# Chemical and Enzymatic Treatment of Hemp Biomass for Bioethanol Production
## 3 Results and Discussion
### Enzymatic Hydrolysis Process  
The complex with the composition--Flashzyme Plus 200, glucosidase and xylanase was selected for the SHF process (1653 mg/g), and the complex with the composition--Flashzyme Plus 200/Celluclast 1.5 L (70/30%) and xylanase for the SSF process (2311 mg/g) according to the enzyme test (Table 3). The optimization of the enzymatic hydrolysis of hemp biomass as the SHF process was carried out according to the RSM (Figure 3). This method is an effective optimization tool which consists of mathematical and statistical techniques, and it is used for the process of optimization [10; 30]. Figure 2: SEM analysis of hemp biomass (**a**) before pretreatment and (**b**) after alkali pretreatment. Figure 3 presents the interaction effect of variables on the glucose yield. The highest was observed at 50 \({}^{\circ}\)C, and in time process between 48 and 72 h, in turn the dose of enzyme was not less than 20 FPU-g\({}^{-1}\). The higher the temperature and the pH of the enzymatic hydrolysis were, the lower the glucose yield occurred. It was found that in the SHF process optimum enzymatic hydrolysis conditions were obtained for the substrate content of 10%, using the following enzymes: Flashzyme 30 FPU g\({}^{-1}\), glucosidase 20 CBU-g\({}^{-1}\) and xylanase 500 XU g\({}^{-1}\), for the process parameters: 50 \({}^{\circ}\)C, pH 4.2, 48 h. These parameters gave the opportunity to obtain a maximum glucose yield which was \(36.9\pm 0.64\) (g/L). Similar research was conducted by [PERSON] [31], during biomass hydrolysis at 50 \({}^{\circ}\)C and 18 FPU g\({}^{-1}\) he obtained the highest glucose yield. [PERSON] et al. 2017 [32] optimized the enzymatic hydrolysis of lignocellulosic biomass using the RSM method. They used a temperature range of 45-60 \({}^{\circ}\)C and a pH of 4.5-6.0. They obtained the highest content of monosaccharides at 45 \({}^{\circ}\)C and pH 6.0. Next, the fermentation process was carried out with _S. cerevisiae_ and the following process conditions: 37 \({}^{\circ}\)C, pH 4.8, 120 h.  
In turn, the hydrolysis and fermentation process for SSF must be carried out under conditions that ensure optimal synergy of enzymes and distillery yeast. To optimize the SSF process acc. to  
Figure 3: Enzymatic hydrolysis process of hemp biomass (RSM). Response surface representing the interaction effect of temperature, time, dose, pH and substrate content on glucose yield. \begin{table}
\begin{tabular}{c c c} \hline \hline
**Enzymes** & \multicolumn{2}{c}{**Reducing Sugars (mg/g)**} \\ \cline{2-3}  & **55 \({}^{\circ}\)C (SHF)** & **38 \({}^{\circ}\)C (SSF)** \\ \hline Flashzyme Plus 200 & 1100 & 1214 \\ Celluclast 1.5 L & 875 & 909 \\ Flashzyme/Celluclast 1.5 L (30/70) & 1197 & 1306 \\ Flashzyme/Celluclast 1.5 L (50/50) & 1265 & 1295 \\ Flashzyme/Celluclast 1.5 L (70/30) & 1258 & 1315 \\ Flashzyme/glucosidase & 1547 & 1905 \\ Flashzyme/xylanase & 1518 & 1914 \\ Flashzyme/glucosidase/xylanase & 1653 & 2005 \\ Flashzyme/Celluclast 1.5 L (70/30)/glucosidase & 1488 & 2035 \\ Flashzyme/Celluclast 1.5 L (70/30)/xylanase & 1507 & 2311 \\ Flashzyme/Celluclast 1.5 L & 1431 & 2289 \\ \hline \hline \end{tabular}
\end{table} 
Table 3: The content of the reducing sugars after the enzyme test. the RSM, the ranges of process parameters were selected: substrate content 5-7% _w/v_, the dose of Flashzyme/Celluclast 1.5 L enzymes 10-30 FPU-g\({}^{-1}\) using _S. cerevisiae_ yeast at 37 \({}^{\circ}\)C, pH 4.8 and 120 h. Then, the tests were carried out using the selected parameters and the amount of ethanol (HPLC) was determined. The optimal conditions of the SSF process for Tygra hemp biomass were selected. The highest ethanol concentration for SSF was observed at substrate content 5% _w/v_ and the dose of enzyme not lower than 20 FPU-g\({}^{-1}\).