Due to the lack of solids supplementation during ex-situ BM, nutrient supply becomes necessary 25. The non-enriched cow manure was used as the liquid nutrient and inoculum in this work that had an initial pH of 7.7. After its introduction to both TBRs, the pH of the liquid increased for most of the time above 8.0. It can be related to the partial dissolving of fed CO2 into liquid phase as HCO3− (pKa = 6.85). The pH values in the counter-current reactor ranged from 7.80 to 8.61 and 7.87 to 8.55 for the concurrent reactor. Similar values were reported by Porté et al. (2019) that used digestate from a biogas plant as a liquid nutrient source reached pH up to 8.6. Additionally, Dahl Jønson et al. (2020) reached pH up to of 8.5 using cow manure. The values >8.6 were indicated as above the optimum level for methanogens, however, no further pH increase was observed, and therefore no pH adjustments were applied in this work.
Further increase in gas loading that led to the lower CH4 production also decreased the pH in the liquid phase of TBRs. The lowest average pH for counter-current reactor was 7.9 ± 0.1 at gas loading rate of 30 m3/m3/d. The CH4 content in the effluent gas decreased as the pH decreased (Fig. 3). Strübing et al. (2017) mentioned potential reasons for pH decline during biomethanation such as buffer capacity reduction due to H2O production and homoacetogenesis. Although, in this work, the sharper decrease was observed for concurrent configuration where the CH4 content <60% was achieved already at pH 8.2, such low CH4 content was reported only once for counter-current configuration at pH 7.9. These observations may indicate the higher performance vulnerability of this configuration, as well as possible differences in the microbiology of these configurations.
In general, either mineral media or pH maintaining systems are used in TBR studies to maintain the process at certain pH range. To achieve the optimal performance of TBR pH values below reported in this work were used. For example, the efficient CH4 production was reported e.g., at pH range 6.8-7.7 yielding in CH4 content >95% 11,21. Although, our results indicate the advantage of using cow manure as a suitable source of liquid phase for ex-situ biomethanation that does not require pH maintenance or mineral supplementation.
3.2.2. Acetic acid and alkalinity
The reactor liquid phase parameters such as alkalinity and acetic acid were influenced by different gas loadings and reactor configurations (Fig. 4). A significant difference in alkalinity level and acetic acid presence was found in both reactors.
The alkalinity and acetic acid decreased over time in the counter-current reactor. Acetic acid presence decreased threefold from 27.7 to 9.2 mg/L even when the liquid phase was periodically exchanged. Similarly, alkalinity decreased 1.6 times from 4051.5 to 2508.6 mgCaCO3/L. This trend can be attributed to the liquid phase dilution, due to the H2O production (second product of hydrogenotrophic methanogenesis, Eq. 1). At higher gas loadings, the volume of produced H2O is increasing that ends up in the liquid phase of TBR. We observed that the level of liquid phase at the reactor bottom increases, especially at gas loadings >10 m3/m3/d.
To equalize the liquid level in TBR, the excess of liquid was removed at the end of each experimental period. For the counter-current reactor low volumes of excess liquid ca. 30, 50 and 70 mL were removed after applied gas loadings of 20, 25 and 30 m3/m3/d, respectively. It can be hypothesized that without excess liquid removal, the dilution would be more severe, and concentrations of acetic acid and alkalinity will be even lower, leading to the process destabilization.
In concurrent configuration, the unstable CH4 production was accompanied by unstable levels of acetic acid and alkalinity. The acetic acid concentrations were found higher than in the counter-current reactor and varied from 33.1 to 63.8 mg/L. The results were consistent with Porte et al. (2019), which observed that the counter-current configuration could affect the process due to the densities of gases passing through the reactor. H2 has a much lower density than CH4 and CO2, and a downward flow could result in a higher H2 partial pressure in the liquid than an upflow process. The alkalinity in concurrent configuration varied from 2775.3 to 3642.0 mgCaCO3/L. Interestingly, the highest levels of acetic acid at 20 m3/m3/d (days 11-25) occurred at the same time as the lowest content of CH4 (average: 47.1 ± 9.6%) and the lowest pH (average: 8.1 ± 0.1) were obtained. The declining pH can be correlated to acetate accumulation, as similarly observed by e.g., Kougias et al. (2017). In this work, other VFAs were also measured, however, their concentrations did not exceed 10 mg/L for both configurations. Variability in acetic acid presence can indicate changes in substrates metabolism and possible homoacetogenesis conducted by autotrophic acetogenic microorganisms (Eq. 3). The fed substrates could be at some degree utilized to produce acetate instead of CH4 27. Consequently, alkalinity reaches its lowest level at the highest acetic acid presence and the lowest CH4 production.
2CO2 + 4H2 → CH3COOH + 2H2O
The free-energy (∆G0’) of hydrogenotrophic methanogenesis is larger (-135.0) than homoacetogenesis (-104.0), therefore, homoacetogenesis is considered thermodynamically unfavorable. However, homoacetogens have been shown to outcompete hydrogenotrophic methanogens at lower temperatures and also when methanogenic activity is inhibited 27,28. Their presence was previously reported at different mesophilic, thermophilic and hyperthermophilic conditions such as AD or dark fermentation 29. Therefore, potentially under unstable CH4 production due to reactor overloading, homoacetogens found a niche for their activity leading to higher acetic acid presence in the liquid phase of concurrent TBR.
The increase in liquid level was also observed at the concurrent configuration, however, only after gas loadings of 15 and 20 m3/m3/d (days 36-45) where ca. 50 mL of excess liquid phase was removed