Direct interspecies electron transfer (DIET), which does not involve mediation by electron carriers, is realized by the addition of conductive materials to an anaerobic digester, which then activates syntrophism between acetogenic and methanogenic microorganisms. This study aimed to investigate the effect of the addition of two conductive materials, zero-valent iron (ZVI) and magnetite, on the methane production and microbial consortium via DIET in the anaerobic digestion of food wastewater. The operation of a batch reactor for food wastewater without the addition of the conductive materials yielded a biochemical methane potential (B_u), maximum methane production rate (R_m), and lag phase time (λ) of 0.380 Nm³ kg⁻¹-VS_added, 15.73 mL day⁻¹, and 0.541 days, respectively. Upon the addition of 1.5% ZVI, B_u and R_m increased significantly to 0.434 Nm³ kg⁻¹-VS_added and 19.63 mL day⁻¹, respectively, and λ was shortened to 0.065 days. Simultaneously, Methanomicrobiales increased from 26.60% to 46.90% and Methanosarcinales decreased from 14.20% to 1.50% as the ZVI input increased from 0% to 1.50%. Magnetite, at an input concentration of 1.00%, significantly increased the B_u and R_m to 0.431 Nm³ kg⁻¹-VS_added and 18.44 mL day⁻¹, respectively. However, although magnetite improves the efficiency of methanogenesis via DIET, the effect thereof on the methanogen community remains unclear.

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 (COD_Cr), total volatile fatty acids (TVFAs), and alkalinity), and methane production (M_p). 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 COD_Cr 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 M_p for T2 was similar to that of the control, with a maximum discharge of magnetite of 3.06%, and the M_p 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.

This study investigated the feasibility of incorporating Chenopodium album L. (CAL) into ruminant feed ingredients through evaluating the effects of harvest time and substitution levels on in vitro rumen fermentation. In the first phase, a sole-substrate experiment was conducted using CAL harvested from June to August, analyzing its chemical composition and total saponins content. The impact of harvest time on fermentation parameters was assessed with CAL as the sole substrate. The second phase involved a mixed-substrate experiment using an early-fattening Hanwoo diet (30% rice straw and 70% concentrate), where increasing proportions of CAL (control: 0%, T1: 5%, T2: 10%, T3: 15%, and T4: 20%) replaced rice straw. Seasonal variations in CAL composition influenced the fermentation characteristics. CAL harvested in July exhibited higher fermentability, with total volatile fatty acids (TVFAs) reaching 103.87 mM at 72 h. In contrast, CAL harvested in August showed lower fermentability and digestibility. However, August-harvested CAL was selected for the subsequent experiment, as it provided a more practical balance of sufficient biomass yield and a higher saponins concentration, aligned with the study’s methane mitigation objectives, while also exhibiting a fiber composition comparable to that of rice straw. We hypothesized that the saponins in CAL contribute to methane reductions. Supplementation with 15% of CAL significantly reduced methane production per gram of inoculated and digested dry matter (p < 0.05), likely due to differences in crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF), and saponins content. However, despite having the lowest fiber content, T4 (20% CAL) exhibited the lowest in vitro dry matter digestibility (IVDMD), suggesting that factors such as saponins, CAL’s chemical composition, or microbial shifts may have hindered digestibility. Ammonia–nitrogen production increased from 0 to 3 h, but it continuously decreased between 3 and 9 h due to microbial growth and nitrogen assimilation, as microbes incorporate ammonia into their biomass (p < 0.05). Fermentation characteristics further revealed that the acetate-to-propionate (A/P) ratio decreased with increasing CAL levels, with T4 showing the lowest ratio (1.55 at 72 h), confirming a shift toward propionate-based fermentation. Notably, T2 (10% CAL) showed an optimized fermentation efficiency, producing the highest TVFA concentration at 24 h (98.28 mM). These findings highlight the potential for using CAL as a functional feed ingredient, with moderate substitution levels (10–15%) enhancing fermentation efficiency while reducing methane production.

This study investigated the feasibility of incorporating Chenopodium album L (CAL) into ruminant feed ingredients by evaluating the effects of harvest time and substitution levels on in vitro rumen fermentation. In the first phase, a sole-substrate experiment was conducted using CAL harvested from June to August, analyzing its chemical composition and total saponin content. The impact of harvest time on fermentation parameters was assessed with CAL as the sole substrate. The second phase involved a mixed-substrate experiment using an early-fattening Hanwoo diet (30% rice straw and 70% concentrate), where increasing proportions of CAL (Control: 0%, T1: 5%, T2: 10%, T3: 15%, and T4: 20%) replaced rice straw. Seasonal variations in CAL composition influenced fermentation characteristics, with July-harvested CAL exhibiting higher fermentability, while August-harvested CAL had lower fermentability. However, August-harvested CAL was selected for the second experiment due to its greater availability. We hypothesized that saponins in CAL contribute to methane reduction. Supplementation with 15% of CAL significantly reduced methane production per gram of digested substrate (p < 0.05), likely due to differences in crude protein (CP), neutral detergent fiber (NDF), acid detergent fiber (ADF), and saponin content. However, despite having the lowest fiber content, T4 (20% CAL) exhibited the lowest in vitro dry matter digestibility (IVDMD), suggesting that factors such as saponins, CAL’s chemical composition, or microbial shifts may have hindered digestibility. Fermentation characteristics further revealed that the acetate-to-propionate (A/P) ratio decreased with increasing CAL levels, with T4 showing the lowest ratio (1.55 at 72 hours), confirming a shift toward propionate-based fermentation. Notably, T2 (10% CAL) optimized fermentation efficiency, producing the highest total volatile fatty acid (VFA) concentration at 24 hours (98.28 mM). These findings highlight the potential of CAL as a functional feed ingredient, with moderate substitution levels (10–15%) enhancing fermentation efficiency while reducing methane production.