## 3 Results and Discussions
### Mass Balance and Chemical Composition of the Untreated and Pretreated Substrate and Effluent
Conversely, the nitrogen content increased to 5.28 \(\pm\) 0.7 in the solid fraction and 2.60%TS in the liquid fraction. A similar trend was observed in previous studies when using bagasse and birch as substrates [44; 45]. The carbon content in the effluent kept increasing until the last operational day 98 (Table 2). The value increased 1.26 times from that on day 0. However, the nitrogen value decreased towards the end of the operation, indicating that undigested carbon was still present in the reactor. A similar protein trend was noted towards the end of the operation (Table 2). More than 50% of the protein was still present in the reactor on day 98, leading to low substrate degradation. The higher carbon and protein amount present inside the reactor indicated that the hydrolysis step was a limiting factor that further affected biogas production [12; 46]. Ammonia is an essential nitrogen source for bacteria, as low ammonia concentrations are valuable to the process [47]. Nitrogenous compounds are commonly proteins converted to ammonia by AD. However, some studies reveal that ammonium inhibition leads to digester upset or failure [48]. The inhibition of the biological process can inhibit methanogenesis when exceeding 10 mM [49]. On day 0, the ammonia value in the effluent was 1.0 g L\({}^{-1}\), which kept decreasing till day 98 (0.1 g L\({}^{-1}\)), indicating no inhibition from ammonia. The degradation of lignin releases multiple phenolic compounds during SE pretreatment, which inhibit AD [15; 50]. The phenolic compounds mainly comprise of vanillin, p-coumaric acid, ferulic acid and syringaldehyde. The phenolic compounds recovered from the pretreated biomass have shown a strong inhibitory effect on the cellulocytic enzyme [29]. A study by [51] shows that the phenolic compounds of 3.5 mg phenolic/mg protein enzyme led to a 20% lower cellulose conversion when sugarcane bagasse was used (liquid hot water pretreatment, SF = 3.83). The phenolic compounds in the SE liquid fraction amounted to 0.03 g L\({}^{-1}\). However, this value changed to 0.07 g L\({}^{-1}\) on day 0 and 0.18 g L\({}^{-1}\) on day 98 in the effluent. The values obtained in previous studies clarify that 3.50 g L\({}^{-1}\) of phenolic compounds can start inhibiting the AD process when wastewater was used as a substrate [52]. However, this study had a much lower value than the inhibiting value proving no inhibition from the phenolic compounds. During the SE pretreatment, the lignin-carbohydrate bonds are broken, and the lignin is partially solubilised, causing the formation of dissolved lignin in the liquid fraction [7]. Dissolved lignin inhibits AD, especially the hydrolysis step, because of its hardly degradable nature [14]. In addition, lignin interferes with the carbohydrate recovery during the pretreatment. Therefore, it can accumulate in the effluent when performing a semi-continuous operation and cause an adverse effect on the AD process [53]. The value of dissolved lignin in the SE substrate was 0.64 g L\({}^{-1}\). However, the value in the effluent on day 0 was 0.27 g L\({}^{-1}\), and it kept increasing until day 98 (0.40 g L\({}^{-1}\)). Therefore, the inhibition of dissolved lignin was not observed because the value was lower than the inhibiting value of >0.50 g L\({}^{-1}\), as reported by [14].