Unravelling ethanol metabolic pathways under different feeding regimes in anaerobic digestion ecosystems

Feeding regimes critically influence the efficiency of anaerobic digestion, yet its impact on ethanol-type anaerobic digestion ecosystems remains inadequately understood. A comprehensive investigation into the effect of feeding regimes on ethanol-type anaerobic digestion was conducted. Sequencing batch reactor (SBR) and continuous-flow reactor (CFR) were adopted for acclimating ethanol-type anaerobic methanogenesis. During long-term operation, the CFR system achieved complete removal of ethanol and volatile fatty acids, while an accumulation of 2250.0 ± 130.0 mg COD/L acetate and 931.0 ± 184.0 mg COD/L butyrate was observed in the SBR system. The difference was probably due to the limited activity of methanogens under acid accumulation conditions in SBR. Notably, the accumulation of acetate and the reduced pH in SBR facilitated ethanol metabolism towards butyrate production by a chain-elongating bacteria: Clostridium . Additionally, acetoclastic methanogenesis was not detected in SBR, and the dominated hydrogenotrophic methanogen was Methanobacterium . Ethanol was exclusively oxidized to acetate by ethanol oxidizers, including Desulfolutivibrio and Desulfomicrobium , facilitating interspecies hydrogen transfer with Methanospirillum in CFR. The electroactive bacteria Geobacter was enriched in CFR. Moreover, the relative abundance of genes related to hydrogen transfer and direct electron transfer increased in CFR, possibly enhancing electron transfer. These results contribute to a deeper understanding of how feeding regimes shape ethanol-type anaerobic ecosystems, providing valuable insights for optimizing anaerobic digestion processes to enhance methane production and electron transfer. • SBR facilitated ethanol metabolism towards butyrate production. • Acetoclastic methanogenesis was inhibited in SBR. • Methanobacterium predominated in SBR under acidic conditions. • CFR exhibited enhanced electron transfer activities.