*3.2. Xylose-Rich Hydrolysates from Rice Straw and Wheat Bran*

In order to produce xylose-rich hydrolysates, acidic pre-treatments of wheat bran and rice straw were investigated. In the case of wheat bran, two-step sulfuric acid hydrolyses, suggested in our previous study [17], were performed, resulting in two types of xylose-rich hydrolysates (Table 1). In the case of rice straw, a phosphoric acid hydrolysis, proposed by Jampatesh et al. [35] as an optimal pre-treatment for succinate production and with high hemicellulose saccharification yield, was used. The effects of the particle size on the efficiency of phosphoric acid pre-treatment were evaluated by using ground (average particle size of 1.42 mm) and fine ground (average particle size of 0.67 mm) rice straw samples (Table 1). In addition, sulfuric acid pre-treatments of ground and fine ground rice straws under mild conditions were also examined (Table 1). In order to predict the applicability of those hydrolysates in xylitol fermentation experiments, the sugar compositions, amount of inhibitory compounds and protein content were evaluated. The sugar composition of the wheat bran hydrolysates were quite similar to that obtained previously [17] (Table 4); however, a higher amount of acetic acid (0.7–1 g/L (Table 4) compared to 0.3 g/L [17]) was observed in this study. Interestingly, only phenolic substances were observed as inhibitory compounds beside acetic acid. Furfural, HMF and formic acid were not detected in the hydrolysates (Table 4). In addition, wheat bran hydrolysates contained relatively high amount of solubilized proteins (7 g/L), which is advantageous in terms of their fermentability. WB1/S contained a slightly higher amount of xylose (22.6 g/L) compared to WB2/S (21.1 g/L) (Table 4); hence, WB1/S was selected for xylitol fermentation experiments. In the case of rice straw, sulfuric acid and phosphoric acid treatments by using ground and fine ground raw material were investigated, resulting in four different types of xylose-rich hydrolysate (Table 1). In the case of using phosphoric acid, glucose concentration of the hydrolysates was not quantified, because of the overlapping peaks of phosphoric acid and glucose in our HPLC analyses. The phosphoric acid treatment of ground and fine ground rice straws yielded relatively low xylose concentrations of 11.1 g/L and 11.0 g/L, respectively. These concentrations correspond to the xylose yields of 69% and 68%, respectively. The total concentration of furfural, formic acid, HMF and acetic acid was around 3 g/L in both cases, which is similar to that obtained by Jampatesh et al. [35] under the same conditions. Besides those inhibitors, GRS/P and FGRS/P also contained considerable amounts of phenolic substances, resulting in total inhibitor concentrations of 4.5 g/L and 4.2 g/L, respectively (Table 4). Comparing the xylose and different inhibitor concentrations of GRS/P and FGRS/P at 0.05 significance level, significant differences were not observed. Thus, it could be concluded that particle size of rice straw does not have a significant effect on the performance of the phosphoric acid hydrolysis within the investigated range. Sulfuric acid treatment of ground and fine ground rice straws resulted in 20.6 g/L and 19.9 g/L xylose, respectively. These values correspond to the xylose yields of 94% and 91%, respectively, which are considerably higher than the xylose yields achieved during the phosphoric acid treatment. That could be partly because of an incomplete degradation of the solubilized xylan part in the case of the phosphoric acid treatment. Thin-layer chromatography analysis showed sharper spots for the xylo-oligosaccharides (DP 2–6) when phosphoric acid treatment was performed, suggesting an incomplete hydrolysis of xylan (Figure S2). When the severity factors of the two treatments were compared, a value of 1.3 was obtained for both treatments, suggesting that sulfuric acid is more efficient in decomposing hemicellulose completely. The total inhibitor concentrations of GRS/P and FGRS/P were 4.5 g/L and 4.2 g/L, respectively (Table 4). Significant differences were not observed when the xylose and different inhibitor concentrations of GRS/S and FGRS/S were compared at 0.05 significance level. Thus, particle size had no significant effect on the quality of the sulfuric acid hydrolysates within the investigated range. The protein content of both the sulfuric acid- and phosphoric acid-catalyzed hydrolysates was very low (below 1 g/L) (Table 4). The hydrolysates from wheat bran seemed to be a better medium for xylitol production due to their higher xylose but lower inhibitor content, compared to the hydrolysates from rice straw. However, in terms of the concentration of other sugars, rice

straw hydrolysates were more advantageous. They contained much less arabinose beside the xylose, which can enhance the purity of the fermented broth. GRS/P contained quite low xylose concentration, which would probably result in decreased xylitol production, thus only GRS/S was tested in xylitol fermentation beside WB1/S.


**Table 4.** Composition of different lignocellulosic hydrolysates.

n.m.: not measured; n.d.: not detected; Standard deviations are calculated from triplicates and presented in parentheses.
