A novel approach to estimate methanogenic pathways in biogas reactors via stable carbon isotope analysis

Two microbial pathways are responsible for most of the methane produced during anaerobic digestion: acetoclastic methanogenesis (AM) and hydrogenotrophic methanogenesis (HM) coupled with syntrophic acetate oxidation (SAO). Identifying the dominant methanogenic pathway active in a system provides the information necessary to manage and optimize productivity, stability, process control, and gas quality in biogas reactors. In this study, a modified method is proposed to estimate methanogenic pathways in different biogas systems via short-term parallel incubations with methyl-labeled acetate (2-13C-acetate). Cavity ring-down spectroscopy was applied to measure the δ13C–CH4 and δ13C–CO2 isotopic signatures of produced biogas. Preliminary experiments demonstrated that longer incubation times led to significant variations in δ13C–CH4 and δ13C–CO2 and consequently interfered with the calculated fraction of CH4 produced from HM (fHM). This variability is likely caused by the dilution of 13CH4 and 13CO2 as 2-13C-acetate is consumed, along with potential changes in organic matter quality and quantity, microbial community composition, and environmental factors such as pH, volatile fatty acid content, and ammonia levels, during longer incubations. We applied this new approach to sludge from six full-scale reactors (three mesophilic and three thermophilic) and validated its potential with consistent estimates of fHM with minimal variation. Mesophilic reactors exhibited AM dominance, while HM was the dominant pathway in thermophilic reactors, aligning with reports in the literature.