*3.3. Biogas Production*

Cumulative biogas production from the AD-MEC treatment was 93.0% higher than the AD-only treatment (Figure 3) during the MEC-inclusion period (days 20–31). The AD-MEC treatment produced 26.0 L of useful gases (CH4 + H2) over the additional 11-day digestion period, with 116% more CH4 (23.6 L) and 51,804% more H2 (2.43 L) than the AD-only treatment, which had 10.9 L CH4 and 0.04 L H2 (Figures 3 and 4). These results are similar to previous studies [34,35] that have shown that MEC inclusion into AD increased biogas and CH4 production by 80–100%. The AD-MEC also reduced the CO2 concentration (37.9% of the total gas) compared to AD-only (40.2% of the total gas), illustrating how MEC incorporation can both decrease CO2 concentration and increase the generation of useful gases (CH4 and H2) in the biogas [4]. Our MEC novel design confined the cathode and the anode inside a single chamber, which reduced the cathode and anode distance, and thus, potential ohmic losses, but still allowed liquid to flow easily between the MEC and AD chambers. Recent studies [36–38] have reported that limiting the distance between the electrodes can increase the gas production, as large distances between the electrodes can inhibit electron flow between the anode and the cathode, increasing ohmic losses. Additionally, the manganese used in the stainless grade 201 could have increased the gas production, as Mg has been shown to enhance electrical energy produced in MFCs by a factor of ten [21,22].

**Figure 3.** Cumulative and daily biogas production for AD-MEC and AD-only for days 20–31 (272 cumulative h) after MEC introduction (days 0–20 not shown, as treatments acted as duplicate treatments).

**Figure 4.** Methane (CH4) and hydrogen (H2) production for AD-MEC and AD-only for days 20–31 (272 cumulative h) after MEC introduction (days 0–20 not shown, as reactors acted as duplicate reactors).

Overall, the AD-MEC produced 149% more daily useful gases (H2 and CH4) (0.26 m<sup>3</sup>/m<sup>3</sup>/d) compared to AD-only (0.11 m<sup>3</sup>/m<sup>3</sup>/d) over the 11 additional days of digestion. The daily production rate of H2 and CH4 in the AD-MEC treatment reached a maximum of 0.16 m3H2/m<sup>3</sup>/<sup>d</sup> and 0.39 m3CH4/m<sup>3</sup>/<sup>d</sup>

within the first 24 h. During the 11-day period, a cumulative H2 + CH4 production rate of 2.87 m<sup>3</sup>/m<sup>3</sup> was observed for the AD-MEC treatment, compared to 1.15 m<sup>3</sup>/m<sup>3</sup> observed for the AD-only treatment.

Hydrogen concentration recorded in the first 24 h of the MEC introduction reached 20% of biogas volume, but was then reduced to 2% H2, as CH4 increased from 50% to 56.9% after 5 days and to 63% CH4 after 11 days. Most MEC studies observed high H2 generation during experimental startup, which would gradually decrease while CH4 concentration increased [13,14,24,29,39]. Recent studies have shown that hydrogenotrophic methanogens can survive in harsh environmental conditions, with continued utilization of the produced H2 with CO2 to form CH4 [40–42]. It has also been shown that hydrogenotrophic methanogens can directly receive electrons from MEC electrodes [43] or H2 [14] to enhance CH4 production [44].
