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%.

Since the observation of direct interspecies electron transfer (DIET) in anaerobic mixed cultures in 2010s, the topic “DIET-stimulation” has been the main route to enhance the performance of anaerobic digestion (AD) under harsh conditions, such as high organic loading rate (OLR) and the toxicants’ presence. In this review article, we tried to answer three main questions: (i) What are the merits and strategies for DIET stimulation? (ii) What are the consequences of stimulation? (iii) What is the mechanism of action behind the impact of this stimulation? Therefore, we introduced DIET history and recent relevant findings with a focus on the theoretical advantages. Then, we reviewed the most recent articles by categorizing how DIET reaction was stimulated by adding conductive material (CM) and/or applying external voltage (EV). The emphasis was made on the enhanced performance (yield and/or production rate), CM type, applied EV, and mechanism of action for each stimulation strategy. In addition, we explained DIET-caused changes in microbial community structure. Finally, future perspectives and practical limitations/chances were explored in detail. We expect this review article will provide a better understanding for DIET pathway in AD and encourage further research development in a right direction.

Various pretreatment methods have been combined and employed for maximizing the solubilization of waste-activated sludge (WAS). However, the question “by changing the series of applied combined pretreatments (CPs), can the solubilization efficiency of WAS be affected?” has never been addressed. In this study, firstly, thermal (T), alkaline (A), and ultrasonic (U) pretreatments were individually applied at broad strengths (T = 80–120 °C for 30 min, A = pH 9–12, and U = 5–60 min at 300 W). Then, pretreatment conditions that caused similar solubilization (13.0%) (120 °C, pH 11, and 30 min for T, A, and U, respectively), were adopted for CP with reverse sequences of T&A, U&A, and T&U. A similar disintegration degree was observed in U→A and A→U, while a meaningful difference was found in T&A and T&U: T→A (28.3%), A→T (42.9%), T→U (22.9%), and U→T (27.1%). The difference in pretreatment series also affected the characteristics of soluble matters, which was analyzed by excitation emission matrix and molecular weight distribution. Due to these differences, the highest methane yield of 68.8% (based on (chemical oxygen demand) COD_input) was achieved at A→T, compared to T→A (62.3%). Our results suggested a simple strategy for increasing solubilization, at the same expense of energy, which might be beneficial in the following treatment process, such as dewatering and transportation.