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The accumulation of butyrate is a recurrent phenomenon in anaerobic digesters utilizing animal manure as the primary feedstock. Despite its prevalence, the factors governing the anaerobic degradation of butyrate remain inadequately explored. In this study, a series of experiments were carried out under different total ammonia concentrations (TAN, 0.18–20 g N/L) and pH conditions (7.0–8.0) to investigate the inhibition of butyrate anaerobic degradation by different ammonia species (NH 4 + and NH 3) and to assess the recoverability following severe ammonia inhibition. The findings indicate that at pH 7.5, butyrate degradation experienced remarkable inhibition when TAN exceeded 8.0 g N/L, while no discernible impact was observed at pH 7.0–8.0 and 4.0 g TAN/L. Additionally, the lag phase for butyrate degradation extended with increasing TAN concentration. Notably, the activity of butyrate-degrading bacteria exhibited full recovery from severe ammonia inhibition (TAN 20 g N/L or NH 3 779.2 mg N/L), provided prolonged adaption time was allowed. The analysis using a modified Monod inhibition model highlighted that NH 4 + contributed more to inhibition than NH 3 at TAN concentrations of 2.0–20.0 g N/L. Therefore, simply reducing pH levels would not adequately counteract ammonia inhibition. Implementing an extended hydraulic retention time emerges as an effective measure to reduce butyrate accumulation in anaerobic digestion systems, particularly the feedstock being nitrogen-rich materials (e.g., animal manure). • Butyrate degradation significantly hindered above 8.0 g TAN/L at pH 7.5. • NH 4 + made a greater contribution to the inhibition of butyrate degradation. • Full recovery from severe ammonia inhibition occurred in butyrate anaerobic degradation. • Lowering pH levels would not adequately relieve ammonia inhibition. [ABSTRACT FROM AUTHOR]

[Display omitted] • The stimulation of AQS and cysteine on methanogenesis was concentration-dependent. • The addition of AQS and cysteine altered the microbial communities differently. • Cysteine promoted the growth of both acetoclastic and hydrogenotrophic methanogens. • Wide spectrum of SFOBs could use AQS as electron acceptor in the early phase. • Reduced AQS donated electrons to hydrogenotrophic methanogens in the late phase. Interspecies electron transfer (IET) between syntrophic fatty-acid oxidizing bacteria (SFOBs) and methanogens decided the performance of anaerobic digestion. Electron shuttles, as potential IET accelerators, were controversial concerning their influences on methanogenesis. In this study, concentration-dependent effects of anthraquinone-2-sulfonate (AQS) and cysteine on glucose digestion were firstly demonstrated: low dosage of AQS and cysteine (50 and 100 µM, respectively) had highest methane yield (133.5% and 148.6%, respectively). Using butyrate as substrate, distinct tendencies towards the enrichment of methanogenic community were further revealed. Cysteine just acted as a reductant which lowered ORP quickly and enriched most methanogens. It benefited methanogenesis right until methanogenic substrates accumulated. AQS, however, showed characteristic features of electron shuttles: it was firstly oxidized by SFOBs and then reduced by hydrogenotrophic methanogens, which accelerated methanogenic butyrate degradation. This study showed wide spectrum of SFOBs and methanogens benefited from the addition of electron shuttles, which laid foundation for future application. [ABSTRACT FROM AUTHOR]