Long-term and multiscale assessment of methanogenesis enhancement mechanisms in magnetite nanoparticle-mediated anaerobic digestion reactor

Magnetite nanoparticles (Fe₃O₄-NPs) have been demonstrated to be involved in direct interspecies electron transfer between syntrophic bacteria, yet a comprehensive assessment of the ability of Fe₃O₄-NPs to cope with process instability and volatile fatty acids (VFAs) accumulation in scaled-up anaerobic reactors is still lacking. Here, we investigated the start-up characteristics of an expanded granular sludge bed (EGSB) with Fe₃O₄-NPs as an adjuvant at high organic loading rate (OLR). The results showed that the methane production rate of R1 (with Fe₃O₄-NPs) was approximately 1.65 folds of R0 (control), and effluent COD removal efficiency was maintained at approximately 98.32% upon 20 kg COD/(m³·d) OLR. The components of volatile fatty acids are acetate and propionate, and the rapid scavenging of propionate accumulation was the difference between R1 and the control. The INT-ETS activity of R1 was consistently higher than that of R0 and R2, and the electron transfer efficiencies increased by 68.78% and 131.44%, respectively. Meanwhile, the CV curve analysis showed that the current of R1 was 40% higher than R3 (temporary addition of Fe₃O₄-NPs), indicating that multiple electron transfer modes might coexist. High-throughput analysis further revealed that it was difficult to reverse the progressive deterioration of system performance with increasing OLR by simply reconfiguring bacterial community structure and abundance, demonstrating that the Fe₃O₄-NPs-mediated DIET pathway is a prerequisite for establishing multiple electron transfer systems. This study provides a long-term and multi-scale assessment of the gaining effect of Fe₃O₄-NPs in anaerobic digestion scale-up devices, and provides technical support for their practical engineering applications.