Sara L. Caldwell Eldridge Elizabeth J. Tomaszewski Matthew S. Varonka Anna Martini Michael Carley James Schramski Haiyan Zheng Elliott P. Barnhart Elisha Kelly Moore 2025 <p><span>The expansion of natural gas production and utilization worldwide has led to the decline of many once-productive wells, eventually resulting in costly well-plugging and unused infrastructure. However, in areas like the Michigan basin, MI, where the majority of natural gas is biogenically produced, microbial communities could potentially be stimulated to generate additional methane, increasing gas supply and reducing the need to drill new wells. In this study, we performed metaproteomic, metagenomic, and geochemical analyses of Antrim Shale formation water from a marginally producing natural gas well to evaluate resident microbial community functions in the context of potential bioenergy production. Functional proteins involved in methanogenesis, degradation/catabolism (including organic matter degradation), biosynthesis, energy utilization, transmembrane transport, and stress response were among the most commonly identified groups. Three metagenome-assembled genomes (MAGs) were characterized, including Methanomicrobiaceae,&nbsp;</span><i>Methanothrix</i><span>, and&nbsp;</span><i>Smithella</i><span>. For each, the identified proteins involved in methanogenesis and the degradation of diverse organic compounds, strongly suggest their role in utilizing shale-derived organic matter. These findings provide an increased understanding of the microorganisms and their metabolisms generating natural gas in the Antrim Shale and establish a foundation for future stimulation efforts aimed at enhancing biogenic methane production in marginal gas wells.</span></p> application/pdf 10.1038/s43247-025-02776-2 en Nature Metaproteomics and metagenomics reveal microbial pathways of organic matter degradation and methanogenesis in a marginally producing natural gas well article