## 3 Results and Discussion
### Evolution of Microbial Metabolites after Initial BAC Exposure  
Once signals were identified in the \({}^{1}\)H NMR spectra, CHENOMX was used to quantify selected fatty acids and amino acids by spectral deconvolution and comparison to an internal concentration standard, DSS, included in each sample at a known concentration. Figure 6 compares the progression of fatty acid concentrations for each microcosm treatment \begin{table}
\begin{tabular}{c c c c c c c} \hline
**ID** & **Compound** & **ID** & **Compound** & **ID** & **Compound** & **ID** & **Compound** \\ \hline  & _Fatty Acids_ & & Amino Acids & & Alcohols & & _Auxins_ \\
1 & Formic Acid & 10 & Glycine & 20 & Ethanol & 28 & Indole-3-Acetate \\
2 & Acetic Acid & 11 & Alanine & 21 & Ethylene Glycol & & _Disinfecting Agents_ \\
3 & Propionic Acid & 12 & Threonine & 22 & Propylene Glycol & 29 & Benzalkonium Chloride \\
4 & Butyric Acid & 13 & Glutamine & & _Amines_ & & \\
5 & Isobutryic Acid & 14 & Methionine & 23 & Methyl Amine & \\
6 & Valeric Acid & 15 & Valine & _Amides_ & & \\
7 & Isovaleric Acid & 16 & Isoleucine & 24 & Acetamide & \\
8 & Succinic Acid & 17 & Tyrosine & & _Pyrimidines_ & \\ _Fatty Acid Metabolites_ & 18 & Phenyalalanine & 25 & Uracil & \\
9 & Phenylacetate & 19 & Tryptophan & & & \\ \hline \end{tabular}
\end{table} 
Table 1: Metabolites identified in microcosm studies using \({}^{1}\)H NMR spectroscopy. Figure 5: \({}^{1}\)H-\({}^{1}\)H TOSY NMR spectra of the functionalized alkyl and alkyl regions of microcosm samples after 18 days of BAC exposure. (**a**) 0 mg/L BAC, (**b**) 16.7 mg/L BAC, (**c**) 33.3 mg/L, (**d**) 66.7 mg/L BAC. Colors denote different classes of compounds; BAC (red), linear fatty acids (green); branched chain fatty acids (blue); amino acids (purple), alcohols (orange). over time. In the control microcosm, only acetic acid and formic acid were observed. The concentrations of these two VFAs were constant throughout, apart from the measurements on day 18 at which acetic acid increased and formic acid decreased. The observation of these two VFAs is consistent with a normal anaerobic digestion process. In the BAC treatment microcosms, the concentrations of most identified VFAs, including both linear and branched VFAs, were observed to increase over the course of the study, indicating disruption of the anaerobic digestion. Figure 7 compares the progression of amino acid concentrations over time for the 33.33 mg/L and 66.7 mg/L BAC microcosms. Amino acid signals were not detected in the 0 or 16.7 mg/L microcosm treatments and as such these treatments are excluded from Figure 7. Here, the measured concentrations of amino acids are observed to decrease to non-detect in the 33.3 mg/L BAC treatment, while the amino acid concentrations are observed to increase over the study period in the 66.7 mg/L BAC treatment. Figure 6: Changes in fatty acid concentrations over time after exposure to BAC as measured using \({}^{1}\)H NMR spectroscopy for (**a**) 0 mg/L BAC, (**b**) 16.7 mg/L BAC, (**c**) 33.3 mg/L BAC, and (**d**) 66.7 mg/L BAC. Measurements of CO\({}_{2}\) and CH\({}_{4}\) in the microcosm headspace were performed by GC-FID immediately before NMR measurements were performed. Figure 8 compares the % contribution of CH\({}_{4}\) to the biogas for each microcosm treatment over the duration of the experiment starting on day 2 as a measure of the relative proportion of CH\({}_{4}\) compared to CO\({}_{2}\). Figure 9 compares the change in the combined CO\({}_{2}\) and CH\({}_{4}\) biogas relative to the day 2, 0 mg/L BAC control microcosm as a measure of total gas production in response to the different BAC treatments. Figure 8: Percent of CH\({}_{4}\) in the microcosm headspace relative to the combined CH\({}_{4}\) and CO\({}_{2}\) after exposure to BAC. Figure 7: Changes in selected amino acid concentrations in the microcosms exposed to BAC as measured using \({}^{1}\)H NMR spectroscopy for (**a**) 33.3 mg/L BAC and (**b**) 66.7 mg/L BAC.