Methane production from acetate, formate and H2/CO2 under high ammonia level: Modified ADM1 simulation and microbial characterization

Highlights: • Formate-utilizing methanogenesis contributed about 30% of the total methanation. • Formate-utilizing methanogenesis was kinetically and thermodynamically favorable. • Both formate-utilizing and hydrogen-utilizing methanogenesis had strong ammonia resistance. • ADM1 was extended with the development of formate-related reactions and modified with ammonia inhibition equations. • Microbial characterization and the modified ADM1 simulations mutually reinforced. This study evaluated the methanogenic performance of typical substrates (acetate, formate, H₂/CO₂, and glucose) under low and high ammonia levels and the Anaerobic Digestion Model No.1 (ADM1) was extended and modified for better simulation and understanding of the process. Formate-utilizing and hydrogen-utilizing methanogenesis showed stronger ammonia resistance than acetate-utilizing methanogenesis (13–23% vs. 34% decrease in methane production (MP)). Model extension, based on foundational experiments fed with three typical precursors (R² > 0.92), was then validated with glucose degradation experiments, and satisfactory predictions of MP and total volatile fatty acids were obtained (R² > 0.91). Based on the modified ADM1, the carbon fluxes of glucose degradation were determined, and formate-utilizing methanogenesis showed its importance with a 28–34% contribution of the total methanation, becoming the dominant pathway under high ammonia level. Formate-utilizing methanogenesis also had a thermodynamic advantage among the three pathways. 16S rRNA sequencing suggested a homology between the hydrogen-utilizing and formate-utilizing methanogens. Methanobacterium and Methanobrevibacter were found to be key methanogens, and their enrichment under high ammonia level confirmed the stronger ammonia tolerance of formate-utilizing and hydrogen-utilizing methanogenesis. The microbial characterization and modified ADM1 simulations supported each other.