1. A Town on Fire! Metagenomic Analysis of Bacterial Communities in Soils Overlying the Centralia, Pennsylvania Coal Mine Fire

2. Case Study Overview  Class 1: Introduction to Centralia and Metagenomic Analysis  Activity #1: Introduction to Linux and QIIME (If you have a Mac, you can download the QIIME program..it takes around ½ hour, so do not try to do it during class). See me for help.  Activity #2: Who lives there?? Proposing and testing hypotheses regarding Centralia’s resident microbes.  Activity #3: Statistical and Phylogenetic Analysis, Part 1  Activity #5: Statistical and Phylogenetic Analysis, Part 2  Final Presentations

3. Overview  Historical Perspective  What Do We Know About the Environment?  What Do We Know About the Microbes?

4. Where is Centralia?

5. A Short History of the Centralia Mine Fire  May, 1962: The Fire Begins

6. A Short History of the Centralia Mine Fire, Continued…  The Fire Continues To Burn…….

7. A Short History of the Centralia Mine Fire, Continued…  1962-1978: Unsuccessful attempts to put out fire  1979-1983 OSM estimates $663 million needed to put out fire  DEP determines fumes and subsidence unsafe  1983 Route 61 collapses  1984 Congress provides $42 million to relocate residents

8. Centralia, Then and Now

9. What Do We Know About the Environment?  Earth and Environmental Sciences:  Composition and Structure of the Anthracite Coal Seams  Predict and Measure the Fire’s Spread  Nature of “Anthracite Smokers”

10. What Do We Know About the Environment?  Chemistry  Temperatures range to over 300°C (60°C Common)  High levels of SOx, NOx, As, CO, Al 2 (SO 4 ) 3, NH 4 Al(SO 4 ) 2, and NH 4 Cl compounds

11. What Do We Know About the Microbes?  Prediction, Exploration, Experimentation … and Serendipity

12. Predictions  Resident Microbes Should Include:  Thermophiles- some similar to those in other geothermal environments, some novel  Nitrogen-Cycling Bacteria  Sulfur-Cycling Bacteria

13. Experimental Approach  Collect Soil From Boreholes Near Active Vents (37°C, 52°C, 65°C)  Culture-Independent Methods  Metagenomic Analysis of 16S rRNA genes, Functional Metagenomics, Transcriptomics  Culture-Dependent Methods  Physiological Assays  Microscopy  Staining  Genome Sequencing

14. Metagenomic Analysis of Bacterial 16S rRNA Genes  What are the 16S rRNA genes?  Why use them?

15. Pyrosequencing

16. Questions?