## 3 Results and Discussions
### Mass Balance and Chemical Composition of the Untreated and Pretreated Substrate and Effluent
\begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multirow{2}{*}{**Parameter**} & \multicolumn{4}{c}{**Substrate**} & \multicolumn{2}{c}{**Effluent**} \\ \cline{2-6}  & **Untreated** & \multicolumn{2}{c}{**Steam Exploded**} & & \\ \cline{2-6}  & **Solid** & **Solid** & **Liquid** & **Day 0** & **Day 98** \\ \hline TS (g L\({}^{-1}\)) & 194.50 & 187.10 & 8.60 & 28.50 & 20.50 \\ VS (g L\({}^{-1}\)) & 166.70 & 162.10 & 5.40 & 15.80 & 15.70 \\ Cellulose (\%TS) & 32.83 \(\pm\) 0.5 & 28.02 \(\pm\) 0.2 & \(-\) & \(-\) & 5.03 \(\pm\) 0.8 \\ Hemicululose (\%TS) & ND & ND & \(-\) & \(-\) & ND \\ Lignin (\%TS) & 25.22 \(\pm\) 4.6 & 29.15 \(\pm\) 0.5 & \(-\) & \(-\) & 17.0 \(\pm\) 1.0 \\ C/N (\%TS) & 22.12 \(\pm\) 1.1 & 8.24 \(\pm\) 0.9 & 10.82 \(\pm\) 0.2 & 7.32 \(\pm\) 0.1 & 14.3 \(\pm\) 0.8 \\ Carbon (\%TS) & 40.51 \(\pm\) 0.0 & 42.90 \(\pm\) 0.5 & 28.03 \(\pm\) 0.5 & 29.63 \(\pm\) 0.0 & 37.33 \(\pm\) 0.9 \\ Nitrogen (\%TS) & 1.83 \(\pm\) 0.1 & 5.28 \(\pm\) 0.7 & 2.60 \(\pm\) 0.0 & 4.04 \(\pm\) 0.0 & 2.61 \(\pm\) 0.1 \\ Protein (\%TS) & 11.48 \(\pm\) 0.6 & 33.0 \(\pm\) 4.3 & 16.19 \(\pm\) 0.0 & 25.3 \(\pm\) 0.2 & 16.3 \(\pm\) 0.7 \\ NH\({}_{4}\)\({}^{+}\) (g L\({}^{-1}\)) & \(-\) & \(-\) & 0.1 \(\pm\) 0.0 & 1.0 \(\pm\) 0.0 & 0.1 \(\pm\) 0.0 \\ Phenolic compounds (g L\({}^{-1}\)) & \(-\) & \(-\) & 0.03 \(\pm\) 0.0 & 0.07 \(\pm\) 0.0 & 0.18 \(\pm\) 0.0 \\ Dissolvedigenin (g L\({}^{-1}\)) & \(-\) & \(-\) & 0.64 & 0.27 & 0.40 \\ \hline \end{tabular} All the parameters were measured in triplicates to evaluate the error outcome. ND = Not detected.  
\end{table} 
Table 2: Chemical composition of the untreated and pretreated substrate and effluent. of 5.03 \(\pm\) 0.8%TS on day 98 proves that SE helped disrupt the lignocellulosic structure, leading to a higher degradation of cellulose when subjected to anaerobic microorganisms during the AD process. Whereas lignin showed a different trend, the lignin value on day 98 (17 \(\pm\) 1.0%TS) proved that more than half of the lignin was still present inside the reactor, indicating lower lignin degradation. This high amount of lignin present inside the CSTR on the last operational day can be a limitation for a stable biogas yield due to its lower degradability. Such a trend of lower lignin degradation, which led to a lower biogas yield in the reactor, was previously observed [39]. The C/N ratio for the untreated _L. grandiflora_ (solid fraction) was 22.12 \(\pm\) 1.1 (Table 2). However, following the SE pretreatment, it suddenly changed to 8.24 \(\pm\) 0.9 in the solid fraction and 10.82 \(\pm\) 0.2 in the liquid fraction. In AD, carbon acts as the energy source and nitrogen for developing of the cell structure, enhancing microbial growth [40]. The optimal C/N ratio varies depending on the type of feedstock to be digested [41; 42]. Various studies recommend a C/N ratio range of 20/1 to 30/1, with optimal being 25/1 for the proper growth of bacteria during AD. A low C/N ratio can cause ammonia accumulation, whereas a high C/N ratio can reduce the biodegradation rate. To possibly improve the C/N ratio, co-digestion with some easily digestible biomass can be a required method. It can produce a combined effect because of the nutrients contribution or the balance of the carbon-to-nitrogen (C/N) ratio, which can further improve the stability of the AD process [43]. The untreated _L. grandiflora_ used in this study had a C/N ratio (22.12 \(\pm\) 1.1) in the optimum range. The C/N ratio on day 0 in the effluent was recorded as 7.3 \(\pm\) 0.1, and towards the end of the reactor, it increased to 14.3 \(\pm\) 0.8, suggesting an increase in either carbon or nitrogen contents in the reactor. The change in the carbon or nitrogen content will be an essential parameter to monitor the situation of microorganisms during AD. The carbon and nitrogen contents in the untreated _L. grandiflora_ were 40.51 and 1.83 \(\pm\) 0.1%TS. These values changed following the SE pretreatment as the carbon content in the pretreated solids and liquid fraction was 42.90 \(\pm\) 0.5 and 28.03 \(\pm\) 0.5, respectively.