Modeling thermophilic syntrophic VFA oxidation using thermodynamic principles: Insights from enrichment cultures

In anaerobic digestion, high ammonia concentration and thermophilic conditions inhibit acetoclastic methanogens, favoring syntrophic oxidation of volatile fatty acids. In the well-known ADM1 model, however, syntrophic oxidation of acetate is not included. In this study, we estimated and validated kinetic parameters of syntrophic acetate oxidizing bacteria (SAOB) and associated syntrophs (syntrophic propionate oxidizing bacteria (SPOB), hydrogenotrophic methanogen (HM)) using data from dedicated enrichment experiments. Syntrophic interactions are inherently constrained by thermodynamics, requiring tight cooperation between partners to make methanogenesis possible. We thus compared a classical ADM1-based approach (M AMD1 ) with a thermodynamically constrained version (M Th ) that includes estimation of growth yields and inhibition directly from thermodynamic principles. Both modeling approaches enabled successful parameter estimation, but M Th had several advantages: by reducing the number of empirical parameters and enforcing thermodynamic feasibility, it improved parameter identifiability and provided more realistic growth yields, although uncertainties in half-saturation constants (K S ) remain relatively high. The analysis further revealed that, unlike SPOB and HM, SAOB cannot generate ATP through substrate oxidation alone yet still exhibit growth. This paradox points to missing or poorly understood metabolic pathways (e.g., alternative electron shuttle or energy conservation mechanisms). Overall, the study provides validated parameter ranges for syntrophic partners under thermophilic and high-ammonia conditions and demonstrates the added value of incorporating thermodynamic constraints in ADM1-type models to improve robustness and reveal knowledge gaps in microbial energy metabolism.