*3.3. Xylitol Fermentation on Xylose-Rich Hydrolysates of Rice Straw and Wheat Bran*

In Section 3.1, the effects of OTR and initial xylose concentration on the xylitol production of *Candida boidinii* were investigated. The applicability and adequacy of the models for maximum xylitol yield (Equation (2)), maximum xylitol volumetric productivity (Equation (3)), and xylitol yield after 24 h (Equation (4)), developed by using semi-defined medium, were also tested in the case of using xylose-rich hydrolysates derived from rice straw (GRS/S) and wheat bran (WB1/S). Xylitol fermentation experiments on the xyloserich hydrolysates were performed under the aeration condition that provided 2.1 mmol O2/(L × h) OTR value in the case of semi-defined medium. However, these conditions resulted in 1.6 mmol O2/(L × h) OTR when WB1/S was used, probably due to the different chemical composition. Xylitol fermentation on WB1/S showed similar profile than that of the fermentation obtained by using semi-defined medium (30 g/L initial xylose, 2.1 mmol O2/(L × h) OTR) (Figures 2A and 3A); however, both the initial xylose concentration (22.1 g/L) and the OTR (1.6 mmol O2/(L × h)) were a bit lower in the case of WB1/S. Maximum xylitol concentration (14.2 g/L) was obtained at 24 h, resulting in the maximum xylitol yield of 60% (spec. xylitol yield: 0.72 g/g) (Figure 3A). The maximum xylitol productivity was also reached after 24 h, and it was 0.58 g/(L × h). Predictions for the maximum xylitol yield, xylitol yield after 24 h, and maximum xylitol volumetric productivity were calculated by the previously fitted models (Equations (2)–(4)) using 22.1 g/L initial xylose concentration and 1.6 mmol O2/(L × h) OTR value as input parameters. The maximum xylitol yield was achieved also in 24 h, thus it was equal to the xylitol yield after 24 h. Due to the fact that different models were fitted to the maximum and 24-h xylitol yields, the models provided slightly different predictions for those response variables but with overlapping predicted intervals. The models predicted 61% maximum xylitol yield with a prediction interval of 58–65%, and 57% xylitol yield after 24 h with a prediction interval of 52–61%. Thus, the experimentally measured xylitol yield (60%) fitted in with the predictions of both models. Maximum xylitol productivity was estimated to be 0.49 g/(L × h) by the model with a prediction interval of 0.35–0.63 g/(L × h), showing a good agreement with the experimentally obtained value (0.58 g/(L × h)). Therefore, the models developed by using semi-defined medium were found appropriate to provide adequate predictions for xylitol fermentations on WB1/S, indicating that wheat bran hydrolysate is a suitable raw material to produce xylitol by using *Candida boidinii* NCAIM Y.01308. Ethanol production was observed in both cases of using a semi-defined medium or WB1/S. The ethanol concentration reached its maximum in 48 h, and then, ethanol was totally

consumed by the end of the fermentation (Figures 2A and 3A). The growth of the cells was also similar in both cases. The cell concentrations increased by about 1 g/L during the fermentations. The profiles of xylose decrease were also similar, except that a small amount of xylose seemed to remain in wheat bran hydrolysate (Figure 3A). It could be explained by the presence of small amount of galactose in the wheat bran hydrolysate [36], which could be detected as xylose due to the overlapping peaks in the HPLC analysis used in this study. Residual galactose during xylitol fermentation on corn fiber hydrolysate by *Candida boidinii* was observed in our previous study [39]. Xylitol fermentations were also performed on GRS/S under the same aeration condition, resulting in similar OTR value (2.1 mmol O2/(L × h)) to that measured in the semi-defined medium. The profile of the xylitol fermentation on GRS/S (Figure 3B) was different from that observed on the semi-defined medium or WB1/S. Although the initial xylose concentration was similar to that in WB1/S, the achieved maximum xylitol yield was significantly lower (20%, spec. xylitol yield: 0.26 g/g), and it was reached latter (72 h). The remaining xylose concentration (3.4 g/L) at this point was similar to that observed in the cases of a semi-defined medium and WB1/S (Figures 2A and 3A,B). However, it could contain small amount of galactose as well, which was not analyzed in this study. The xylitol yield after 24 h (19%) was similar to the maximum xylitol yield (20%), but at 24 h, the remaining xylose concentration was much higher (8 g/L). This is almost half of the initial xylose concentration and nearly twice of the xylose concentration measured in semi-defined medium and WB1/S after 24 h of fermentation (Figures 2A and 3A,B). The maximum xylitol volumetric productivity obtained in 24 h was also very low (0.14 g/(L × h)) compared to the previous fermentations. In the case of GRS/S, a higher amount of ethanol was produced (8.4 g/L after 72 h) compared to the previous cases. Moreover, ethanol production exceeded xylitol production (Figure 3B).

**Figure 3.** Profiles of the xylitol fermentations on WB1/S (**A**) GRS/S (**B**) GRS/S supplemented with 2 g/L peptone (**C**) and GRS/S treated by activated carbon and supplemented with 2 g/L peptone (**D**) using *Candida boidinii* NCAIM Y.01308. Standard deviations are calculated from duplicates.

Since GRS/S hydrolysate had a low protein content (Table 4), supplementations with 2 g/L of inorganic (ammonium sulphate) or organic (peptone) nitrogen sources were investigated with the aim of enhancing xylitol production. In the case of supplementation with ammonium-sulphate, the maximum xylitol yield was 15% (spec. xylitol yield: 0.22 g/g

at this point), and it was achieved after 48 h (data not shown). The xylitol yield after 24 h and the maximum xylitol productivity were 10% and 0.08 g/(L × h), respectively (data not shown). All of these results are lower than that achieved without supplementation. In contrast, the addition of peptone resulted in a small increase in maximum xylitol yield (25%, (spec. xylitol yield: 0.30 g/g at this point)), xylitol yield after 24 h (15%) and maximum xylitol volumetric productivity (0.15 g/(L × h)) (Figure 3C) as well. Hence, xylitol fermentation was slightly enhanced by adding peptone; however, ethanol concentration was also considerably increased. The ethanol concentration was 10 g/L at 48 h beside only 5 g/L of xylitol (Figure 3C).

To improve xylitol fermentation on GRS/S, activated carbon treatment was tested in addition to the supplementation with peptone. After the activated carbon treatment, detectable amount of phenols and HMF were not present in GRS/S. On the other hand, the amount of organic acids (0.9 g/L formic acid and 2.4 g/L acetic acid) were not reduced significantly compared to the GRS/S without detoxification (1.1 g/L formic acid and 2.4 g/L acetic acid). Moreover, the activated carbon treatment caused significant xylose loss, resulting in 15.4 g/L initial xylose concentration. After the detoxification step, GRS/S was also supplemented by 2 g/L peptone. Due to the activated carbon treatment, the maximum xylitol yield increased to 30% (spec. xylitol yield: 0.33 g/g), and it was reached after 48 h. The xylitol yield after 24 h and the maximum xylitol productivity were 29% and 0.19 g/(L × h), respectively. All of these results are lower compared to those achieved on WB1/S or a semi-defined medium under similar aeration conditions. The ethanol production was reduced due to the detoxification step, resulting in only 4.7 g/L ethanol concentration at the point of the maximum xylitol yield. This was a half of the ethanol produced in the hydrolysate without detoxification. The maximum specific xylitol yields were reached after 24 h in all fermentations on lignocellulosic hydrolysates. The specific xylitol yields were 0.35 g/g, 0.24 g/g, 0.35 g/g, and 0.38 g/g in the cases of GRS/S, GRS/S supplemented with ammonium-sulphate, GRS/S supplemented with peptone, GRS/S clarified by activated carbon and supplemented with peptone, respectively. The cell concentrations increased by 1–1.8 g/L during the fermentations, except in the cases of GRS/S supplemented with peptone and detoxified GRS/S with peptone supplementation, where the cell mass did not change. Small amount of glucose (3.5–5 g/L) was also present in the rice straw hydrolysates, which was totally consumed after 24 h (Figure 3B–D).
