PUTATIVE METHANOGEN MOLECULAR EVIDENCE IN PALEOCENE FORT UNION COALS IN THE POWDER RIVER BASIN, WYOMING AND MONTANA

Understanding the sources, biogeochemistry, and factors influencing possible replenishment/stimulation of coalbed methane (CBM) in low-rank coals is of increasing interest in relation to managing this globally abundant and increasingly economically important energy resource. In this study, coal samples from the Wyodak-Anderson coal beds of the Paleocene Fort Union Formation, which produced 65 percent of the total CBM in the Powder River Basin in Wyoming and Montana as of 2004, and deeper coal beds of the Fort Union were examined for the possible presence of methanogen-related molecular sequences. Twenty-five 3-inch diameter core samples were collected and maintained under cooled/frozen conditions until subsampled.

All subsampling was carried out using flame-sterilized tools in a sterile, laminar-flow hood; the final powder-form coal samples used in the molecular analyses were recovered from freshly exposed core-sample faces using a sterile, flame-sterilized minidrill system. Positive controls [mixed sewage sludge and Methanococcus maripaludis deoxyribonucleic acid (DNA) and negative (E. coli DNA)] were used in these experiments, together with appropriate abiotic and polymerase chain reaction (PCR) controls. The FastDNA kit gave better extraction results than a traditional phenol-chloroform extraction method. The nested PCR system selected for use in these experiments included U341F as the forward primer in both reactions, and 1204R and Meth860R as reverse primers in the initial and nested PCR reactions, respectively.

DNA from three of the Wyodak-Anderson coal samples were cloned and sequenced based on final PCR product size and differentiation from control results and phylogenetic comparisons were made. Seven methanogen-primer based sequences were clearly separated from the other cloned PCR products that clustered with sequences derived from extraction blanks, considered to be PCR artifacts, as well as from positive M. matipaludis controls. These seven methanogen primer-derived sequences formed a separate deeply rooted group that did not give significant matches with known methanogenic database sequences in the National Center for Biotechnological Information Basic Local Alignment Search Tool, implying that these might represent molecular remnants of undescribed methanogens.