Methane Stimulation and Searching Endemic Methanogens in Montana Coal Beds Joel Vargas Muniz 1, Elizabeth J.P. Jones 2, William H. Orem 2 Department of Biology, University of Puerto Rico, Mayaguez, PR 1, U.S. Geological Survey, National Center, Reston, VA The coal bed methane in Montana represents an important renewable energy source that could reduce the high dependence on coal to produce electricity in United States. The methane generation and accumulation in coal beds is controlled by several factors that include methanogenesis. However few things about microorganisms that create methanogenesis and their capacity to decompose organic complex compounds in coal to produce methane are known. The main purpose of this research is to evaluate the Montana coal potential to produce methane under different treatments and to determine the presence of endemic methanogens. A series of treatments with Montana’s coal samples were developed. The treatments included the addition of a known microorganism’s culture (WBC-2) with the presence of methanogens and the supply of certain conditions that benefit the microbial endemic activity in coal. It was observed that in all the treatments methane was produced, though the treatments with the culture WBC-2 presented a higher production. When some organic complex compounds were added to the treatments with WBC-2, higher levels of methane was produced. The treatment with WBC-2, in groundwater no indicates the presence of mcrA gene. This study indicates that methane production in Montana coal can be stimulated adding some complex organic compounds, that exists endemic methanogens in Montana coal and that the groundwater can inhibit the mcrA gene expression in molecular analysis. The demonstration of endemic activity in Montana coal and the stimulation of methane production provide a platform for anthropogenic manipulation. Research is in progress to determine the microorganism biodiversity. The coal bed methane in Montana represents an important renewable energy source that could reduce the high dependence on coal to produce electricity in United States. The methane generation and accumulation in coal beds is controlled by several factors that include methanogenesis. However few things about microorganisms that create methanogenesis and their capacity to decompose organic complex compounds in coal to produce methane are known. The main purpose of this research is to evaluate the Montana coal potential to produce methane under different treatments and to determine the presence of endemic methanogens. A series of treatments with Montana’s coal samples were developed. The treatments included the addition of a known microorganism’s culture (WBC-2) with the presence of methanogens and the supply of certain conditions that benefit the microbial endemic activity in coal. It was observed that in all the treatments methane was produced, though the treatments with the culture WBC-2 presented a higher production. When some organic complex compounds were added to the treatments with WBC-2, higher levels of methane was produced. The treatment with WBC-2, in groundwater no indicates the presence of mcrA gene. This study indicates that methane production in Montana coal can be stimulated adding some complex organic compounds, that exists endemic methanogens in Montana coal and that the groundwater can inhibit the mcrA gene expression in molecular analysis. The demonstration of endemic activity in Montana coal and the stimulation of methane production provide a platform for anthropogenic manipulation. Research is in progress to determine the microorganism biodiversity. Abstract Introduction Methods Objectives Results Future work Acknowledgements References Discussion Coal is a non renewable fuel that is mainly used in United States to produce electricity. The 72% of all coal produced in the United States comes from Wyoming, West Virginia, Kentucky, Pennsylvania and Montana. However Montana’s coal contains a high methane level called coal bed methane (CBM). CBM is a form of natural gas that represents a renewable energy source. Trapped methane in coal layers is produced by chemical reactions and microbial processes. The most important microbial activity in coal is methanogenesis, made in an anaerobic form by a microorganism community. The microbial coal methanogenesis study’s got a particular interest since it represents a way to renew CBM and use the methane as an energy source. On the other hand, few things about the methanogenesis microbial community and its ability to decompose organic complex compounds in coal to produce methane are known. There are several possible factors that could influence the generation and accumulation of methane in coal beds, including the bioavailability of carbon in the coal, the presence of a microbial community that is able to utilize the coal carbon, and environmental conditions that support microbial growth and methanogenesis, such as availability of nutrients and a lack of toxic or inhibitory factors. The main purpose of this research is to evaluate the Montana coal potential to produce methane under different treatments and to determine the presence of endemic methanogens.  Evaluate the Montana coal potential to produce methane under different treatments and to determine the presence of endemic methanogens.  To stimulate the methane production through the use of several different organic complex compounds.  Evaluate the Montana coal potential to produce methane under different treatments and to determine the presence of endemic methanogens.  To stimulate the methane production through the use of several different organic complex compounds. This research shows that methane production in Montana’s coal can be stimulated adding some organic complex compounds like humic acid, glycerol, acetate, malate and fumarate. It is demonstrated that there’s an endemic methanogens community in coal that produces methane, which suggests that its growth and microbial activity can be simulated with anthropogenic manipulation. On the other hand, due to the absence of mcrA gene in coal’s samples with culture WBC-2 treatment’s addition in groundwater, it’s inferred that the mcrA gene’s expression in molecular analysis can be inhibited by the groundwater. Make a TRFLP with the Montana’s coal samples with the different treatments and determine the microorganism biodiversity in these ones. Also to make experiments to confirm the inhibition in molecular analysis of the mcrA gene’s expression by the groundwater.  Jones, E. J. P., M. A. Voytek, M. D. Corum, W. H. Orem Stimulation of methane generation from nonproductive coal by addition of nutrients or microbial consortium. Applied and Environmental Microbiology. 76:  Jones, E. J. P., M. A. Voytek, P. D. Warwick, M. D. Corum, A. Cohn, J. E. Bunnell, A. C. Clark, and W. H. Orem Bioassay for estimating the biogenic methane-generating potential of coal samples. Int. J. Coal Geol. 76:  Jones, E. J. P., M. A. Voytek, M. D. Corum, W. H. Orem Stimulation of methane generation from nonproductive coal by addition of nutrients or microbial consortium. Applied and Environmental Microbiology. 76:  Jones, E. J. P., M. A. Voytek, P. D. Warwick, M. D. Corum, A. Cohn, J. E. Bunnell, A. C. Clark, and W. H. Orem Bioassay for estimating the biogenic methane-generating potential of coal samples. Int. J. Coal Geol. 76: Eastern Energy Team USGS For give me the opportunity to be part of the team in the summer Kaylene Charles For give me the training in the laboratory and help me in the communication in English. Eastern Energy Team USGS For give me the opportunity to be part of the team in the summer Kaylene Charles For give me the training in the laboratory and help me in the communication in English FG3E 2- FG3F 3- FG3G 4- FG3H 5- FG3I 6- FG3J 7- FG3K 8- FG3L 9- FG8E 10- FG8F 11- FG8G 12- FG8H 13- FG8I 14- FG8J 15- FG8K 16- FG8L 17- FG9B 18- FG9C 19- FG9D 20- FG9E 21- FG9F 22- FG11A 23- FG11B Figure 1. Treatment with bicarb buffer, nutrients, coal and culture WBC -2. Figure 2. Treatment with groundwater, nutrients, coal and culture WBC -2. Figure 3. Treatment with groundwater, nutrients and coal. Figure 4. Treatment with groundwater and coal. Figure 5. DNA Extraction Figure 8. PCR using mcrAf and mcrAr primers. Figure 10. qPCR using mcrAf and mcrAr primers FG3E 2- FG3F 3- FG3G 4- FG3H 5- FG3I 6- FG3J 7- FG3K 8- FG3L 9- FG8E 10- FG8F 11- FG8G 12- FG8H 13- FG8I 14- FG8J 15- FG8K 16- FG8L 17- FG9B 18- FG9C 19- FG9D 20- FG9E 21- FG9F 22- FG11A 23- FG11B 1- FG3K 2- FG3L 3- FG8E 4- FG8F 5- FG8G 6- FG8H 7- FG8I 8- FG8J 9- FG8K 10- FG8L FG3K 2- FG3L 3- FG8E 4- FG8F 5- FG8G 6- FG8H 7- FG8I 8- FG8J 9- FG8K 10- FG8L Figure 6. PCR using universal 16S rRNA gene bacterial primers FAM- 46f and 519r. Figure 7. PCR using 46 universal 16S rRNA gene bacterial primers FAM-46f and 51with diluted DNA. 1/10 1/50 Figure 9. PCR using mcrAf and mcrAr primers with diluted DNA. 1- FG 9B 2- FG 9C 3- FG 9D 4- FG 9E 5- FG 9F 6- FG 11 A 7- FG 11 B Coal sample sites Anaerobic chamber with coal and treatments Incubation in dark HPLC- GC DNA extraction PCR reaction qPCR reaction FG3E 2- FG3F 3- FG3G 4- FG3H 5- FG3I 6- FG3J 7- FG3K 8- FG3L 9- FG8E 10- FG8F 11- FG8G 12- FG8H 13- FG8I 14- FG8J 15- FG8K 16- FG8L 17- FG9B 18- FG9C 19- FG9D 20- FG9E 21- FG9F 22- FG11A 23- FG11B FG3L 2- FG8E 3- FG8F 4- FG8G 5- FG8H 6- FG8I 7- FG8J 8- FG8K 9- FG9B 10- FG9C 11- FG9D 12- FG9E 13- FG9F 14- FG11A 15- FG11B 1/10 1/50