Tracing Methanogenesis Pathways via Stable Carbon Isotopes for Sustainable Biogas Production in Continuous-Flow Open Systems

The common products of acidogenesis, the key stage in the process of anaerobic digestion, are lactate, butyrate, propionate, and acetate. They were decomposed in the Up-flow Anaerobic Sludge Blanket bioreactors working in continuous-flow open systems. A comprehensive analysis of variations in both isotopic ratios and concentrations of organic acids in the effluents was conducted to enhance comprehension of methanogenic processes. The analysis of carbon isotope fractionation in the CO 2 -CH 4 system, as evidenced by the α 13 C CO2-CH4 factor, has indicated that acetate decarboxylation has occurred. Furthermore, a decline in CO 2 levels was observed, accompanied by the predominance of butyrate and propionate, despite the presence of acetic acid in the effluents from all the bioreactors. Butyric acid demonstrated the greatest resistance to decomposition, resulting in 13 C-enrichment of DIC. Lactic acid was utilised almost entirely. The observations presented above were subsequently validated through statistical analysis. A comparative analysis of the δ 13 C(CH 4 ) and δ 13 C(CO 2 ) values of our study with those of other natural substrates (detritic lignite, xylite, maize silage, and cattle manure) was undertaken, and it was found that isotope fractionation differs significantly in closed (potential thermodynamic processes) and open systems (expected Rayleigh processes). In the context of open systems, the isotope fractionation factor α 13 C CO2-CH4 during methaneogenesis has been observed to attain values that are consistent with those observed in CH 4 oxidation. The study revealed that the presence of acetate in the substrate (i.e., the M4 bioreactor) led to the generation of CO 2 with a higher proportion of light carbon isotopes. This, in turn, resulted in a shift in the isotope fractionation factor (i.e., α 13 C CO2-CH4 ) to values below 1.03. Our results suggest that methanogenic pathway signatures in open, continuous-flow systems may only be partially apparent. This is because substrate depletion drives Rayleigh-type isotope enrichment, while the dominance of a single substrate and its constant inflow stabilise pathway expression and shift control towards substrate dynamics rather than intrinsic microbial changes. Our finding suggests that isotope-based diagnostics could enhance process control in biogas plants by identifying substrate-driven limitations and facilitating more efficient and stable CH 4 production.