This study was conducted in order to examine the impact of magnetite (Fe3O4), a conductive material capable of promoting direct interspecies electron transfer (DIET) among microorganisms, on the efficiency of anaerobic digestion in a plug flow reactor (PFR) using food wastewater (FW) as the substrate. The effects of recovering and replenishing magnetite discharged along with the digestate during continuous operation of the PFR were also evaluated. A PFR with a total volume of 17 L was utilized as the reactor for anaerobic digestion. The inoculum was obtained from Icheon Biogas Research Facility, which operated with a mixture of pig slurry and FW in a 7:3 (w/w) ratio. FW was used as the substrate (volatile solids (VS) content of 85,865 mg-VS/L). The PFR was set for operation at 39 °C, and after a stabilization period of approximately 82 days, the hydraulic retention time (HRT) was set at 40 days. The study was conducted in three stages: stage 1 (83~122 days), stage 2 (123~162 days), and stage 3 (163~202 days). For the maintenance of an organic loading rate of 2.12 kg-VS/m3/d, 0.3 L/d of substrate was added every 24 h, and analysis of an equal amount of discharged digestate was performed. The experimental treatments included a control without the addition of magnetite after the stabilization period, treatment (T1) with addition of magnetite (20 mM in digestate) and subsequent recovery and replenishment of magnetite on the discharge of digestate, and treatment (T2) with addition of magnetite (20 mM) without the replenishment of magnetite. Analytical parameters included the characteristics of the discharged digestate (pH, NH4+-N, chemical oxygen demand (CODCr), total volatile fatty acids (TVFAs), and alkalinity), and methane production (Mp). During the period of operation of the PFR after the stabilization period, no significant differences in pH and NH4+-N, based on the recovery and replenishment of magnetite, were observed, and a stably functioning PFR was observed. However, in stage 2, due to the increased degradation of organic matter caused by DIET, the CODCr of T1 and T2 decreased by 9.42% compared with the control. In stage 3, the magnetite content in the reactor in T2 decreased by a maximum of 9.42% compared to T1. In stage 3, the Mp for T2 was similar to that of the control, with a maximum discharge of magnetite of 3.06%, and the Mp decreased by 5.40% compared to T1. Regarding the ratio of methanogens in the community, the results of an analysis of the digestate from stage 3 showed an increase in the community of acetotrophic methanogens, specifically Methanosarcina. The findings of this study confirm that DIET was effectively promoted by maintaining the concentration of 20 mM magnetite in the PFR while using FW as a substrate.
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