## 3 Results and Discussion
### HSQC-NMR Determination  
The determination of C-H bonds in the lignin structure was conducted using HSQC-NMR for qualitative and semi-quantitative analysis of the fundamental structural units and primary connecting bonds of lignin. Following a method described in the literature, the guaiacyl, syringyl, p-hydroxyphenyl structural units, and major linkage bonds such as aryl ether bonds in lignin were analysed semi-quantitatively [32]. The HSQC-NMR spectra of lignin encompassed the benzene ring region (100-150/6.0-8.0 ppm) and the side chain region (50-90/2.5-6.0 ppm) (Figure 3). Within the side chain region, the signal peaks corresponding to the C\(\alpha\)-H\(\alpha\) and C\(\beta\)-H\(\beta\) bonds in the aryl ether bond (\(\beta\)-O-4) were observed at 71.7/4.83 ppm and 86.3/4.04 ppm, respectively. The signal peaks attributed to the resin (\(\beta\)-\(\beta\)) were detected at 84.9/4.65 ppm and 53.5/3.06 ppm for the C\(\alpha\)-H\(\alpha\) and C\(\beta\)-H\(\beta\) bonds, respectively. Additionally, the signal peaks associated with \(\beta\)-5 were observed at 86.9/5.46 ppm for the C\(\alpha\)-H\(\alpha\) bond and 53.8/3.51 ppm for the C\(\beta\)-H\(\beta\) bond. Lastly, the signal corresponding to methoxy was detected at 55.5/3.7 ppm [26]. The findings of the semi-quantitative analysis are shown in Table 3. The aryl ether bond, being the primary linkage bond in lignin, experienced a slight increase in the number of bonds after ozone pretreatment of MWL. In the region of the benzene ring, the syringyl unit (S) exhibited a signal at 104.5/6.67 ppm, while the oxidized syringyl unit (S\({}^{\prime}\)) displayed a signal at 101.4/7.22 ppm. The signals at 111.0/6.98 ppm, 115.6/6.71 ppm, and 119.5/6.78 ppm were assigned to the C2-H2, C5-H5, and C6-H6 bonds, respectively, in the guaiacyl units (G2, G5, G6). The C2,6-H2,6 in p-hydroxyphenyl units (H) were characterized by signal peaks at 128.0/7.18 ppm [32; 33]. Additionally, the signals corresponding to p-coumaric acid (p-CA) C2/6-H2/6, C\(\alpha\)-H\(\alpha\), and C\(\beta\)-H\(\beta\) were detected at 130.6/7.48 ppm, 145.1/7.45 ppm, and 114.2/6.28 ppm, respectively. Similarly, the signals for ferulate (FA) C\(\alpha\)-H\(\alpha\) and C\(\beta\)-H\(\beta\) were observed at 145.2/7.26 ppm and 114.2/6.14 ppm, respectively [34]. Following ozone pretreatment, the relative content of S-type structural units was lower than that in MWL, while the relative content of other structural units increased slightly. On one hand, the observed phenomenon can be attributed to the elimination of methoxy groups from S-type phenyl propane and the subsequent formation of G-type or H-type phenyl propane as a consequence of ozone pretreatment. On the other hand, it can be attributed to the preferential degradation of S-type structural units by ozone molecules [35; 36], resulting in a reduction in the S/G ratio of lignin from 3.1 (MWL) to 3.0 (OL60) and 2.7 (OL120). The S-type structural unit contains two methoxyl groups, and a decrease in the S/G ratio often signifies a decrease in the hydrophobic nature of the lignin [31]. Furthermore, p-CA is primarily linked to lignin or carbohydrates via ester bonds, while FA is bound to lignin through ether bonds and serves as a connecting agent between lignin and carbohydrates through ester bonds. The application of ozone pretreatment resulted in an augmentation of p-CA and FA levels within the MWL. Additionally, the augmentation of hydrophilic groups, such as coumarate and ferulate, is advantageous for reducing the hydrophobicity of lignin. \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Lignin Structure** & \multicolumn{3}{c}{**Lignin Samples (mmol/g)**} \\ \cline{2-3}  & **MWL** & **OL60** & **OL120** \\ \hline Aliphatic hydroxyl & 2.13 & 1.88 & 1.75 \\
5-substituted-OH & 1.08 & 0.88 & 0.79 \\ G-OH & 0.49 & 0.43 & 0.44 \\ H-OH & 0.69 & 0.66 & 0.65 \\ Total phenol hydroxyl & 2.26 & 1.97 & 1.88 \\ COOH & 0.21 & 0.30 & 0.47 \\ \hline \hline \end{tabular}
\end{table}