# Exploring the Bioethanol Production Potential of _Miscanthus_ cultivars
## 2 Materials and Methods
### \(\alpha\)-Cellulose and Hemicellulose Analysis  
First, 1 g of _Miscanthus_ sample was placed in a 50 mL Ehrlenmeyer flask. Deionised water (80 mL), glacial acetic acid (0.5 mL) and sodium chloride solution (10 mL, 15% \(w/v\)) were added. A small conical flask was inverted in the neck of the Erhlenmeyer flasks to contain the liberated chlorine gas. The flasks were then placed in a water bath set at 75 \({}^{\circ}\)C. The temperature was maintained for one hour, and the flasks were swirled gently periodically. After one hour, glacial acetic acid (0.5 mL) and sodium chlorite (10 mL) were added; this step was repeated twice subsequently. The samples were then cooled to below 10 \({}^{\circ}\)C by placing the flask in an ice bath. Once cooled, the suspension was filtrated through  
\begin{table}
\begin{tabular}{l l l l} \hline \hline
**GENOTYPE** & **Samples** & & \\  & **Code** & **Harvest Point** & **Reference** \\ \hline _Miscanthus_ Cultivar A M. \(\times\)_giante_s & BE01 & Early season * & \\ _Miscanthus_ Cultivar A M. \(\times\)_sigameteus_ & BE07 & Late season * & \\ _Miscanthus_ Cultivar B M. \(\times\)_sigameteus_ & BE04 & Early season * & \\ _Miscanthus_ Cultivar B M. \(\times\)_sigameteus_ & BE10 & Late season * & \\ _Miscanthus_ Cultivar C (CNT3) & BE13 & Early season * & [15] \\ _Miscanthus_ Cultivar C (CNT3) & BE16 & Late season * & [15] \\ _Miscanthus_ Cultivar C (CNT3) & Various harvest points ** & \\ _Miscanthus_ Cultivar D (CNT5) & BE19 & Early season * & [16] \\ _Miscanthus_ Cultivar D (CNT5) & BE22 & Late season * & [16] \\ _Miscanthus_ Cultivar D (CNT5) & Various harvest points ** & \\ _Miscanthus_ Cultivar E & Various harvest points ** & \\ _Miscanthus_ Cultivar F & Various harvest points ** & \\ _Miscanthus_ Cultivar G (GNT14) & BE25 & Early season * & [14] \\ _Miscanthus_ Cultivar G (GNT14) & Various harvest points ** & \\ \hline \hline \end{tabular}  
* Harvested in the 2016–2017 season; ** Harvested in the 2018–2019 season, specifically on 1 November 2018, 20 November 2018, 14 December 2018, 16 January 2019, 5 February 2019 and 7 February 2019.  
\end{table}
Table 1: _Miscanthus_ cultivars analysed in this study.  
a pre-weighed sintered crucible and washed with ethanol (50 mL), ice water (50 mL) and, finally, acetone (50 mL) before being placed in an oven at 50 \({}^{\circ}\)C to dry overnight. The sinter crucibles were reweighed to determine the quantity of holocellulose in each sample.  
The holocellulose samples from the previous reaction (approximately 0.7 g) were placed in a 250 mL Ehrlenmeyer flask. Sodium hydroxide solution (20 mL, 17.5% \(w/v\)) was added to the flask, which was then sealed with parafilm. The sample was incubated for 2 h at room temperature with occasional agitation. The solids were vacuum-filtered through a pre-weighed sintered crucible and washed consecutively with deionised water (50 mL), methanol (50 mL) and acetone (50 mL). The sinter crucibles were dried overnight at 100 \({}^{\circ}\)C and reweighed to determine the quantity of \(\alpha\)-cellulose.  
The hemicellulose content was calculated by subtracting the \(\alpha\)-cellulose weight from the holocellulose weight.