Overcoming Extreme Ammonia Inhibition on Methanogenesis by Artificially Constructing a Synergistically Community with Acidogenic Bacteria and Hydrogenotrophic Archaea.

High total ammonia nitrogen (TAN) inhibits anaerobic digestion (AD) and cannot be completely eliminated by merely enhancing a stage of AD. This study incorporates TAN-tolerant inoculum into substrates hydrolyzed by Rhizopus mixed agents to simultaneously enhance hydrolysis-acidogenesis-methanogenesis. The results show a 16.46-fold increase in CH4 production under TAN-inhibited (6870.97 mg L-1) conditions, even exceeding the AD without TAN by 21.10%. Model substrates sodium acetate and mixed H2 confirm hydrogenotrophic methanogenesis is the main pathway, with reduced TAN inhibition. Furthermore, a synergistic metabolic microbial community dominated by hydrolytic bacteria JAAYGG01 sp. and DTU014 sp., acidogenic bacteria DTU015 sp., DTU013 sp., and JAAYLO01 sp., and methanogens Methanosarcina mazei and an unclassified species in the Methanoculleus is reconstructed to resist TAN inhibition. Metagenomic combined with metatranscriptomic sequencing identifies that this microbial community carries xynD and bglB to regulate substrate hydrolysis, leading to acetate production through glycolysis, butyrate, and pyruvate metabolism with high acetate kinase activity, thereby CH4 produced primarily via hydrogenotrophic methanogenesis with high coenzyme F420 activity, facilitated by efficient mass transfer processes and quorum sensing regulation. This cleaner strategy obtains higher economic benefit (US$149.02) than conventional AD and can increase 154.64-fold energy production of a 24 000 m3 biogas plant, guided by machine learning.