Generally, researchers conduct in-depth research on the mechanisms of DIET in defined co-culture systems using ethanol as a substrate (Table 1). Compared with indirect electron transfer, which relies on H₂ and formate as electron carriers (Figure 2A), biological structure mediating DIET achieves electrical connection via e-pili and OmcS of electron-donating microorganisms (Summers et al., 2010). E-pili, also known as nanowires, are protein filaments produced by microorganisms during electron transfer under appropriate conditions (Malvankar and Lovley, 2012). Malvankar et al. (2011) found that the conductivity of pili protein extracted from Geobacter sulfurreducens was approximately 5 μS/cm, and exhibited temperature dependence similar to that of disordered metals. OmcS is a type of Fe(III) redox protein that is related to the process of electron transfer from cell surface to extracellular electron acceptor. Leang et al. (2010) observed that the OmcS was mainly distributed along nanowires or exists on the outer surface of cells using scanning electron microscopy. Shrestha et al. (2013) found that the key genes encoding e-pili and OmcS were highly expressed in the co-culture system of G eobacter metallireducens and G. sulfurreducens. Rotaru et al. (2014b) confirmed that co-culture of microorganisms cannot reproduce without the key genes encoding e-pili and OmcS. Moreover, e-pili- or OmcS-deficient strains cannot metabolize ethanol to produce methane in the defined co-cultures (Rotaru et al., 2014a,b). These results indicate that e-pili and OmcS play an important role in establishing DIET. The possible related mechanisms are summarized in Figure 2B.

The mechanism of methanogens to accept electrons may be similar to that of electron donation microorganisms (Li et al., 2021). However, methanogens, including Methanosarcina barkeri, and Methanosarcina harundinaceae have been confirmed to participate in DIET without OmcS on their outer surface (Holmes et al., 2018; Yee and Rotaru, 2020). In addition, Walker et al. (2019) reported Methanospirillum hungatei could secrete e-pili. However, M. hungatei could not conduct the DIET with G. metallireducens in the co-culture system (Rotaru et al., 2014b). The study on extracellular electron transfer mechanism of methanogens has just begun, which is far less than the understanding with Geobacter species. Therefore, it is necessary to deeply study the molecular targets related to biological pathway of methanogens to accept electrons.

Conductive carbon materials have been proven to significantly improve anaerobic digestion performance (Lee et al., 2016; Lin et al., 2017; Li J. et al., 2018). Liu et al. (2012) thought that the reason why active carbon promoted DIET was that the conductivity of active carbon was higher than that of methanogenic aggregates. However, the promotion effect of biochar is almost the same as that of active carbon even though the conductivity of biochar is one-thousandth that of activated carbon (Chen et al., 2014b). Therefore, it can be inferred that any conductivity greater than a certain threshold is sufficient to trigger the DIET methanogenic pathway (Barua and Dhar, 2017).

Direct interspecies electron transfer promoted by conductive carbon materials is not completely dependent on e-pili or OmcS as the media for electron transfer (Zhao and Zhang, 2019). Rather, the electrical connection was realized through carbon materials, not intercellular contact, because there were no aggregates of G. metallireducens and M. harundinaceae on the surface of carbon materials (Chen et al., 2014b). Liu et al. (2012) confirmed that e-pili- and OmcS-deficient strains could metabolize ethanol and reduce fumarate to succinate with addition of conductive carbon materials. Park et al. (2018b) found that conductive carbon materials made the abundance of key genes encoding e-pili and OmcS in wild-type strains decrease compared with the control, indicating that conductive carbon materials replaced the functions of e-pili and OmcS. Therefore, establishing DIET by adding conductive carbon materials might consume less energy to satisfy the growth of syntrophic partners and metabolize more substrates as the microorganisms did not need to highly express the key genes encoding e-pili and OmcS (Zhao et al., 2015).

Furthermore, conductive carbon materials have large specific surface areas and porosities (Figueiredo et al., 1999; Chen et al., 2014b; Zhao et al., 2016a), which play a vital role in the adsorption, fixation, and enrichment of microorganisms involved in DIET (Qi et al., 2021). Meanwhile, the redox groups on the surface of conductive carbon materials, such as quinone or quinone hydrogen, may be closely related to DIET (Wang et al., 2020b). The possible related mechanisms are shown in Figure 2C.

Conductive iron materials also promote DIET formation (Kato et al., 2012; Zhu et al., 2017). Currently, researches are mainly focused on the promotion of DIET by magnetite. Ueki et al. (2018) found that magnetite could not replace conductive carbon materials as adding magnetite did not promote DIET in a co-culture system of e-pili-deficient strains. However, Liu et al. (2015) found that magnetite enabled OmcS-deficient strains to have the ability to realize extracellular electron transfer. They found that the expression of wild-type OmcS gene was inhibited because of the presence of magnetite. Moreover, transmission electron microscopy showed that magnetite, similar to OmcS, was dispersed between cells and distributed along the e-pili (Liu et al., 2015). These results indicated that magnetite replaced the role of OmcS rather than that of e-pili (Lovley, 2017). The stimulating effect of magnetite is different from that of conductive carbon materials probably because of their size and structural differences. Magnetite particles are generally 20–50 nm in size, which is smaller than microbial cells, while conductive carbon materials have a significantly bigger size and provide a large surface area for microbial attachment (Lovley, 2017; Yin and Wu, 2019; Zhang et al., 2020).

In addition, many studies have shown that conductive iron materials can enrich microorganisms with extracellular electron transfer abilities, which may be involved in DIET (Yamada et al., 2015; Yang et al., 2015b). The possible related mechanisms are shown in Figure 2D.

Lag period is the time required for inoculum to adapt to the new environment. A long lag period means that a longer start-up time is needed for anaerobic digestion. In a practical start-up process, microorganisms must firstly recover from dormancy. The growth rate of methanogens in the original anaerobic sludge is much slower than that of bacteria. Usually, anaerobic digestion reactors start with short hydraulic retention time, low organic load rate (OLR), and low feeding concentration. These factors lead to a longer start-up cycle, which affects anaerobic digestion efficiency and causes an increase in operating cost. Studies have shown that DIET mediated by conductive materials can significantly shorten lag time of anaerobic digestion reactor with different types of substrates, such as glucose (Luo et al., 2015; Yan et al., 2017), phenol (Wang et al., 2020a), benzoate (Zhuang et al., 2015), and mixed organic carbon (Yin et al., 2017b; Wang G. et al., 2018). Moreover, establishing DIET by adding conductive materials significantly shortened the lag period of anaerobic digestion system even under conditions of high hydrogen partial pressure, high concentration of ammonia nitrogen, and sulfur inhibition. Wang et al. (2020b) found that DIET stimulation shortened the lag time of acetate, propionate, butyrate, and valerate by 29.2, 20.5, 9.1, and 10.5%, respectively. Therefore, the promotion on syntrophic metabolism of volatile fatty acids (VFAs) may be the main reason why DIET shortens the lag period.

As DIET is more efficient than IHT, the establishment of DIET mediated by conductive materials has been widely recognized to promote methane yield. Park et al. (2018b) found that methane yield with GAC addition increased by 31% compared to control without GAC addition. Similarly, Zhang et al. (2020) observed that the methane yield in GAC-amended systems increased by 3.9 times. Li et al. (2020a) reported that the methane yield with biochar increased by about 44% and the effluent concentration of total organic substrate decreased compared with control. Besides, improvement in the methane yield has been also repeatedly reported and verified with other conductive materials, such as graphite (Tan et al., 2015), magnetite (Ye et al., 2018), and carbon cloth (Zhao et al., 2017c). Studies showed that the increase in methane yield is mainly because that DITE mediated by conductive materials facilitated the conversion of interspecies metabolites to methane (Tian et al., 2017; Jiang et al., 2020; Sun et al., 2020).

Direct interspecies electron transfer mediated by conductive materials plays an important role in maintaining the stability of anaerobic digestion system. To achieve efficient energy production, anaerobic digestion systems are generally maintained at high OLRs (Appels et al., 2011). However, high OLRs often cause the acidogenesis rate to be faster than methanogenesis rate, resulting in a sharp drop in pH and VFA accumulation (Babel et al., 2004). Nevertheless, DIET can still improve anaerobic digestion performances under high OLR conditions (Dang et al., 2016, 2017). Moreover, a high hydrogen partial pressure is another factor that leads to VFA accumulation. However, DIET-based syntrophic relationships can enable anaerobic digestion systems to overcome the limitation of hydrogen partial pressure (Nagarajan et al., 2013; Zhao et al., 2016b; Zhao Z. et al., 2018). Organic matters containing nitrogen (protein, amino acids, and urea) can release high concentration of ammonia in an anaerobic digestion system. The ammonia can exist in the form of ammonium ion or free ammonia, which inhibit microbial activity and cell growth (Kayhanian, 1999). However, Lu et al. (2016) found that the methane yield increased with the addition of biochar under an ammonia inhibition environment. This may be because that the establishment of DIET resisted the toxic inhibition of ammonia and maintained the stability of syntrophic metabolism. In addition, supplemental magnetite significantly increased the conversion rate of acetate to methane under high ammonium concentrations (Zhuang et al., 2018). Sulfate in the anaerobic digestion system can stimulate the growth of sulfate-reducing bacteria which compete with methanogens for acetate, H₂, and intermediates, such as propionate, butyrate, and ethanol (Li Q. et al., 2015; Qian et al., 2019). Hydrogen sulfide, the product of sulfate reduction, can significantly inhibit the activities of methanogens, which decreases the anaerobic digestion performances (Chen et al., 2008). However, some studies have demonstrated that DIET mediated by conductive materials was expected to promote methane production in anaerobic digestion system treating organic wastewater with a high concentrations of sulfate (Li et al., 2017; Jin et al., 2019). The potential mechanism may be that methanogens through DIET metabolic pathway are more competitive to substrates than sulfate-reducing bacteria (Liu et al., 2019).

Study on the biological pathway of DIET is helpful for understanding microbial syntrophic metabolism. Figure 3 shows the model of DIET methanogenic pathways between G. metallireducens and M. barkeri. However, the electron transfer biological pathway in DIET between microorganisms remains unclear.

To date, it is unclear how electron donating bacteria transfer electrons outside of the cell. Geobacter is often used as a model microorganism to study the mechanism of extracellular electron transfer because of its completely available genome sequence and skilled gene engineering methods (Coppi et al., 2001). In general, it is believed that the e-pili of Geobacter transfer electrons to OmcS and then to extracellular insoluble electron acceptors such as iron oxides and electrodes. However, the mechanism of electron transfer to extracellular e-pili is unclear. Currently, cytochromes (OmaB, OmaC, OmcB, and OmcC) and porins (OmbB and OmbC) are considered to play essential roles in the transfer of electrons to e-pili (Costa et al., 2018). Besides, it remains unclear how electron-accepting microorganisms directly accept extracellular electrons from their syntrophic partners. Studies on extracellular electron transfer showed that OmcS and pili nanowires were indispensable during the process of extracellular electron transfer. However, unlike Geobacter species, M. barkeri, and M. harundinaceae without outer surface e-pili and OmcS have also been found to be involved in DIET (Holmes et al., 2018). Therefore, it is necessary to further find the differential expression of genes in electroactive methanogens under different methanogenic conditions, and then verify the functions of related genes through gene mutations, and finally reveal the extracellular electron transfer mechanism of electron-donating microorganisms. Finally, it is unclear how methanogens utilize extracellular electrons and reduce CO₂ through DIET. Thauer et al. (2008) proposed that the CO₂ reduction pathways of methanogens with and without cytochromes might vary. Recent research showed that DIET promoted transcripts of F₄₂₀H₂ dehydrogenase (Fpo) and heterodisulfide reductase (HdrABC) (Holmes et al., 2018). Electrons transferred via the DIET pathway were first employed to reduce methanophenazine (MP), and then the generated MPH₂ could be used as the electron donor for Fpo to reduce coenzyme F₄₂₀. Half of the produced F₄₂₀H₂ in this model was regarded as the electron donor for HdrABC bifurcation reaction to further yield reduced ferredoxin for CO₂ reduction (Holmes et al., 2018). Simultaneously, a bifurcation reaction facilitated the reduction of coenzyme B-coenzyme M heterodisulfide (CoB-S-S-CoM), which is necessary for CH₄ production. However, how electrons are transferred to MP should be further studied (Holmes et al., 2018).

Recently, Holmes et al. (2021) proposed a new model of DIET methanogenic pathways, which was confirmed in the defined co-culture of G. metallireducens and Methanosarcina acetivorans. Multiheme membrane-bound c-type cytochrome A (MmcA) in M. acetivorans played an important role in electron transfer. GAC did not make the co-culture system with MmcA-deficient strains produce methane rapidly, which was mainly because that MmcA was embedded in the cell membrane of M. acetivorans (Ferry, 2020). Researches show that MmcA may exchange electrons with MP or rhodobacter nitrogen (Rnf) complex (Suharti et al., 2014; Ferry, 2020; Holmes et al., 2021). The function of the Rnf is to accept electrons to produce the reduced ferredoxin, which is required for the first step in the reduction of CO₂ (Holmes et al., 2021). The model in M. acetivorans is significantly different from that in M. barkeri, because M. Barkeri lack genes for MmcA and Rnf (Zhou et al., 2021). Therefore, Methanogen growth via DIET may adopt different electron-acceptance strategies to accommodate these physiological differences (Holmes et al., 2021).

The positive effect of conductive materials on anaerobic digestion performances has been confirmed. Therefore, most studies speculated that performance improvement might be attributed to DIET existing in complex anaerobic digestion systems based on microorganisms already known to take part in DIET. However, the relative abundance of Geobacter in anaerobic sludge is low (Dang et al., 2016; Peng et al., 2018; Li et al., 2020b) or even undetectable (Wang et al., 2020b). In addition, several studies have demonstrated that conductive materials would enrich microorganisms with extracellular electron transfer ability, such as Desulfuromonas (Barua et al., 2018), Pseudomonas (Barua and Dhar, 2017), Azovibrio (Ruiz et al., 2014), and Syntrophomonas (Chen Y. et al., 2020), which may participate in DIET. However, it has not been proven that these microorganisms can conduct DIET in defined co-cultures with methanogens. Therefore, it is insufficient to infer that DIET occurred in anaerobic digestion systems based on the anaerobic digestion performance improvement and microbial community structure.

In addition to DIET, other stimulatory mechanisms of conductive materials in anaerobic digestion cannot be ignored. The related mechanisms are illustrated in Figure 4. Conductive carbon materials not only promote metabolism of inhibitory intermediates by fixing and enriching specific microorganisms but also alleviate inhibition of toxic substances via adsorption effect (Martins et al., 2018; Jung et al., 2020). The pH buffering capacity of conductive carbon materials also creates a favorable environment for methanogenesis (Wang et al., 2017, 2020b). Simultaneously, conductive carbon materials can improve anaerobic digestion performances through other mechanisms, such as function of released ions and surface redox groups, which may affect the redox potential of anaerobic digestion systems. In general, it is believed that a redox potential between −200 and −400 mV is beneficial for methane production (Hirano et al., 2013). Salvador et al. (2017) confirmed that a more negative redox potential occurred with an increase in the carbon nanotube dosage in co-culture systems. Similarly, the effects of non-DIET mechanisms cannot be neglected with addition of conductive iron materials. The iron ions released from conductive iron materials can decrease the negative charge on the surface of extracellular polymeric substances, thereby reducing the repulsive force between cells and increasing the stability of microbial aggregates. Furthermore, the release of iron ions might be regarded as a trace element that promotes microorganism activity.

Therefore, whether the improvement of anaerobic digestion performances is caused by DIET mediated by conductive materials and the DIET is the dominant mechanism in a complex anaerobic digestion system requires in-depth exploration and analysis.

Ethanol has been proven to participate in DIET methanogenesis in a defined co-culture (Rotaru et al., 2014a,b). Although acetate has not been proven to be a substrate that participates in DIET, Zhuang et al. (2018) found that the degradation of acetate in bioreactor containing magnetite was much less affected by hydrogen partial pressure, which may be because that the acetate oxidation metabolism was not completely dependent on IHT. Propionate and butyrate are important intermediate metabolites in anaerobic digestion. Thus, limiting propionate and butyrate accumulation is crucial for improving anaerobic digestion performances. Studies showed that magnetite significantly increased propionate and butyrate degradation, suggesting that propionate and butyrate may be converted to methane via DIET metabolic pathway (Cruz Viggi et al., 2014; Li H. et al., 2015; Jing et al., 2017). Similarly, Barua et al. (2018) confirmed that carbon fibers promoted the conversion of propionate and butyrate to methane. In a syntrophic propionate metabolism, Cruz Viggi et al. (2014) confirmed that conductive materials provided 10⁶ times higher electron carrier flux than that of IHT. In a syntrophic butyrate metabolism, Xu et al. (2018) found that the conversion rate of butyrate into acetate increased by 2.5–7.0 times when conductive material dosage increased from 0.5 to 25 g/L, which may be because that the increase in conductive material dosage provided more carriers for syntrophic partners to exchange electron (Yin and Wu, 2019). Zhuang et al. (2015) found that the addition of magnetite and hematite promoted the anaerobic digestion performances of benzoate. Similar studies demonstrated that adding magnetite or carbon nanotubes to an anaerobic digestion system accelerated the degradation of phenol and reduced its inhibitory effect on biological activity of methanogens (Yan et al., 2018). In addition, glucose and sucrose have been widely reported as substrates of DIET methanogenesis. However, in addition to ethanol as a substrate that participates in DIET, the establishment of DIET metabolic pathway in an anaerobic digestion system with other organics as substrates lacks direct biological evidence.

Compared with simple organics, complex organics must undergo hydrolysis and acidification before methanogenesis. Therefore, the DIET mechanisms of complex organics may be more complicated than that of simple organics. Yang et al. (2017) and Sun et al. (2020) confirmed that addition of conductive materials improved the conversion rate of sludge to methane. Similarly, although the anaerobic digestion of leachate from municipal solid waste is limited by high OLRs and toxic substances, some studies found that establishing DIET could increase the digestibility of leachate and methane production (Lei et al., 2016, 2018). However, Zhao et al. (2017b) added magnetite and GAC to hydrolysis-acidogenesis and methanogenesis reactors, respectively, and found that the supplemental GAC had little effect on syntrophic metabolism of alcohols and VFAs when hydrolysis-acidogenesis was ineffective or did not work. After magnetite promoted acidogenesis efficiency, the syntrophic metabolism of alcohols and VFAs in methanogenesis phase was markedly enhanced by GAC. Anaerobic digestion modified by magnetite can enrich iron-reducing bacteria, which play an important role in the hydrolysis-acidogenesis of complex organics (Peng et al., 2018). The increase in methane production in anaerobic digestion with GAC is probably caused by DIET between syntrophic bacteria and methanogens (Peng et al., 2018). Therefore, establishing DIET may not change the rate-limiting step of anaerobic digestion with complex organic matters because the confirmed DIET mainly occurs in the methanogenic stage. Thus, further researches are necessary to promote the hydrolysis and acidogenesis of complex organics via DIET metabolic pathway.

There are few successful practical application of DIET in anaerobic digestion via adding conductive materials. This is mainly because that the establishment of DIET does not achieve satisfactory results in amplification experiments and demonstration projects. The complexity of reactor operation control makes it difficult to ensure that conductive materials take effect in the engineered anaerobic digestion system. In addition to considering the positive effect of DIET mediated by conductive materials, it is also important to consider the economic cost and benefit. Although researchers usually add conductive materials to anaerobic digestion, this method is not economically scalable (Zhao et al., 2020). There is loss of conductive materials in the digester discharge (Yin et al., 2017a; Zhang et al., 2018). For example, Zhang et al. (2018) found that the cost required to maintain the level of GAC in the anaerobic digestion accounted for 38.3–81.8% of the revenue from methane production each day. The labor for supplement of conductive materials also increases the economic cost on a daily basis (Zhao et al., 2020). Furthermore, the impacts of conductive material discharge on ecological environment and downstream processing should be valued. Currently, there are few studies on the influence of adding conductive materials to anaerobic digestion system on sludge dewatering (Nguyen et al., 2021). Obviously, addition of conductive materials significantly increases the amount of biosolids (Chen S. et al., 2020; Yuan et al., 2021), which increases the cost of solid waste transportation and treatment in the later stage. Recycling and reuse of conductive materials may be an effective strategy, but the complex technical operation and high investment cannot be ignored. Some studies have suggested that conductive materials designed as packed beds inside the reactor should be considered to continually promote DIET. However, this approach usually incurs considerable cost for the maintenance of equipment and construction of reactor (Tatara et al., 2008; Zhao and Zhang, 2019).

Some researchers have also found that conductive materials may have an inhibitory effect on anaerobic digestion performances. For example, Tian et al. (2017) found that the methane yield at a graphene dosage of 30 mg/L was 14.3% higher than that of the control after long-term operation at room temperature, while the methane yield at a graphene dosage of 120 mg/L exhibited a slight inhibitory effect. Salvador et al. (2017) found that carbon nanotubes of 5 g/L inhibited methane production compared with dosages of 0.5 and 1 g/L. These results indicated that excessive dosage of conductive materials could inhibit the microbial metabolism. The potential inhibitory effect may be attributed to the increased resistance to mass transfer between substrates and microorganisms. In addition, studies showed that the supplementation of carbon black nanoparticles resulted in a significant inhibitory effect on an anaerobic digestion system treating glucose, which might be because that carbon black nanoparticles possessed antibacterial properties (Martins et al., 2018; Fujinawa et al., 2019). Similar to conductive carbon materials, conductive iron materials, such as ferrihydrite, also exhibit an inhibitory effect on methane production (Zhou et al., 2014; Martins et al., 2018). Therefore, the dosage and type of conductive material may have unpredictable effect on the anaerobic digestion system. Further, the surface structure of conductive materials, such as the specific surface area, pore size, and roughness, affects the adhesion and colonization of relevant microorganisms in anaerobic digestion process. These factors limit the practical application of DIET mediated by conductive materials in anaerobic digestion.