# Chemical and Enzymatic Treatment of Hemp Biomass for Bioethanol Production
## 3 Results and Discussion  
### Chemical Pretreatment of Hemp Biomass  
The alkaline pretreatment is a helpful method of removing lignin from materials with lignocellulosic and it leads to an increase in the accessibility of biomass structure. The main advantages of the alkaline treatment are high efficiency in lignin removal as well as effective removal of acetyl groups and uronic substitutions from hemicellulose. The type of reagent used has a significant effect on the performance of the alkaline pretreatment. Sodium hydroxide is one of the most popular alkaline reagents used in this process. The reagent concentration, reaction time and temperature also affect the biomass delignification [22]. The optimization of alkaline treatment conditions for hemp biomass ground on a knife mill with a mesh size of 2 mm was carried out with 1.5-3% sodium hydroxide. In order to determine the efficiency of the chemical processing of the hemp biomass, the content of reducing sugars was measured by [PERSON]'s method in the enzymatic test. The results allow to conclude that the Tygra variety is susceptible to alkaline treatment at an optimum concentration of 2% NaOH. The content of reducing sugars for 2% NaOH was about 13% higher than for 1.5%. In turn, for 3% sodium hydroxide, the content of reducing sugars was at a similar level. Thus, the choice of time and temperature treatment proved to be effective. [PERSON] et al. [23] showed a similar result and they stated that the sodium hydroxide pretreatment of waste plant biomass resulted in the highest level of delignification at 2% NaOH. Subsequently, determination of the chemical composition of hemp biomass after NaOH treatment was performed and compared to the chemical composition of the biomass before pretreatment (Table 1). The analysis of chemical composition confirmed an increase in cellulose content and partial degradation of hemicellulose. The results obtained during the analysis of the chemical composition of hemp biomass were confirmed by FTIR spectra. The major bands observed in the FTIR spectra of hemp biomass and their assignments to vibrations of chemical group and molecules are summarized in Table 2. FTIR spectroscopy is the most useful tool in providing information about molecular fragments, the presence or absence of specific functional groups. Figure 1 shows the changes in FTIR spectra after alkali treatment of hemp biomass in between 600 cm\({}^{-1}\) and 4000 cm\({}^{-1}\). \begin{table}
\begin{tabular}{c c c c} \hline \hline
**Samples** & **Cellulose (\%)** & **Hemicellulose (\%)** & ** Lignin (\%)** \\ \hline BP & \(50.82\pm 0.12\) & \(27.79\pm 0.33\) & \(14.68\pm 0.46\) \\ AP & \(62.70\pm 0.09\) & \(20.16\pm 0.16\) & \(15.12\pm 0.22\) \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Chemical composition of hemp biomass (% of dry matter); BP—before pretreatment; AP—after pretreatment. \begin{table}
\begin{tabular}{c c c} \hline \hline
**Vibration of Function Group** & **Source** & **Wavenumber (cm\({}^{-1}\))** \\ \hline OH stretching & polysaccharides & 3340 \\ C-H symmetrical stretching & polysaccharides & 2897 \\ C = O unconjugated stretching & hemicellulose & 1733 \\ OH (water) & cellulose & 1636 \\ C = C symmetrical stretching of the aromatic ring & lignin & 1507 \\ CH\({}_{2}\) bending & cellulose & 1422 \\ glycosidic bonds symmetric ring-stretching mode & polysaccharides & 896 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Characteristic absorption bands in infrared (A-amorphous; C-crystalline) according to [PERSON] et al. (2017). Figure 1: FTIR spectra of hemp biomass before and after the chemical treatment. The spectrum shows typical cellulose absorption peaks at 3300 cm\({}^{-1}\), 2900 cm\({}^{-1}\), 1610 cm\({}^{-1}\) and 1420 cm\({}^{-1}\). At the wave number of 1420 cm\({}^{-1}\), stretching vibrations of methylene functional groups (CH2) occur.