Abstract Anaerobic co-digestion of protein-rich substrates is a prominent strategy for converting valuable feedstocks into methane, but it releases ammonia, which can inhibit methanogenesis. This study developed a cutting-edge combined culturomic and metagenomic approach to investigate the microbial composition of an ammonia-tolerant biogas plant. Newly-isolated microorganisms were used for bioaugmentation of stressed batch reactors fed with casein, maize silage and their combination. A co-culture enriched with proteolytic bacteria was isolated, selected and compared with the proteolytic collection strain Pseudomonas lundensis DSM6252. The co-culture and P. lundensis were combined with the ammonia-resistant archaeon Methanoculleus bourgensis MS2 to boost process stability. A microbial population pre-adapted to casein was also tested for evaluating the digestion of protein-rich feedstock. The promising results suggest combining proteolytic bacteria and M. bourgensis could exploit microbial co-cultures to improve anaerobic digestion stability and ensure stable productivity even under the harshest of ammonia conditions. Graphical abstract Highlights - High ammonia release from protein-rich substrates inhibits anaerobic digestion - Newly isolated bacterial strains from anaerobic digester were obtained - Proper bioaugmentation alleviates stress in casein and maize silage co-digestion - Bioaugmentation with a hydrolytic/hydrogenotrophic co-culture enhances CH 4 yields

Bioaugmentation strategies based on bacterial and methanogenic cultures to relieve stress in anaerobic digestion of protein-rich substrates