Enhanced acidogenic gas utilization in two-stage co-digestion via biogas recirculation: Metagenomics analysis

Acidogenic gas (H2 and CO2) from acidogenic reactors is often ignored in two-stage anaerobic digestion due to its high CO2 content. While biogas recirculation improves methane production and substrate utilization, the underlying metabolic mechanisms remain unclear. This study explores these mechanisms using metagenomics in a novel two-stage system utilizing acidogenic gas. Biogas recirculation in the methanogenic stage increased average methane yield from 554 to 608 mL/g VS as the flow rate rose from 0 to 0.4 L/min, with a peak of 696 mL/g VS at 0.4 L/min. However, the methane yield decreased to 586 mL/g VS at 0.8 L/min. Recirculation enriched fermentative bacteria, boosting soluble metabolite production but slightly reducing organic matter removal. Although dominant microbial communities were significantly unaltered, syntrophic bacteria such as norank_f__norank_o__MBA03 (8.8–12.2 %) were enriched, strengthening microbial networks. Different methanogenic genera emerged, enabling rapid metabolite consumption via hydrogenotrophic, acetoclastic, and methylotrophic pathways. Metagenomic analysis revealed that recirculation upregulated key functions like signal transduction, cell motility, aromatic degradation, methanogenesis, and possible methane oxidation. This promoted carbon substrate availability and methane production while highlighting potential for valuable biochemical recovery from volatile fatty acids, supporting the circular economy and enhancing the cost-effectiveness of biogas systems.