Determination of Methanogenic Pathways through Carbon Isotope (δ13C) Analysis for the Two-Stage Anaerobic Digestion of High-Solids Substrates

This study used carbon isotope (δ13C)-based calculations to quantify the specific methanogenic pathways in a two-stage experimental biogas plant composed of three thermophilic leach bed reactors (51–56 °C) followed by a mesophilic (36.5 °C) anaerobic filter. Despite the continuous dominance of the acetoclastic Methanosaeta in the anaerobic filter, the methane (CH4) fraction derived from carbon dioxide reduction (CO2), f mc, varied significantly over the investigation period of 200 days. At organic loading rates (OLRs) below 6.0 gCOD L–1d–1, the average f mc value was 33%, whereas at higher OLRs, with a maximum level of 17.0 gCOD L–1d–1, the f mc values reached 47%. The experiments allowed for a clear differentiation of the isotope fractionation related to the formation and consumption of acetate in both stages of the plant. Our data indicate constant carbon isotope fractionation for acetate formation at different OLRs within the thermophilic leach bed reactors as well as a negligible contribution of homoacetogenesis. These results present the first quantification of methanogenic pathway (f mc values) dynamics for a continually operated mesophilic bioreactor and highlight the enormous potential of δ13C analysis for a more comprehensive understanding of the anaerobic degradation processes in CH4-producing biogas plants.