Anaerobic digestion is a sustainable bioprocess that stabilizes organic waste, while recovering methane as a useful by-product. In anaerobic digestion, extracellular hydrolytic enzymes first break down complex organic matter into monomers. Acidogenic bacteria ferment the monomers to intermediates, including acetate, hydrogen, and formic acid, and methanogenic archaea convert the intermediates to methane [1
]. Thus, anaerobic digestion is a kind of indirect interspecies electron transfer (IIET) process, in which intermediates shuttle electrons between acidogenic bacteria and methanogenic archaea [2
]. However, the IIET involved in methane production is a series of multi-step enzymatic reactions with significant electron losses [1
]. The enzymatic reactions cannot fully transfer electrons thermodynamically from the substrate to methane [4
]. Therefore, the methane yield that can be obtained from organic matter does not reach its theoretical value of 350 mL/g CODr
. In addition, the physiological properties of acidogenic bacteria, such as their growth rate and susceptibility to environmental conditions, are different from those of the methanogenic archaea [4
]. Therefore, the anaerobic digestion process can easily be destabilized by the imbalance between the IIET steps, even with a small external shock [7
However, direct interspecies electron transfer (DIET), without any electron shuttle from a microbial species to another species, can be more thermodynamically and kinetically advantageous over the IIET [4
]. DIET can be a breakthrough to address the limitations in the anaerobic digestion based on IIET. The microbial species involved in DIET from organic matter to methane are electroactive microorganisms, including exoelectrogenic bacteria (EEB) and electrotrophic methanogenic archaea (EMA) [2
]. The electroactive microorganisms are the microbial species with conductive proteins including cytochrome C over-expressed to the outer membrane of the cell and conductive pili as an appendage connecting microbial species [5
]. EEB releases electrons derived from the oxidation of low molecular organics, while EMA directly reduces carbon dioxide using those electrons to produce methane [9
]. In anaerobic digestion, electroactive microorganisms are generally abundant in anaerobic microbial aggregates, conductive material surfaces, and polarized electrode surfaces [7
]. These microbial species can directly transfer electrons for their syntrophic metabolism through the electrical connection by physical contact with each other (biological DIET), or through the mediation by conductive materials (cDIET) or polarized electrodes (eDIET) [6
]. In bioelectrochemical anaerobic digesters with polarized electrodes such as microbial electrolysis cells, the potential difference between the electrode surface and the bulk solution causes the faradaic current for methane production through eDIET. However, electrical energy is required in proportion to the amount of methane produced through eDIET [2
]. Interestingly, electroactive microorganisms can also be abundant in the bulk solution of bioelectrochemical anaerobic digesters with polarized electrodes, and improve methane production [4
]. It is worth noting that the bulk solution around the polarized electrode is exposed to an electric field. This indicates that the electric field formed by polarized electrode enriches the bulk solution with electroactive microorganisms, and significantly promotes biological DIET [16
]. Meanwhile, the faradaic current for eDIET can be blocked by insulating the electrode surface with a dielectric material. This indicates that polarizing the insulated electrodes creates the electric field in the bulk solution, which improves methane production through biological DIET without the consumption of electric energy.
In general, both DIET and IIET can simultaneously contribute to methane production in bioelectrochemical anaerobic digesters with polarized electrodes [4
]. As the relative contribution of DIET increases, the anaerobic digestion process becomes more robust, and methane production from organic matter further increases, improving the performance of anaerobic digestion [2
]. In the electric power supply sector, the share of renewable energy such as wind and solar power is increasing significantly. However, wind and solar power are fluctuating and intermittent energies have to be balanced through long-term storage and reserve production to stabilize the power grid [19
]. Power to gas (PtG) technology that converts excess renewable energy to hydrogen or methane might contribute to mitigating the fluctuation and intermittence of renewable energy [19
]. Bioelectrochemical anaerobic digestion that improves methane production with small electric power has great potential as a PtG technology for intermittent renewable energy in the near future.
However, DIET and IIET can compete for electrons to produce methane in bioelectrochemical anaerobic digesters with polarized electrodes [16
]. In thermodynamics, the equilibrium constant (K) for the interspecies electron transfers, including IIET and DIET, depends on the free energy change (∆G = −RT ln K) [22
]. The free energy (G) is a function of the enthalpy (H), entropy (S), and temperature (G = H−TS) [23
]. This indicates that the substrate type can affect the relative contribution of DIET and IIET to methane production in anaerobic digesters with polarized electrodes. Among organic substrates, acetate is a simple and non-fermentable substrate. Acetate can be easily converted to methane by mainly acetoclastic or syntrophic acetate oxidation pathway in anaerobic digestion [1
]. Acetate is also a suitable substrate for EEB [21
]. In anaerobic digesters with polarized electrodes, this implies that acetate can be converted to methane in the bulk solution by biological DIET between EEB and EMA. However, as of yet, there is little information reporting the transfer rate and conservation of electrons associated with DIET for methane production from acetate. Unlike acetate, glucose is a fermentable substrate [21
]. In anaerobic digestion, one of the main pathways for electron transfer that produces methane from glucose is IIET between acidogenic bacteria and methanogenic archaea through the intermediates, such as acetate and hydrogen. However, EEB can metabolize glucose, as well as acetate, and release electrons outside the cell [25
]. In anaerobic reactors with polarized electrodes, it seems that DIET and IIET can compete or cooperate to produce methane from glucose. In the case of a substrate mixture of acetate and glucose, the routes for the electron transfer for methane production would be similar to those of glucose. However, it is believed that the contribution of DIET and IIET to methane production depends on the relative fraction of acetate to glucose in the mixture. The characteristics of methane production from the mixture of acetate and glucose would be slightly different from those of the acetate or the glucose alone.
The purpose of this study was to investigate the bioelectrochemical methane production depending on the substrate type, including non-fermentable, fermentable, and mixed substrates, in the anaerobic batch reactor in which the bulk solution was exposed to an electric field. For this, the yield and production rate of methane from acetate were compared with those of glucose and their mixture and also discussed based on the electron balance and the contribution of DIET to methane production. In addition, the microbial community and electrochemical activity of the bulk solution depending on the substrate type were analyzed.