Similar Methanogenic Shift but Divergent Syntrophic Partners in Anaerobic Digesters Exposed to Direct versus Successive Ammonium Additions

During anaerobic digestion (AD) of protein-rich wastewater, ammonium (NH ) is released by amino acid degradation. High NH concentrations disturb the AD microbiome balance, leading to process impairments. The sensitivity of the AD microbiome to NH and the inhibition threshold depend on multiple parameters, especially the previous microbial acclimation to ammonium stress. However, little is known about the effect of different NH acclimation strategies on the differential expression of key active microbial taxa. Here, we applied NH inputs of increasing intensity (from 1.7 to 15.2 g N-NH liters ) in batch assays fed with synthetic wastewater, according to two different strategies: (i) direct independent inputs at a unique target concentration and (ii) successive inputs in a stepwise manner. In both strategies, along the NH gradient, the active methanogens shifted from acetoclastic to and eventually hydrogenotrophic . Despite shorter latency times, the successive input modality led to lower methane production rate, lower soluble chemical oxygen demand (sCOD) removal efficiency, and lower half maximal inhibitory concentration, together with higher volatile fatty acid (VFA) accumulation, compared to the independent input modality. These differential performances were associated with a drastically distinct succession pattern of the active bacterial partners in both experiments. In particular, the direct exposure modality was characterized by a progressive enrichment of VFA producers (mainly ) and syntrophic VFA oxidizers (mainly ) with increasing NH concentration, while the successive exposure modality was characterized by a more dynamic succession of VFA producers (mainly , , ) and syntrophic VFA oxidizers (mainly , ). These results bring relevant insights for improved process management through inoculum adaptation, bioaugmentation, or community-driven optimization. Anaerobic digestion (AD) is an attractive biotechnological process for wastewater bioremediation and bioenergy production in the form of methane-rich biogas. However, AD can be inhibited by ammonium generated by protein-rich effluent, commonly found in agro-industrial activities. Insights in the microbial community composition and identification of AD key players are crucial for anticipating process impairments in response to ammonium stress. They can also help in defining an optimal microbiome adapted to high ammonium levels. Here, we compared two strategies for acclimation of AD microbiome to increa