Methane Stimulation and Searching Endemic Methanogens in Montana Coal Beds

1. Methane Stimulation and Searching Endemic Methanogens in Montana Coal Beds Joel Vargas Muniz1, Elizabeth J.P. Jones2, William H. Orem2 Department of Biology, University of Puerto Rico, Mayaguez, PR1, U.S. Geological Survey, National Center, Reston, VA 201922 Methods Discussion Abstract 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. 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. Incubation in dark HPLC- GC Anaerobic chamber with coal and treatments Coal sample sites Future work 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. PCR reaction DNA extraction qPCR reaction References Results Introduction • Jones, E. J. P., M. A. Voytek, M. D. Corum, W. H. Orem. 2010. Stimulation of methane generation from nonproductive coal by addition of nutrients or microbial consortium. Applied and Environmental Microbiology. 76: 7013-7022. • 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. 2008. Bioassay for estimating the biogenic methane-generating potential of coal samples. Int. J. Coal Geol. 76: 138-150. 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. Figure 4. Treatment with groundwater and coal. 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. 1- 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 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 1 2 3 4 5 6 7 8 9 10 Acknowledgements 1/10 1- FG3K 2- FG3L 3- FG8E 4- FG8F 5- FG8G 6- FG8H 7- FG8I 8- FG8J 9- FG8K 10- FG8L 1 2 3 4 5 6 7 8 9 10 11 Eastern Energy Team USGS For give me the opportunity to be part of the team in the summer 2011. Kaylene Charles For give me the training in the laboratory and help me in the communication in English. 1 2 3 4 5 6 7 8 9 10 + 1- 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 16 17 18 19 20 21 22 23 + - 1- FG 9B 2- FG 9C 3- FG 9D 4- FG 9E 5- FG 9F 6- FG 11 A 7- FG 11 B 1/50 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- 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 2 3 4 5 6 7 8 9 10 11 12 13 14 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 12 13 14 15 16 17 18 19 20 21 22 23 Objectives 1- 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 2 3 4 5 6 7 8 9 10 11 12 13 14 15 + Figure 10. qPCR using mcrAf and mcrAr primers. Figure 5. DNA Extraction 15 16 17 18 19 20 21 22 23 + - • 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. 1/50 Figure 9. PCR using mcrAf and mcrAr primers with diluted DNA . Figure 8. PCR using mcrAf and mcrAr primers.