Thermodynamic restrictions determine ammonia tolerance of functional floras during anaerobic digestion

[Display omitted] •Differential ammonia tolerance of functional flora was observed.•Ammonia prolonged lag phases and reduced CH4 production rates and yields.•Thermodynamic restrictions were the key factor determining the ammonia tolerance.•Functional flora with higher energy availability is more resisted to ammonia stress. Ammonia inhibition is a major challenge in anaerobic digestion processes, affecting the activity and performance of functional floras, including syntrophic butyrate oxidation (FSBO), syntrophic propionate oxidation (FSPO), acetoclastic methanogenesis (FAMs), and hydrogenotrophic methanogenesis (FHMs). FHMs was more tolerant to ammonia, with a half maximal inhibitory concentration (IC50) of 18.80 g/L, followed by FSBO (IC50 = 14.26 g/L) and FSPO (IC50 = 10.47 g/L), and FAMs was the most sensitive to ammonia with the lowest IC50 (1.74 g/L). The order of ammonia tolerance (IC50) was found to be correlated with the Gibbs free energy of metabolic reactions in each functional flora. The results suggested the functional flora with higher energy availability (exergonic capacity) exhibited enhanced ammonia resistance ability. These findings provide insights into the thermodynamic restrictions and ammonia tolerance mechanisms of functional floras, which can guide the optimization and operation of anaerobic digestion systems for efficient methane production.