A statistical framework for identifying microbial indicators of ammonia-induced process instability in food waste anaerobic digestion

Ammonia is an essential nutrient for anaerobic digestion (AD) but becomes inhibitory at elevated concentrations, leading to process instability. Although numerous microbial taxa and functional genes have been proposed as indicators of ammonia stress, most lack systematic validation across defined inhibitory thresholds. In this study, batch anaerobic digestion assays were conducted under increasing total ammonia nitrogen concentrations to experimentally characterize ammonia-induced inhibition. Methane yields obtained from batch tests were fitted using a Hill model to define non-inhibitory, inhibitory, and minimum inhibitory ammonia levels. Shotgun metagenomic sequencing was applied to samples representative of each inhibition level, and a statistical framework integrating differential abundance analysis, network topology, redundancy analysis, and metabolic relevance was used to identify robust microbial indicators. Key taxa, including Anaerolinea, Methanomassiliicoccus, and Syntrophobacter, along with functional genes involved in acetate and propionate metabolism (e.g., acs and fhs), showed consistent and threshold-dependent responses to ammonia stress. These microbial indicators provide mechanistic insight into ammonia-induced AD instability and offer a promising basis for early-warning monitoring and microbial management strategies to improve the operational stability of anaerobic digesters treating food waste.