 A-1 A-1 A-1 A-1 DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Nov03-FPD24.chr Nov03-FPD62.chr Nov03-FPD107.chr Nov03-FPD159.chr Nov03-FPD198.chr Nov03-FPD238.chr Nov03-FPD283.chr Nov03-FPD369.chr Nov03-FPD429.chr A-2 A-2 A-2 A-2 A-2 A-2 A-2 A-2 A-2 Nov03-FPD25.chr Nov03-FPD63.chr Nov03-FPD108.chr Nov03-FPD160.chr Nov03-FPD199.chr Nov03-FPD239.chr Nov03-FPD284.chr Nov03-FPD370.chr Nov03-FPD430.chr 100 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/4/2003 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/1/2003 12/2/2003 12/4/2003 21:00:52 2:57:06 8:34:36 17:02:35 12:55:22 12:48:46 11:09:46 19:27:24 18:58:12 17:58:55 H2S H2S H2S H2S H2S H2S H2S H2S H2S H2S 0 0 2.616 2.633 2.616 0 0 2.625 2.6 2.608 0 0 50.75 34.74 7.79 0 0 8.06 10.42 17.36 COS COS COS COS COS COS COS COS COS COS 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm 20:54:50 2:35:47 8:24:58 16:50:56 12:48:29 12:33:54 10:49:20 18:43:54 17:42:59 H2S H2S H2S H2S H2S H2S H2S H2S H2S 0 2.625 2.583 2.583 2.616 2.583 2.575 2.616 2.441 0 12.4 26130.2 2871.07 7784.12 1503.09 14.6 422.18 93429.38 COS COS COS COS COS COS COS COS COS 0 4.075 0 4.258 0 4.191 4.058 0 0 0 3.77 0 8.79 0 2.74 2.39 0 0 1 1 1 6.672 1 3.109 2.877 1 1 ppm ppm ppm ppm ppm ppm ppm ppm ppm CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH 0 7.225 7.433 7.225 0 7.433 7.408 7.433 7.408 0 0 0 0 0 7.425 0 7.408 7.416 7.433 0 5.32 2.41 6.06 0 23.45 56.4 237.01 394.24 0 0 0 0 0 12.14 0 66.7 18.54 24.58 A-3 A-3 A-3 A-3 A-3 A-3 A-3 A-3 A-3 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/4/2003 20:49:46 2:29:01 8:15:11 16:40:21 12:40:16 12:18:57 10:32:23 18:27:04 17:24:32 H2S H2S H2S H2S H2S H2S H2S H2S H2S 0 0 2.625 2.608 2.6 2.6 2.575 2.6 2.516 0 0 1076.3 765.96 6248.68 19663.39 16.27 465.77 80749.2 10.2 10.163 9.737 1 1 1 1 1 1 1 1 1 19.858 95.79 22.375 10.682 61.82 28.45 1 1 1 97.617 204.82 348.37 998.706 COS COS COS COS COS COS COS COS COS 0 0 0 0 0 0 0 4.191 0 0 0 0 0 0 0 0 2.68 0 1 1 1 1 1 1 1 3.071 1 ppm ppm ppm ppm ppm ppm ppm ppm ppm CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH 0 0 0 0 0 7.416 7.408 7.433 7.433 0 0 0 0 0 5.56 12.77 129.59 224.71 1 1 1 1 1 11.183 15.159 41.767 54.103 1 1 6.775 11.092 19.482 28.53 55.488 70.851 1 1 1 1 1 14.861 ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm 30.779 ppm 17.63 ppm 19.854 ppm DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 5 1 1 1 1 1 1 1 1 5 1 1 1 1 1 1 1 1 1 1 ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 14.666 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 24.73 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 313 100 100 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/1/2003 12/2/2003 12/4/2003 17:12:45 13:04:06 13:04:19 11:18:06 20:05:10 19:13:33 18:08:45 H2S H2S H2S H2S H2S H2S H2S 2.55 2.591 2.591 2.625 2.441 2.425 2.458 11/23/2003 21:05:55 11/24/2003 3:06:11 11/24/2003 8:44:25 654.82 5522.48 754.84 125.22 111725.16 120501.83 122594.42 H2S H2S H2S 2.7 2.6 2.591 2.22 10.1 14141.16 COS COS COS ppm ppm ppm ppm ppm ppm ppm COS COS COS COS COS COS COS 0 0 0 0 4.15 3.95 4.2 4.175 4.158 4.2 0 0 0 0 6.62 2.58 3.06 8.38 4.32 5.47 1 ppm 1 ppm 1 ppm 1 5.68 3.001 3.319 6.486 4.157 4.921 ppm ppm ppm ppm ppm ppm ppm 100 11/23/2003 21:11:08 11/24/2003 3:12:17 11/24/2003 8:54:50 11/24/03 5:12 PM 11/25/03 1:04 PM 11/26/03 1:04 PM 11/28/03 11:18 AM 12/1/03 8:05 PM 12/2/03 7:13 PM 12/4/03 6:08 PM 11/23/03 8:00 PM 11/23/03 9:11 PM 11/24/03 3:12 AM 11/24/03 8:54 AM H2S H2S H2S 0 2.616 2.616 1697.00 2898.00 4338.00 7832.00 12682.00 14047.00 15911.00 1.00 71.00 431.00 1494.00 189.05 313.164 81.317 32.706 5461.798 5887.285 5988.732 1 0 1 27.36 16.583 26220.69 3291.4975 ppm ppm ppm COS COS COS 0 0 0 0 0 0 1 ppm 1 ppm 1 ppm Nov03-FPD26.chr Nov03-FPD64.chr Nov03-FPD109.chr Nov03-FPD161.chr Nov03-FPD200.chr Nov03-FPD240.chr Nov03-FPD285.chr Nov03-FPD371.chr Nov03-FPD431.chr A-4 A-4 A-4 A-4 A-4 A-4 A-4 A-4 A-4 Nov03-FPD27.chr Nov03-FPD65.chr Nov03-FPD110.chr Nov03-FPD162.chr Nov03-FPD201.chr Nov03-FPD241.chr Nov03-FPD286.chr Nov03-FPD331.chr Nov03-FPD372.chr Nov03-FPD432.chr A-5 A-5 A-5 A-5 A-5 A-5 A-5 A-5 A-5 A-5 Nov03-FPD28.chr Nov03-FPD66.chr Nov03-FPD111.chr A-6 A-6 A-6 cum time (min) 11/23/03 8:00 PM 11/24/03 2:15 AM 11/24/03 8:04 AM 11/24/03 4:26 PM 11/25/03 12:24 PM 11/26/03 12:04 PM 12/2/03 5:53 PM 12/4/03 5:04 PM 11/23/03 8:00 PM 11/23/03 8:44 PM 11/24/03 2:23 AM 11/24/03 8:09 AM 11/24/03 4:31 PM 11/25/03 12:30 PM 11/26/03 12:08 PM 11/28/03 10:21 AM 12/2/03 6:07 PM 12/4/03 5:09 PM 11/23/03 8:00 PM 11/23/03 8:49 PM 11/24/03 2:29 AM 11/24/03 8:15 AM 11/24/03 4:40 PM 11/25/03 12:40 PM 11/26/03 12:18 PM 11/28/03 10:32 AM 12/2/03 6:27 PM 12/4/03 5:24 PM 11/23/03 8:00 PM 11/23/03 8:54 PM 11/24/03 2:35 AM 11/24/03 8:24 AM 11/24/03 4:50 PM 11/25/03 12:48 PM 11/26/03 12:33 PM 11/28/03 10:49 AM 12/2/03 6:43 PM 12/4/03 5:42 PM 11/23/03 8:00 PM 11/23/03 9:00 PM 11/24/03 2:57 AM 11/24/03 8:34 AM 11/24/03 5:02 PM 11/25/03 12:55 PM 11/26/03 12:48 PM 11/28/03 11:09 AM 12/1/03 7:27 PM 12/2/03 6:58 PM 12/4/03 5:58 PM 11/23/03 8:00 PM 11/23/03 9:05 PM 11/24/03 3:06 AM 11/24/03 8:44 AM ppm ppm ppm ppm ppm ppm ppm 63.507 ppm 3960.104 ppm 1 1 ppm 12.796 ppm ppm 184.626 ppm 422.807 ppm 118.307 ppm 13.396 ppm 60.407 ppm 4574.831 ppm 1 1 ppm 1 ppm 21.389 ppm 18.235 ppm 11.536 ppm 1 ppm 1 ppm 11.607 ppm 12.254 ppm 14.153 ppm 1 9.616 ppm 12.165 ppm ppm CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH 0 0 0 0 7.416 7.416 7.425 7.4 7.416 7.425 0 0 0 0 0 0 0 27.18 8.56 482.94 2863.3 3739.37 4330.08 0 0 0 1 ppm 1 ppm 1 ppm 1 20.71 13.012 78.927 295.653 375.417 429.2 ppm ppm ppm ppm ppm ppm ppm 1 ppm 1 ppm 1 ppm DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS 0 0 0 0 10.333 10.316 0 10.325 10.325 10.325 0 0 0 0 0 0 0 7.43 8 0 47.55 47.73 52.15 0 0 0 1 ppm 1 ppm 1 ppm 1 ppm 10.334 ppm ppm ppm 23.527 ppm 23.571 ppm 24.553 ppm 1 ppm 1 ppm 1 ppm cum time (min) 1.00 375.00 1444.00 1950.00 2852.00 4270.00 13976.00 15847.00 1.00 44.00 383.00 1449.00 1954.00 2858.00 4274.00 7775.00 13981.00 15852.00 1.00 49.00 389.00 1455.00 1965.00 2868.00 4284.00 7786.00 14001.00 15867.00 1.00 54.00 395.00 1464.00 1675.00 2876.00 4299.00 7803.00 14017.00 15885.00 1.00 60.00 417.00 1474.00 1687.00 2889.00 4314.00 7823.00 12650.00 14032.00 15901.00 1.00 65.00 425.00 1484.00 DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS 0 0 0 0 0 0 0 0 0 14.57 0 0 0 0 0 0 0 0 0 0 0 0 72 0 0 0 0 0 0 0 0 0 0 0 0 Nov03-FPD163.chr Nov03-FPD202.chr Nov03-FPD242.chr Nov03-FPD287.chr Nov03-FPD332.chr Nov03-FPD373.chr Nov03-FPD433.chr A-6 A-6 A-6 A-6 A-6 A-6 A-6 Nov03-FPD29.chr Nov03-FPD67.chr Nov03-FPD112.chr A-7 A-7 A-7 0 0 0 SAMPLE 11/24/2003 17:24:07 11/24/2003 11/25/2003 11/25/2003 11/26/2003 11/28/2003 12/1/2003 12/2/2003 12/4/2003 11/23/2003 21:16:22 11/24/2003 3:24:51 D2 250 250 250 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/1/2003 12/2/2003 12/4/2003 9:04:57 17:42:06 13:39:50 13:38:59 12:00:51 20:50:59 19:48:37 18:58:56 H2S H2S H2S H2S H2S H2S H2S H2S 2.608 2.625 2.608 2.566 2.633 0 0 2.533 6.63 43.26 17.09 9.54 7.93 0 0 9.47 ppm ppm ppm ppm ppm ppm ppm ppm COS COS COS COS COS COS COS COS 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 ppm ppm ppm ppm ppm ppm ppm ppm CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH 0 0 7.416 7.391 7.416 7.433 7.425 7.408 0 0 3.23 3.46 19.87 414.12 345.82 139.3 0 0 9.29 9.481 18.147 72.661 66.443 43.2 ppm ppm ppm ppm ppm ppm ppm ppm DMS DMS DMS DMS DMS DMS DMS DMS 0 0 0 0 0 0 10.33 0 0 0 0 0 0 0 2.69 0 1 1 1 1 1 1 6.9 1 ppm ppm ppm ppm ppm ppm ppm ppm DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS H2S H2S 0 2.7 0 2.51 COS COS 0 0 0 0 1 ppm 1 ppm CH3SH CH3SH 0 0 0 0 0 ppm 0 ppm DMS DMS 0 0 0 0 1 ppm 1 ppm 17:35:58 13:25:07 13:30:28 13:24:18 11:37:50 20:27:08 19:30:48 18:40:21 H2S H2S H2S H2S H2S H2S H2S H2S 2.625 2.691 2.641 2.458 2.491 2.5 2.458 2.491 19347.1 2.56 21067.82 88644.86 94740.1 101224.29 105060.58 97804.95 COS COS COS COS COS COS COS COS 0 0 0 4.158 4.191 4.2 4.166 4.183 0 0 0 6.33 7.63 12.61 11.38 16.74 1 1 1 5.49 6.141 8.414 7.851 9.875 ppm ppm ppm ppm ppm ppm ppm ppm CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH 0 0 0 7.425 7.425 7.425 7.425 7.425 0 0 0 221.2 865.72 1534.54 1766.45 1384.68 1 1 1 53.696 113.778 174.673 195.788 161.028 ppm ppm ppm ppm ppm ppm ppm ppm DMS DMS DMS DMS DMS DMS DMS DMS 0 0 0 0 10.325 10.325 10.316 10.291 0 1 ppm 0 1 ppm 0 1 ppm 0 1 ppm 4.23 8.272 ppm 9.35 11.432 ppm 9.61 11.573 ppm 8.67 11.035 ppm COS 0 0 1 ppm CH3SH 0 0 1 ppm DMS 0 0 1 ppm DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS date time gas reten area conc units gas reten area conc units gas reten area conc units gas reten 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 11.4 0 0 area 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 112 0 0 conc 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Nov03-FPD164.chr 50 50 50 A-7 INJECT SIZE 100 cum time (min) 11/24/03 5:24 PM cum time gas reten area conc units (min) 1709.00 H2S 2.541 43385.81 5371.8825 ppm Nov03-FPD165.chr Nov03-FPD203.chr Nov03-FPD204.chr Nov03-FPD243.chr Nov03-FPD288.chr Nov03-FPD333.chr Nov03-FPD374.chr Nov03-FPD434.chr A-7 A-7 A-7 A-7 A-7 A-7 A-7 A-7 Nov03-FPD30.chr Nov03-FPD68.chr A-8 A-8 11/24/03 5:35 PM 11/25/03 1:25 PM 11/25/03 1:30 PM 11/26/03 1:24 PM 11/28/03 11:37 AM 12/1/03 8:27 PM 12/2/03 7:30 PM 12/4/03 6:40 PM 11/23/03 8:00 PM 11/23/03 9:16 PM 11/24/03 3:24 AM 1720.00 2919.00 2924.00 4364.00 7851.00 12714.00 14064.00 15943.00 1.00 76.00 443.00 9833.72 ppm ppm 10667.92 ppm 4342.881 ppm 4638.373 ppm 4952.723 ppm 5138.704 ppm 4786.955 ppm 1 1 ppm 9.717 ppm Nov03-FPD113.chr Nov03-FPD166.chr Nov03-FPD205.chr Nov03-FPD244.chr Nov03-FPD289.chr Nov03-FPD334.chr Nov03-FPD375.chr Nov03-FPD435.chr A-8 A-8 A-8 A-8 A-8 A-8 A-8 A-8 Nov03-FPD31.chr Nov03-FPD69.chr Nov03-FPD114.chr Nov03-FPD167.chr Nov03-FPD206.chr Nov03-FPD245.chr Nov03-FPD290.chr Nov03-FPD376.chr Nov03-FPD436.chr 100 50 50 A-9 A-9 A-9 A-9 A-9 A-9 A-9 A-9 A-9 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/4/2003 21:22:57 3:31:17 9:10:45 17:48:55 13:49:38 13:51:43 12:21:00 20:03:36 19:13:00 H2S H2S H2S H2S H2S H2S H2S H2S H2S 0 2.625 2.625 2.616 2.6 2.45 2.5 2.458 2.45 0 557.08 721.96 11641.38 23189.37 88643.38 93667.81 108564.95 104552.85 ppm ppm ppm ppm ppm ppm ppm ppm ppm COS COS COS COS COS COS COS COS COS 0 0 0 0 0 4.15 4.2 4.2 4.158 0 0 0 0 0 4.62 6.77 11.33 9.99 1 1 1 1 1 4.354 5.752 7.829 7.217 ppm ppm ppm ppm ppm ppm ppm ppm ppm CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH 0 0 0 0 0 7.441 7.425 7.425 7.425 0 0 0 0 0 23.8 75.93 389.89 418.46 1 1 1 1 1 19.598 32.659 70.455 73.056 ppm ppm ppm ppm ppm ppm ppm ppm ppm DMS DMS DMS DMS DMS DMS DMS DMS DMS 0 0 0 0 0 0 0 10.308 10.316 0 0 0 0 0 0 0 5.16 5.37 1 1 1 1 1 1 1 9.045 9.168 ppm ppm ppm ppm ppm ppm ppm ppm ppm DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS BIG 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 314 250 250 11/24/2003 3:41:07 11/24/2003 9:20:03 11/24/2003 17:59:10 12/4/2003 19:33:04 11/24/2003 2:09:21 11/24/2003 7:57:51 11/24/2003 20:15:37 11/28/2003 9:06:10 12/2/2003 9:25:26 H2S H2S H2S H2S H2S 0 0 2.808 2.408 0 0 0 4.73 2.62 0 ppm ppm ppm ppm ppm COS COS COS COS COS 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 H2S H2S H2S H2S 0 0 2.575 0 0 0 9.42 0 ppm ppm ppm ppm COS COS COS COS 0 0 0 0 0 0 0 0 1 1 1 1 ppm ppm ppm ppm ppm ppm ppm ppm ppm 250 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/3/2003 21:29:21 3:46:32 9:25:12 18:04:13 14:26:43 14:20:41 12:58:33 10:06:51 H2S H2S H2S H2S H2S H2S H2S H2S 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ppm ppm ppm ppm ppm ppm ppm ppm COS COS COS COS COS COS COS COS 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 ppm ppm ppm ppm ppm ppm ppm ppm 250 11/23/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 21:35:22 3:53:32 18:17:30 14:32:50 14:27:14 13:02:22 10:31:52 10:15:04 H2S H2S H2S H2S H2S H2S H2S H2S 0 0 0 2.625 0 2.633 0 2.633 0 0 0 28.54 0 329.87 0 31.22 ppm ppm ppm ppm ppm ppm ppm ppm COS COS COS COS COS COS COS COS 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 ppm ppm ppm ppm ppm ppm ppm ppm 100 11/23/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 21:50:17 4:09:55 18:27:56 14:48:21 14:41:01 11/23/03 8:00 PM 11/23/03 9:29 PM 11/24/03 3:46 AM 11/24/03 9:25 AM 11/24/03 6:04 PM 11/25/03 2:26 PM 11/26/03 2:20 PM 11/28/03 12:58 PM 12/3/03 10:06 AM 11/23/03 8:00 PM 11/23/03 9:35 PM 11/24/03 3:53 AM 11/24/03 6:17 PM 11/25/03 2:32 PM 11/26/03 2:27 PM 11/28/03 1:02 PM 12/2/03 10:31 AM 12/3/03 10:15 AM 11/23/03 8:00 PM 11/23/03 9:50 PM 11/24/03 4:09 AM 11/24/03 6:27 PM 11/25/03 2:48 PM 11/26/03 2:41 PM H2S H2S H2S H2S H2S 0 2.608 0 0 2.625 0 2016.86 0 0 30.39 ppm ppm ppm ppm ppm COS COS COS COS COS 1.00 89.00 465.00 1529.00 1743.00 2983.00 4420.00 7932.00 15012.00 1.00 94.00 472.00 1756.00 2990.00 4427.00 7936.00 13631.00 15021.00 1.00 109.00 488.00 1766.00 3006.00 4441.00 1 1 10.496 9.757 1 1 1 1 1 1 1 1 1 1 1 1 1 1 16.846 1 53.268 1 17.446 1 1 143.214 1 1 17.261 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 ppm ppm ppm ppm ppm Nov03-FPD70.chr Nov03-FPD115.chr Nov03-FPD168.chr Nov03-FPD437.chr A-10 A-10 A-10 A-10 11/24/03 9:04 AM 11/24/03 5:42 PM 11/25/03 1:39 PM 11/26/03 1:38 PM 11/28/03 12:00 PM 12/1/03 8:50 PM 12/2/03 7:48 PM 12/4/03 6:58 PM 11/23/03 8:00 PM 11/23/03 9:22 PM 11/24/03 3:31 AM 11/24/03 9:10 AM 11/24/03 5:48 PM 11/25/03 1:49 PM 11/26/03 1:51 PM 11/28/03 12:21 PM 12/2/03 8:03 PM 12/4/03 7:13 PM 11/23/03 8:00 PM 11/24/03 3:41 AM 11/24/03 9:20 AM 11/24/03 5:59 PM 12/4/03 7:33 PM CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 7.441 7.425 7.425 7.408 7.433 0 0 0 0 7.425 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 25.64 35.37 60.78 132.35 163.72 0 0 0 0 27.05 1 1 1 1 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 20.201 23.203 29.514 42.174 46.61 1 1 1 1 20.667 ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm 1504.00 1721.00 2938.00 4378.00 7874.00 12737.00 14076.00 15961.00 1.00 82.00 450.00 1510.00 1727.00 2948.00 4391.00 7895.00 14081.00 15976.00 1.00 460.00 1524.00 1738.00 15996.00 11.165 19.969 14.078 12.014 11.572 1 1 11.995 1 1 69.601 198.745 6098.04 11696.43 4342.809 4586.39 5308.593 5114.089 1 1 1 11.979 1 DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS 0 0 0 0 0 0 0 10.491 7.675 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3.39 3.16 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 0 0 0 7.55 9.228 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm DMDS DMDS DMDS DMDS DMDS DMDS DMDS CS2 DMS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS CS2 DMS DMS 1 1 1 1 ppm 1 ppm DMDS DMDS DMDS CS2 CS2 0 0 0 0 0 0 0 11.783 10.125 0 0 0 0 0 0 0 0 0 0 0 0 0 11.525 10.408 10.3 0 0 0 0 11.433 0 0 0 0 0 0 0 13.18 6.46 0 0 0 0 0 0 0 0 0 0 0 0 0 2.04 5.77 3.54 0 0 0 0 3.47 0 0 0 0 0 0 0 0 9.781 0 0 0 0 0 0 0 0 0 0 0 0 0 0 9.393 7.683 0 0 0 0 0 Nov03-FPD60.chr Nov03-FPD105.chr Nov03-FPD189.chr Nov03-FPD279.chr Nov03-FPD335.chr AIR AIR AIR AIR AIR Nov03-FPD32.chr Nov03-FPD71.chr Nov03-FPD116.chr Nov03-FPD169.chr Nov03-FPD209.chr Nov03-FPD247.chr Nov03-FPD292.chr Nov03-FPD382.chr B-1 B-1 B-1 B-1 B-1 B-1 B-1 B-1 Nov03-FPD33.chr Nov03-FPD72.chr Nov03-FPD171.chr Nov03-FPD210.chr Nov03-FPD248.chr Nov03-FPD293.chr Nov03-FPD337.chr Nov03-FPD383.chr B-2 B-2 B-2 B-2 B-2 B-2 B-2 B-2 Nov03-FPD34.chr Nov03-FPD74.chr Nov03-FPD172.chr Nov03-FPD212.chr Nov03-FPD249.chr B-3 B-3 B-3 B-3 B-3 SAMPLE 11/28/2003 13:24:29 12/2/2003 10:51:22 12/3/2003 10:30:07 H2S H2S H2S 2.558 2.575 2.591 46700 3.21 42.72 COS COS COS 0 0 0 0 0 0 1 ppm 1 ppm 1 ppm CH3SH CH3SH CH3SH 7.425 7.416 7.433 82.58 96.52 132.68 33.914 ppm 36.436 ppm 42.223 ppm DMS DMS DMS 0 0 0 0 0 0 1 1 1 DMDS DMS DMS INJECT SIZE date time gas reten area conc units gas reten area conc units gas reten area conc units gas reten area conc units gas reten 0 10.325 10.358 area 0 2.96 2.94 conc 0 7.177 7.168 Nov03-FPD294.chr Nov03-FPD338.chr Nov03-FPD384.chr B-3 B-3 B-3 Nov03-FPD35.chr Nov03-FPD75.chr Nov03-FPD119.chr Nov03-FPD173.chr Nov03-FPD213.chr Nov03-FPD250.chr Nov03-FPD295.chr Nov03-FPD339.chr Nov03-FPD385.chr 250 100 B-4 B-4 B-4 B-4 B-4 B-4 B-4 B-4 B-4 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 21:55:52 4:19:01 9:50:00 18:38:08 14:58:11 15:01:03 13:40:14 11:11:49 10:46:15 H2S H2S H2S H2S H2S H2S H2S H2S H2S 0 2.6 2.625 2.575 2.583 2.591 2.625 0 2.65 0 250.58 12294.77 821.38 282.53 82.63 23.59 0 478.47 COS COS COS COS COS COS COS COS COS 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 ppm ppm ppm ppm ppm ppm ppm ppm ppm CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH 0 0 0 0 7.408 7.416 7.433 7.416 7.433 0 0 0 0 13.57 30.84 72.62 180 222.29 1 1 1 1 15.538 21.911 31.985 48.72 53.823 ppm ppm ppm ppm ppm ppm ppm ppm ppm DMS DMS DMS DMS DMS DMS DMS DMS DMS 0 0 0 0 0 0 0 0 10.325 0 0 0 0 0 0 0 0 2.93 1 1 1 1 1 1 1 1 7.157 ppm DMDS DMDS DMDS DMDS DMDS DMS DMS DMS DMS 0 0 0 0 0 10.333 10.391 10.525 10.325 0 0 0 0 0 2.68 3.27 3.07 2.93 0 0 0 0 0 6.939 7.446 7.273 7.157 Nov03-FPD36.chr Nov03-FPD76.chr Nov03-FPD174.chr Nov03-FPD251.chr Nov03-FPD296.chr Nov03-FPD340.chr Nov03-FPD386.chr 250 B-5 B-5 B-5 B-5 B-5 B-5 B-5 11/23/2003 11/24/2003 11/24/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 22:01:45 4:24:39 18:48:52 15:14:27 13:58:38 11:50:54 11:01:11 H2S H2S H2S H2S H2S H2S H2S 0 0 0 0 0 0 0 0 0 0 0 0 0 0 COS COS COS COS COS COS COS 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 ppm ppm ppm ppm ppm ppm ppm CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH 0 0 0 7.416 7.416 7.191 7.45 0 0 0 11.22 9.63 3.81 6.62 1 1 1 14.422 13.663 9.777 11.831 ppm ppm ppm ppm ppm ppm ppm DMS DMS DMS DMS DMS DMS DMS 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ppm 0 ppm 0 DMDS DMDS DMDS DMDS DMS CS2 DMS 0 0 0 0 10.325 0 10.341 0 0 0 0 2.82 0 3.31 0 0 0 0 7.064 0 7.484 Nov03-FPD37.chr Nov03-FPD77.chr Nov03-FPD121.chr Nov03-FPD215.chr Nov03-FPD252.chr Nov03-FPD297.chr Nov03-FPD341.chr Nov03-FPD387.chr 250 B-6 B-6 B-6 B-6 B-6 B-6 B-6 B-6 11/23/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 22:06:59 4:30:32 10:04:16 15:35:21 15:34:17 14:17:56 12:10:16 11:15:21 H2S H2S H2S H2S H2S H2S H2S H2S 0 2.625 2.65 2.625 2.625 2.625 2.483 2.45 0 60.26 60.26 34.68 1046.35 4.29 75465.76 90361.91 COS COS COS COS COS COS COS COS 0 0 0 0 0 0 4.183 4.125 0 0 0 0 0 0 2.54 4.28 1 1 1 1 1 1 2.972 4.13 ppm ppm ppm ppm ppm ppm ppm ppm CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH 0 0 0 7.408 7.433 7.425 7.416 7.433 0 0 0 4.52 21.21 46.05 86.89 147.94 1 1 1 10.382 18.67 26.06 34.716 44.433 ppm ppm ppm ppm ppm ppm ppm ppm DMS DMS DMS DMS DMS DMS DMS DMS 0 0 0 0 0 0 10.3 10.341 0 0 0 0 0 0 3.78 3.69 0 0 0 0 0 0 7.884 ppm 7.804 ppm DMDS DMDS DMDS CS2 DMDS DMS DMS DMS 0 0 0 11.35 0 10.333 10.3 10.341 0 0 0 2.69 0 2.74 3.78 3.69 0 0 0 0 0 6.993 7.884 7.804 315 100 100 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 22:13:58 4:36:07 10:13:54 19:05:41 17:02:20 15:49:52 14:31:55 12:28:26 11:35:06 H2S H2S H2S H2S H2S H2S H2S H2S H2S 0 2.608 2.591 2.625 2.6 2.566 2.583 2.616 2.508 0 319.67 4715.74 183.98 87.56 1278.34 10.93 386.34 72655.36 COS COS COS COS COS COS COS COS COS 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ppm ppm ppm ppm ppm ppm ppm ppm ppm 250 250 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 22:18:59 4:41:38 10:24:56 19:17:25 17:24:57 16:03:02 14:52:58 12:48:22 11:50:27 H2S H2S H2S H2S H2S H2S H2S H2S H2S 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 COS COS COS COS COS COS COS COS COS 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ppm ppm ppm ppm ppm ppm ppm ppm ppm 250 100 50 100 100 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/28/2003 12/2/2003 12/3/2003 11/24/2003 11/24/2003 11/24/2003 11/28/2003 12/3/2003 5:06:54 10:52:55 19:24:49 15:27:31 12:22:59 H2S H2S H2S H2S H2S 2.525 2.5 2.625 2.583 2.6 5.27 5.19 5.38 12.18 18.86 22:24:45 4:47:35 4:57:48 5:02:22 10:31:41 17:39:08 15:08:22 13:00:26 12:04:14 H2S H2S H2S H2S H2S H2S H2S H2S H2S 0 2.533 2.566 2.641 2.533 2.458 2.416 2.508 2.391 0 60018.5 37842.06 8693 67171.91 105144.81 117171.04 95692.66 132370.1 COS COS COS COS COS COS COS COS COS COS COS COS COS COS 0 0 0 0 0 4.05 4.175 4.191 4.158 0 0 0 0 0 0 0 0 0 0 4 20.9 6.89 25.81 0 0 0 0 0 1 1 1 1 1 3.942 11.321 14.5125 12.706 1 1 1 1 1 ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm 250 250 250 Nov03-FPD38.chr Nov03-FPD78.chr Nov03-FPD122.chr Nov03-FPD176.chr Nov03-FPD216.chr Nov03-FPD253.chr Nov03-FPD298.chr Nov03-FPD342.chr Nov03-FPD388.chr B-7 B-7 B-7 B-7 B-7 B-7 B-7 B-7 B-7 CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH 0 0 0 0 0 7.425 7.433 7.425 7.425 0 0 0 0 0 0 0 7.408 7.433 0 0 0 0 0 7.416 7.416 7.45 7.425 0 0 0 0 0 0 0 0 0 0 15.12 44.04 103.46 204.55 0 0 0 0 0 0 0 2.88 5.22 0 0 0 0 0 22.7 78.54 14.63 110.26 0 0 0 0 0 1 1 1 1 1 16.272 25.553 37.624 51.755 1 1 1 1 1 1 1 8.989 10.975 1 1 1 1 1 19.236 33.163 40.105 38.755 1 1 1 1 1 ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 ppm 1 1 ppm 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ppm 1 1 1 1 1 1 1 1 DMDS DMDS CS2 DMDS DMS DMDS DMDS DMDS DMS DMDS DMDS DMDS DMDS DMDS DMDS DMS DMDS DMDS DMDS DMDS DMDS DMDS DMDS CS2 DMS DMDS DMS DMDS DMDS DMDS DMS DMDS 0 0 0 0 10.575 0 0 0 10.283 0 0 0 0 0 0 10.283 0 0 0 0 0 0 0 11.625 10.35 0 10.35 0 0 0 10.366 0 0 0 0 0 3.95 0 0 0 2.36 0 0 0 0 0 0 7.13 0 0 0 0 0 0 0 6.84 4.07 0 4.86 0 0 0 6.63 0 0 0 0 0 8.032 0 0 0 6.664 0 0 0 0 0 0 10.164 0 0 0 0 0 0 0 0 8.132 0 8.815 0 0 0 9.879 0 Nov03-FPD39.chr Nov03-FPD79.chr Nov03-FPD123.chr Nov03-FPD178.chr Nov03-FPD217.chr Nov03-FPD254.chr Nov03-FPD299.chr Nov03-FPD343.chr Nov03-FPD389.chr B-8 B-8 B-8 B-8 B-8 B-8 B-8 B-8 B-8 Nov03-FPD40.chr Nov03-FPD80.chr Nov03-FPD81.chr Nov03-FPD82.chr Nov03-FPD124.chr Nov03-FPD218.chr Nov03-FPD300.chr Nov03-FPD344.chr Nov03-FPD390.chr B-9 B-9 B-9 B-9 B-9 B-9 B-9 B-9 B-9 Nov03-FPD83.chr Nov03-FPD126.chr Nov03-FPD180.chr Nov03-FPD301.chr Nov03-FPD391.chr B-10 B-10 B-10 B-10 B-10 cum time (min) 11/28/03 1:24 PM 12/2/03 10:51 AM 12/3/03 10:30 AM 11/23/03 8:00 PM 11/23/03 9:55 PM 11/24/03 4:19 AM 11/24/03 9:50 AM 11/24/03 6:38 PM 11/25/03 2:58 PM 11/26/03 3:01 PM 11/28/03 1:40 PM 12/2/03 11:11 AM 12/3/03 10:46 AM 11/23/03 8:00 PM 11/23/03 10:01 PM 11/24/03 4:24 AM 11/24/03 6:48 PM 11/26/03 3:14 PM 11/28/03 1:58 PM 12/2/03 11:50 AM 12/3/03 11:01 AM 11/23/03 8:00 PM 11/23/03 10:06 PM 11/24/03 4:30 AM 11/24/03 10:04 AM 11/25/03 3:35 PM 11/26/03 3:34 PM 11/28/03 2:17 PM 12/2/03 12:10 PM 12/3/03 11:15 AM 11/23/03 8:00 PM 11/23/03 10:13 PM 11/24/03 4:36 AM 11/24/03 10:13 AM 11/24/03 7:05 PM 11/25/03 5:02 PM 11/26/03 3:49 PM 11/28/03 2:31 PM 12/2/03 12:28 PM 12/3/03 11:35 AM 11/23/03 8:00 PM 11/23/03 10:18 PM 11/24/03 4:41 AM 11/24/03 10:24 AM 11/24/03 7:17 PM 11/25/03 5:24 PM 11/26/03 4:03 PM 11/28/03 2:52 PM 12/2/03 12:48 PM 12/3/03 11:50 AM 11/23/03 8:00 PM 11/23/03 10:24 PM 11/24/03 4:47 AM 11/24/03 4:57 AM 11/24/03 5:02 AM 11/24/03 10:31 AM 11/25/03 5:39 PM 11/28/03 3:08 PM 12/2/03 1:00 PM 12/3/03 12:04 PM 11/23/03 8:00 PM 11/24/03 5:06 AM 11/24/03 10:52 AM 11/24/03 7:24 PM 11/28/03 3:27 PM 12/3/03 12:22 PM ppm 9.963 ppm 19.867 ppm 1 1 ppm 46.304 ppm 641.48 ppm 212.285 ppm 49.228 ppm 26.794 ppm 15.737 ppm 1 ppm 64.404 ppm 1 1 ppm 1 ppm 1 ppm 1 ppm 1 ppm 1 ppm 1 ppm 1 1 ppm 23.142 ppm 23.142 ppm 18.222 ppm 96.164 ppm 10.343 ppm 3703.967 ppm 4426.122 ppm 1 1 ppm 52.404 ppm 685.135 ppm 99.0125 ppm 27.51 ppm 107.411 ppm 12.392 ppm 57.723 ppm 3567.721 ppm 1 1 ppm 1 ppm 1 ppm 1 ppm 1 ppm 1 ppm 1 ppm 1 ppm 1 ppm 1 1 ppm 2955.094 ppm 4699.9875 ppm 4668.69 ppm 8254.7175 ppm 5142.787 ppm 5725.81 ppm 11711.383 ppm 6462.65 ppm 10.7 10.688 ppm 10.659 ppm 10.727 ppm 12.734 ppm 14.563 ppm cum time (min) 7968.00 13651.00 15036.00 1.00 114.00 498.00 867.00 1777.00 3016.00 4461.00 7984.00 13671.00 15042.00 1.00 120.00 503.00 1787.00 4474.00 8002.00 13710.00 15057.00 1.00 125.00 509.00 897.00 3053.00 4494.00 8011.00 13730.00 15071.00 1.00 132.00 515.00 906.00 1799.00 3209.00 4650.00 8026.00 13744.00 15091.00 1.00 137.00 520.00 917.00 1811.00 3231.00 4664.00 8047.00 13764.00 15106.00 1.00 143.00 526.00 536.00 541.00 924.00 3246.00 8063.00 13776.00 15120.00 1.00 545.00 945.00 1818.00 8074.00 15138.00 SAMPLE INJECT SIZE 11/23/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 11/23/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 23:03:13 5:45:32 11:49:00 18:07:09 17:05:07 16:03:04 13:30:58 12:45:31 H2S H2S H2S H2S H2S H2S H2S H2S 0 0 2.65 2.591 2.6 2.633 2.65 2.658 0 0 11.1 23.82 13.89 568.01 340.19 23.31 1 1 12.441 15.788 13.203 70.293 54.091 15.674 ppm ppm ppm ppm ppm ppm ppm ppm COS COS COS COS COS COS COS COS 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 ppm ppm ppm ppm ppm ppm ppm ppm CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ppm ppm ppm ppm ppm ppm ppm ppm DMS DMS DMS DMS DMS DMS DMS DMS 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 22:55:01 5:39:49 11:44:47 17:57:28 16:54:11 15:46:37 13:24:47 12:37:05 H2S H2S H2S H2S H2S H2S H2S H2S 0 0 0 0 2.566 2.641 0 0 0 0 0 0 12.08 5.74 0 0 1 1 1 1 1 1 1 1 1 ppm ppm ppm ppm ppm ppm ppm ppm COS COS COS COS COS COS COS COS 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 ppm ppm ppm ppm ppm ppm ppm ppm CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ppm ppm ppm ppm ppm ppm ppm ppm DMS DMS DMS DMS DMS DMS DMS DMS 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMS DMDS DMDS DMDS date time gas reten area conc units gas reten area conc units gas reten area conc units gas reten area conc units gas reten area conc Nov03-FPD45.chr Nov03-FPD89.chr Nov03-FPD132.chr Nov03-FPD219.chr Nov03-FPD257.chr Nov03-FPD302.chr Nov03-FPD346.chr Nov03-FPD392.chr C-1 C-1 C-1 C-1 C-1 C-1 C-1 C-1 0 0 0 0 0 0 0 0 0 0 0 0 10.183 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3.04 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 7.249 0 0 0 Nov03-FPD46.chr Nov03-FPD90.chr Nov03-FPD133.chr Nov03-FPD220.chr Nov03-FPD258.chr Nov03-FPD303.chr Nov03-FPD347.chr Nov03-FPD393.chr C-2 C-2 C-2 C-2 C-2 C-2 C-2 C-2 Nov03-FPD47.chr Nov03-FPD91.chr Nov03-FPD134.chr Nov03-FPD183.chr Nov03-FPD221.chr Nov03-FPD259.chr Nov03-FPD304.chr Nov03-FPD348.chr Nov03-FPD394.chr 100 C-3 C-3 C-3 C-3 C-3 C-3 C-3 C-3 C-3 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 23:08:28 5:50:54 11:53:05 19:36:15 18:16:38 17:20:05 16:19:14 13:44:53 12:57:12 H2S H2S H2S H2S H2S H2S H2S H2S H2S 0 0 0 0 2.591 2.658 2.625 2.65 2.625 0 0 0 0 5.63 4.6 338.91 63.34 21.09 ppm ppm ppm ppm ppm ppm ppm ppm ppm COS COS COS COS COS COS COS COS COS 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 ppm ppm ppm ppm ppm ppm ppm ppm ppm CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ppm ppm ppm ppm ppm ppm ppm ppm ppm DMS DMS DMS DMS DMS DMS DMS DMS DMS 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 316 12/2/2003 13:56:15 11/23/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 11/23/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 12/3/2003 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 1.00 H2S H2S H2S 0 0 0 0 0 0 11/24/2003 0:02:26 11/24/2003 6:18:38 11/24/2003 12:18:02 23:53:44 6:13:17 12:14:16 19:28:41 18:58:39 17:52:21 17:17:51 14:20:25 13:40:37 H2S H2S H2S H2S H2S H2S H2S H2S H2S 0 0 0 0 0 0 2.566 2.616 2.658 0 0 0 0 0 0 192.5 4435.59 2066.67 23:46:26 6:07:46 12:10:09 18:45:45 17:44:09 13:32:48 H2S H2S H2S H2S H2S H2S 0 0 0 0 0 0 0 0 0 0 0 0 ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm COS COS COS COS COS COS COS COS COS COS COS COS COS COS COS COS COS COS 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 23:40:24 6:02:27 12:05:29 18:34:10 17:37:58 16:48:52 14:05:59 13:18:55 H2S H2S H2S H2S H2S H2S H2S H2S 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ppm ppm ppm ppm ppm ppm ppm ppm COS COS COS COS COS COS COS COS 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 12/2/03 1:56 AM H2S 2.641 107.36 30.385 ppm COS 0 0 1 ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm 1 1 1 1 1 1 1 1 1 ppm ppm ppm ppm ppm ppm ppm ppm ppm 1 ppm 1 ppm 1 ppm 13832.00 1.00 219.00 601.00 1018.00 3299.00 4754.00 8163.00 13841.00 15193.00 1.00 225.00 606.00 1023.00 3310.00 4761.00 15207.00 1.00 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 11/23/03 11:40 PM 11/24/03 6:02 AM 11/24/03 12:05 PM 11/25/03 6:34 PM 11/26/03 5:37 PM 11/28/03 4:48 PM 12/2/03 2:05 PM 12/3/03 1:18 PM 11/23/03 8:00 PM 11/23/03 11:46 PM 11/24/03 6:07 AM 11/24/03 12:10 PM 11/25/03 6:45 PM 11/26/03 5:44 PM 12/3/03 1:32 PM 11/23/03 8:00 PM 11/23/03 11:53 PM 11/24/03 6:13 AM 11/24/03 12:14 PM 11/24/03 7:28 PM 11/25/03 6:58 PM 11/26/03 12:00 AM 11/28/03 5:17 PM 12/2/03 2:20 PM 12/3/03 1:40 PM 11/23/03 8:00 PM 11/24/03 12:02 AM 11/24/03 6:18 AM 11/24/03 12:18 PM 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 40.52 260.473 145.629 1 1 1 1 Nov03-FPD48.chr Nov03-FPD92.chr Nov03-FPD135.chr Nov03-FPD182.chr Nov03-FPD222.chr Nov03-FPD260.chr Nov03-FPD305.chr CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH C-4 C-4 C-4 C-4 C-4 C-4 C-4 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 23:21:02 5:56:34 11:58:02 19:32:48 18:26:20 17:28:05 16:33:18 cum time (min) 11/23/03 8:00 PM 11/23/03 10:55 PM 11/24/03 5:29 AM 11/24/03 11:44 AM 11/25/03 5:57 PM 11/26/03 4:54 PM 11/28/03 3:46 PM 12/2/03 1:24 PM 12/3/03 12:37 PM 11/23/03 8:00 PM 11/23/03 11:03 PM 11/24/03 5:45 AM 11/24/03 11:49 AM 11/25/03 6:07 PM 11/26/03 5:05 PM 11/28/03 4:03 PM 12/2/03 1:30 PM 12/3/03 12:45 PM 11/23/03 8:00 PM 11/23/03 11:08 PM 11/24/03 5:50 AM 11/24/03 11:53 AM 11/24/03 7:36 PM 11/25/03 6:16 PM 11/26/03 5:20 PM 11/28/03 4:19 PM 12/2/03 1:44 PM 12/3/03 12:57 PM 11/23/03 8:00 PM 11/23/03 11:21 PM 11/24/03 5:56 AM 11/24/03 11:58 AM 11/24/03 7:32 PM 11/25/03 6:26 PM 11/26/03 5:28 PM 11/28/03 4:33 PM H2S H2S H2S H2S H2S H2S H2S 0 0 0 0 0 2.625 2.625 0 0 0 0 0 83.77 184.34 1 1 1 1 10.812 10.452 53.988 59.1175 15.173 1 1 1 1 1 1 26.96 39.644 ppm ppm ppm ppm ppm ppm ppm COS COS COS COS COS COS COS 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 ppm ppm ppm ppm ppm ppm ppm CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm 1 1 1 1 1 1 1 1 1 ppm ppm ppm ppm ppm ppm ppm ppm ppm 1 ppm 1 ppm 1 ppm DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 ppm 1 1 1 1 1 1 ppm 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ppm 1 1 1 1 1 1 DMS DMDS DMDS DMDS DMDS CS2 DMDS DMDS DMDS DMDS DMDS CS2 DMDS DMDS DMDS DMS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS CS2 DMDS DMDS DMDS DMDS DMDS DMDS cum time (min) 1.00 174.00 568.00 997.00 3262.00 4715.00 8101.00 13800.00 15153.00 1.00 182.00 584.00 1002.00 3272.00 4722.00 8118.00 13806.00 15160.00 1.00 187.00 589.00 1006.00 1830.00 3281.00 4737.00 8134.00 13820.00 15172.00 1.00 200.00 595.00 1011.00 1834.00 3291.00 4745.00 8148.00 10.458 0 0 0 0 0 0 0 0 0 0 0 0 0 0 10.275 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3.43 0 0 0 0 0 0 0 0 0 0 0 0 0 0 3.46 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 7.682 0 0 0 0 0 0 0 0 0 0 0 0 0 0 7.614 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Nov03-FPD349.chr C-4 Nov03-FPD49.chr Nov03-FPD93.chr Nov03-FPD136.chr Nov03-FPD223.chr Nov03-FPD261.chr Nov03-FPD306.chr Nov03-FPD350.chr Nov03-FPD396.chr C-5 C-5 C-5 C-5 C-5 C-5 C-5 C-5 Nov03-FPD50.chr Nov03-FPD94.chr Nov03-FPD137.chr Nov03-FPD224.chr Nov03-FPD262.chr Nov03-FPD397.chr C-6 C-6 C-6 C-6 C-6 C-6 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 Nov03-FPD51.chr Nov03-FPD95.chr Nov03-FPD138.chr Nov03-FPD181.chr Nov03-FPD225.chr Nov03-FPD263.chr Nov03-FPD308.chr Nov03-FPD352.chr Nov03-FPD398.chr C-7 C-7 C-7 C-7 C-7 C-7 C-7 C-7 C-7 Nov03-FPD52.chr Nov03-FPD96.chr Nov03-FPD139.chr C-8 C-8 C-8 SAMPLE 11/26/2003 11/28/2003 12/2/2003 12/3/2003 11/24/2003 11/24/2003 11/24/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 11/24/2003 11/24/2003 11/26/2003 11/28/2003 12/3/2003 12/3/2003 12/4/2003 12/3/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/25/2003 pH 6:29:28 12:27:25 20:17:14 17:48:53 14:18:36 9:00:13 9:31:22 9:54:23 12:38:09 14:49:08 20:24:13 9:57:40 10:55:17 COS COS COS COS COS COS COS COS COS COS COS COS COS 0 0 0 0 0 4.308 3.991 4.225 4.175 4.166 4.183 4.158 4.191 0 0 0 0 0 4.24 7.05 7.12 717.25 565.64 457.26 346.02 140.75 1 1 1 1 1 4.105 5.88 5.909 70.999 66.523 59.629 51.611 32.209 ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH 0 0 0 0 0 7.516 7.466 0 7.391 7.416 7.416 7.4 7.416 0 0 0 0 0 3.38 3.6 0 570.2 359.42 327.77 190.95 108 1 1 1 1 1 9.415 9.599 1 68.509 68.556 65.567 50.575 38.396 ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS 0:09:34 6:24:00 12:23:53 19:51:11 17:38:06 14:39:31 14:06:58 COS COS COS COS COS COS COS 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 ppm ppm ppm ppm ppm ppm ppm CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 ppm ppm ppm ppm ppm ppm ppm DMS DMS DMS DMS DMS DMS DMS 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 ppm 0 0 1 ppm 0 0 1 0 0 1 0 0 1 10.3 425.89 69.319 ppm 0 0 1 0 0 1 18:03:42 17:27:40 14:30:57 13:55:01 COS COS COS COS 0 0 0 0 0 0 0 0 1 1 1 1 ppm ppm ppm ppm CH3SH CH3SH CH3SH CH3SH 0 0 0 0 0 0 0 0 1 1 1 1 ppm ppm ppm ppm DMS DMS DMS DMS 0 0 0 0 0 0 0 0 1 1 1 1 DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS INJECT SIZE date time gas reten area conc units gas reten area conc units gas reten area conc units gas reten 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 11.391 10.366 0 10.291 10.3 11.35 10.316 10.308 area 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 4 2.28 0 1372.63 498.29 2.25 58.98 171.66 conc 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 6.597 0 74.985 74.997 0 25.677 43.131 Nov03-FPD264.chr Nov03-FPD309.chr Nov03-FPD353.chr Nov03-FPD399.chr C-8 C-8 C-8 C-8 Nov03-FPD53.chr Nov03-FPD97.chr Nov03-FPD140.chr Nov03-FPD273.chr Nov03-FPD310.chr Nov03-FPD354.chr Nov03-FPD400.chr C-9 C-9 C-9 C-9 C-9 C-9 C-9 Nov03-FPD98.chr Nov03-FPD141.chr Nov03-FPD275.chr Nov03-FPD311.chr Nov03-FPD401.chr Nov03-FPD377.chr Nov03-FPD413.chr Nov03-FPD379.chr Nov03-FPD143.chr Nov03-FPD150.chr Nov03-FPD190.chr Nov03-FPD193.chr Nov03-FPD196.chr C-10 C-10 C-10 C-10 C-10 DRY BLANK DRY BLANK h2s STAND INTAKE TUBE INTAKE TUBE INTAKE TUBE INTAKE TUBE INTAKE TUBE cum time (min) 11/26/03 6:03 PM 11/28/03 5:27 PM 12/2/03 2:30 PM 12/3/03 1:55 PM 11/23/03 8:00 PM 11/24/03 12:09 AM 11/24/03 6:24 AM 11/24/03 12:23 PM 11/26/03 7:51 PM 11/28/03 5:38 PM 12/2/03 2:39 PM 12/3/03 2:06 PM 11/23/03 8:00 PM 11/24/03 6:29 AM 11/24/03 12:27 PM 11/26/03 8:17 PM 11/28/03 5:48 PM 12/3/03 2:18 PM 317 7.14 7.06 7.12 7.02 6.74 6.65 6.64 6.53 6.36 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/1/2003 12/2/2003 12/3/2003 22:36:08 5:17:29 11:07:27 15:50:32 20:36:28 18:11:08 20:45:29 14:35:26 16:55:29 16:12:55 COS COS COS COS COS COS COS COS COS COS 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 D5 7.33 1 1 1 1 1 1 1 1 1 1 ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm 7.02 6.95 6.92 6.8 6.78 6.53 6.48 6.41 11/23/2003 22:48:21 11/24/2003 5:28:36 11/24/2003 11:27:04 11/24/2003 16:10:56 H2S H2S H2S H2S 0 0 2.633 2.65 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 12/1/2003 12/2/2003 12/3/2003 0 0 1249.04 33.59 22:42:37 5:22:48 11:17:31 16:00:06 19:56:29 20:45:04 18:18:02 14:53:57 17:11:20 16:23:07 H2S H2S H2S H2S H2S H2S H2S H2S H2S H2S 0 0 2.6 2.616 2.7 2.608 0 0 2.616 0 0 0 3078.81 16705.94 17.75 201.3 0 0 68.42 0 ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm COS COS COS COS COS COS COS COS COS COS COS COS COS COS 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm 11/23/03 8:00 PM 11/23/03 10:42 PM 11/24/03 5:22 AM 11/24/03 11:17 AM 11/24/03 4:00 PM 11/24/03 7:56 PM 11/25/03 8:45 PM 11/26/03 6:18 PM 12/1/03 2:53 PM 12/2/03 5:11 PM 12/3/03 4:23 PM 11/23/03 8:00 PM 11/23/03 10:48 PM 11/24/03 5:28 AM 11/24/03 11:27 AM 11/24/03 4:10 PM 1.00 162.00 565.00 920.00 1197.00 1421.00 3450.00 4775.00 12172.00 13827.00 15379.00 1.00 168.00 571.00 930.00 1207.00 1 1 1 194.697 855.331 14.26 41.465 1 1 24.483 1 1 1 1 105.991 17.978 Nov03-FPD41.chr Nov03-FPD84.chr Nov03-FPD127.chr Nov03-FPD153.chr Nov03-FPD184.chr Nov03-FPD231.chr Nov03-FPD271.chr Nov03-FPD318.chr Nov03-FPD325.chr Nov03-FPD363.chr Nov03-FPD408.chr CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH M-1 M-1 M-1 M-1 M-1 M-1 M-1 M-1 M-1 M-1 M-1 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/1/2003 12/2/2003 12/3/2003 22:30:09 5:12:09 10:57:13 15:45:50 19:40:39 20:24:22 19:26:02 20:34:08 14:21:22 16:50:51 16:06:49 COS COS COS COS COS COS COS COS COS COS COS 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 7.425 7.441 7.425 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 11.19 21.19 34.88 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 14.41 18.659 23.062 ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 ppm 1 1 1 1 1 1 1 1 1 1 1 1 ppm 1 1 1 1 1 1 DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Nov03-FPD42.chr Nov03-FPD85.chr Nov03-FPD128.chr Nov03-FPD154.chr Nov03-FPD232.chr Nov03-FPD265.chr Nov03-FPD319.chr Nov03-FPD326.chr Nov03-FPD364.chr Nov03-FPD409.chr M-2 M-2 M-2 M-2 M-2 M-2 M-2 M-2 M-2 M-2 11/23/03 8:00 PM 11/23/03 10:30 PM 11/24/03 5:12 AM 11/24/03 10:57 AM 11/24/03 3:45 PM 11/24/03 7:40 PM 11/25/03 8:24 PM 11/26/03 7:26 PM 11/28/03 8:34 PM 12/1/03 2:21 PM 12/2/03 4:50 PM 12/3/03 4:06 PM 11/23/03 8:00 PM 11/23/03 10:36 PM 11/24/03 5:17 AM 11/24/03 11:07 AM 11/24/03 3:50 PM 11/25/03 8:36 PM 11/26/03 6:11 PM 11/28/03 8:45 PM 12/1/03 2:35 PM 12/2/03 4:55 PM 12/3/03 4:12 PM cum time gas reten area conc units (min) H2S 2.525 15.45 13.629 ppm H2S 2.591 2.71 9.787 ppm H2S 0 0 1 ppm H2S 0 0 1 ppm 1.00 1 H2S 0 0 1 ppm H2S 0 0 1 ppm H2S 0 0 1 ppm H2S 2.508 3.92 10.214 ppm H2S 2.591 1026 95.178 ppm H2S 2.65 10919.29 574.798 ppm H2S 2.633 29295.99 1465.688 ppm 1.00 1 628.00 H2S 0 0 1 ppm 1040.00 H2S 0 0 1 ppm H2S 0 0 1 ppm 8223.00 H2S 0 0 1 ppm 15253.00 H2S 0 0 1 ppm H2S 2.8 4.44 10.396 ppm H2S 2.416 2.8 9.819 ppm H2S 2.625 774.53 82.399 ppm H2S 2.625 5.44 10.747 ppm H2S 0 0 1 ppm H2S 2.491 8.88 11.832 ppm H2S 2.8 2.46 9.702 ppm H2S 0 0 1 ppm 1.00 H2S 41.1 150.00 H2S 2.608 198.69 41.185 ppm 555.00 H2S 2.616 104.84 30.019 ppm 900.00 H2S 2.666 43.56 20.025 ppm 1182.00 H2S 2.625 16.96 14.044 ppm 1507.00 H2S 2.683 7.08 11.322 ppm 3431.00 H2S 0 0 1 ppm 4813.00 H2S 0 0 1 ppm 8035.00 H2S 0 0 1 ppm 12136.00 H2S 0 0 1 ppm 13806.00 H2S 0 0 1 ppm 15362.00 H2S 0 0 1 ppm 1.00 1 156.00 H2S 0 0 1 ppm 560.00 H2S 0 0 1 ppm 910.00 H2S 2.608 5551.21 314.557 ppm 1187.00 H2S 2.641 106.54 30.265 ppm 3441.00 H2S 2.616 3.62 10.108 ppm 4768.00 H2S 0 0 1 ppm 8046.00 H2S 0 0 1 ppm 12150.00 H2S 0 0 1 ppm 13811.00 H2S 2.666 226.19 43.951 ppm 15368.00 H2S 2.65 1108.11 99.159 ppm Nov03-FPD43.chr Nov03-FPD86.chr Nov03-FPD129.chr Nov03-FPD155.chr Nov03-FPD186.chr Nov03-FPD233.chr Nov03-FPD266.chr Nov03-FPD327.chr Nov03-FPD365.chr Nov03-FPD410.chr M-3 M-3 M-3 M-3 M-3 M-3 M-3 M-3 M-3 M-3 CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH 0 0 0 0 0 0 0 0 7.433 7.366 0 0 0 0 0 0 0 0 0 0 0 0 12.94 28.37 0 0 0 0 1 1 1 1 1 1 1 1 15.241 21.1 1 1 1 1 ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 ppm DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Nov03-FPD44.chr Nov03-FPD87.chr Nov03-FPD130.chr Nov03-FPD156.chr M-4 M-4 M-4 M-4 SAMPLE 11/24/2003 11/25/2003 11/28/2003 12/1/2003 12/2/2003 12/3/2003 20:00:55 20:54:40 21:26:05 18:33:14 17:27:13 16:35:02 H2S H2S H2S H2S H2S H2S 2.65 2.6 0 0 2.6 2.65 8353.19 9.8 0 0 9.56 8.04 ppm ppm ppm ppm ppm ppm COS COS COS COS COS COS 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 ppm ppm ppm ppm ppm ppm CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH 0 0 7.416 7.433 7.425 7.458 0 0 14.22 35.43 42.8 58.5 1 1 15.847 23.221 25.243 29.002 ppm ppm ppm ppm ppm ppm DMS DMS DMS DMS DMS DMS 0 0 0 0 0 0 0 0 0 0 0 0 1 1 ppm 1 1 1 1 DMDS DMDS DMDS DMDS DMDS DMDS INJECT SIZE date time gas reten area conc units gas reten area conc units gas reten area conc units gas reten area conc units gas reten 0 0 0 0 0 0 area 0 0 0 0 0 0 conc 0 0 0 0 0 0 Nov03-FPD187.chr Nov03-FPD234.chr Nov03-FPD321.chr Nov03-FPD328.chr Nov03-FPD366.chr Nov03-FPD411.chr M-4 M-4 M-4 M-4 M-4 M-4 cum time (min) 11/24/03 8:00 PM 11/25/03 8:54 PM 11/28/03 9:26 PM 12/1/03 6:33 PM 12/2/03 5:27 PM 12/3/03 4:35 PM 11/23/03 8:00 PM 11/24/03 5:34 AM 11/24/03 11:37 AM 11/24/03 4:20 PM 11/24/03 8:11 PM 11/25/03 9:06 PM 11/26/03 7:00 PM 11/28/03 9:48 PM 12/1/03 6:53 PM 12/2/03 5:43 PM 12/3/03 4:52 PM ppm cum time (min) 1424.00 3459.00 8087.00 12392.00 13843.00 15391.00 1.00 174.00 940.00 1217.00 1435.00 1490.00 4817.00 8109.00 12412.00 13859.00 15408.00 ppm ppm ppm ppm ppm ppm ppm Nov03-FPD88.chr Nov03-FPD131.chr Nov03-FPD157.chr Nov03-FPD188.chr Nov03-FPD235.chr Nov03-FPD269.chr Nov03-FPD322.chr Nov03-FPD329.chr Nov03-FPD367.chr Nov03-FPD412.chr Nov03-FPD151.chr Nov03-FPD191.chr Nov03-FPD211.chr Nov03-FPD144.chr Nov03-FPD152.chr Nov03-FPD192.chr Nov03-FPD194.chr Nov03-FPD195.chr Nov03-FPD236.chr Nov03-FPD280.chr Nov03-FPD281.chr Nov03-FPD323.chr Nov03-FPD324.chr Nov03-FPD336.chr 12/3/2003 11/24/2003 11/24/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 11/24/2003 11/24/2003 11/24/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 0:55:51 7:02:11 19:32:27 19:32:41 18:46:16 15:20:38 15:04:16 H2S H2S H2S H2S H2S H2S H2S 0 2.6 2.591 2.608 2.633 2.391 2.458 0 64.72 160.78 156.47 41.22 100270.95 103325.71 1 23.874 37.03 36.523 19.582 4906.505 5054.598 ppm ppm ppm ppm ppm ppm ppm COS COS COS COS COS COS COS 0 0 0 0 4.258 4.133 4.175 0:34:08 6:49:12 13:18:44 19:06:18 18:29:29 15:00:50 14:43:55 H2S H2S H2S H2S H2S H2S H2S 0 2.591 0 2.591 2.575 2.5 2.483 0 40.63 0 37590.4 397.96 106446.95 111671.57 1 ppm 19.471 ppm ppm ppm 58.593 ppm 5205.913 ppm 5459.2 ppm COS COS COS COS COS COS COS 0 0 4.141 4.275 4.158 4.191 4.191 0 0 3.62 2.95 3.24 4.68 4.6 0 0 0 0 3.68 4.23 5.38 1 1 4.612 3.249 3.441 4.393 4.3 1 1 1 1 3.728 4.096 4.856 0:16:11 6:35:47 18:41:59 18:11:26 16:35:19 14:25:33 H2S H2S H2S H2S H2S H2S 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 ppm ppm ppm ppm ppm ppm COS COS COS COS COS COS 0 0 0 0 0 0 0 0 0 0 0 0 1 1 1 1 1 1 ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm 9:20:47 H2S 0 0 0 ppm COS 4.15 472.49 60.631 ppm CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH 7.425 7.4 7.425 7.416 7.433 7.441 7.425 7.408 7.416 7.425 7.425 7.408 7.425 7.433 7.45 7.425 7.425 7.425 7.416 7.4 7.416 361.13 195.95 244.04 98.12 37.27 10.16 8.95 4.78 14.95 66.8 911.73 1845.83 3314.53 3968.38 4.03 21.58 195.19 720.23 1395.15 2347.12 2675.2 ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS 0 10.3 10.308 10.316 10.308 0 398.56 452.69 429.16 320.99 M-5 M-5 M-5 M-5 M-5 M-5 M-5 M-5 M-5 M-5 MIDPOINT MIDPOINT MIX 33 STAND? OUTTAKE TUBE OUTTAKE TUBE OUTTAKE TUBE OUTTAKE TUBE s-GAS STAND s-GAS STAND s-GAS STAND s-GAS STAND s-GAS STAND s-GAS STAND s-GAS STAND 11/24/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/1/2003 12/2/2003 12/3/2003 11/24/2003 11/24/2003 11/25/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/25/2003 11/26/2003 11/28/2003 11/28/2003 12/1/2003 12/1/2003 12/2/2003 5:34:09 11:37:39 16:20:30 20:11:16 21:06:57 19:00:32 21:48:05 18:53:40 17:43:03 16:52:34 15:08:06 20:46:42 14:42:38 12:57:58 15:30:52 21:09:15 10:24:12 10:39:45 11:43:48 9:26:27 9:40:55 13:40:47 13:58:56 9:56:01 H2S H2S H2S H2S H2S H2S H2S H2S H2S H2S H2S H2S H2S H2S H2S H2S H2S H2S H2S H2S H2S H2S H2S H2S 0 0 0 0 0 0 0 0 0 0 2.583 0 0 0 0 0 0 0 2.525 2.391 2.408 2.433 2.758 2.85 0 0 0 0 0 0 0 0 0 0 4.16 0 0 0 0 0 0 0 12.13 3.48 7.72 2.44 11.68 2.67 450.395 12.085 1 1 12.019 11.604 1 1 1 1 1 1 1 1 1 1 1 10.296 0 0 0 0 0 0 0 12.721 10.058 11.514 9.692 12.599 9.775 ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm COS COS COS COS COS COS COS COS COS COS COS COS COS COS COS COS COS COS COS COS COS COS COS COS 0 0 0 0 0 0 0 0 0 0 4.158 4.2 0 4.175 4.2 4.208 4.191 4.183 4.125 4.208 4.183 4.258 4.175 4.175 0 0 0 0 0 0 0 0 0 0 524.86 427.02 0 588.13 521.64 449.13 89.57 479.59 439.45 6.08 334.31 2.14 533.34 410.33 1 1 1 1 1 1 1 1 1 1 64.007 57.546 0 64.251 63.805 59.069 25.328 61.099 58.402 5.325 50.698 2.602 64.541 56.376 ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH 0 0 0 0 0 0 0 0 0 0 7.425 7.425 0 7.416 7.441 7.433 7.425 7.416 7.375 7.283 7.416 7.333 7.425 7.416 0 0 0 0 0 0 0 0 0 0 120.21 168.6 0 18.24 29.47 81.28 115.9 369.15 299.97 3.93 271.27 5.18 365.97 281.82 1 1 1 1 1 1 1 1 1 1 40.612 47.662 0 12.46 21.298 33.8 39.91 68.567 62.071 9.883 59.163 10.947 68.277 60.264 ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS DMS 0 0 0 0 0 0 0 0 0 0 10.308 0 0 0 10.316 0 10.308 10.308 10.283 7.516 0 10.308 0 10.308 0 0 0 0 0 0 0 0 0 0 455.71 0 0 0 482.42 0 339.23 537.5 196.24 8.92 0 3.03 0 391.2 1 1 1 1 1 1 1 1 1 1 71.71 0 0 0 73.776 0 61.841 75.035 45.999 13.227 0 7.237 0 64.248 0 70 65.997 70.209 67.213 58.356 DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS 0 0 0 0 0 0 0 0 0 0 11.558 10.316 0 10.308 11.375 10.325 11.525 11.416 11.466 0 10.308 10.458 10.325 11.541 10.325 10.3 10.308 10.316 11.716 10.3 11.733 10.3 10.316 10.308 10.316 10.316 10.233 11.416 10.308 10.316 10.308 10.308 11.6 10.316 10.308 0 0 0 0 0 0 0 0 0 0 2.2 398.79 0 522.11 2.04 422.41 2.35 2.56 3.31 0 363.2 3.09 438.64 3.1 516.1 398.56 452.69 429.16 3.94 521.91 2.33 67.25 98.09 116.42 129.97 118.28 673.87 3.64 78.01 89.13 116.65 119.6 3.6 111.33 119.5 0 0 0 0 0 0 0 0 0 0 0 67.064 0 46.276 0 69.041 0 0 0 0 61.969 7.296 67.934 0 73.544 65.997 70.209 67.213 0 73.948 0 28.096 33.592 21.893 37.748 36.116 84.534 0 30.159 32.093 35.878 36.301 0 35.068 36.286 318 100 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 11/24/2003 1:32:57 H2S 0 1:09:26 7:16:12 13:52:16 19:47:29 19:57:46 19:15:45 15:41:11 15:21:17 H2S H2S H2S H2S H2S H2S H2S H2S 0 2.6 2.6 2.608 2.616 2.633 2.483 2.45 0 319.22 17202.31 50.65 308.59 348.2 83083.34 95023.72 0 1 ppm 52.366 ppm ppm 21.371 ppm 51.46 ppm 54.729 ppm 4073.262 ppm 4652.123 ppm 1 ppm COS COS COS COS COS COS COS COS COS 0 0 0 0 4.166 4.191 4.183 4.133 0 0 0 0 0 2.82 3.58 4.33 3.92 0 1 1 1 1 3.161 3.668 4.165 3.888 ppm ppm ppm ppm ppm ppm ppm ppm 1 ppm 11/23/03 8:00 PM 11/24/03 12:16 AM 11/24/03 6:35 AM 11/26/03 6:41 PM 11/28/03 6:11 PM 12/2/03 4:35 PM 12/3/03 2:25 PM 11/23/03 8:00 PM 11/24/03 12:34 AM 11/24/03 6:49 AM 11/24/03 1:18 PM 11/25/03 7:06 PM 11/28/03 6:29 PM 12/2/03 3:00 PM 12/3/03 2:43 PM 11/23/03 8:00 PM 11/24/03 12:55 AM 11/24/03 7:02 AM 11/25/03 7:32 PM 11/26/03 7:32 PM 11/28/03 6:46 PM 12/2/03 3:20 PM 12/3/03 3:04 PM 11/23/03 8:00 PM 11/24/03 1:09 AM 11/24/03 7:16 AM 11/24/03 1:52 PM 11/25/03 7:47 PM 11/26/03 7:57 PM 11/28/03 7:15 PM 12/2/03 3:41 PM 12/3/03 3:21 PM 11/23/03 8:00 PM 11/24/03 2:38 AM 1.00 256.00 667.00 4237.00 7087.00 13781.00 15341.00 1.00 274.00 681.00 1019.00 2783.00 7069.00 13686.00 15359.00 1.00 295.00 694.00 2809.00 4249.00 7086.00 13706.00 15380.00 1.00 309.00 708.00 1053.00 2824.00 4274.00 7115.00 13727.00 15397.00 1.00 340.00 Nov03-FPD378.chr s-GAS STAND Nov03-FPD54.chr Nov03-FPD99.chr Nov03-FPD268.chr Nov03-FPD312.chr Nov03-FPD362.chr Nov03-FPD402.chr SOX-1 SOX-1 SOX-1 SOX-1 SOX-1 SOX-1 Nov03-FPD55.chr Nov03-FPD100.chr Nov03-FPD145.chr Nov03-FPD270.chr Nov03-FPD313.chr Nov03-FPD357.chr Nov03-FPD403.chr SOX-2 SOX-2 SOX-2 SOX-2 SOX-2 SOX-2 SOX-2 Nov03-FPD56.chr Nov03-FPD101.chr Nov03-FPD228.chr Nov03-FPD272.chr Nov03-FPD314.chr Nov03-FPD358.chr Nov03-FPD404.chr SOX-3 SOX-3 SOX-3 SOX-3 SOX-3 SOX-3 SOX-3 Nov03-FPD57.chr Nov03-FPD102.chr Nov03-FPD147.chr Nov03-FPD229.chr Nov03-FPD274.chr Nov03-FPD315.chr Nov03-FPD359.chr Nov03-FPD405.chr SOX-4 SOX-4 SOX-4 SOX-4 SOX-4 SOX-4 SOX-4 SOX-4 CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH 7.4 7.416 7.416 7.425 7.416 7.416 7.425 7.416 7.408 3.17 15.31 8.14 130.8 582.18 1114.53 1614.77 1855.84 5.19 67.836 56 55.416 61.745 36.709 23.745 13.921 13.246 1 9.282 15.883 23.636 117.967 203.015 336.736 396.268 1 8.627 18.941 50.61 100.532 161.981 248.656 278.527 1 7.727 16.072 ppm ppm ppm ppm ppm ppm ppm ppm 41.946 87.962 136.432 181.978 203.926 1 9.635 ppm DMS DMS DMS DMS DMS DMS DMS DMS DMS ppm ppm ppm ppm ppm ppm ppm ppm ppm DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS 10.325 10.308 11.616 11.408 10.308 10.308 10.325 11.716 10.316 78.67 102.77 3.16 3.14 104.43 107.38 122.58 2.59 106.98 30.274 34.332 0 0 34.015 34.465 36.716 0 34.996 Nov03-FPD58.chr SOX-5 10.3 294.85 56.002 70 10.3 67.25 28.096 10.316 98.09 33.592 10.308 116.42 10.316 129.97 37.748 10.316 118.28 36.116 10.233 673.87 10.316 152.62 40.764 70 10.308 78.01 30.159 10.316 89.13 32.093 10.308 116.65 35.878 10.308 119.6 36.301 10.316 109.86 34.844 10.316 111.33 35.068 10.308 119.5 36.286 70 10.325 78.67 30.274 10.308 102.77 34.332 10.308 23.11 10.3 86.71 31.146 10.308 104.43 34.015 10.308 107.38 34.465 10.325 122.58 36.716 10.316 140.41 39.191 70 10.316 106.98 34.996 SAMPLE INJECT SIZE 100 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 11/24/2003 11/24/2003 11/24/2003 11/26/2003 11/28/2003 12/3/2003 12/3/2003 12/3/2003 1:49:14 7:44:04 14:29:15 20:43:23 19:59:35 15:52:06 10:00:58 10:04:19 H2S H2S H2S H2S H2S H2S H2S H2S 2.575 2.6 0 0 0 0 0 0 4622.72 218.73 0 0 0 0 0 0 COS COS COS COS COS COS COS COS 4.175 0 0 0 0 0 0 0 3.59 0 0 0 0 0 0 0 ppm ppm ppm ppm ppm ppm ppm ppm CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 DMS DMS DMS DMS DMS DMS DMS DMS 10.316 10.308 10.308 10.316 10.333 10.325 0 0 450.58 234.31 202.9 151.66 118.31 114.09 0 0 7:29:34 14:07:34 20:09:47 20:23:06 19:36:47 16:08:56 15:37:16 11/24/03 7:29 AM 11/24/03 2:07 PM 11/25/03 8:09 PM 11/26/03 8:23 PM 11/28/03 7:36 PM 12/2/03 4:08 PM 12/3/03 3:37 PM 11/23/03 8:00 PM 11/24/03 1:49 AM 11/24/03 7:44 AM 11/24/03 2:29 PM 11/26/03 8:43 PM 11/28/03 7:59 PM 12/3/03 3:52 PM 721.00 1068.00 2846.00 4310.00 7136.00 13754.00 15403.00 1.00 340.00 721.00 1068.00 7136.00 13754.00 15403.00 H2S H2S H2S H2S H2S H2S H2S 2.6 2.6 2.633 2.625 2.608 2.508 2.433 6.59 5502.59 83.19 4329.38 12774.36 102164.49 111627.62 COS COS COS COS COS COS COS 0 0 0 0 0 0 0 0 0 0 0 0 0 0 ppm ppm ppm ppm ppm ppm ppm CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH CH3SH 7.4 7.408 7.425 7.433 7.425 7.433 7.416 12.33 7.83 121.44 318.86 763.77 1245.94 1675.86 DMS DMS DMS DMS DMS DMS DMS 10.316 10.3 10.316 10.308 10.316 10.316 10.316 114.93 20.68 120.86 123.48 101.74 117.89 151.7 DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS DMDS date time cum time (min) cum time gas reten (min) area conc units gas reten area conc units gas reten area conc units gas reten area conc units gas reten 10.316 10.3 10.316 10.308 10.316 10.316 11.566 10.316 10.308 10.308 11.658 10.333 10.325 0 0 area 114.93 20.68 120.86 123.48 101.74 117.89 2.94 450.58 234.31 202.9 3.58 118.31 114.09 0 0 conc 36.232 9.284 36.476 36.842 33.605 36.062 0 70.05 51.004 28.878 0 36.121 35.489 0 0 Nov03-FPD103.chr Nov03-FPD148.chr Nov03-FPD230.chr Nov03-FPD276.chr Nov03-FPD316.chr Nov03-FPD360.chr Nov03-FPD406.chr SOX-5 SOX-5 SOX-5 SOX-5 SOX-5 SOX-5 SOX-5 Nov03-FPD59.chr Nov03-FPD104.chr Nov03-FPD149.chr Nov03-FPD277.chr Nov03-FPD317.chr Nov03-FPD407.chr Nov03-FPD380.chr Nov03-FPD381.chr SOX-6 SOX-6 SOX-6 SOX-6 SOX-6 SOX-6 1000PPM co2 98.6 ch4 11.152 ppm ppm 26.876 ppm 255.324 ppm 664.731 ppm 4998.303 ppm 5457.069 ppm 269.544 269.544 ppm 43.214 ppm 1 ppm 1 ppm 1 ppm 1 ppm 0 ppm 0 ppm 1 1 1 1 1 1 1 3.2 3.184 1 1 1 1 1 0 0 14.483 ppm ppm 40.526 ppm 63.909 ppm 104.496 ppm 148.396 ppm 187.539 ppm 1 1 ppm 1 ppm 1 ppm 1 ppm 1 ppm 1 ppm 0 ppm 0 ppm 36.232 ppm ppm 36.476 ppm 36.842 ppm 33.605 ppm 36.062 ppm 40.646 ppm 70.05 70.05 ppm 51.004 ppm ppm 40.64 ppm 36.121 ppm 35.489 ppm 0 0 319 Appendix D - Chapter 5 Gas chromatography data from Nov 2003 TCD Results SAMPLE Nov03-TCD61.chr Nov03-TCD106.chr Nov03-TCD158.chr Nov03-TCD197.chr Nov03-TCD237.chr Nov03-TCD282.chr Nov03-TCD368.chr Nov03-TCD428.chr Nov03-TCD24.chr Nov03-TCD62.chr Nov03-TCD107.chr Nov03-TCD159.chr Nov03-TCD198.chr Nov03-TCD238.chr Nov03-TCD283.chr Nov03-TCD369.chr Nov03-TCD429.chr Nov03-TCD25.chr Nov03-TCD63.chr Nov03-TCD108.chr Nov03-TCD160.chr Nov03-TCD199.chr Nov03-TCD239.chr Nov03-TCD284.chr Nov03-TCD330.chr Nov03-TCD370.chr Nov03-TCD430.chr Nov03-TCD26.chr Nov03-TCD64.chr Nov03-TCD109.chr Nov03-TCD161.chr Nov03-TCD200.chr Nov03-TCD240.chr Nov03-TCD285.chr Nov03-TCD371.chr Nov03-TCD431.chr Nov03-TCD27.chr Nov03-TCD65.chr Nov03-TCD110.chr Nov03-TCD162.chr Nov03-TCD201.chr Nov03-TCD241.chr Nov03-TCD286.chr Nov03-TCD331.chr Nov03-TCD372.chr Nov03-TCD432.chr Nov03-TCD28.chr Nov03-TCD66.chr Nov03-TCD111.chr Nov03-TCD163.chr Nov03-TCD202.chr Nov03-TCD242.chr Nov03-TCD287.chr Nov03-TCD332.chr Nov03-TCD373.chr Nov03-TCD433.chr Nov03-TCD29.chr Nov03-TCD67.chr Nov03-TCD112.chr Nov03-TCD164.chr Nov03-TCD165.chr Nov03-TCD204.chr Nov03-TCD243.chr Nov03-TCD288.chr Nov03-TCD333.chr Nov03-TCD374.chr Nov03-TCD434.chr Nov03-TCD30.chr Nov03-TCD68.chr Nov03-TCD113.chr Nov03-TCD166.chr Nov03-TCD205.chr Nov03-TCD244.chr A-1 A-1 A-1 A-1 A-1 A-1 A-1 A-1 A-2 A-2 A-2 A-2 A-2 A-2 A-2 A-2 A-2 A-3 A-3 A-3 A-3 A-3 A-3 A-3 A-3 A-3 A-3 A-4 A-4 A-4 A-4 A-4 A-4 A-4 A-4 A-4 A-5 A-5 A-5 A-5 A-5 A-5 A-5 A-5 A-5 A-5 A-6 A-6 A-6 A-6 A-6 A-6 A-6 A-6 A-6 A-6 A-7 A-7 A-7 A-7 A-7 A-7 A-7 A-7 A-7 A-7 A-7 A-8 A-8 A-8 A-8 A-8 A-8 250 250 250 100 100 50 50 100 100 100 250 InJ SIZE date 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/4/2003 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/4/2003 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/1/2003 12/2/2003 12/4/2003 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/4/2003 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/1/2003 12/2/2003 12/4/2003 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/1/2003 12/2/2003 12/4/2003 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/1/2003 12/2/2003 12/4/2003 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 time 2:15:30 8:04:27 16:26:23 12:24:20 12:04:10 10:01:11 17:53:08 17:04:53 20:44:03 2:23:06 8:09:44 16:31:05 12:30:36 12:08:54 10:21:19 18:07:16 17:09:45 20:49:46 2:29:01 8:15:11 16:40:21 12:40:16 12:18:57 10:32:23 19:07:14 18:27:04 17:24:32 20:54:50 2:35:47 8:24:58 16:50:56 12:48:29 12:33:54 10:49:20 18:43:54 17:42:59 21:00:52 2:57:06 8:34:36 17:02:35 12:55:22 12:48:46 11:09:46 19:27:24 18:58:12 17:58:55 21:05:55 3:06:11 8:44:25 17:12:45 13:04:06 13:04:19 11:18:06 20:05:10 19:13:33 18:08:45 21:11:08 3:12:17 8:54:50 17:24:07 17:35:58 13:30:28 13:24:18 11:37:50 20:27:08 19:30:48 18:40:21 21:16:22 3:24:51 9:04:57 17:42:06 13:39:50 13:38:59 cum time 11/23/03 20:00 11/24/03 2:15 11/24/03 8:04 11/24/03 16:26 11/25/03 12:24 11/26/03 12:04 11/28/03 10:01 12/2/03 17:53 12/4/03 17:04 11/23/03 20:00 11/23/03 20:44 11/24/03 2:23 11/24/03 8:09 11/24/03 16:31 11/25/03 12:30 11/26/03 12:08 11/28/03 10:21 12/2/03 18:07 12/4/03 17:09 11/23/03 20:00 11/23/03 20:49 11/24/03 2:29 11/24/03 8:15 11/24/03 16:40 11/25/03 12:40 11/26/03 12:18 11/28/03 10:32 12/1/03 19:07 12/2/03 18:27 12/4/03 17:24 11/23/03 20:54 11/24/03 2:35 11/24/03 8:24 11/24/03 16:50 11/25/03 12:48 11/26/03 12:33 11/28/03 10:49 12/2/03 18:43 12/4/03 17:42 11/23/03 21:00 11/24/03 2:57 11/24/03 8:34 11/24/03 17:02 11/25/03 12:55 11/26/03 12:48 11/28/03 11:09 12/1/03 19:27 12/2/03 18:58 12/4/03 17:58 11/23/03 21:05 11/24/03 3:06 11/24/03 8:44 11/24/03 17:12 11/25/03 13:04 11/26/03 13:04 11/28/03 11:18 12/1/03 20:05 12/2/03 19:13 12/4/03 18:08 11/23/03 21:11 11/24/03 3:12 11/24/03 8:54 11/24/03 17:24 11/24/03 17:35 11/25/03 13:30 11/26/03 13:24 11/28/03 11:37 12/1/03 20:27 12/2/03 19:30 12/4/03 18:40 11/23/03 21:16 11/24/03 3:24 11/24/03 9:04 11/24/03 17:42 11/25/03 13:39 11/26/03 13:38 cum minutes gas CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 reten 1.975 1.975 1.983 1.958 1.958 1.975 2.008 1.966 1.958 1.975 1.975 1.975 1.958 1.991 1.958 1.975 1.975 1.95 1.958 1.966 1.958 1.958 1.958 1.941 1.941 1.95 1.941 1.966 1.966 1.958 1.941 1.958 1.95 1.933 1.958 1.941 1.95 1.975 1.975 1.975 1.958 1.941 1.966 1.933 1.933 1.95 1.966 1.966 1.95 1.866 1.933 1.958 1.958 1.916 1.933 1.958 1.916 1.925 1.95 1.916 1.958 1.966 1.925 1.95 1.958 1.941 1.941 1.916 1.908 1.925 1.941 1.925 1.916 area 2.8 3.31 3.28 3.59 3.02 3.18 4.45 4.74 2.2 4.2 7.31 10.9 15.24 18.05 25.06 54.38 71.67 2.43 5.21 8.82 5.02 18.74 24.65 56.47 95.83 96.56 103.45 2.57 5.8 10.57 14.18 19.8 31.99 66.52 99.08 100.1 1.6 5.53 13.52 17.08 33.17 42.85 75.72 114.45 98.88 106.9 2.59 8.22 6.5 1.47 43.99 17.83 86.8 136.83 133.5 136.24 156.95 139.3 52.08 48.77 28.4 19.81 129.46 134.97 136.25 139.95 125.5 155.36 143.91 123.87 111.72 118.85 113.49 conc 0.1 0.359 %vol 0.419 %vol 0.415 %vol 0.452 %vol 0.325 %vol 0.339 %vol 0.457 %vol 0.484 %vol 0.1 0.288 %vol 0.524 %vol 0.891 %vol 1.313 %vol 1.825 %vol 1.718 %vol 2.368 %vol 5.088 %vol 6.692 %vol 0.1 0.315 %vol 0.643 %vol 1.069 %vol 1.55 %vol 2.237 %vol 2.331 %vol 5.282 %vol 8.933 %vol 9.001 %vol 9.64 %vol 0.1 0.332 %vol 0.713 %vol 1.275 %vol 1.7 %vol 2.362 %vol 3.012 %vol 6.214 %vol 9.235 %vol 9.329 %vol 0.1 0.218 %vol 0.68 %vol 1.622 %vol 2.041 %vol 3.936 %vol 4.019 %vol 7.067 %vol 10.66 %vol 9.216 %vol 9.96 %vol 0.1 0.335 %vol 0.997 %vol 1.99 %vol 0.505 %vol 5.211 %vol %vol 8.095 %vol 12.736 %vol 12.427 %vol 12.681 %vol 18.6 18.517 %vol 16.438 %vol 15.41 %vol 5.774 %vol 33.75 %vol 23.63 %vol 12.052 %vol 12.564 %vol 12.683 %vol 13.026 %vol 11.685 %vol 18.6 18.329 %vol 16.981 %vol 14.621 %vol 13.19 %vol 14.03 %vol 10.571 %vol units 1 375 1444 1950 2852 4270 7755 13976 15847 1 44 383 1449 1954 2858 4274 7775 13981 15852 1 49 389 1455 1965 2868 4284 7786 12653 14001 15867 1 54 395 1464 1675 2876 4299 7803 14017 15885 1 60 417 1474 1687 2889 4314 7823 12650 14032 15901 1 65 425 1484 1697 2898 4338 7832 12682 14047 15911 1 71 431 1494 1709 1720 2919 4364 7851 12714 14064 15943 1 76 443 1504 1721 2938 4378 320 SAMPLE Nov03-TCD289.chr Nov03-TCD334.chr Nov03-TCD375.chr Nov03-TCD435.chr Nov03-TCD31.chr Nov03-TCD69.chr Nov03-TCD114.chr Nov03-TCD167.chr Nov03-TCD206.chr Nov03-TCD207.chr Nov03-TCD245.chr Nov03-TCD376.chr Nov03-TCD436.chr Nov03-TCD70.chr Nov03-TCD115.chr Nov03-TCD168.chr Nov03-TCD208.chr Nov03-TCD246.chr Nov03-TCD291.chr Nov03-TCD437.chr Nov03-TCD60.chr Nov03-TCD105.chr Nov03-TCD189.chr Nov03-TCD32.chr Nov03-TCD71.chr Nov03-TCD116.chr Nov03-TCD169.chr Nov03-TCD209.chr Nov03-TCD247.chr Nov03-TCD292.chr Nov03-TCD382.chr Nov03-TCD33.chr Nov03-TCD72.chr Nov03-TCD117.chr Nov03-TCD170.chr Nov03-TCD210.chr Nov03-TCD248.chr Nov03-TCD293.chr Nov03-TCD337.chr Nov03-TCD383.chr Nov03-TCD34.chr Nov03-TCD74.chr Nov03-TCD118.chr Nov03-TCD172.chr Nov03-TCD212.chr Nov03-TCD249.chr Nov03-TCD294.chr Nov03-TCD338.chr Nov03-TCD384.chr Nov03-TCD35.chr Nov03-TCD75.chr Nov03-TCD119.chr Nov03-TCD173.chr Nov03-TCD213.chr Nov03-TCD250.chr Nov03-TCD295.chr Nov03-TCD339.chr Nov03-TCD385.chr Nov03-TCD36.chr Nov03-TCD76.chr Nov03-TCD120.chr Nov03-TCD174.chr Nov03-TCD214.chr Nov03-TCD251.chr Nov03-TCD296.chr Nov03-TCD340.chr Nov03-TCD386.chr Nov03-TCD37.chr Nov03-TCD77.chr Nov03-TCD121.chr Nov03-TCD175.chr Nov03-TCD215.chr Nov03-TCD252.chr Nov03-TCD297.chr Nov03-TCD341.chr Nov03-TCD387.chr A-8 A-8 A-8 A-8 A-9 A-9 A-9 A-9 A-9 A-9 A-9 A-9 A-9 A-10 A-10 A-10 A-10 A-10 A-10 A-10 AIR AIR AIR B-1 B-1 B-1 B-1 B-1 B-1 B-1 B-1 B-2 B-2 B-2 B-2 B-2 B-2 B-2 B-2 B-2 B-3 B-3 B-3 B-3 B-3 B-3 B-3 B-3 B-3 B-4 B-4 B-4 B-4 B-4 B-4 B-4 B-4 B-4 B-5 B-5 B-5 B-5 B-5 B-5 B-5 B-5 B-5 B-6 B-6 B-6 B-6 B-6 B-6 B-6 B-6 B-6 INJECT S date 11/28/2003 12/1/2003 12/2/2003 12/4/2003 11/23/2003 11/24/2003 time 12:00:51 20:50:59 19:48:37 18:58:56 21:22:57 3:31:17 9:10:45 17:48:55 13:49:38 14:01:58 13:51:43 20:03:36 19:13:00 3:41:07 9:20:03 17:59:10 14:15:28 14:04:15 12:49:41 19:33:04 2:09:21 7:57:51 20:15:37 21:29:21 3:46:32 9:25:12 18:04:13 14:26:43 14:20:41 12:58:33 10:06:51 21:35:22 3:53:32 9:28:55 18:12:15 14:32:50 14:27:14 13:02:22 10:31:52 10:15:04 21:50:17 4:09:55 9:39:45 18:27:56 14:48:21 14:41:01 13:24:29 10:51:22 10:30:07 21:55:52 4:19:01 9:50:00 18:38:08 14:58:11 15:01:03 13:40:14 11:11:49 10:46:15 22:01:45 4:24:39 10:00:03 18:48:52 15:15:28 15:14:27 13:58:38 11:50:54 11:01:11 22:06:59 4:30:32 10:04:16 18:53:58 15:35:21 15:34:17 14:17:56 12:10:16 11:15:21 cum time 11/28/03 12:00 12/1/03 20:50 12/2/03 19:48 12/4/03 18:58 11/23/03 21:22 11/24/03 3:31 11/24/03 9:10 11/24/03 17:48 11/25/03 13:49 11/25/03 14:01 11/26/03 13:51 12/2/03 20:03 12/4/03 19:13 11/24/03 3:41 11/24/03 9:20 11/24/03 17:59 11/25/03 14:15 11/26/03 14:04 11/28/03 12:49 12/4/03 19:33 cum minutes gas CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 reten 1.95 1.941 1.941 1.916 1.908 1.925 1.958 1.95 1.941 1.925 1.908 1.95 1.941 1.933 1.975 1.933 1.958 1.958 1.975 1.95 1.95 1.966 1.991 1.958 1.958 1.975 1.975 1.975 1.958 1.975 1.991 1.958 1.925 1.975 1.933 1.958 1.958 1.958 1.941 1.966 1.941 1.958 1.941 1.958 1.966 1.966 1.958 1.941 1.95 1.983 1.958 1.975 1.95 1.958 1.958 1.958 1.95 1.983 1.966 1.941 1.983 1.975 1.958 1.958 1.933 1.916 1.975 1.966 1.966 1.983 1.975 1.958 1.958 1.95 1.933 1.925 area 125.63 151.88 160.3 141.01 162.66 140.8 52.56 31.35 23.67 121.33 111.19 132.37 128 23.99 22.28 20.16 19.05 17.66 18.17 18.17 0.6 0.7 0.95 1.82 2.79 2.78 2.08 2.81 2.64 2.69 2.62 2.95 8.65 4.99 1.19 29.4 36.86 62.55 89.34 98.56 2.83 8.85 5.62 6.81 18.57 43.95 69.66 85.96 94.44 2.57 8.53 13.08 3.87 29.9 38.51 56.91 79.72 84.08 2.2 9.43 14.65 16.03 28.83 40.87 53.75 69.56 74.41 2.6 8.65 15 21.6 34.12 43.99 86.73 96.26 112.98 conc units 11.697 %vol 14.132 %vol 14.913 %vol 13.123 %vol 19.5 19.189 %vol 16.615 %vol 15.55 %vol 37.22 %vol 28.17 %vol 14.322 %vol 10.357 %vol 12.323 %vol 11.917 %vol 2.9 2.855 %vol 2.654 %vol 2.404 %vol 2.273 %vol 1.682 %vol 1.73 %vol 1.729 %vol 0.1 %vol 0.111 %vol 0.141 %vol 0.244 0.244 %vol 0.358 %vol 0.356 %vol 0.275 %vol 0.361 %vol 0.289 %vol 0.294 %vol 0.287 %vol 0.244 0.376 %vol 1.048 %vol 1.5425 %vol 1.7 %vol 3.492 %vol 3.464 %vol 5.846 %vol 8.331 %vol 9.186 %vol 0.244 0.362 %vol 1.072 %vol 1.7275 %vol 2.0775 %vol 1.767 %vol 4.121 %vol 6.505 %vol 8.017 %vol 8.804 %vol 0.244 0.332 %vol 1.034 %vol 1.57 %vol 1.215 %vol 2.817 %vol 3.617 %vol 5.323 %vol 7.439 %vol 7.843 %vol 0.244 0.289 %vol 1.14 %vol 1.755 %vol 1.917 %vol 2.719 %vol 3.835 %vol 5.03 %vol 6.497 %vol 6.946 %vol 0.244 0.335 %vol 1.048 %vol 1.796 %vol 2.573 %vol 3.209 %vol 4.125 %vol 8.089 %vol 8.973 %vol 10.523 %vol 100 50 50 250 11/24/2003 11/24/2003 11/25/2003 11/25/2003 11/26/2003 12/2/2003 12/4/2003 11/24/2003 250 250 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/4/2003 11/24/2003 11/24/2003 11/24/2003 11/23/2003 11/24/2003 7874 12737 14076 15961 1 82 450 1510 1727 2948 4391 7895 14081 15976 1 460 1524 1738 2965 4699 7923 15996 11/23/03 21:29 11/24/03 3:46 11/24/03 9:25 11/24/03 18:04 11/25/03 14:26 11/26/03 14:20 11/28/03 12:58 12/3/03 10:06 11/23/03 20:00 11/23/03 21:35 11/24/03 3:53 11/24/03 9:28 11/24/03 18:12 11/25/03 14:32 11/26/03 14:27 11/28/03 13:02 12/2/03 10:31 12/3/03 10:15 11/23/03 20:00 11/23/03 21:50 11/24/03 4:09 11/24/03 9:39 11/24/03 18:27 11/25/03 14:48 11/26/03 14:41 11/28/03 13:24 12/2/03 10:51 12/3/03 10:30 11/23/03 20:00 11/23/03 21:55 11/24/03 4:19 11/24/03 9:50 11/24/03 18:38 11/25/03 14:58 11/26/03 15:01 11/28/03 13:40 12/2/03 11:11 12/3/03 10:46 11/23/03 20:00 11/23/03 22:01 11/24/03 4:24 11/24/03 10:00 11/24/03 18:48 11/25/03 15:15 11/26/03 15:14 11/28/03 13:58 12/2/03 11:50 12/3/03 11:01 11/23/03 20:00 11/23/03 22:06 11/24/03 4:30 11/24/03 10:04 11/24/03 18:53 11/25/03 15:35 11/26/03 15:34 11/28/03 14:17 12/2/03 12:10 12/3/03 11:15 250 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/3/2003 11/23/2003 11/24/2003 100 50 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 11/23/2003 11/24/2003 100 100 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 11/23/2003 11/24/2003 250 100 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 11/23/2003 11/24/2003 250 250 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 11/23/2003 11/24/2003 250 250 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 1 89 465 1529 1743 2983 4420 7932 15012 1 94 472 1756 2990 4427 7936 13631 13641 15021 1 109 488 1766 3006 4441 7968 13651 13661 15036 1 113 498 867 1777 3016 4461 7984 13671 15042 1 120 503 877 1797 3033 4474 8002 13710 15057 1 125 509 881 1802 3053 4494 8011 13730 15071 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 321 SAMPLE Nov03-TCD38.chr Nov03-TCD78.chr Nov03-TCD122.chr Nov03-TCD253.chr Nov03-TCD298.chr Nov03-TCD342.chr Nov03-TCD388.chr Nov03-TCD39.chr Nov03-TCD79.chr Nov03-TCD123.chr Nov03-TCD178.chr Nov03-TCD217.chr Nov03-TCD254.chr Nov03-TCD299.chr Nov03-TCD343.chr Nov03-TCD389.chr Nov03-TCD40.chr Nov03-TCD80.chr Nov03-TCD81.chr Nov03-TCD82.chr Nov03-TCD124.chr Nov03-TCD179.chr Nov03-TCD218.chr Nov03-TCD255.chr Nov03-TCD300.chr Nov03-TCD344.chr Nov03-TCD390.chr Nov03-TCD83.chr Nov03-TCD126.chr Nov03-TCD180.chr Nov03-TCD301.chr Nov03-TCD345.chr Nov03-TCD391.chr Nov03-TCD41.chr Nov03-TCD84.chr Nov03-TCD127.chr Nov03-TCD153.chr Nov03-TCD184.chr Nov03-TCD231.chr Nov03-TCD271.chr Nov03-TCD318.chr Nov03-TCD325.chr Nov03-TCD363.chr Nov03-TCD408.chr Nov03-TCD42.chr Nov03-TCD85.chr Nov03-TCD128.chr Nov03-TCD154.chr Nov03-TCD185.chr Nov03-TCD232.chr Nov03-TCD265.chr Nov03-TCD319.chr Nov03-TCD326.chr Nov03-TCD364.chr Nov03-TCD409.chr Nov03-TCD43.chr Nov03-TCD86.chr Nov03-TCD129.chr Nov03-TCD155.chr Nov03-TCD186.chr Nov03-TCD233.chr Nov03-TCD266.chr Nov03-TCD320.chr Nov03-TCD327.chr Nov03-TCD365.chr Nov03-TCD410.chr Nov03-TCD44.chr Nov03-TCD87.chr Nov03-TCD130.chr Nov03-TCD156.chr Nov03-TCD187.chr Nov03-TCD234.chr Nov03-TCD267.chr Nov03-TCD321.chr Nov03-TCD328.chr Nov03-TCD366.chr Nov03-TCD411.chr Nov03-TCD88.chr Nov03-TCD131.chr Nov03-TCD157.chr B-7 B-7 B-7 B-7 B-7 B-7 B-7 B-8 B-8 B-8 B-8 B-8 B-8 B-8 B-8 B-8 B-9 B-9 B-9 B-9 B-9 B-9 B-9 B-9 B-9 B-9 B-9 B-10 B-10 B-10 B-10 B-10 B-10 M-1 M-1 M-1 M-1 M-1 M-1 M-1 M-1 M-1 M-1 M-1 M-2 M-2 M-2 M-2 M-2 M-2 M-2 M-2 M-2 M-2 M-2 M-3 M-3 M-3 M-3 M-3 M-3 M-3 M-3 M-3 M-3 M-3 M-4 M-4 M-4 M-4 M-4 M-4 M-4 M-4 M-4 M-4 M-4 M-5 M-5 M-5 INJECT S date 11/23/2003 11/24/2003 time 22:13:58 4:36:07 10:13:54 15:49:52 14:31:55 12:28:26 11:35:06 22:18:59 4:41:38 10:24:56 19:17:25 17:24:57 16:03:02 14:52:58 12:48:22 11:50:27 22:24:45 4:47:35 4:57:48 5:02:22 10:31:41 19:20:41 17:39:08 16:14:50 15:08:22 13:00:26 12:04:14 5:06:54 10:52:55 19:24:49 15:27:31 13:16:52 12:22:59 22:30:09 5:12:09 10:57:13 15:45:50 19:40:39 20:24:22 19:26:02 20:34:08 14:21:22 16:50:51 16:06:49 22:36:08 5:17:29 11:07:27 15:50:32 19:52:19 20:36:28 18:11:08 20:45:29 14:35:26 16:55:29 16:12:55 22:42:37 5:22:48 11:17:31 16:00:06 19:56:29 20:45:04 18:18:02 21:07:46 14:53:57 17:11:20 16:23:07 22:48:21 5:28:36 11:27:04 16:10:56 20:00:55 20:54:40 18:34:23 21:26:05 18:33:14 17:27:13 16:35:02 5:34:09 11:37:39 16:20:30 cum time 11/23/03 22:13 11/24/03 4:36 11/24/03 10:13 11/26/03 15:49 11/28/03 14:31 12/2/03 12:28 12/3/03 11:35 11/23/03 20:00 11/23/03 22:18 11/24/03 4:41 11/24/03 10:24 11/24/03 19:17 11/25/03 17:24 11/26/03 16:03 11/28/03 14:52 12/2/03 12:48 12/3/03 11:50 11/23/03 20:00 11/23/03 22:24 11/24/03 4:47 11/24/03 4:57 11/24/03 5:02 11/24/03 10:31 11/24/03 19:20 11/25/03 17:39 11/26/03 16:14 11/28/03 15:08 12/2/03 13:00 12/3/03 12:04 11/23/03 20:00 11/24/03 5:06 11/24/03 10:52 11/24/03 19:24 11/28/03 15:27 12/2/03 13:16 12/3/03 12:22 11/23/03 20:00 11/23/2003 22:30 11/24/2003 5:12 11/24/2003 10:57 11/24/2003 15:45 11/24/2003 19:40 11/25/2003 20:24 11/26/2003 19:26 11/28/2003 20:34 12/1/2003 14:21 12/1/2003 16:50 12/3/2003 16:06 11/23/03 20:00 11/23/2003 22:36 11/24/2003 5:17 11/24/2003 11:07 11/24/2003 15:50 11/24/2003 19:52 11/25/2003 20:36 11/26/2003 18:11 11/28/2003 20:45 12/1/2003 14:35 12/2/2003 16:55 12/3/2003 16:12 11/23/03 20:00 11/23/2003 22:42 11/24/2003 5:22 11/24/2003 11:17 11/24/2003 16:00 11/24/2003 19:56 11/25/2003 20:45 11/26/2003 18:18 11/28/2003 21:07 12/1/2003 14:53 12/2/2003 17:11 12/3/2003 16:23 11/23/03 20:00 11/23/2003 22:48 11/24/2003 5:28 11/24/2003 11:27 11/24/2003 16:10 11/24/2003 20:00 11/25/2003 20:54 11/26/2003 18:34 11/28/2003 21:26 12/1/2003 18:33 12/2/2003 17:27 12/3/2003 16:35 11/23/03 20:00 11/24/2003 5:34 11/24/2003 11:37 11/24/2003 16:20 cum minutes gas CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 reten 1.908 1.908 1.933 1.908 1.925 1.933 1.925 1.925 1.908 1.941 1.95 1.95 1.933 1.933 1.95 1.958 1.933 1.908 1.916 1.966 1.95 1.941 1.925 1.925 1.925 1.983 1.916 1.95 1.983 1.966 1.95 1.966 1.975 1.958 1.95 2 1.975 1.966 1.941 1.925 1.95 2 1.975 1.966 1.95 1.966 1.975 1.991 1.95 1.958 1.95 1.958 1.966 2.008 1.991 1.966 1.95 1.966 1.975 2 1.966 1.95 1.983 1.966 1.975 1.966 1.95 1.941 1.983 1.991 1.983 1.966 1.941 1.975 1.95 1.975 1.975 1.933 1.966 1.975 area 164.85 151.92 55.46 134.34 141.99 153.2 186.88 162.58 142.5 138.97 113.09 129.03 126.55 137.96 146.81 162.38 162.84 152.21 62.37 25.72 58.91 59.94 133.25 126.82 145.33 56.43 162.88 22.03 19.77 16.84 16.03 15.97 16.12 1.1 1.6 1.47 1.35 0.87 0.83 1.16 1.07 0.62 1.1 0.78 1.67 3.72 5.09 5.45 3.17 7.21 9.62 10.17 12.22 13.2 14.87 1.43 3.31 5.4 6.44 5.38 8.02 9.85 11.22 15.83 18.79 20.17 1.25 3.78 4.75 5.21 6.4 5.88 9.22 14.83 14.95 12.87 14.18 3.94 2.55 1.88 conc 19.5 units 19.447 %vol 17.925 %vol 16.405 %vol 12.505 %vol 13.215 %vol 14.254 %vol 17.379 %vol 19.5 19.18 %vol 16.815 %vol 16.399 %vol 13.35 %vol 12.012 %vol 11.783 %vol 12.841 %vol 13.662 %vol 15.106 %vol 19.5 19.211 %vol 17.959 %vol 18.4425 %vol 30.58 %vol 17.4225 %vol 17.725 %vol 12.404 %vol 11.808 %vol 13.524 %vol 13.1975 %vol 15.153 %vol 2.65 2.624 %vol 2.358 %vol 2.013 %vol 1.531 %vol 1.526 %vol 1.54 0.15 0.158 0.218 0.203 0.188 0.132 0.121 0.152 0.143 0.101 0.146 0.117 0.22 0.226 0.468 0.629 0.671 0.403 0.713 0.937 0.988 1.178 1.269 1.424 0.17 0.198 0.419 0.666 0.788 0.663 0.789 0.958 1.085 1.513 1.787 1.915 0.17 0.177 0.475 0.589 0.643 0.783 0.59 0.899 1.42 1.431 1.238 1.36 0.5 0.494 0.33 0.251 %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol 100 11/24/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 11/23/2003 11/24/2003 250 250 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 11/23/2003 11/24/2003 100 50 100 100 11/24/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 100 12/2/2003 12/3/2003 250 250 250 250 11/24/2003 11/24/2003 11/24/2003 11/28/2003 12/2/2003 12/3/2003 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/1/2003 12/2/2003 12/3/2003 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/1/2003 12/2/2003 12/3/2003 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/1/2003 12/2/2003 12/3/2003 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/1/2003 12/2/2003 12/3/2003 11/24/2003 11/24/2003 11/24/2003 1 132 515 906 4650 8026 13744 15091 1 137 520 917 1811 3231 4664 8047 13764 15106 1 143 526 536 541 924 1814 3246 4675 8063 13776 15120 1 545 945 1818 8074 13789 15138 1 150 555 900 1182 1507 3431 4813 8035 12136 13806 15362 1 156 560 910 1187 3441 4768 8046 12150 13811 15368 1 162 565 920 1197 1421 3450 4775 12172 13827 15379 1 168 571 930 1207 1424 3459 8087 12392 13843 15391 1 940 1217 250 322 SAMPLE Nov03-TCD188.chr Nov03-TCD322.chr Nov03-TCD329.chr Nov03-TCD367.chr Nov03-TCD412.chr Nov03-TCD151.chr Nov03-TCD191.chr Nov03-TCD211.chr Nov03-TCD144.chr Nov03-TCD152.chr Nov03-TCD192.chr Nov03-TCD194.chr Nov03-TCD195.chr Nov03-TCD236.chr Nov03-TCD280.chr Nov03-TCD281.chr Nov03-TCD323.chr Nov03-TCD324.chr Nov03-TCD336.chr Nov03-TCD378.chr Nov03-TCD54.chr Nov03-TCD99.chr Nov03-TCD142.chr Nov03-TCD226.chr Nov03-TCD268.chr Nov03-TCD312.chr Nov03-TCD356.chr Nov03-TCD402.chr Nov03-TCD100.chr Nov03-TCD55.chr Nov03-TCD145.chr Nov03-TCD227.chr Nov03-TCD270.chr Nov03-TCD313.chr Nov03-TCD357.chr Nov03-TCD403.chr Nov03-TCD56.chr Nov03-TCD101.chr Nov03-TCD146.chr Nov03-TCD228.chr Nov03-TCD272.chr Nov03-TCD314.chr Nov03-TCD358.chr Nov03-TCD404.chr Nov03-TCD57.chr Nov03-TCD102.chr Nov03-TCD147.chr Nov03-TCD229.chr Nov03-TCD274.chr Nov03-TCD315.chr Nov03-TCD359.chr Nov03-TCD405.chr Nov03-TCD58.chr Nov03-TCD103.chr Nov03-TCD148.chr Nov03-TCD230.chr Nov03-TCD276.chr Nov03-TCD316.chr Nov03-TCD360.chr Nov03-TCD406.chr Nov03-TCD59.chr Nov03-TCD104.chr Nov03-TCD149.chr Nov03-TCD277.chr Nov03-TCD317.chr Nov03-TCD361.chr Nov03-TCD407.chr M-5 M-5 M-5 M-5 M-5 MIDPOINT MIDPOINT MIX 33 STAND? OUTTAKE TUBE OUTTAKE TUBE OUTTAKE TUBE OUTTAKE TUBE s-GAS STAND s-GAS STAND s-GAS STAND s-GAS STAND s-GAS STAND s-GAS STAND s-GAS STAND s-GAS STAND SOX-1 SOX-1 SOX-1 SOX-1 SOX-1 SOX-1 SOX-1 SOX-1 SOX-2 SOX-2 SOX-2 SOX-2 SOX-2 SOX-2 SOX-2 SOX-2 SOX-3 SOX-3 SOX-3 SOX-3 SOX-3 SOX-3 SOX-3 SOX-3 SOX-4 SOX-4 SOX-4 SOX-4 SOX-4 SOX-4 SOX-4 SOX-4 SOX-5 SOX-5 SOX-5 SOX-5 SOX-5 SOX-5 SOX-5 SOX-5 SOX-6 SOX-6 SOX-6 SOX-6 SOX-6 SOX-6 SOX-6 INJECT S date 11/24/2003 11/28/2003 12/1/2003 12/2/2003 12/3/2003 11/24/2003 11/24/2003 11/25/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/25/2003 11/26/2003 11/28/2003 11/28/2003 12/1/2003 12/1/2003 12/2/2003 12/3/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 11/24/2003 11/24/2003 11/24/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 time 20:11:16 21:48:05 18:53:40 17:43:03 16:52:34 15:08:06 20:46:42 14:42:38 12:57:58 15:30:52 21:09:15 10:24:12 10:39:45 11:43:48 9:26:27 9:40:55 13:40:47 13:58:56 9:56:01 9:20:47 0:16:11 6:35:47 12:31:13 19:11:41 18:41:59 18:11:26 14:55:07 14:25:33 6:49:12 0:34:08 13:18:44 19:18:09 19:06:18 18:29:29 15:00:50 14:43:55 0:55:51 7:02:11 13:33:31 19:32:27 19:32:41 18:46:16 15:20:38 15:04:16 1:09:26 7:16:12 13:52:16 19:47:29 19:57:46 19:15:45 15:41:11 15:21:17 1:32:57 7:29:34 14:07:34 20:09:47 20:23:06 19:36:47 16:08:56 15:37:16 1:49:14 7:44:04 14:29:15 20:43:23 19:59:35 16:27:38 15:52:06 cum time 11/24/2003 20:11 11/28/2003 21:48 12/1/2003 18:53 12/2/2003 17:43 12/3/2003 16:52 1435 8109 12412 13859 15408 gas CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 CO2 reten 1.95 1.975 1.941 1.983 2 0 0 1.958 0 0 0 0 0 0 0 0 0 0 0 0 1.95 1.95 1.966 1.941 1.966 1.975 1.983 1.983 1.941 1.958 1.95 1.975 1.95 1.908 1.958 1.975 1.983 1.95 1.975 1.95 1.933 1.958 1.925 1.958 1.966 1.958 1.975 1.95 1.95 1.95 1.95 1.941 1.975 1.966 1.966 1.983 1.958 1.95 1.966 1.941 1.941 1.966 1.983 1.966 1.991 1.975 1.975 area 5.38 2.96 3.99 4.11 4.37 0 0 10.3 0 0 0 0 0 0 0 0 0 0 0 0 0.76 0.61 0.72 2.32 1.47 1.38 1.39 1.54 8.88 2.6 15.29 42.82 66.17 100.84 113.44 120.19 2.92 7.95 6.14 34.81 57.6 83.14 107.57 111.86 2.03 7.1 5.26 31.61 54.89 81.12 101.72 109.89 2.97 8.34 5.82 40.67 54.69 85.79 104.99 119.15 18.53 17.66 16.8 15.53 14.14 12.91 13.44 conc 0.662 0.319 0.415 0.426 0.449 0 0 1.243 0 0 0 0 0 0 0 0 0 0 0 0 0.1 0.119 0.101 0.114 0.259 0.181 0.172 0.173 0.187 0.5 1.076 0.336 1.831 4.016 6.182 9.398 10.566 11.193 0.3 0.374 0.966 0.753 3.274 5.387 7.756 10.022 10.42 0.2 0.269 0.865 0.649 2.976 5.136 7.568 9.479 10.237 0.3 0.379 1.011 0.715 3.817 5.117 8.002 9.782 11.096 2.22 2.212 2.109 2.008 1.485 1.355 1.242 1.291 units %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol 100 100 100 11/23/03 20:00 11/24/2003 0:16 11/24/2003 6:35 11/24/2003 12:31 11/25/2003 19:11 11/26/2003 18:41 11/28/2003 18:11 12/2/2003 14:55 12/3/2003 14:25 11/23/03 20:00 11/24/2003 6:49 11/24/2003 11:34 11/24/2003 13:18 11/25/2003 19:18 11/26/2003 19:06 11/28/2003 18:29 12/2/2003 15:00 12/3/2003 14:43 11/23/03 20:00 11/24/2003 0:55 11/24/2003 7:02 11/24/2003 13:33 11/25/2003 19:32 11/26/2003 19:32 11/28/2003 18:46 12/2/2003 15:20 12/3/2003 15:04 11/23/03 20:00 11/24/2003 1:09 11/24/2003 7:16 11/24/2003 13:52 11/24/2003 19:47 11/26/2003 19:57 11/28/2003 19:15 12/2/2003 15:41 12/3/2003 15:21 11/23/03 20:00 11/24/2003 1:31 11/24/2003 7:29 11/24/2003 14:07 11/25/2003 20:09 11/26/2003 20:23 11/28/2003 19:36 12/2/2003 16:08 12/3/2003 15:37 11/23/03 20:00 11/24/2003 1:49 11/24/2003 7:44 11/24/2003 14:29 11/26/2003 20:43 11/28/2003 19:59 12/2/2003 16:27 12/3/2003 15:52 1 256 651 1007 2727 4077 6927 12622 15260 1 665 944 1055 2734 4102 6945 12627 15278 1 295 678 1077 2748 4128 6962 12647 15299 1 309 682 1096 4143 6991 12668 15316 1 331 695 1111 2785 4169 7012 12695 15332 1 349 710 1133 4189 7035 12714 15347 323 Appendix D - Chapter 5 Gas chromatography data from Nov 2003 FID Results Nov03-FID381.chr Nov03-FID61.chr Nov03-FID106.chr Nov03-FID158.chr Nov03-FID197.chr Nov03-FID237.chr Nov03-FID282.chr Nov03-FID368.chr Nov03-FID428.chr Nov03-FID24.chr Nov03-FID62.chr Nov03-FID107.chr Nov03-FID159.chr Nov03-FID198.chr Nov03-FID238.chr Nov03-FID283.chr Nov03-FID369.chr Nov03-FID429.chr Nov03-FID25.chr Nov03-FID63.chr Nov03-FID108.chr Nov03-FID160.chr Nov03-FID199.chr Nov03-FID239.chr Nov03-FID284.chr Nov03-FID330.chr Nov03-FID370.chr Nov03-FID430.chr Nov03-FID26.chr Nov03-FID64.chr Nov03-FID109.chr Nov03-FID161.chr Nov03-FID200.chr Nov03-FID240.chr Nov03-FID285.chr Nov03-FID371.chr Nov03-FID431.chr Nov03-FID27.chr Nov03-FID65.chr Nov03-FID110.chr Nov03-FID162.chr Nov03-FID201.chr Nov03-FID241.chr Nov03-FID286.chr Nov03-FID331.chr Nov03-FID372.chr Nov03-FID432.chr Nov03-FID28.chr Nov03-FID66.chr Nov03-FID111.chr Nov03-FID163.chr Nov03-FID202.chr Nov03-FID242.chr Nov03-FID287.chr Nov03-FID332.chr Nov03-FID373.chr Nov03-FID433.chr Nov03-FID29.chr Nov03-FID67.chr Nov03-FID112.chr Nov03-FID164.chr Nov03-FID165.chr Nov03-FID203.chr SAMPLE 98.6 ch4 A-1 A-1 A-1 A-1 A-1 A-1 A-1 A-1 A-2 A-2 A-2 A-2 A-2 A-2 A-2 A-2 A-2 A-3 A-3 A-3 A-3 A-3 A-3 A-3 A-3 A-3 A-3 A-4 A-4 A-4 A-4 A-4 A-4 A-4 A-4 A-4 A-5 A-5 A-5 A-5 A-5 A-5 A-5 A-5 A-5 A-5 A-6 A-6 A-6 A-6 A-6 A-6 A-6 A-6 A-6 A-6 A-7 A-7 A-7 A-7 A-7 A-7 250 INJECT date 12/3/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/4/2003 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/4/2003 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/1/2003 12/2/2003 12/4/2003 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/4/2003 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/1/2003 12/2/2003 12/4/2003 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/1/2003 12/2/2003 12/4/2003 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 time 10:04:19 2:15:30 8:04:27 16:26:23 12:24:20 12:04:10 10:01:11 17:53:08 17:04:53 20:44:03 2:23:06 8:09:44 16:31:05 12:30:36 12:08:54 10:21:19 18:07:16 17:09:45 20:49:46 2:29:01 8:15:11 16:40:21 12:40:16 12:18:57 10:32:23 19:07:14 18:27:04 17:24:32 20:54:50 2:35:47 8:24:58 16:50:56 12:48:29 12:33:54 10:49:20 18:43:54 17:42:59 21:00:52 2:57:06 8:34:36 17:02:35 12:55:22 12:48:46 11:09:46 19:27:24 18:58:12 17:58:55 21:05:55 3:06:11 8:44:25 17:12:45 13:04:06 13:04:19 11:18:06 20:05:10 19:13:33 18:08:45 21:11:08 3:12:17 8:54:50 17:24:07 17:35:58 13:25:07 cum time 11/23/03 20:00 11/24/2003 2:15 11/24/2003 8:04 11/24/2003 16:26 11/25/2003 12:24 11/26/2003 12:04 11/28/2003 10:01 12/2/2003 17:53 12/4/2003 17:04 11/23/03 20:00 11/23/2003 20:44 11/24/2003 2:23 11/24/2003 8:09 11/24/2003 16:31 11/25/2003 12:30 11/26/2003 12:08 11/28/2003 10:21 12/2/2003 18:07 12/4/2003 17:09 11/23/03 20:00 11/23/2003 20:49 11/24/2003 2:29 11/24/2003 8:15 11/24/2003 16:40 11/25/2003 12:40 11/26/2003 12:18 11/28/2003 10:32 12/1/2003 19:07 12/2/2003 18:27 12/4/2003 17:24 11/23/03 20:00 11/23/2003 20:54 11/24/2003 2:35 11/24/2003 8:24 11/24/2003 16:50 11/25/2003 12:48 11/26/2003 12:33 11/28/2003 10:49 12/2/2003 18:43 12/4/2003 17:42 11/23/03 20:00 11/23/2003 21:00 11/24/2003 2:57 11/24/2003 8:34 11/24/2003 17:02 11/25/2003 12:55 11/26/2003 12:48 11/28/2003 11:09 12/1/2003 19:27 12/2/2003 18:58 12/4/2003 17:58 11/23/03 20:00 11/23/2003 21:05 11/24/2003 3:06 11/24/2003 8:44 11/24/2003 17:12 11/25/2003 13:04 11/26/2003 13:04 11/28/2003 11:18 12/1/2003 20:05 12/2/2003 19:13 12/4/2003 18:08 11/23/03 20:00 cum min 1.00 375.00 1444.00 1950.00 2852.00 4270.00 7755.00 13976.00 15847.00 1.00 44.00 383.00 1449.00 1954.00 2858.00 4274.00 7775.00 13981.00 15852.00 1.00 49.00 389.00 1455.00 1965.00 2868.00 4284.00 7786.00 12653.00 14001.00 1.00 54.00 395.00 1464.00 1675.00 2876.00 4299.00 7803.00 14017.00 15885.00 1.00 60.00 417.00 1474.00 1687.00 2889.00 4314.00 7823.00 12650.00 14032.00 15901.00 1.00 65.00 425.00 1484.00 1697.00 2898.00 4338.00 7832.00 12682.00 14047.00 15911.00 1.00 71.00 431.00 1494.00 1709.00 1720.00 1720.00 gas Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane conc units 101.34 ppm 13.2 ppm ppm 16.33 ppm 17 ppm 16.2 ppm ppm ppm 16.7 ppm ppm 9.6 ppm ppm 11.4 ppm 8.6 ppm 7.78 ppm ppm ppm 12.3 ppm ppm 9.3 ppm ppm ppm 10.46 ppm 10.6 ppm ppm 10.7 ppm ppm 13.2 ppm ppm 8.2 ppm ppm 9.7 ppm 10.7 ppm 8.9 ppm ppm ppm 15.4 ppm ppm 8.4 ppm ppm 12.6 ppm 17.5 ppm 17.55 ppm ppm 20.26 ppm ppm 20.4 ppm ppm 9.2 ppm ppm ppm 11.05 ppm ppm ppm 14.3 ppm ppm 16.3 ppm ppm 7.05 ppm ppm ppm ppm ppm 100 100 100 100 100 50 50 324 Nov03-FID243.chr Nov03-FID288.chr Nov03-FID333.chr Nov03-FID374.chr Nov03-FID434.chr Nov03-FID30.chr Nov03-FID68.chr Nov03-FID113.chr Nov03-FID166.chr Nov03-FID205.chr Nov03-FID244.chr Nov03-FID289.chr Nov03-FID334.chr Nov03-FID375.chr Nov03-FID435.chr Nov03-FID31.chr Nov03-FID69.chr Nov03-FID114.chr Nov03-FID167.chr Nov03-FID207.chr Nov03-FID245.chr Nov03-FID290.chr Nov03-FID376.chr Nov03-FID436.chr Nov03-FID70.chr Nov03-FID115.chr Nov03-FID168.chr Nov03-FID208.chr Nov03-FID246.chr Nov03-FID291.chr Nov03-FID437.chr Nov03-FID32.chr Nov03-FID71.chr Nov03-FID116.chr Nov03-FID169.chr Nov03-FID209.chr Nov03-FID247.chr Nov03-FID292.chr Nov03-FID382.chr Nov03-FID33.chr Nov03-FID72.chr Nov03-FID117.chr Nov03-FID170.chr Nov03-FID171.chr Nov03-FID210.chr Nov03-FID248.chr Nov03-FID293.chr Nov03-FID337.chr Nov03-FID383.chr Nov03-FID34.chr Nov03-FID74.chr Nov03-FID118.chr Nov03-FID172.chr Nov03-FID212.chr Nov03-FID249.chr Nov03-FID294.chr Nov03-FID338.chr Nov03-FID384.chr Nov03-FID35.chr Nov03-FID75.chr Nov03-FID119.chr Nov03-FID173.chr Nov03-FID213.chr Nov03-FID250.chr SAMPLE A-7 A-7 A-7 A-7 A-7 A-8 A-8 A-8 A-8 A-8 A-8 A-8 A-8 A-8 A-8 A-9 A-9 A-9 A-9 A-9 A-9 A-9 A-9 A-9 A-10 A-10 A-10 A-10 A-10 A-10 A-10 B-1 B-1 B-1 B-1 B-1 B-1 B-1 B-1 B-2 B-2 B-2 B-2 B-2 B-2 B-2 B-2 B-2 B-2 B-3 B-3 B-3 B-3 B-3 B-3 B-3 B-3 B-3 B-4 B-4 B-4 B-4 B-4 B-4 INJECT date 11/26/2003 11/28/2003 12/1/2003 12/2/2003 12/4/2003 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/1/2003 12/2/2003 12/4/2003 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/4/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/4/2003 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/3/2003 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 time 13:24:18 11:37:50 20:27:08 19:30:48 18:40:21 11/23/03 20:00 21:16:22 3:24:51 9:04:57 17:42:06 13:39:50 13:38:59 12:00:51 20:50:59 19:48:37 18:58:56 11/23/03 20:00 21:22:57 3:31:17 9:10:45 17:48:55 14:01:58 13:51:43 12:21:00 20:03:36 19:13:00 3:41:07 9:20:03 17:59:10 14:15:28 14:04:15 12:49:41 19:33:04 21:29:21 3:46:32 9:25:12 18:04:13 14:26:43 14:20:41 12:58:33 10:06:51 21:35:22 3:53:32 9:28:55 18:12:15 18:17:30 14:32:50 14:27:14 13:02:22 10:31:52 10:15:04 21:50:17 4:09:55 9:39:45 18:27:56 14:48:21 14:41:01 13:24:29 10:51:22 10:30:07 21:55:52 4:19:01 9:50:00 18:38:08 14:58:11 15:01:03 11/23/03 20:00 11/24/2003 3:41 11/24/2003 9:20 11/24/2003 17:59 11/25/2003 14:15 11/26/2003 14:04 11/28/2003 12:49 12/4/2003 19:33 11/23/03 20:00 11/23/2003 21:29 11/24/2003 3:46 11/24/2003 9:25 11/24/2003 18:04 11/25/2003 14:26 11/26/2003 14:20 11/28/2003 12:58 12/3/2002 10:06 11/23/03 20:00 11/23/2003 21:35 11/24/2003 3:52 11/24/2003 9:28 11/24/2003 18:12 11/24/2003 18:17 11/25/2003 14:32 11/26/2003 14:27 11/28/2003 13:02 12/2/2003 10:31 12/3/2003 10:15 11/23/03 20:00 11/23/2003 21:50 11/24/2003 4:09 11/24/2003 9:39 11/24/2003 18:27 11/25/2003 14:48 11/26/2003 14:41 11/28/2003 13:24 12/2/2003 10:51 12/3/2003 10:30 11/23/03 20:00 11/23/2003 21:55 11/24/2003 4:19 11/24/2003 9:50 11/24/2003 18:38 11/25/2003 14:58 11/26/2003 15:01 gas 4364.00 Methane 7851.00 12714.00 14064.00 15943.00 1.00 76.00 443.00 1504.00 1721.00 2938.00 4378.00 7874.00 12737.00 14076.00 15961.00 1.00 82.00 450.00 1510.00 1727.00 2948.00 4391.00 7895.00 14081.00 15976.00 1.00 460.00 1524.00 1738.00 2952.00 4699.00 7923.00 15996.00 1.00 89.00 465.00 1529.00 1743.00 2983.00 4420.00 7932.00 15012.00 1.00 94.00 472.00 1532.00 1756.00 1761.00 2990.00 4427.00 7936.00 13631.00 15021.00 1.00 109.00 488.00 1543.00 1766.00 3006.00 4441.00 7968.00 13661.00 15028.00 1.00 113.00 498.00 867.00 1777.00 3016.00 4461.00 Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane conc units 9.75 ppm ppm 15 ppm ppm 8.1 ppm ppm 7.29 ppm ppm 13.9 ppm 18.32 ppm 22.3 ppm ppm 28.1 ppm ppm 28 ppm ppm 7.94 ppm ppm ppm 9.9 ppm 13.4 ppm 8.1 ppm ppm 11 ppm 11.71 ppm ppm 14.2 ppm 11.66 ppm 14 ppm ppm 10.89 ppm ppm 12.9 ppm ppm 13.5 ppm 14.7 ppm 24.4 ppm ppm 17.9 ppm ppm 8.3 ppm ppm ppm 10 ppm 10.3 ppm 9.7 ppm ppm 9.75 ppm 20.4 ppm ppm 6.5 ppm ppm 6.98 ppm 7.2 ppm 11.1 ppm ppm 12.9 ppm 10.5 ppm 10.5 ppm 6.9 ppm ppm ppm 7.97 ppm 15.2 ppm 250 250 250 100 50 250 250 250 250 100 50 250 100 100 250 100 325 Nov03-FID295.chr Nov03-FID339.chr Nov03-FID385.chr Nov03-FID36.chr Nov03-FID76.chr Nov03-FID120.chr Nov03-FID174.chr Nov03-FID214.chr Nov03-FID251.chr Nov03-FID296.chr Nov03-FID340.chr Nov03-FID386.chr Nov03-FID37.chr Nov03-FID77.chr Nov03-FID121.chr Nov03-FID175.chr Nov03-FID215.chr Nov03-FID252.chr Nov03-FID297.chr Nov03-FID341.chr Nov03-FID387.chr Nov03-FID38.chr Nov03-FID78.chr Nov03-FID122.chr Nov03-FID176.chr Nov03-FID216.chr Nov03-FID253.chr Nov03-FID298.chr Nov03-FID342.chr Nov03-FID388.chr Nov03-FID39.chr Nov03-FID79.chr Nov03-FID123.chr Nov03-FID177.chr Nov03-FID178.chr Nov03-FID217.chr Nov03-FID254.chr Nov03-FID299.chr Nov03-FID343.chr Nov03-FID389.chr Nov03-FID40.chr Nov03-FID80.chr Nov03-FID81.chr Nov03-FID82.chr Nov03-FID124.chr Nov03-FID125.chr Nov03-FID179.chr Nov03-FID218.chr Nov03-FID255.chr Nov03-FID300.chr Nov03-FID344.chr Nov03-FID390.chr Nov03-FID83.chr Nov03-FID126.chr Nov03-FID180.chr Nov03-FID256.chr Nov03-FID301.chr Nov03-FID345.chr Nov03-FID391.chr Nov03-FID45.chr Nov03-FID89.chr Nov03-FID132.chr Nov03-FID219.chr Nov03-FID257.chr Nov03-FID302.chr Nov03-FID346.chr SAMPLE B-4 B-4 B-4 B-5 B-5 B-5 B-5 B-5 B-5 B-5 B-5 B-5 B-6 B-6 B-6 B-6 B-6 B-6 B-6 B-6 B-6 B-7 B-7 B-7 B-7 B-7 B-7 B-7 B-7 B-7 B-8 B-8 B-8 B-8 B-8 B-8 B-8 B-8 B-8 B-8 B-9 B-9 B-9 B-9 B-9 B-9 B-9 B-9 B-9 B-9 B-9 B-9 B-10 B-10 B-10 B-10 B-10 B-10 B-10 C-1 C-1 C-1 C-1 C-1 C-1 C-1 INJECT date 11/28/2003 12/2/2003 12/3/2003 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 11/24/2003 11/24/2003 11/24/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 11/23/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 time 13:40:14 11:11:49 10:46:15 22:01:45 4:24:39 10:00:03 18:48:52 15:15:28 15:14:27 13:58:38 11:50:54 11:01:11 22:06:59 4:30:32 10:04:16 18:53:58 15:35:21 15:34:17 14:17:56 12:10:16 11:15:21 22:13:58 4:36:07 10:13:54 19:05:41 17:02:20 15:49:52 14:31:55 12:28:26 11:35:06 22:18:59 4:41:38 10:24:56 19:15:45 19:17:25 17:24:57 16:03:02 14:52:58 12:48:22 11:50:27 22:24:45 4:47:35 4:57:48 5:02:22 10:31:41 10:46:35 19:20:41 17:39:08 16:14:50 15:08:22 13:00:26 12:04:14 5:06:54 10:52:55 19:24:49 16:37:22 15:27:31 13:16:52 12:22:59 22:55:01 5:39:49 11:44:47 17:57:28 16:54:11 15:46:37 13:24:47 11/24/2003 5:06 11/24/2003 10:52 11/24/2003 19:24 11/26/2003 16:37 11/28/2003 15:27 12/2/2003 13:16 12/3/2003 12:22 11/28/2003 13:40 12/2/2003 11:11 12/3/2003 10:46 11/23/03 20:00 11/23/2003 22:01 11/24/2003 11/24/2003 10:00 11/24/2003 18:48 11/25/2003 15:15 11/26/2003 15:14 11/28/2003 13:58 12/2/2003 11:50 12/3/2003 11:01 11/23/03 20:00 11/23/2003 22:06 11/24/2003 4:30 11/24/2003 10:04 11/24/2003 18:53 11/25/2003 15:35 11/26/2003 15:34 11/28/2003 14:17 12/2/2003 12:10 12/3/2003 11:15 7984.00 13671.00 15042.00 1.00 120.00 503.00 877.00 1797.00 3033.00 4474.00 8002.00 13710.00 15057.00 1.00 125.00 509.00 881.00 1802.00 3053.00 4494.00 8011.00 13730.00 15071.00 1.00 132.00 515.00 906.00 1814.00 3140.00 4650.00 8026.00 13744.00 15091.00 1.00 137.00 520.00 917.00 1811.00 1813.00 3231.00 4664.00 8047.00 13764.00 15106.00 1.00 143.00 526.00 536.00 541.00 924.00 929.00 1814.00 3246.00 4675.00 8063.00 13776.00 15120.00 1.00 545.00 945.00 1818.00 4698.00 8074.00 13789.00 15138.00 gas Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane conc units ppm 11.2 ppm 12 ppm ppm 6.8 ppm ppm 8.3 ppm 9.9 ppm 16.6 ppm ppm 13.1 ppm 11.2 ppm 250 250 250 250 ppm 6.8 ppm ppm 8.6 ppm 10.97 ppm 10.96 ppm ppm 10.1 ppm 8.7 ppm ppm 6.7 ppm ppm ppm 9.48 ppm 15.1 ppm ppm 17.6 ppm 9.2 ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm 100 100 250 250 250 6.5 6.97 7.2 7.4 9.4 100 50 100 50 100 100 ppm 6.3 ppm ppm ppm ppm ppm ppm 9.58 ppm ppm ppm ppm 8.1 ppm 12.7 ppm ppm 9.2 ppm 7.1 ppm ppm 10.8 ppm 10.5 ppm ppm 14.2 ppm ppm 17.5 ppm 25 ppm ppm 18.9 ppm 250 250 250 250 326 Nov03-FID392.chr Nov03-FID98.chr Nov03-FID141.chr Nov03-FID275.chr Nov03-FID311.chr Nov03-FID355.chr Nov03-FID401.chr Nov03-FID46.chr Nov03-FID90.chr Nov03-FID133.chr Nov03-FID220.chr Nov03-FID258.chr Nov03-FID303.chr Nov03-FID347.chr Nov03-FID393.chr Nov03-FID47.chr Nov03-FID91.chr Nov03-FID134.chr Nov03-FID183.chr Nov03-FID221.chr Nov03-FID259.chr Nov03-FID304.chr Nov03-FID348.chr Nov03-FID394.chr Nov03-FID48.chr Nov03-FID92.chr Nov03-FID135.chr Nov03-FID182.chr Nov03-FID222.chr Nov03-FID260.chr Nov03-FID305.chr Nov03-FID349.chr Nov03-FID395.chr Nov03-FID49.chr Nov03-FID93.chr Nov03-FID136.chr Nov03-FID223.chr Nov03-FID261.chr Nov03-FID306.chr Nov03-FID350.chr Nov03-FID396.chr Nov03-FID50.chr Nov03-FID94.chr Nov03-FID137.chr Nov03-FID224.chr Nov03-FID262.chr Nov03-FID307.chr Nov03-FID351.chr Nov03-FID397.chr Nov03-FID51.chr Nov03-FID95.chr Nov03-FID138.chr Nov03-FID181.chr Nov03-FID225.chr Nov03-FID263.chr Nov03-FID308.chr Nov03-FID352.chr Nov03-FID398.chr Nov03-FID52.chr Nov03-FID96.chr Nov03-FID139.chr Nov03-FID264.chr Nov03-FID309.chr Nov03-FID353.chr Nov03-FID399.chr Nov03-FID53.chr Nov03-FID97.chr Nov03-FID140.chr Nov03-FID273.chr Nov03-FID310.chr Nov03-FID354.chr Nov03-FID400.chr SAMPLE C-1 C-10 C-10 C-10 C-10 C-10 C-10 C-2 C-2 C-2 C-2 C-2 C-2 C-2 C-2 C-3 C-3 C-3 C-3 C-3 C-3 C-3 C-3 C-3 C-4 C-4 C-4 C-4 C-4 C-4 C-4 C-4 C-4 C-5 C-5 C-5 C-5 C-5 C-5 C-5 C-5 C-6 C-6 C-6 C-6 C-6 C-6 C-6 C-6 C-7 C-7 C-7 C-7 C-7 C-7 C-7 C-7 C-7 C-8 C-8 C-8 C-8 C-8 C-8 C-8 C-9 C-9 C-9 C-9 C-9 C-9 C-9 INJECT date 12/3/2003 11/24/2003 11/24/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 11/23/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 11/23/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 11/23/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 11/24/2003 11/24/2003 11/24/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 11/24/2003 11/24/2003 11/24/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 time 12:37:05 6:29:28 12:27:25 20:17:14 17:48:53 14:51:33 14:18:36 23:03:13 5:45:32 11:49:00 18:07:09 17:05:07 16:03:04 13:30:58 12:45:31 23:08:28 5:50:54 11:53:05 19:36:15 18:16:38 17:20:05 16:19:14 13:44:53 12:57:12 23:21:02 5:56:34 11:58:02 19:32:48 18:26:20 17:28:05 16:33:18 13:56:15 13:12:59 23:40:24 6:02:27 12:05:29 18:34:10 17:37:58 16:48:52 14:05:59 13:18:55 23:46:26 6:07:46 12:10:09 18:45:45 17:44:09 17:03:37 14:09:32 13:32:48 23:53:44 6:13:17 12:14:16 19:28:41 18:58:39 17:52:21 17:17:51 14:20:25 13:40:37 0:02:26 6:18:38 12:18:02 18:03:42 17:27:40 14:30:57 13:55:01 0:09:34 6:24:00 12:23:53 19:51:11 17:38:06 14:39:31 14:06:58 100 gas Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane conc units 20.1 ppm 13.59 ppm ppm ppm ppm 12.3 ppm 11.9 ppm ppm 12.04 ppm ppm 12.79 ppm 15.8 ppm ppm 14 ppm 12.9 ppm ppm 11.25 ppm ppm 10.76 ppm 13 ppm 13.9 ppm ppm 7.5 ppm 11.5 ppm ppm ppm ppm 10 ppm 10.7 ppm 14.3 ppm ppm 17.6 ppm 11.1 ppm ppm 10.2 ppm ppm 12.3 ppm 10.1 ppm ppm 12.6 ppm 11.99 ppm ppm 8.8 ppm ppm 10.96 ppm 12.1 ppm ppm 8.4 ppm 10.5 ppm ppm 7.6 ppm ppm 7.9 ppm 9.5 ppm 8 ppm ppm 9.5 ppm 9.3 ppm ppm 8.7 ppm ppm 11 ppm ppm 9 ppm 7.6 ppm ppm 6.7 ppm ppm ppm ppm 7.4 ppm ppm 327 Nov03-FID41.chr Nov03-FID84.chr Nov03-FID127.chr Nov03-FID153.chr Nov03-FID184.chr Nov03-FID231.chr Nov03-FID271.chr Nov03-FID318.chr Nov03-FID325.chr Nov03-FID363.chr Nov03-FID408.chr Nov03-FID42.chr Nov03-FID85.chr Nov03-FID128.chr Nov03-FID154.chr Nov03-FID185.chr Nov03-FID232.chr Nov03-FID265.chr Nov03-FID319.chr Nov03-FID326.chr Nov03-FID364.chr Nov03-FID409.chr Nov03-FID43.chr Nov03-FID86.chr Nov03-FID129.chr Nov03-FID155.chr Nov03-FID186.chr Nov03-FID233.chr Nov03-FID266.chr Nov03-FID320.chr Nov03-FID327.chr Nov03-FID365.chr Nov03-FID410.chr Nov03-FID44.chr Nov03-FID87.chr Nov03-FID130.chr Nov03-FID156.chr Nov03-FID187.chr Nov03-FID234.chr Nov03-FID267.chr Nov03-FID321.chr Nov03-FID328.chr Nov03-FID366.chr Nov03-FID411.chr Nov03-FID88.chr Nov03-FID131.chr Nov03-FID157.chr Nov03-FID188.chr Nov03-FID235.chr Nov03-FID269.chr Nov03-FID322.chr Nov03-FID329.chr Nov03-FID367.chr Nov03-FID412.chr Nov03-FID151.chr Nov03-FID191.chr Nov03-FID144.chr Nov03-FID152.chr Nov03-FID192.chr Nov03-FID194.chr Nov03-FID195.chr Nov03-FID236.chr Nov03-FID280.chr Nov03-FID281.chr Nov03-FID323.chr Nov03-FID324.chr Nov03-FID336.chr Nov03-FID378.chr Nov03-FID54.chr Nov03-FID99.chr Nov03-FID142.chr SAMPLE INJECT date time M-1 11/23/2003 22:30:09 M-1 250 11/24/2003 5:12:09 M-1 11/24/2003 10:57:13 M-1 11/24/2003 15:45:50 M-1 11/24/2003 19:40:39 M-1 11/25/2003 20:24:22 M-1 11/26/2003 19:26:02 M-1 11/28/2003 20:34:08 M-1 12/1/2003 14:21:22 M-1 12/2/2003 16:50:51 M-1 12/3/2003 16:06:49 M-2 11/23/2003 22:36:08 M-2 11/24/2003 5:17:29 M-2 11/24/2003 11:07:27 M-2 11/24/2003 15:50:32 M-2 11/24/2003 19:52:19 M-2 11/25/2003 20:36:28 M-2 11/26/2003 18:11:08 M-2 11/28/2003 20:45:29 M-2 12/1/2003 14:35:26 M-2 12/2/2003 16:55:29 M-2 12/3/2003 16:12:55 M-3 11/23/2003 22:42:37 M-3 11/24/2003 5:22:48 M-3 11/24/2003 11:17:31 M-3 11/24/2003 16:00:06 M-3 11/24/2003 19:56:29 M-3 11/25/2003 20:45:04 M-3 11/26/2003 18:18:02 M-3 11/28/2003 21:07:46 M-3 12/1/2003 14:53:57 M-3 12/2/2003 17:11:20 M-3 12/3/2003 16:23:07 M-4 11/23/2003 22:48:21 M-4 11/24/2003 5:28:36 M-4 11/24/2003 11:27:04 M-4 11/24/2003 16:10:56 M-4 11/24/2003 20:00:55 M-4 11/25/2003 20:54:40 M-4 11/26/2003 18:34:23 M-4 11/28/2003 21:26:05 M-4 12/1/2003 18:33:14 M-4 12/2/2003 17:27:13 M-4 12/3/2003 16:35:02 M-5 11/24/2003 5:34:09 M-5 11/24/2003 11:37:39 M-5 11/24/2003 16:20:30 M-5 11/24/2003 20:11:16 M-5 11/25/2003 21:06:57 M-5 11/26/2003 19:00:32 M-5 11/28/2003 21:48:05 M-5 12/1/2003 18:53:40 M-5 12/2/2003 17:43:03 M-5 12/3/2003 16:52:34 MIDPOINT 11/24/2003 15:08:06 MIDPOINT 11/24/2003 20:46:42 OUTTAKE TUBE 11/24/2003 12:57:58 OUTTAKE TUBE 11/24/2003 15:30:52 OUTTAKE TUBE 11/24/2003 21:09:15 OUTTAKE TUBE 11/25/2003 10:24:12 s-GAS STAND 11/25/2003 10:39:45 s-GAS STAND 11/26/2003 11:43:48 s-GAS STAND 11/28/2003 9:26:27 s-GAS STAND 11/28/2003 9:40:55 s-GAS STAND 12/1/2003 13:40:47 s-GAS STAND 12/1/2003 13:58:56 s-GAS STAND 12/2/2003 9:56:01 s-GAS STAND 12/3/2003 9:20:47 SOX-1 11/24/2003 0:16:11 SOX-1 11/24/2003 6:35:47 SOX-1 11/24/2003 12:31:13 gas Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane conc 7.8 7.2 6.8 10.8 7.8 7.99 7.8 7.8 9.7 7.3 8.37 8.6 8.88 9.2 9.5 9.6 10.8 6.66 9.76 6.7 6.56 9 7.7 7.3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 8.9 8.9 units ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm ppm 328 Nov03-FID226.chr Nov03-FID268.chr Nov03-FID312.chr Nov03-FID356.chr Nov03-FID362.chr Nov03-FID402.chr Nov03-FID55.chr Nov03-FID100.chr Nov03-FID145.chr Nov03-FID227.chr Nov03-FID270.chr Nov03-FID313.chr Nov03-FID357.chr Nov03-FID403.chr Nov03-FID56.chr Nov03-FID101.chr Nov03-FID146.chr Nov03-FID228.chr Nov03-FID272.chr Nov03-FID314.chr Nov03-FID358.chr Nov03-FID404.chr Nov03-FID57.chr Nov03-FID102.chr Nov03-FID147.chr Nov03-FID229.chr Nov03-FID274.chr Nov03-FID315.chr Nov03-FID359.chr Nov03-FID405.chr Nov03-FID58.chr Nov03-FID103.chr Nov03-FID148.chr Nov03-FID230.chr Nov03-FID276.chr Nov03-FID316.chr Nov03-FID360.chr Nov03-FID406.chr Nov03-FID59.chr Nov03-FID104.chr Nov03-FID149.chr Nov03-FID277.chr Nov03-FID317.chr Nov03-FID407.chr SAMPLE SOX-1 SOX-1 SOX-1 SOX-1 SOX-1 SOX-1 SOX-2 SOX-2 SOX-2 SOX-2 SOX-2 SOX-2 SOX-2 SOX-2 SOX-3 SOX-3 SOX-3 SOX-3 SOX-3 SOX-3 SOX-3 SOX-3 SOX-4 SOX-4 SOX-4 SOX-4 SOX-4 SOX-4 SOX-4 SOX-4 SOX-5 SOX-5 SOX-5 SOX-5 SOX-5 SOX-5 SOX-5 SOX-5 SOX-6 SOX-6 SOX-6 SOX-6 SOX-6 SOX-6 INJECT date 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/2/2003 12/3/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 11/26/2003 11/28/2003 12/2/2003 12/3/2003 11/24/2003 11/24/2003 11/24/2003 11/26/2003 11/28/2003 12/3/2003 time 19:11:41 18:41:59 18:11:26 14:55:07 16:35:19 14:25:33 0:34:08 6:49:12 13:18:44 19:18:09 19:06:18 18:29:29 15:00:50 14:43:55 0:55:51 7:02:11 13:33:31 19:32:27 19:32:41 18:46:16 15:20:38 15:04:16 1:09:26 7:16:12 13:52:16 19:47:29 19:57:46 19:15:45 15:41:11 15:21:17 1:32:57 7:29:34 14:07:34 20:09:47 20:23:06 19:36:47 16:08:56 15:37:16 1:49:14 7:44:04 14:29:15 20:43:23 19:59:35 15:52:06 100 100 100 gas Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane Methane conc units 9.9 ppm ppm ppm 9.5 ppm ppm ppm ppm 11.3 ppm 7.3 ppm ppm 8.8 ppm ppm 10.9 ppm 10.7 ppm ppm 10.2 ppm ppm 6.3 ppm 9.2 ppm ppm 14.3 ppm 7.3 ppm ppm 7.3 ppm ppm 10.75 ppm 6.7 ppm ppm 8.1 ppm 7.6 ppm ppm 7.1 ppm ppm 9.04 ppm 7.2 ppm ppm 9.4 ppm 9.9 ppm ppm 7.07 ppm 6.7 ppm 9.1 ppm ppm 7 ppm 329 Appendix D - Chapter 5 Gas chromatography data from Nov 2003 HP-TCD Results SAMPLE date time time cum minutes gas reten area conc units gas reten area conc units 100 MICRO-L INJECTS A-1 A-1 A-1 A-1 A-1 A-1 A-2 A-2 A-2 A-2 A-2 A-2 A-3 A-3 A-3 A-3 A-3 A-3 A-4 A-4 A-4 A-4 A-4 A-4 A-5 A-5 A-5 A-5 A-5 A-5 A-6 A-6 A-6 A-6 A-6 A-6 A-7 A-7 A-7 A-7 11/23/2003 11/24/2003 11/24/2003 11/24/2003 21:05:48 3:06:41 8:38:51 17:17:29 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 12/4/2003 21:00:40 3:00:15 8:29:08 17:06:28 12:58:38 13:25:11 O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar 0.908 0.908 0.925 0.908 0.958 1.008 0.916 0.9 0.916 0.891 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 12/4/2003 20:54:48 2:51:59 8:22:58 16:56:19 12:49:52 13:20:47 O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar 0.916 0.908 0 0.916 0.95 1.008 6399.4045 6010.037 0 5739.417 5309.045 1225.805 6383.5835 6020.0188 5672.559 5461.2275 5019.5918 709.9075 3928.4915 3688.3035 3345.188 3323.7345 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 12/4/2003 20:49:18 2:30:27 8:16:56 16:37:02 12:42:40 13:16:26 O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar 0.916 0.908 0.925 0.916 0.958 1.008 6436.567 6137.004 6052.7513 5585.2025 5280.75 1148.7438 %vol %vol %vol %vol %vol %vol 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 12/4/2003 20:43:31 2:23:34 8:09:59 16:31:48 12:34:17 13:10:55 O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar 0.916 0.916 0.925 0.925 0.95 1.008 6345.6467 6140.283 6046.6165 6011.8353 4745.4828 1212.2842 %vol %vol %vol %vol %vol %vol Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen 1.708 1.708 1.725 1.716 1.766 1.775 1.708 1.7 1.725 1.708 1.775 1.766 1.708 1.708 0 1.708 1.766 1.766 1.708 1.7 1.716 1.7 1.775 1.775 1.716 1.708 1.725 1.691 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 12/4/2003 20:37:06 2:17:24 8:04:40 16:25:12 12:26:43 13:05:23 O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar 0.925 0.916 0.925 0.925 0.95 1.008 6268.8295 5992.8325 6300.809 4628.213 5412.637 2928.2768 %vol %vol %vol %vol %vol %vol Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen 1.716 1.716 1.716 1.725 1.766 1.775 25814.957 24928.814 26435.691 19723.8765 23490.1682 26193.9698 26119.646 25528.574 25440.8795 25715.3418 21132.4392 27199.6195 26473.852 25495.8518 25572.3678 24146.183 24000.966 26123.8305 26347.583 25178.1375 0 25263.887 25817.545 26493.2155 26350.4635 25400.1813 24583.661 25351.4653 26562.0405 25117.183 16221.895 15448.073 14194.979 14335.588 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/25/2003 12/4/2003 20:29:53 2:09:04 7:56:51 16:14:34 12:20:45 13:00:04 O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar 0.916 0.916 0.925 0.925 0.95 1.008 6372.4435 6195.0685 5794.721 5203.4348 3548.783 5573.0185 %vol %vol %vol %vol %vol %vol Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen 1.708 1.708 1.716 1.716 1.775 1.766 26211.733 25669.079 24076.7835 21759.224 14844.501 23088.3142 76.3095 74.7297 70.0941 63.347 43.2164 67.2164 75.1544 72.5746 76.9615 57.4216 68.3863 76.2578 76.0414 74.3206 74.0653 74.8644 61.5223 79.1855 77.0726 74.2254 74.4481 70.2961 69.8733 76.0536 76.705 73.3004 0 73.5501 75.1619 77.129 76.7134 73.9468 71.5697 73.805 77.3293 73.123 47.2264 44.9735 41.3255 41.7348 %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol 03NOV-HPTCD-22.CHR 03NOV-HPTCD-61.CHR 03NOV-HPTCD-104.CHR 03NOV-HPTCD-152.CHR 03NOV-HPTCD-180.CHR 03NOV-HPTCD-209.CHR 03NOV-HPTCD-23.CHR 03NOV-HPTCD-62.CHR 03NOV-HPTCD-105.CHR 03NOV-HPTCD-153.CHR 03NOV-HPTCD-181.CHR 03NOV-HPTCD-210.CHR 03NOV-HPTCD-24.CHR 03NOV-HPTCD-63.CHR 03NOV-HPTCD-106.CHR 03NOV-HPTCD-154.CHR 03NOV-HPTCD-182.CHR 03NOV-HPTCD-211.CHR 330 03NOV-HPTCD-25.CHR 03NOV-HPTCD-64.CHR 03NOV-HPTCD-107.CHR 03NOV-HPTCD-155.CHR 03NOV-HPTCD-183.CHR 03NOV-HPTCD-212.CHR 03NOV-HPTCD-26.CHR 03NOV-HPTCD-65.CHR 03NOV-HPTCD-108.CHR 03NOV-HPTCD-156.CHR 03NOV-HPTCD-184.CHR 03NOV-HPTCD-213.CHR 19.6 19.5679 19.0232 17.7939 15.9782 10.8973 17.1131 19.3 19.2497 18.4022 19.3479 14.2119 16.6206 8.9919 19.5 19.4856 18.855 18.5674 18.4606 14.572 3.7226 19.8 19.7648 18.8449 18.5862 17.1505 16.2156 3.5275 19.7 19.6507 18.455 03NOV-HPTCD-27.CHR 03NOV-HPTCD-66.CHR 03NOV-HPTCD-109.CHR 03NOV-HPTCD-157.CHR 03NOV-HPTCD-185.CHR 03NOV-HPTCD-214.CHR 03NOV-HPTCD-28.CHR 03NOV-HPTCD-67.CHR 03NOV-HPTCD-110.CHR 03NOV-HPTCD-158.CHR 11/23/03 20:00 11/23/03 20:29 11/24/03 2:09 11/24/03 7:56 11/24/03 16:14 11/25/03 12:20 12/4/03 13:00 11/23/03 20:00 11/23/03 20:37 11/24/2003 2:17 11/24/2003 8:04 11/24/2003 16:25 11/25/2003 12:26 12/4/2003 13:05 11/23/03 20:00 11/23/2003 20:43 11/24/2003 2:23 11/24/2003 8:09 11/24/2003 16:31 11/25/2003 12:34 12/4/2003 13:10 11/23/03 20:00 11/23/2003 20:49 11/24/2003 2:30 11/24/2003 8:16 11/24/2003 16:37 11/25/2003 12:42 12/4/2003 13:16 11/23/03 20:00 11/23/2003 20:54 11/24/2003 2:51 11/24/2003 8:22 11/24/2003 16:56 11/25/2003 12:49 12/4/2003 13:20 11/23/03 20:00 11/23/2003 21:00 11/24/2003 3:00 11/24/2003 8:29 11/24/2003 17:06 11/25/2003 12:58 12/4/2003 13:25 11/23/03 20:00 11/23/2003 21:05 11/24/2003 3:06 11/24/2003 8:38 11/24/2003 17:17 %vol %vol %vol 17.6241 %vol 16.3025 %vol 3.7641 %vol 19.61 19.6021 %vol 18.4857 %vol 17.4188 %vol 16.7698 %vol 15.4137 %vol 2.1799 %vol 12.1 12.0632 %vol 11.3257 %vol 10.2721 %vol 10.2062 %vol 1 29 369 1434 1936 2848 15603 1 37 377 1444 1947 2854 15608 1 42 383 1449 1953 2864 15613 1 48 390 1456 1959 2872 15619 1 53 411 1462 1978 2879 15623 1 59 420 1469 1988 2888 15628 1 64 426 1478 1999 03NOV-HPTCD-186.CHR 03NOV-HPTCD-215.CHR O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen 1.725 1.725 1.7 1.766 1.766 1.708 1.725 1.766 0 1.775 1.775 1.7 1.716 1.725 1.7 1.758 1.775 1.708 1.708 1.725 1.7 1.7 1.775 1.708 1.708 1.725 1.716 1.758 0 1.766 %vol %vol %vol %vol %vol %vol Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen 1.716 1.733 1.741 1.716 1.783 1.783 14863.6177 13927.6228 14832.7925 15185.2925 12765.0015 15032.7618 25221.0745 24074.739 25886.9975 24862.6195 24385.3023 25261.609 23799.475 25581.5663 0 20177.9475 18006.6285 26146.025 25868.326 22779.3335 25711.862 25446.6195 23951.874 26350.516 25444.959 22705.738 25776.3105 24523.8805 27734.17 25663.724 23895.488 24310.8363 23146.1545 24655.34 0 26214.4125 %vol %vol %vol %vol %vol %vol Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen 1.716 1.716 1.725 1.733 1.783 1.775 15284.905 14361.946 13774.7728 6263.814 15244.8375 17638.2948 44.4985 41.8115 40.1021 18.2357 44.3819 51.3499 43.272 40.5471 43.1823 44.2085 37.1624 43.7645 73.4254 70.0881 75.3641 72.3819 70.9923 73.5434 69.2868 74.4749 0 58.7435 52.4222 76.1182 75.3097 66.3168 74.8542 74.082 69.7304 76.7135 74.0772 66.1026 75.0419 71.3957 80.7417 74.7141 69.5663 70.7755 67.3848 71.7784 0 76.3173 gas O2 + Ar O2 + Ar %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol units %vol %vol gas Nitrogen Nitrogen reten area conc units 1.783 15053.205 43.824 %vol 1.783 14844.3685 43.216 %vol 03NOV-HPTCD-29.CHR 03NOV-HPTCD-68.CHR 03NOV-HPTCD-111.CHR 03NOV-HPTCD-159.CHR 03NOV-HPTCD-187.CHR 03NOV-HPTCD-216.CHR 03NOV-HPTCD-30.CHR 03NOV-HPTCD-69.CHR 03NOV-HPTCD-112.CHR 03NOV-HPTCD-160.CHR 03NOV-HPTCD-188.CHR 03NOV-HPTCD-217.CHR 03NOV-HPTCD-70.CHR 03NOV-HPTCD-113.CHR 03NOV-HPTCD-161.CHR 03NOV-HPTCD-189.CHR 03NOV-HPTCD-218.CHR 03NOV-HPTCD-60.CHR 03NOV-HPTCD-103.CHR 03NOV-HPTCD-190.CHR 03NOV-HPTCD-200.CHR 03NOV-HPTCD-205.CHR 03NOV-HPTCD-206.CHR 2908 15631 1 70 438 1485 2018 2918 15365 1 76 445 1499 2038 2931 15369 1 452 1506 2047 2945 15372 331 1 83 461 1516 2053 2987 15379 1 89 467 1521 2061 2991 15384 1 102 477 1530 2074 3008 4459 15388 03NOV-HPTCD-32.CHR 03NOV-HPTCD-71.CHR 03NOV-HPTCD-114.CHR 03NOV-HPTCD-162.CHR 03NOV-HPTCD-191.CHR 03NOV-HPTCD-219.CHR 03NOV-HPTCD-33.CHR 03NOV-HPTCD-72.CHR 03NOV-HPTCD-115.CHR 03NOV-HPTCD-163.CHR 03NOV-HPTCD-192.CHR 03NOV-HPTCD-220.CHR 03NOV-HPTCD-34.CHR 03NOV-HPTCD-73.CHR 03NOV-HPTCD-116.CHR 03NOV-HPTCD-164.CHR 03NOV-HPTCD-193.CHR 03NOV-HPTCD-201.CHR 03NOV-HPTCD-221.CHR SAMPLE date time time A-7 11/25/2003 13:18:41 11/25/2003 13:18 A-7 12/4/2003 13:28:42 12/4/2003 13:28 11/23/03 20:00 A-8 11/23/2003 21:11:05 11/23/2003 21:11 A-8 11/24/2003 3:18:36 11/24/2003 3:18 A-8 11/24/2003 8:45:45 11/24/2003 8:45 A-8 11/24/2003 17:36:17 11/24/2003 17:36 A-8 11/25/2003 13:28:25 11/25/2003 13:28 A-8 12/4/2003 13:32:30 12/4/2003 13:32 11/23/03 20:00 A-9 11/23/2003 21:17:27 11/23/2003 21:17 A-9 11/24/2003 3:25:47 11/24/2003 3:25 A-9 11/24/2003 8:59:09 11/24/2003 8:59 A-9 11/24/2003 17:43:24 11/24/2003 17:43 A-9 11/25/2003 13:41:29 11/25/2003 13:41 A-9 12/4/2003 13:36:22 12/4/2003 13:36 11/23/03 20:00 A-10 11/24/2003 3:32:57 11/24/2003 3:32 A-10 11/24/2003 9:06:35 11/24/2003 9:06 A-10 11/24/2003 17:52:44 11/24/2003 17:52 A-10 11/25/2003 13:55:29 11/25/2003 13:55 A-10 12/4/2003 13:39:46 12/4/2003 13:39 AIR 11/24/2003 2:04:15 AIR 11/24/2003 7:44:22 AIR 11/25/2003 14:14:42 AIR 11/26/2003 14:46:18 AIR 12/4/2003 12:35:13 AIR 12/4/2003 12:40:36 11/23/03 20:00 B-1 11/23/2003 21:24:21 11/23/2003 21:24 B-1 11/24/2003 3:41:06 11/24/2003 3:41 B-1 11/24/2003 9:16:08 11/24/2003 9:16 B-1 11/24/2003 17:58:42 11/24/2003 17:58 B-1 11/25/2003 14:23:00 11/25/2003 14:23 B-1 12/4/2003 13:46:16 12/4/2003 13:46 11/23/03 20:00 B-2 11/23/2003 21:30:20 11/23/2003 21:30 B-2 11/24/2003 3:47:56 11/24/2003 3:47 B-2 11/24/2003 9:21:34 11/24/2003 9:21 B-2 11/24/2003 18:06:34 11/24/2003 18:06 B-2 11/25/2003 14:27:35 11/25/2003 14:27 B-2 12/4/2003 13:51:16 12/4/2003 13:51 11/23/03 20:00 B-3 11/23/2003 21:43:36 11/23/2003 21:43 B-3 11/24/2003 3:57:20 11/24/2003 3:57 B-3 11/24/2003 9:32:24 11/24/2003 9:32 B-3 11/24/2003 18:23:45 11/24/2003 18:23 B-3 11/25/2003 14:44:34 11/25/2003 14:44 B-3 11/26/2003 14:55:31 11/26/2003 14:55 B-3 12/4/2003 13:55:40 12/4/2003 13:55 reten area conc 0.958 3403.0655 10.4498 1.016 2623.498 8.056 11.4 0.916 3700.7635 11.3639 0.916 3430.9972 10.5356 0.925 3255.0385 9.9953 0.925 1446.568 4.442 0.958 3413.768 10.4827 1 362.2887 1.1125 11.1 0.916 3590.2253 11.0245 0.933 3304.6332 10.1476 0.933 3472.6768 10.6636 0.916 3487.292 10.7084 0.958 2858.0507 8.7762 1.016 2639.2848 8.1045 17.9 0.933 5809.707 17.8399 0.933 5565.06 17.0887 0.908 5890.327 18.0875 0.95 5617.3975 17.2494 1.008 5451.4083 16.7397 0.908 6160.159 18.916 0.925 5777.087 17.7397 0.95 6212.341 19.0763 0 0 0 1.008 5001.955 15.3595 1.008 4470.7825 13.7285 19.5 0.908 6344.385 19.4817 0.925 6243.012 19.1704 0.925 5485.106 16.8431 0.908 6176.254 18.9654 0.941 6145.759 18.8718 1.016 5892.8425 18.0952 19.7 0.916 6392.2215 19.6286 0.916 6049.5525 18.5764 0.925 5328.569 16.3625 0.908 5809.9173 17.8405 0.883 5154.509 15.828 1.008 1406.967 4.3204 19.2 0.916 6224.673 19.1141 0.916 5711.7665 17.5391 0.925 5740.327 17.6268 0.925 5307.3435 16.2973 0.941 5314.239 16.3185 0 0 1.008 1553.3185 4.7698 03NOV-HPTCD-221.CHR O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen %vol %vol %vol %vol %vol Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen 1.708 1.716 1.725 1.716 1.791 1.725 1.716 1.733 1.725 1.491 1.783 1.725 1.716 1.733 1.733 1.783 1.725 1.725 1.733 1.725 1.783 1.7 1.716 1.716 1.766 1.708 1.716 1.716 1.716 1.725 1.766 %vol %vol %vol %vol %vol Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen 1.716 1.716 1.725 1.716 1.766 26348.358 20036.626 23194.4083 23131.2038 23929.1055 25828.213 24551.459 22874.414 24328.531 27848.4827 14101.2498 13795.521 13425.1213 15092.819 13479.8755 17854.216 14580.044 13157.247 13756.154 12812.92 14810.3018 14614.34 14566.538 13388.8353 12315.855 13466.113 23097.337 23078.1865 21889.1712 25669.836 26516.294 24372.0098 24422.053 24793.374 22793.322 27589.1445 %vol %vol %vol %vol %vol Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen 1.708 1.708 1.725 1.708 1.775 26076.852 23481.011 22654.1575 24147.6947 27261.6427 75.9168 68.3596 65.9524 70.3005 79.3661 76.7072 58.3321 67.5252 67.3412 69.6641 75.193 71.476 66.5936 70.827 81.0745 41.0526 40.1625 39.0842 43.9393 39.2436 51.9785 42.4465 38.3043 40.0479 37.3019 43.1168 42.5463 42.4072 38.9785 35.8548 39.2035 67.2426 67.1869 gas O2 + Ar %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol 63.7253 %vol 74.7319 %vol 77.1962 70.9536 71.0992 72.1803 66.3576 80.3195 %vol %vol %vol %vol %vol %vol units %vol gas Nitrogen reten area conc units 1.766 26214.4125 76.3173 %vol 03NOV-HPTCD-35.CHR 03NOV-HPTCD-75.CHR 03NOV-HPTCD-117.CHR 03NOV-HPTCD-165.CHR 03NOV-HPTCD-222.CHR 03NOV-HPTCD-36.CHR 03NOV-HPTCD-76.CHR 03NOV-HPTCD-118.CHR 03NOV-HPTCD-166.CHR 03NOV-HPTCD-223.CHR 03NOV-HPTCD-37.CHR 03NOV-HPTCD-77.CHR 03NOV-HPTCD-119.CHR 03NOV-HPTCD-167.CHR 03NOV-HPTCD-224.CHR 03NOV-HPTCD-38.CHR 03NOV-HPTCD-78.CHR 03NOV-HPTCD-120.CHR 03NOV-HPTCD-168.CHR 03NOV-HPTCD-202.CHR 03NOV-HPTCD-225.CHR 332 03NOV-HPTCD-39.CHR 03NOV-HPTCD-79.CHR 03NOV-HPTCD-121.CHR 03NOV-HPTCD-169.CHR 03NOV-HPTCD-226.CHR 03NOV-HPTCD-40.CHR 03NOV-HPTCD-80.CHR 03NOV-HPTCD-122.CHR 03NOV-HPTCD-170.CHR 03NOV-HPTCD-227.CHR 03NOV-HPTCD-81.CHR 03NOV-HPTCD-123.CHR 03NOV-HPTCD-171.CHR 03NOV-HPTCD-228.CHR 03NOV-HPTCD-41.CHR 03NOV-HPTCD-82.CHR 03NOV-HPTCD-124.CHR 03NOV-HPTCD-147.CHR 03NOV-HPTCD-175.CHR 03NOV-HPTCD-245.CHR SAMPLE date time time B-3 12/4/2003 13:55:40 12/4/2003 13:55 11/23/03 20:00 B-4 11/23/2003 21:50:37 11/23/2003 21:50 B-4 11/24/2003 4:13:29 11/24/2003 4:13 B-4 11/24/2003 9:43:18 11/24/2003 9:43 B-4 11/24/2003 18:29:27 11/24/2003 18:29 B-4 12/4/2003 14:02:33 12/4/2003 14:02 11/23/03 20:00 B-5 11/23/2003 21:56:15 11/23/2003 21:56 B-5 11/24/2003 4:19:13 11/24/2003 4:19 B-5 11/24/2003 9:52:53 11/24/2003 9:52 B-5 11/24/2003 18:45:02 11/24/2003 18:45 B-5 12/4/2003 14:06:58 12/4/2003 14:06 11/23/03 20:00 B-6 11/23/2003 22:01:43 11/23/2003 22:01 B-6 11/24/2003 4:25:18 11/24/2003 4:25 B-6 11/24/2003 9:58:39 11/24/2003 9:58 B-6 11/24/2003 18:53:42 11/24/2003 18:53 B-6 12/4/2003 14:10:19 12/4/2003 14:10 11/23/03 20:00 B-7 11/23/2003 22:07:59 11/23/2003 22:07 B-7 11/24/2003 4:30:55 11/24/2003 4:30 B-7 11/24/2003 10:05:54 11/24/2003 10:05 B-7 11/24/2003 19:01:30 11/24/2003 19:01 B-7 11/26/2003 15:54:56 11/26/2003 15:54 B-7 12/4/2003 14:16:16 12/4/2003 14:16 11/23/03 20:00 B-8 11/23/2003 22:13:50 11/23/2003 22:13 B-8 11/24/2003 4:36:29 11/24/2003 4:26 B-8 11/24/2003 10:11:49 11/24/2003 10:11 B-8 11/24/2003 19:10:15 11/24/2003 19:10 B-8 12/4/2003 14:20:45 12/4/2003 14:20 11/23/03 20:00 B-9 11/23/2003 22:19:28 11/23/2003 22:19 B-9 11/24/2003 4:42:11 11/24/2003 4:42 B-9 11/24/2003 10:18:57 11/24/2003 10:18 B-9 11/24/2003 19:16:27 11/24/2003 19:16 B-9 12/4/2003 14:25:25 12/4/2003 14:25 B-10 11/24/2003 5:01:27 11/24/2003 5:01 B-10 11/24/2003 10:26:17 11/24/2003 10:26 11/23/03 20:00 B-10 11/24/2003 19:23:12 11/24/2003 19:23 B-10 12/4/2003 14:30:50 12/4/2003 14:30 11/23/03 20:00 M-1 11/23/2003 22:24:52 11/23/2003 22:24 M-1 11/24/2003 5:06:53 11/24/2003 5:06 M-1 11/24/2003 10:31:46 11/24/2003 10:31 M-1 11/24/2003 15:35:36 11/24/2003 15:35 M-1 11/24/2003 19:55:32 11/24/2003 19:55 M-1 12/4/2003 17:03:40 12/4/2003 17:03 15388 1 109 493 1550 2080 15395 1 115 499 1559 2096 15399 1 120 499 1565 2104 15403 1 126 504 1562 2112 4518 15409 1 132 508 1568 2121 15414 1 138 524 1575 2127 15419 535 1588 1 2134 15423 1 143 540 1593 1897 2211 15456 reten area conc 1.008 1553.3185 4.7698 19.5 0.916 6331.624 19.4425 0.916 5587.7305 17.1583 0.925 5306.9515 16.2961 0.916 5457.7127 16.759 1.016 2378.334 7.3032 19.6 0.916 6381.5743 19.5959 0.916 4718.9835 14.4906 0.925 5379.4378 16.5187 0.925 5127.3562 15.7446 1.008 2579.266 7.9202 19.3 0.916 6269.315 19.2512 0.916 5848.2185 17.9581 0.933 5377.797 16.5136 0.925 5448.8888 16.7319 1.008 1127.2893 3.4616 10.5 0.925 3412.0053 10.4773 0.908 3278.882 10.0685 0.933 3122.7753 9.5891 0.925 3398.8525 10.4369 0 0 0 1 356.335 1.0942 10.9 0.916 3525.6903 10.8264 0.916 3153.3565 9.683 0.933 3268.291 10.036 0.925 2998.2935 9.2069 1.008 1331.9778 4.0901 10.9 0.916 3532.2733 10.8466 0.925 3460.498 10.6262 0.933 3123.812 9.5923 0.925 2794.618 8.5814 1.016 2325.518 7.141 0.9 5388.5585 16.5467 0.925 5377.345 16.5122 16.6 0.925 5081.1708 15.6028 1.008 6085.134 18.6856 19.8 0.916 6442.0927 19.7818 0.925 5904.5285 18.1311 0.925 5887.648 18.0792 0.916 5979.7785 18.3621 0.933 5504.84 16.9037 1.008 6841.472 21.0081 SAMPLE date M-2 M-2 M-2 M-2 M-2 M-2 M-2 M-3 M-3 M-3 M-3 M-3 M-3 M-4 M-4 M-4 M-4 M-4 M-4 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/24/2003 12/4/2003 22:42:36 5:23:20 11:20:15 16:00:21 20:28:14 17:20:44 O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar 0.916 0.908 0.925 0.925 0.916 1.008 6363.0715 5766.313 5342.94 5710.426 6114.2275 6260.0563 Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen 1.708 1.708 1.725 1.716 1.708 1.766 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/24/2003 12/4/2003 22:37:28 5:17:39 10:56:00 15:49:28 20:18:30 17:13:17 O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar 0.916 0.916 0.933 0.916 0.925 1.016 6338.988 5993.1188 5615.3685 6088.7148 2262.35 5270.659 Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen 1.708 1.708 1.733 1.708 1.733 1.775 26112.07 24903.757 23655.4175 25724.136 9658.599 22782.859 26195.587 23987.0887 22454.116 24014.227 25733.075 26644.4048 11/23/2003 11/24/2003 11/24/2003 11/24/2003 11/24/2003 11/26/2003 12/4/2003 22:30:46 5:12:10 10:48:46 15:41:57 20:05:19 18:08:49 17:09:52 O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar 0.925 0.908 0.925 0.925 0.925 0 1.016 6285.711 5632.678 5187.4047 5718.879 6110.4395 0 5310.0725 19.4 19.3016 17.2963 15.929 17.561 18.7634 Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen 1.716 1.708 1.716 1.716 1.716 1.5 1.775 25872.8925 23396.5262 21696.3858 24088.078 25761.9895 8590.3295 21910.0968 75.323 68.1137 63.1641 70.127 75.0002 25.0088 63.7863 76.0193 72.5016 68.8674 74.89 28.1188 66.3271 76.2625 69.8329 65.37 69.912 74.916 77.5691 %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol time time gas reten area conc units gas reten area conc units 03NOV-HPTCD-42.CHR 03NOV-HPTCD-83.CHR 03NOV-HPTCD-125.CHR 03NOV-HPTCD-148.CHR 03NOV-HPTCD-176.CHR 03NOV-HPTCD-204.CHR 03NOV-HPTCD-246.CHR 03NOV-HPTCD-43.CHR 03NOV-HPTCD-84.CHR 03NOV-HPTCD-126.CHR 03NOV-HPTCD-149.CHR 03NOV-HPTCD-177.CHR 03NOV-HPTCD-247.CHR 03NOV-HPTCD-44.CHR 03NOV-HPTCD-85.CHR 03NOV-HPTCD-127.CHR 03NOV-HPTCD-150.CHR 03NOV-HPTCD-178.CHR 03NOV-HPTCD-248.CHR 03NOV-HPTCD-86.CHR 03NOV-HPTCD-128.CHR 03NOV-HPTCD-151.CHR 03NOV-HPTCD-179.CHR 03NOV-HPTCD-249.CHR 03NOV-HPTCD-141.CHR SOX-1 SOX-1 SOX-1 SOX-1 SOX-2 SOX-2 SOX-2 SOX-2 SOX-3 SOX-3 SOX-3 SOX-3 SOX-3 SOX-4 SOX-4 SOX-4 SOX-4 11/24/2003 11/24/2003 11/24/2003 11/25/2003 12/4/2003 11/24/2003 11/24/2003 11/24/2003 12/4/2003 0:50:43 6:56:04 13:17:16 19:30:58 15:27:32 1:02:58 7:08:54 13:22:27 15:31:12 O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar 11/24/2003 11/24/2003 11/24/2003 12/4/2003 0:27:28 6:42:57 13:03:45 15:24:04 O2 + Ar O2 + Ar O2 + Ar O2 + Ar 0.916 0.9 0.925 1 0.916 0.925 0.925 0.95 1.008 0.908 0.916 0.933 1.008 11/24/2003 11/24/2003 11/24/2003 12/4/2003 0:09:20 6:30:27 12:31:55 15:19:58 O2 + Ar O2 + Ar O2 + Ar O2 + Ar 0.916 0.908 0.925 1.008 5979.884 5904.5885 5517.309 6436.2473 6121.3975 5875.46 5217.5922 956.4325 6249.548 5896.148 5232.758 4802.71 977.072 6088.5797 6281.786 4785.231 1037.5573 M-5 M-5 M-5 M-5 M-5 OUTTAKE 11/24/2003 11/24/2003 11/24/2003 11/24/2003 12/4/2003 11/24/2003 5:28:49 11:28:07 16:06:50 20:42:42 17:26:37 12:51:07 11/23/03 20:00 11/23/2003 22:30 11/24/2003 5:12 11/24/2003 10:48 11/24/2003 15:41 11/24/2003 20:05 11/26/2003 18:08 12/4/2003 17:09 11/23/03 20:00 11/23/2003 22:37 11/24/2003 5:17 11/24/2003 10:56 11/24/2003 15:49 11/24/2003 20:18 12/4/2003 17:13 11/23/03 20:00 11/23/2003 22:42 11/24/2003 5:23 11/24/2003 11:20 11/24/2003 16:00 11/24/2003 20:28 12/4/2003 17:20 11/23/03 20:00 11/24/2003 5:28 11/24/2003 11:28 11/24/2003 16:06 11/24/2003 20:42 12/4/2003 17:26 O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar 0.916 0.925 0.925 0.916 1.008 0.933 5725.0423 5784.22 5404.796 6158.451 6280.155 2060.334 Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen Nitrogen 1.708 1.716 1.716 1.708 1.766 1.8 1.708 1.7 1.725 1.766 1.708 1.691 1.725 1.766 1.708 1.716 1.725 1.766 1.775 1.7 1.708 1.725 1.775 23867.291 24055.082 22401.4735 25523.645 25135.873 31354.618 24779.341 24681.9195 22426.5255 26302.6853 25350.529 24896.234 22822.0907 24688.643 25878.911 24931.193 22314.8168 24263.642 23920.724 25184.0333 26469.823 20248.747 24779.7048 69.4842 70.0309 65.2168 74.3063 73.1774 91.2818 72.1394 71.8558 65.2897 76.5743 73.8023 72.4797 66.4413 71.8754 75.3406 72.5815 64.9645 70.6381 69.6397 73.3176 77.0609 58.9496 72.1405 1 149 546 1610 1903 2221 4760 15462 1 156 551 1610 1911 2234 15666 1 161 557 1641 1920 2244 15612 1 166 1649 1926 2258 15618 333 11/23/03 20:00 11/24/2003 0:09 11/24/2003 6:30 11/24/2003 12:31 12/4/2003 15:19 11/23/03 20:00 11/24/2003 0:27 11/24/2003 6:42 11/24/2003 13:03 12/4/2003 15:24 11/23/03 20:00 11/24/2003 0:50 11/24/2003 6:56 11/24/2003 13:17 11/25/2003 19:30 12/4/2003 15:27 11/24/2003 1:02 11/24/2003 7:08 11/24/2003 13:22 12/4/2003 15:31 11/23/03 20:00 1 249 646 1007 15314 1 269 658 1039 15319 1 292 672 2032 2746 15322 304 684 2037 15326 1 %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol %vol 03NOV-HPTCD-54.CHR 03NOV-HPTCD-97.CHR 03NOV-HPTCD-139.CHR 03NOV-HPTCD-239.CHR 03NOV-HPTCD-55.CHR 03NOV-HPTCD-98.CHR 03NOV-HPTCD-142.CHR 03NOV-HPTCD-240.CHR 03NOV-HPTCD-56.CHR 03NOV-HPTCD-99.CHR 03NOV-HPTCD-143.CHR 03NOV-HPTCD-199.CHR 03NOV-HPTCD-241.CHR 03NOV-HPTCD-57.CHR 03NOV-HPTCD-100.CHR 03NOV-HPTCD-144.CHR 03NOV-HPTCD-242.CHR %vol %vol %vol %vol %vol %vol 16.3057 %vol 19.5 19.4652 %vol 18.4031 %vol 17.2431 %vol 18.6966 %vol %vol 16.1846 %vol 19.6 19.5391 %vol 17.7066 %vol 16.4066 %vol 17.535 %vol 18.775 %vol 19.2228 %vol 17.6 17.5799 %vol 17.7616 %vol 16.5965 %vol 18.9108 %vol 19.2845 %vol 6.3267 %vol 18.4 18.3625 %vol 18.1312 %vol 16.942 %vol 19.7638 %vol 18.8 18.797 %vol 18.0418 %vol 16.0217 %vol 2.9369 %vol 19.2 19.1905 %vol 18.1053 %vol 16.0683 %vol 14.7477 %vol 3.0003 %vol 18.6962 %vol 19.2895 %vol 14.694 %vol 3.186 %vol 18.9 reten 0.9 0.925 0.925 1 0.916 0.925 0.933 1.016 1 1.016 0.941 0.941 9457.303 5907.526 4989.735 2906.063 29.0406 18.1403 15.322 8.9237 %vol %vol %vol %vol Nitrogen Nitrogen Nitrogen Nitrogen 1.825 1.775 1.758 1.766 36355.533 26365.0505 24331.901 13132.159 Nitrogen Nitrogen Nitrogen Nitrogen 1.708 1.725 1.733 1.775 25068.775 24079.1885 11699.2315 24744.987 72.982 70.1011 34.0596 72.0394 105.8409 76.7558 70.8368 38.2313 1.7 1.716 1.716 1.766 25529.983 25464.8662 22818.3603 25386.557 reten area 03NOV-HPTCD-58.CHR 03NOV-HPTCD-101.CHR 03NOV-HPTCD-145.CHR 03NOV-HPTCD-243.CHR O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar O2 + Ar gas O2 + Ar O2 + Ar O2 + Ar O2 + Ar %vol %vol %vol %vol %vol %vol %vol %vol gas Nitrogen Nitrogen Nitrogen Nitrogen conc 74.3247 74.1352 66.4305 73.9072 units %vol %vol %vol %vol 03NOV-HPTCD-59.CHR 03NOV-HPTCD-102.CHR 03NOV-HPTCD-146.CHR 03NOV-HPTCD-244.CHR STAND STAND 12/4/2003 12/4/2003 11/25/2003 11/25/2003 12:47:56 12:53:45 15:07:18 17:48:10 SAMPLE date time time SOX-5 11/24/2003 1:21:47 11/24/2003 1:21 SOX-5 11/24/2003 7:20:31 11/24/2003 7:20 SOX-5 11/24/2003 13:29:23 11/24/2003 13:29 SOX-5 12/4/2003 15:34:37 12/4/2003 15:34 11/23/03 20:00 SOX-6 11/24/2003 1:42:21 11/24/2003 1:42 SOX-6 11/24/2003 7:34:15 11/24/2003 7:34 SOX-6 11/24/2003 14:04:35 11/24/2003 14:04 SOX-6 12/4/2003 15:39:29 12/4/2003 15:39 323 696 2044 15329 1 345 710 3079 15334 area conc units 6139.1925 18.8516 %vol 6006.2172 18.4433 %vol 5308.872 16.302 %vol 953.292 2.9273 %vol 18.1 5880.251 18.0565 %vol 5612.537 17.2344 %vol 2703.701 %vol 5758.275 17.682 %vol 03NOV-HPTCD-207.CHR 03NOV-HPTCD-208.CHR 03NOV-HPTCD-194.CHR 03NOV-HPTCD-195.CHR 334 APPENDIX E Chapter 6 Supplement Appendix E Table of Contents ............................................................................ 335 Scanning electron photomicrographs of calcite from in situ microcosms ......... 336 335 File microcosm10-chip1.doc Comments: Pics from calcite chip from Date May 3, 2002 microcosm at 203 m Sample upper spring white mat Description chip from microcosm at 203 m Calcite chip deeply corroded white mats at Site 3 and severely etched where filaments were covering surface Duration in place 4 months Date emplaced Aug 2002 Rare, strange quartz within Date retrieved Dec 2002 biofilms.... Don't know if this Method of preservation gluteraldehyde, ethanol series is from the wall crusts that fell into mats and was incorporated Coating material gold into mat, or if the euhedral Time of coating 2x 15 sec quartz formed within the mat Accelerating voltage 30 kv authigenically (saw only 2 Are bugs present? Singles? Groups? crystals within the mat) No. types present lots cocci rods branchy Radius of bugs Length Etching? Secondary minerals? 336 File microcosm10-chip2.doc Date May 3, 2002 Sample upper spring white mat Sample # Description chip2 from microcosm at 203 m white mats at Site 3 Duration in place 4 months Date emplaced Aug 2002 Date retrieved Dec 2002 Method of preservation gluteraldehyde, ethanol series Coating material gold Time of coating 2x 15 sec Accelerating voltage 30 kv Are bugs present? Singles? Groups? No. types present lots cocci rods branchy Etching? Secondary minerals? Comments: Pics from calcite chip 2 from microcosm at 203 m Calcite chip deeply corroded and severely etched where filaments were covering surface Filaments covering surface- crosscutting cleavage surfaces embedded filaments and cocci in dissolving calcite Two types of filaments one with septa and another without. Cocci very small and attached to filaments, or embedding within glycocaylx lot of slime on surfaces 337 File ESEMmicro_chip3.doc Date Aug 29, 2002 Sample upper spring white mat- ESEM Description "Chip3" from microcosm at 203 m white mats at Site 3 Duration in place 4-5 months Date emplaced March 2002 Date retrieved Aug 2002 Method of preservation samples collected and frozen Coating material nothing Time of coating -----Accelerating voltage several stages Are bugs present? Etching? Singles? rods Groups? branchy No. types present lots cocci Comments: ESEM Pics from calcite chip 3 from microcosm at 203 m (aken by Phil and Libby) Calcite chip corroded where filaments were covering surface Filaments covering surfacecrosscutting cleavage surfaces embedded filaments in dissolving calcite Dissolution under sulfurfilaments less than dissolution under non-sulfur-filaments Two types of filaments one with septa and another without, and one with intracellular sulfur and another without. Secondary minerals? 338 APPENDIX F Chapter 7 Supplement Appendix E Table of Contents ............................................................................ 339 Scanning electron photomicrographs of calcite from in situ microcosms ......... 340 339 Date 2-1-01 Sample wall crust Sample # 175 m Description red crust on wall pH 0-1 Date retrieved 9-00 trip Method of preservation bag-glut-ethanol Coating material gold Time of coating 30 sec Accelerating voltage 30 Kv Are bugs present? Singles? rods Groups? branchy No. types present 1 cocci No. photomicrographs 2 File names wall-1 Comments: Problem with charging, so only took 2 pictures Nice Si xtals no etching or signs of weathering no pitting... embedded in biofilm matrix. Most of the xtals are gypsum (Ca and S) long tabular gypsum needles/xtals Cells are mixed with matrix- if there are any real cells it's hard to tell Radius of bugs ~1 um Length ~1um File names wall-2 (in order below) wall biofilm, no descrip (some cells) gypsum (big xtal in back) and Si (nice xtal in front) with biofilm 340 Date 2-2-01 Sample wall mat Sample # 175 m Description red crust on gysum walls, pH 0-1 site Date retrieved 9-00 trip Method of preservation bag glut-ethanol Coating material gold Time of coating 30 sec Accelerating voltage 30 Kv Are bugs present? Singles? rods Groups? branchy No. types present 1 cocci Etching? EDAX Comments: Si xtals on and within biofilms. Biofilm made up of rods and filaments embedded in matrix (Ca,Si, Fe, S composition, see EDS). Quartz xtals range in size from 200 nm to about 5-10 um, and some xtals may be bigger (about 20 um). Most show nice clean surfaces, no etching or pitting... and very awesome euhedral shapes (see below). Cells hard to measure because the shapes are hidden in the biofilm matrix, but most cells appear to be about 1 um long, but others look like cocci (short and stubby).... Only a few "fat" rods >1 um Radius of bugs <1 um Length <1 um hard to measure Secondary minerals? File names WallEDS2-1 (Si, CaS, SiS,etc.) 140 120 100 s p e c t ru m 80 60 40 20 0 1 82 163 244 325 406 487 568 649 730 811 892 973 channel 341 File SEM5701acid Date 5-7-01 Sample wall mat, acid crust near Up Spg Sample # lkb-01m-007 Description gyp and rusty-colored crust on wall, acid Date retrieved 3-01 trip Method of preservation glut Coating material gold Time of coating 30 sec Accelerating voltage 30 KV Are bugs present? Singles? Groups? No. types present cocci rods branchy Radius of bugs small, <1 m) Length 1 m Etching? Secondary minerals? Quartz Comments: Acid wall crust, more pictures of beautiful quartz crystals.... And strange thin filaments that grow around and near some of them (Folk thinks that it may be palygorskite?). Quartz ranges from about 1mm long to about 5 mm long, some having doubly-terminated ends (quite euhedral and longer than wide), while others are round. Some faces appear to be coated maybe with a biofilm.... Cells few and far between, although where they are present, and are clumped in a biofilm hard to get size, but avg 1 m long as rods. 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Watanabe, K., Kodama, Y., and Kaku, N., 2002, Diversity and abundance of bacteria in an underground oil-storage cavity: BMC Microbiology, v. 2, p.23 [online]. Watanabe, K., Watanabe, K., Kodama, Y., Syutsubo, K., and Harayama, S., 2000, Molecular characterization of bacterial populations in petroleumcontaminated groundwater discharged from underground crude oil storage cavities: Applied and Environmental Microbiology, v. 66, p. 4803-4809. Watts, S.F., 2000, The mass budgets of carbonyl sulfide, dimethyl sulfide, carbon disulfide, and hydrogen sulfide: Atmospheric Environment, v. 34, p. 761779. White, W., 1988, Geomorphology and Hydrology of Karst Terrains: New York, Oxford University Press, 464 p. 373 Widdel, F., and Bak, F., 1992, Gram-negative mesophilic sulfate-reducing bacteria, in Balows, A., Truper, H., Dworkin, M., Harder, W., and Schleifer, K.-H., eds., The Prokaryotes: New York, Springer-Verlag, p. 3352-3378. Wilson, W.H., 1960, Radioactive mineral deposits of Wyoming: Laramie, Geological Survey of Wyoming, Report of Investigations 7, p. 41. Wind, T., Stubner, S., and Conrad, R., 1999, Sulfate-reducing bacteria in rice field soil and on rice roots: Systematic and Applied Microbiology, v. 22, p. 269279. Wirsen, C.O., Sievert, S.M., Cavanaugh, C.M., Molyneaux, S.J., Ahmad, A.T., L.T., DeLong, E.F., and Taylor, C.D., 2002, Characterization of an autotrophic sulfide-oxidizing marine Arcobacter sp. that produces filamentous sulfur: Applied and Environmental Microbiology, v. 68, p. 316325. 374 Vita Annette Summers Engel, born Annette Marie Summers in Akron, Ohio, on December 21, 1972, was the first child to Ronald Lee and Marie Alice Summers. After graduating from Marlington High School in Alliance, Ohio, in 1991, she attended Wittenberg University in Springfield, Ohio, from 1991-1995. She majored in Geology, went caving nearly every weekend, and graduated cum laude with a Bachelor of Arts degree in May, 1995. Annette met Scott Allen Engel at a Wittenberg caving function, and they were married November 4, 1995. Scott and Annette moved to Cincinnati, Ohio, where Annette attended graduate school at the University of Cincinnati from 1995-1999. She received a Masters of Science from the Department of Geology in 1997, with the thesis "The Speleogenesis of Movile Cave, Southern Dobrogea, Romania," and followed that degree with a Masters of Science from the Department of Biological Sciences in 1999 for completing the thesis work entitled "The Geomicrobiology of Sulfidic Cave Systems." Annette entered the University of Texas at Austin in the Fall of 1999 where she began her doctoral research in the Department of Geological Sciences with Philip C. Bennett. She and Scott look forward to the future when they will have more time to explore wild places. Permanent address: 2400 Stone River Drive, Austin, Texas 78745 This dissertation was typed by the author. 375

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