Methanogenesis in biogas reactors under inhibitory ammonia concentration requires community-wide tolerance.

Ammonia (NH 3 ) inhibition represents a major limitation to methane production during anaerobic digestion of organic material in biogas reactors. This process relies on co-operative metabolic interactions between diverse taxa at the community-scale. Despite this, most investigations have focused singularly on how methanogenic Archaea respond to NH 3 stress. With a high-NH 3 pre-adapted and un-adapted community, this study investigated responses to NH 3 inhibition both at the community-scale and down to individual taxa. The pre-adapted community performed methanogenesis under inhibitory NH 3 concentrations better than the un-adapted. While many functionally important phyla were shared between the two communities, only taxa from the pre-adapted community were robust to NH 3 . Functionally important phyla were mostly comprised of sensitive taxa (≥ 50%), yet all groups, including methanogens, also possessed tolerant individuals (10-50%) suggesting that potential mechanisms for tolerance are non-specific and widespread. Hidden Markov Model-based phylogenetic analysis of methanogens confirmed that NH 3 tolerance was not restricted to specific taxonomic groups, even at the genus level. By reconstructing covarying growth patterns via network analyses, methanogenesis by the pre-adapted community was best explained by continued metabolic interactions (edges) between tolerant methanogens and other tolerant taxa (nodes). However, under non-inhibitory conditions, sensitive taxa re-emerged to dominate the pre-adapted community, suggesting that mechanisms of NH 3 tolerance can be disadvantageous to fitness without selection pressure. This study demonstrates that methanogenesis under NH 3 inhibition depends on broad-scale tolerance throughout the prokaryotic community. Mechanisms for tolerance seem widespread and non-specific, which has practical significance for the development of robust methanogenic biogas communities. KEY POINTS: • Ammonia pre-adaptation allows for better methanogenesis under inhibitory conditions. • All functionally important prokaryote phyla have some ammonia tolerant individuals. • Methanogenesis was likely dependent on interactions between tolerant individuals.