## 3 Results and Discussion  
### Raw BSG Characterization and Pretreatment  
The raw BSG used presented a moisture of ca. 72% (\(w/w\)) that, after drying, decreased to 8.6 \(\pm\) 0.4% (\(w/w\)). The dried BSG was composed of ca. 46% (\(w/w\)) carbohydrates, ca. 27% (\(w/w\)) proteins, ca. 12% (\(w/w\)) lipids and ca. 3% (\(w/w\)) inorganic salts content (Table 1). The composition of the raw BSG used in this study is in the range of values reported for other BSG samples described in the literature (Table 1). The observed minor differences are probably related to the BSG source, the brewery's operating conditions and ingredients used for brewing. The dried raw BSG was mostly constituted by particles with an average size above 0.250 mm (ca. 94% \(w/w\)), with the largest fraction higher than 0.710 mm (ca. 55% \(w/w\)) (Figure 2). A grinding step was applied to the dried BSG aiming at reducing the particle size, thus improving the particle surface area per unit volume. This facilitates the contact of the acid with the particles and boosts the rate of acidic hydrolysis [11]. Results show that the BSG average particle-size was clearly reduced, with around 80% (\(w/w\)) below 0.5 mm (Figure 2). The ground BSG was then submitted to two sequential acid hydrolysis steps to extract simple sugars from BSG's carbohydrate fraction. After the first hydrolysis, a second step was performed to the remaining solid residue to increase the total amount of sugars extracted from BSG. The extract resulting from the first hydrolysis had a sugars concentration of 32.4 \(\pm\) 2.6 g L\({}^{-1}\), while the second hydrolysis had a lower concentration, 16.4 \(\pm\) 2.9 g L\({}^{-1}\) (Table 2). Considering the dried BSG carbohydrates content (46.2 \(\pm\) 0.06% \(w/w\)), in the first hydrolysis 56.0% of the sugars were extracted, whereas in the second hydrolysis 64.6% were extracted (Table 2). The global efficiency of the sequential hydrolysis was 84.5%. [PERSON] and [PERSON] [22] who studied acid and alkaline pretreatment of lignocellulosic biomass reported an efficiency of extraction of reducing sugars of 49, 45 and 40% for rice waste, corn cobs and barley straw, respectively, after acid hydrolysis under 3% H\({}_{2}\)SO\({}_{4}\) (\(v/v\)) and 121 \({}^{\circ}\)C, for 30 min. These values are lower than those obtained in our study for the first hydrolysis step of BSG. \begin{table}
\begin{tabular}{l c c c c} \hline \hline
**Components** & \multirow{2}{*}{**This Study**} & **[PERSON] et al.** & **[PERSON] et al.** & **[PERSON] et al.** \\ (\%, \(w/w\))** & & **[**20**]** & **[**21**]** & **[**18**]** \\ \hline
**Moisture** & 72.2 \(\pm\) 0.1 & 80 & n.a. & n.a. \\
**Ash**\({}^{\text{a}}\) & 3.40 \(\pm\) 0.04 & 1.2 & 2.2 & 2.4 \\
**Protein**\({}^{\text{a}}\) & 26.9 \(\pm\) 0.1 & 24.6 & 39.1 & 24.0 \\
**Lipids**\({}^{\text{a}}\) & 11.5 \(\pm\) 0.03 & n.a. & 10.5 & 10.6 \\
**Carbohydrates**\({}^{\text{a}}\) & 46.2 \(\pm\) 0.06 \({}^{\text{b}}\) & 51.5 & 36.2 & 47.2 \\
**Lignin**\({}^{\text{a}}\) & 12 \({}^{\text{b}}\) & 21.7 & 25.6 & 11.9 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Chemical composition of the BSG used in this study and comparison with literature data. Figure 2: BSG-particle size distribution before the grinding process and after the grinding process. The first and the second hydrolyses were mixed and the resulting hydrolysate was characterized before neutralization and after neutralization (Table 3). The hydrolysate after neutralization was used as the feed for following fermentation experiments. The main sugar in the hydrolysate was xylose (12.4 \(\pm\) 2.8 g L\({}^{-1}\)) followed by arabinose (6.53 \(\pm\) 0.60 g L\({}^{-1}\)) and glucose (3.47 \(\pm\) 0.19 g L\({}^{-1}\)) (Table 3), which is in agreement with the reported values for BSG sugar composition [20; 23]. Indeed, the xylose concentration is about 55.5% of the total extracted sugars and the sugars extracted account for 41.9% (gCOD gCOD\({}^{-1}\)) of the total COD in the combined hydrolyses after neutralization.