## 3 Results
### PHA Identification and Characterization
#### 3.7.4 NMR and ESI-MS Results  
The \({}^{1}\)H-NMR of PHAs produced from the samples, PS0, PS1, and PS2, as an additional carbon source in TSB media are shown in Figure 9.  
The \({}^{1}\)H-NMR spectra of PSx-PHA revealed the presence of characteristic signals corresponding to the protons of 3-hydroxybutyrate (3-HB) and 3-hydroxyvalerate (3-HV) repeating units. From the integration of the signals of methyl groups derived from 3-HB (1.28 ppm) and 3-HV (0.9 ppm), the chemical compositions of PHA obtained were calculated and are presented in Table 6. Unfortunately, on the NMR spectra, there are broad signals at 1.5, 1.8, and 7.1 ppm showing likely \begin{table}
\begin{tabular}{c c c c} \hline
**Sample Name** & \(\mathbf{T_{max}}\) (\({}^{\circ}\)C) & **Residue after First Step (\%)** & **Residue after Second Step (\%) (Inorganic Part)** \\ \hline PHA from PS0 & 295/435 & 51.8 & 2.3 \\ PHA from PS1 & 294/435 & 27.0 & 1.5 \\ PHA from PS2 & 289/435 & 10.0 & 1.7 \\ \hline \end{tabular}
\end{table} 
Table 5: The thermogravimetric parameters for the PHA samples produced after 48 h fermentation with PS0–2 as an additional carbon source in TSB media. Figure 9: \({}^{1}\)H-NMR of PHAs produced from (**a**) PS0; (**b**) PS1; and (**c**) PS2 as an additional carbon source in TSB media. contamination from PS (see Figure 9c); these results are consistent with the previous TGA data. Moreover, the \({}^{1}\)H-NMR results observed for the PHA produced from PS2 samples present a smaller amount of contamination from PS, which is also consistent with the previously reported TGA results. Table 6 also illustrates that sample composition change was not due to thermal degradation, and any differences in HV unit content might be due to the bacterial synthesis. \({}^{13}\)C-NMR was also performed, which corroborated these \({}^{1}\)H-NMR results, but offered no additional information (data not shown). The results from the partial degradation of the PHA low molar mass PHA oligomers (below 2000 Da) needed for ESI-MS/MS analysis were found to possess nearly the same content of HB units as revealed by \({}^{1}\)H-NMR (Table 6). The expanded region of the ESI-MS spectrum of the low molar mass PHA oligomer (resulting from the partial degradation of PHA obtained using PS2 as a carbon source) acquired in the positive ion mode is presented in Figure 10a. The most intense ions of the analyzed cluster were potassium adduct ions at _m/z_ 555, 569, 583, and 597. The expanded spectral range at _m/z_ 500 to 650 indicates the presence of ions with a mass difference between the _m/z_ values equal to 14 Da (i.e., _m/z_ 555, 569, 583, and 597), which corresponds to the difference between the molar mass of the individual co-monomeric units with longer side chains. The general structure of the ions presented in the ESI-MS spectrum is shown in Scheme 1. \begin{table}
\begin{tabular}{c c} \hline \hline \multirow{2}{*}{**Sample Name**} & **Sample Composition** \\ \cline{2-3}  & **Content of Non-HB Units (\%)** \\ \hline PS1-PHA0 & 8.2 \\ PS1-PHAD & 8.3 \\ PS2-PHA0 & 12.1 \\ PS2-PHAD & 11.5 \\ \hline \hline \end{tabular}
\end{table} 
Table 6: The chemical composition of PSx-PHA samples before (0) and after partial depolymerization for ESI-MS/MS analysis (D) (determined from \({}^{1}\)H-NMR analysis). Figure 10: (**a**) The expanded spectral range at _m/z_ 500 to 650 of the ESI-MS spectrum of low molar mass PHA oligomer; (**b**) the ESI-MS/MS spectrum of selected potassium adduct ions of oligomers [HB\({}_{6}\) + K]\({}^{+}\) at _m/z_ 555. The ESI-MS/MS fragmentation experiments were conducted for selected cluster ions. The ESI-MS/MS product ions spectrum for the selected potassium adduct of the oligomer, [HB\({}_{6}\) + K]\({}^{+}\), at _m_/_z_ 555 is presented in Figure 10b. The fragmentation of this ion, which was obtained from the random breakage of ester bonds along both sides of the oligomer chain, led to the formation of only one set of oligo(3-hydroxybutyrate) product ions at _m_/_z_ 469 and 383, terminated by crotonate and carboxyl end groups.