Target the Enhancement of DIET-Based CO₂‑Dependent Methanogenesis via Synergy of Magnetite Addition and Inorganic Carbon Recirculation

In anaerobic systems, CO2-dependent methanogens as terminal electron acceptors determine the feasibility of organic degradation. In this study, targeting the enhancement of CO2-dependent methanogenesis, a strategy coupling the addition of magnetite and inorganic carbon recirculation was proposed. The results showed that the strategy facilitated the enrichment of Methanosaeta and Methanosarcina to the greatest extent (abundance of 42.9 and 38.8%, respectively), and metaproteomic analysis revealed that their enzymes coding for CO2-dependent methanogenesis were maximally expressed, compared with the single strategy. Combined with electrochemical analysis, magnetite was speculated to trigger direct interspecies electron transfer (DIET)-based syntrophy with extracellular redox-active mediators (Cyt C and flavins) in anaerobic sludge. The ability of Methanosaeta and Methanosarcina to accept electrons was significantly improved, as evidenced by the upregulated electron transfer proteins, such as F420H2 dehydrogenase, coenzyme F420 hydrogenase, and CoB-CoM heterodisulfide reductase. Meanwhile, Methanosaeta and Methanosarcina enhanced HCO3– takeup into cells via an ABC-type transport system under the condition of inorganic carbon recirculation, thus thermodynamically inducing electron consumption and inorganic carbon reduction. In summary, the coupling strategy had a great synergistic effect on the enhancement of DIET-based CO2-dependent methanogenesis and endowed the up-flow anaerobic sludge bed reactor with superior methanogenic performance for stable anaerobic wastewater treatment