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This study investigated the impact of stimulating direct interspecies electron transfer (DIET), by supplementing nano-sized magnetite (nFe3O4, 0.5 g Fe/g VSS) and carbon nanotubes (CNT, 1 g/L), in anaerobic digestion of oleic acid (OA) at various concentrations (0.10–4.00 g chemical oxygen demand(COD)/L). Both supplementations could enhance CH4 production, and its beneficial impact increased with increased OA concentration. The biggest improvements of 114% and 165% compared to the control were achieved by nFe3O4 and CNT, respectively, at OA of 4 g COD/L. The enhancement can be attributed to the increased sludge conductivity: 7.1 ± 0.5 (control), 12.5 ± 0.8 (nFe3O4-added), and 15.7 ± 1.1 µS/cm (CNT-supplemented). Dissolved iron concentration, released from nFe3O4, seemed to have a negligible role in improving CH4 production. The excretion of electron shuttles, i.e., humic-like substances and protein-like substances, were found to be stimulated by supplementing nFe3O4 and CNT. Microbial diversity was found to be simplified under DIET-stimulating conditions, whereby five genera accounted for 88% of the total sequences in the control, while more than 82% were represented by only two genera (Methanotrix concilli and Methanosarcina flavescens) by supplementing nFe3O4 and CNT. In addition, the abudance of electro-active bacteria such as Syntrophomonas zehnderi was significantly increased from 17% to around 45%.

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