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Aims: The aim of the present study was to design and test a method allowing the detection and quantification of methanogenic consortia in organic-rich rocks to determine the potential of methane biotransformation. Methods and Results: Methanogen numbers in the rock are often below the detection levels of quantification methods. Biostimulation was tested as a means to specifically increase bacterial and archaeal numbers above the detection levels in microcosms. Biostimulation reveals the presence of active heterotrophic and syntrophic bacterial consortia, methane accumulation and methanogens in one of four rock samples. Syntrophs and heterotrophs were dominated by Firmicutes, whereas archaeal diversity was limited to methanogens. Methane-producing microcosms were characterized by a higher Firmicutes diversity. Conclusions: Biostimulation is a reliable tool for detection of methanogenic consortia in organic-rich rocks. For routine and large scale experimentation, methane accumulation monitoring after biostimulation appears as the most time, work and cost efficient approach to detect the presence of active methanogenic consortia. Significance and Impact of the Study: We report for the first time the presence of live methanogenic consortia in organic-rich shales and their ability to mineralize the rock into methane. This approach will be instrumental to quantify the potential of these rocks to produce methane as a novel energy source.

Hydrogen peroxide (H 2 O 2 ), superoxide (O •−2 ), and hydroxyl radicals (OH • ) are produced in natural waters via ultraviolet (UV) light-induced reactions between dissolved oxygen (O 2 ) and organic carbon, and further reaction of H 2 O 2 and Fe(II) (i.e., Fenton chemistry). The temporal and spatial dynamics of H 2 O 2 and other dissolved compounds [Fe(II), Fe(III), H 2 S, O 2 ] were measured during a diel cycle (dark/light) in surface waters of three acidic geothermal springs (Beowulf Spring, One Hundred Springs Plain, and Echinus Geyser Spring; pH = 3-3.5, T = 68-80 • C) in Norris Geyser Basin, Yellowstone National Park. In situ analyses showed that H 2 O 2 concentrations were lowest (ca. 1 µM) in geothermal source waters containing high dissolved sulfide (and where oxygen was below detection) and increased by 2-fold (ca. 2-3 µM) in oxygenated waters corresponding to Fe(III)-oxide mat formation down the water channel. Small increases in dissolved oxygen and H 2 O 2 were observed during peak photon flux, but not consistently across all springs sampled. Iron-oxide microbial mats were sampled for molecular analysis of ROS gene expression in two primary autotrophs of acidic Fe(III)-oxide mat ecosystems: Metallosphaera yellowstonensis (Archaea) and Hydrogenobaculum sp. (Bacteria). Expression (RT-qPCR) assays of specific stress-response genes (e.g., superoxide dismutase, peroxidases) of the primary autotrophs were used to evaluate possible changes in transcription across temporal, spatial, and/or seasonal samples. Data presented here documented the presence of H 2 O 2 and general correlation with dissolved oxygen. Moreover, two dominant microbial populations expressed ROS response genes throughout the day, but showed less expression of key genes during peak sunlight. Oxidative stress response genes (especially external peroxidases) were highly-expressed in microorganisms within Fe(III)-oxide mat communities, suggesting a significant role for these proteins during survival and growth in situ.