## 3 Results and Discussion
### Evolution of Microbial Metabolites after Initial BAC Exposure
In the microcosms prepared with 0 mg/L BAC, the formation and persistence of additional fatty acids, aside from acetic acid and formic acid, were not observed in the NMR spectra throughout the study (see Figure 6), even though a low-level of BAC was observed throughout due to previous contamination. For these microcosms, the initial composition of CH\({}_{4}\) in the headspace was \(\sim\)65 %, which increases to \(\sim\)70% after 5 days (see Figure 8). This level remains constant for the duration of the study. The combined CO\({}_{2}\) and CH\({}_{4}\) in the headspace also increase significantly in these microcosms over the duration of the study (see Figure 9). The gradual increase in CH\({}_{4}\) and CO\({}_{2}\) in the microcosm headspace, and the higher proportion of CH\({}_{4}\) after initial construction, is likely due to the establishment of equilibrium after the microbial community was disturbed during the construction of the microcosms, resulting in possible exposure to oxygen [31]. The inclusion of 16.7 mg/L BAC does not affect the CH\({}_{4}\) and CO\({}_{2}\) in the microcosm headspace, with the levels of these gases in the 0 mg/L and 16.7 mg/L BAC microcosms being consistent throughout the experiment (see Figures 8 and 9). Nevertheless, the fatty acid profile of these two microcosms treatments do vary from each other (see Figure 6). While the formic acid levels in the 0 and 16.7 mg/L BAC microcosms are similar, the acetic acid levels in the 16.7 mg/L treatments increase after 5 days by levels that are 100 times those observed in the 0 mg/L BAC microcosms. Additionally, isovalerate and isobutryate are observed in the microcosms with 16.7 mg/L BAC but not in the 0 mg/L control. This change indicates that fatty acid metabolism is being affected by the presence of increased levels of BAC even though the amount and quality of the biogas production remains unchanged. As the measurement of biogas composition does not show a significant difference between the 0 and 16.7 mg/L BAC microcosms, this suggests that the NMR measurements are able to detect and diagnose metabolic stress due to BAC exposure, as well as detecting BAC itself, before the biogas production is affected. Significant deviations from optimal conditions are observed for the microcosms with 33.3 and 66.7 mg/L BAC. In the 33.3 mg/L microcosm, the total acetic acid concentration is now two times that of the 16.7 mg/L microcosm, while propionic acid, butyric acid, valeric acid, isobutyric acid, and isovalerate are also measured at increased levels. Obligate H\({}_{2}\)-producing acetogenic bacteria are responsible for oxidizing VFAs to acetate, CO\({}_{2}\), and H\({}_{2}\)[32; 33]. The buildup of VFA concentrations indicates that these syntrophic acetogenic bacteria are unable to facilitate further degradation in order to form acetate and hydrogen [3; 34; 35; 36]. In the microcosms with 66.7 mg/L BAC, the fatty acid concentrations are lower than in the microcosms with 33.3 mg/L BAC, with only acetic acid and formic acid concentrations. Figure 9: The change in combined CH\({}_{4}\) and CO\({}_{2}\) in the microcosm headspace after exposure to BAC relative to the 0 mg/L microcosm on day 2.  
acid being observed at significant concentrations. Succinic acid is also observed in the 66.7 mg/L BAC microcosms indicating a significant disturbance in the metabolism of the microbial communities. Succinic acid is a key intermediate in the citric acid cycle and an integral part of the electron transport chain, from which oxidative phosphorylation produces ATP [37,38]. The connection between succinic acid metabolism and ATP synthase indicates that the higher BAC concentration directly inhibits regular cellular activity [37,38]. Amino acids are measured in the 33.3 mg/L BAC microcosms up until 10 days, after which these metabolites are no longer observed (see Figure 7). In the 66.7 mg/L BAC microcosms, the concentration of amino acids increases over the course of the experiment.