• Substrate type and microbial activity-controlled carbon isotopic fractionation. • Corn extract promoted hydrogenotrophic methanogenesis with higher fractionation. • Molasses substrates favored acetoclastic pathway under nutrient-limited conditions. • Established a reference isotopic framework for sustainable CBM recovery strategies. Stable isotopic analysis distinguishes gas types by origin. By utilizing plant-based substrates to enhance biogas regeneration in coalbeds, the Biomass Carbon Removal and Storage plus Renewable Natural Gas (BiCRS + RNG) process—currently commercialized by Cowboy Clean Fuels-injects biomass into unmineable coal seams to generate methane (CH 4 ) and carbon dioxide (CO 2 ). Because coal generally exhibits a greater affinity for CO 2 adsorption, the CH 4 can be selectively produced as Low-Carbon Renewable Natural Gas (LCRNG) while the CO 2 remains permanently sequestered in the formation. This LCRNG is isotopically distinct from coalbed methane (CBM) due to its contemporary biological origin, enabling its classification and preferential valuation in decarbonized energy markets. This study introduced a novel stable isotope analytical technique, Gas Chromatograph-Elemental Analyzer-Isotope Ratio Mass Spectrometer (GC-EA-IRMS), enabling precise separation and isotopic analysis of CH 4 and CO 2 . Coal and produced water (PW) samples were collected from coal seams within Wyoming’s Powder River basin (PRB). Corn extract (CE) and molasses desugarized solubles (MDS) feedstocks, were employed under varying nutrient and inoculum conditions to evaluate their impact on enhanced CH 4 pathways and efficiency. Isotopic analysis revealed significant isotopic fractionation for CE-amended substrates (εC: 50–70%, αCO 2 -CH 4 : 1.04–1.07), strongly indicating a hydrogenotrophic methanogenesis and biogenic origin with δ 13 C-CH 4 values ranging from −37 to −49.4‰ and positive δ 13 C-CO 2 values ranging from 10.31‰ to 19.25‰. Conversely, MDS-amended substrate exhibited moderate fractionation (εC: 40–50%, αCO 2 -CH 4 : 1.02–1.04), suggesting predominantly acetoclastic methanogenesis with δ 13 C-CH 4 values: −47.2 to −53.5‰. The δ 13 C-CO 2 values in MDS (−5.2 to 2.4‰) are more depleted in 13 C relative to the CE-amended systems and are consistent with acetate fermentation processes. The advanced GC-EA-IRMS method enabled accurate pathway delineation, offering crucial insights into microbial interactions and metabolic processes, underscoring the influence of substrate composition on gas origin and methanogenic pathways.
Budhathoki et al. (Wed,) studied this question.