*3.1. Investigating the Effects of OTR and Initial Xylose Concentration on Xylitol Production by Candida boidinii*

In order to investigate the effects of initial xylose concentration and OTR on xylitol production, statistical analysis of the results of the designed experiments was performed (Table 2).

Three dependent parameters of the fermentation were evaluated: the maximum xylitol yield, the maximum xylitol volumetric productivity and the xylitol yield after 24 h (Table 2). Maximum xylitol yields were between 34–58, and the fitted model showed that the maximum xylitol yield was affected by the linear and quadratic terms of initial xylose concentration and by the linear term of OTR (Equation (2)). The maximum xylitol yields were reached at different times depending on the initial xylose concentration (Table 2). In general, they were obtained after 24, 72, and 96 h in the cases of 30, 55, and 80 g/L initial xylose concentrations, respectively, independent of the OTR applied. There was only one exception (1.1 mmol O2/(L × h) of OTR and 30 g/L initial xylose concentration) where the maximum xylitol yield was reached in 48 h. The highest maximum xylitol yield (58%) was obtained in the case of 30 g/L initial xylose concentration with 2.1 mmol O2/(L × h) OTR after 24 h. The fitted surface (Figure 1A) clearly shows that high xylitol yields (58–63%) can be achieved at low initial xylose concentrations (20–30 g/L) and OTR values (1.1–1.5 mmol O2/(L × h)). When 30 and 55 g/L initial xylose concentrations were applied, only small amounts of xylose were remaining at the points of the maximum xylitol yields (Table 2). Maximum xylitol volumetric productivities were obtained after 24 h in all of the cases, and the values varied between 0.49 and 0.93 g/(L × h) (Table 2). The statistical analysis showed that all of the terms (linear and quadratic terms of initial xylose concentration, linear and quadratic terms of OTR, and the interaction between the linear terms of initial xylose concentration and OTR) had a significant effect on the maximum xylitol volumetric productivity (Equation (3)). The fitted model predicted a highest value (0.96 g/L × h) for maximum xylitol volumetric productivity in the case of 71.1 g/L initial xylose concentration and 2.7 mmol O2/(L × h) OTR (Figure 1B). Xylitol yields after 24 h of fermentation were also included in the evaluation in order to investigate the effects of the two independent factors at the time points of the maximum xylitol productivities. The maximum xylitol yield and the xylitol yield after 24 h were equal when the maximum xylitol yield was reached at 24 h. However, this occurred in only two cases (initial xylose concentration 30 g/L and OTR 3.1 or 2.1 mmol O2/(L × h)). All of the terms of the independent factors had significant effect on xylitol yield after 24 h, except the term of the interaction between the linear terms of initial xylose concentration and OTR (Equation (4)). The fitted surface (Figure 1C) clearly shows that the optimal OTR range in terms of the xylitol yield after 24 h is between 2.0–2.5 mmol O2/(L × h). Hence, it is different than the optimal OTR range for the maximum xylitol yield (1.0–1.5 mmol O2/(L × h)). The fitted surface area (Figure 1C) also shows that the lower initial xylose concentration resulted in higher xylitol yield after 24 h which was similar to that experienced in the case of maximum xylitol yields. At the end of the fermentations, xylose was completely consumed in most of the cases, except in the cases of 80 g/L initial xylose concentration, where 30.3, 19.8, and 9.7 g/L xylose remained at OTR 1.1, 2.1 and 3.1 mmol O2/(L × h), respectively (Figure S1). A small amount of xylose (5.3 g/L) also remained in the case of 60 g/L initial xylose concentration at 1.1 mmol O2/(L × h) OTR (Figure S1). The cell concentrations increased by 0.5–1.9 g/L throughout the fermentations, and they showed an increasing tendency by increasing the OTR (Figure S1). At the end of the fermentations, different ethanol concentrations were observed depending on the initial xylose concentration and OTR values applied (Table 2). The ethanol production was increased by increasing the initial xylose concentration and OTR. The highest ethanol concentration was 7.8 g/L at 80 g/L initial xylose concentration and 3.1 mmol O2/(L × h) OTR (Table 2).

$$\text{Maximum xylitel yield} = 65.80 + 6.37 \times \text{OTR} - 2.44 \times \text{OTR}^2 - 0.37 \times \text{I\'{XC}} \tag{2}$$

Maximum xylitol yield = 65.80 + 6.37 ൈ OTR െ 2.44 ൈ OTR<sup>ଶ</sup> െ 0.37 ൈ IXC

$$\text{Maximum xylitel volumetric productivity} = -0.48 + 0.59 \times \text{OTR} - 0.14 \times \text{OTR}^2 + 0.02 \times \text{INC} - 0.0002 \times \text{INC}^2 + 0.002 \times \text{OTR} \times \text{BIC} \tag{5}$$

Xylitol yield after 24 h = 34.04 + 40.84 × OTR − 8.65 × OTR<sup>2</sup> − 1.02 × IXC + 0.005 × IXC<sup>2</sup> (4) Xylitol yield after 24 h = 34.04 + 40.84 ൈ OTR െ 8.65 ൈ OTR<sup>ଶ</sup> െ 1.02 ൈ IXC + 0.005 ൈ IXC<sup>ଶ</sup>

**Figure 1.** The fitted surface areas and Pareto charts of the evaluation of designed xylitol fermentation experiments using *Candia boidinii* NCAIM Y.01308. (**A**): maximum xylitol yield, (**B**): maximum xylitol volumetric productivity, (**C**): xylitol yield after 24 h. (OTR: oxygen transfer rate; IXC: initial xylose concentration).

In order to check the reproducibility of the fermentations and validate the fitted models, experiments were performed under two different conditions. In the first case, fermentations were performed under the conditions where the highest maximum xylitol yield was obtained during the designed experiments (30 g/L initial xylose concentration, 2.1 mmol O2/(L × h) OTR). In the second case, a previously unmeasured point was selected, namely, 71 g/L initial xylose concentration and 2.1 mmol O2/(L × h) OTR. This was near to the point where the highest maximum xylitol volumetric productivity was predicted by the model (71.1 g/L initial xylose concentration, 2.7 mmol O2/(L × h)). Fermentation profiles of the validation experiments are shown in Figure 2. In the first

case, 55% maximum xylitol yield and 0.75 g/(L × h) maximum xylitol productivity were achieved. The xylitol concentration reached its maximum value at 24 h, and then, it slightly decreased until the end of the fermentation (Figure 2A). A small amount of ethanol was produced in the first 48 h (4.3 g/L); however, that was completely consumed by the cells until the end of the fermentation (Figure 2). The maximum productivity was also obtained after 24 h. A small amount of xylose (4.7 g/L) remained in the broth at this point, but it was completely depleted after 48 h (Figure 2A). The achieved xylitol yields and volumetric productivity are in accord with the ones predicted by the fitted model and previously obtained during the designed experiments (Tables 2 and 3), indicating the good reproducibility of the fermentations. All the results fitted into the predicted intervals of the models (Table 3). In the second case, 46% maximum xylitol yield, 32% 24-h xylitol yield, and 1.01 g/(L × h) maximum xylitol productivity were achieved. These values are similar to that predicted by the fitted models, and all of them are in the prediction intervals (Table 3). The fermentation profile observed was quite different to that of the first case (Figure 2B). The xylitol concentration continuously increased, meanwhile the xylose concentration decreased until the end of the fermentation. In addition, a significant amount of ethanol was produced by the end of the fermentation (13.8 g/L). In both cases, a small increase in the cell concentration (0.8–1 g/L) was also observed. Based on the results of the verification experiment, it can be concluded that the fitted models are adequate and suitable for good predictions within the experimental range.

**Figure 2.** Profiles of fermentations performed at 2.1 mmol O2/(L × h) OTR on semi-defined medium containing an initial xylose concentration of 30 g/L (**A**) and 71 g/L (**B**) using *Candida boidinii* Y.01308. Standard deviations are calculated from duplicates.

**Table 3.** Maximum xylitol yield, maximum xylitol volumetric productivity, and xylitol yield after 24 h of model predictions and experiments. (OTR: oxygen transfer rate, IXC: initial xylose concentration.)


Standard deviations are calculated from duplicates and presented in parentheses.
