**4. Discussion**

The effects of initial xylose concentration and aeration (OTR) on maximum xylitol yield, maximum xylitol volumetric productivity, and xylitol yield after 24 h were investigated first in this study, by using *Candida boidinii* NCAIM Y.01308 in a semi-defined xylose medium. Vandeska et al. [45] investigated the effect of the initial xylose concentration on the achievable xylitol yield during fermentation on a model xylose medium by *C. boidinii* NRRL Y17213. They varied the initial xylose concentration between 20 and 200 g/L under the same aeration conditions (50 mL medium in 125 mL-flask, 150 rpm shaking), and concluded that the xylitol yield of 14 days fermentation continuously increased from 6% to the maximum value of 52% by increasing the initial xylose concentration from 20 g/L to 150 g/L. Interestingly, initial xylose concentration of 200 g/L significantly inhibited the xylitol fermentation. Vongsuvanlert and Tani [46] reported that a xylose concentration of 150 g/L resulted in lower xylitol production than that of 100 g/L, when *C. boidinii* no. 2201 was used on xylose basal medium. They performed the fermentations in 500 mL-flasks filled with 100 mL medium at 100 rpm shaking. Those studies showed that under given conditions of aeration, increasing xylose concentration increased the xylitol production until a certain value above which it had a negative effect. Osmotic stress on the cells is often hypothesized as a possible reason for the negative effect of high xylose concentration [46,47]. However, in our study, a decreasing tendency in the xylitol yield was observed by increasing the xylose concentration under the investigated range of xylose concentration and aeration. Oxygen availability during xylitol fermentation is a key factor due to its

influence on the intracellular redox balance. The key enzymes for assimilation of D-xylose in yeasts are the xylose reductase (XR), which catalyses the reduction of D-xylose to xylitol requiring NAD(P)H as cofactor, and the NAD-dependent xylitol dehydrogenase (XDH) catalysing the xylitol conversion to xylulose [48]. Xylitol accumulation under microaerobic conditions is the result of a deficient NAD regeneration by the respiratory chain which leads to diminished XDH activity [45,48]. In addition, formation of certain by-products during the xylitol fermentation can also contribute to maintaining the intracellular cofactor balance [49]. Winkelhausen et al. [50] investigated the effect of different kLa values on the xylitol production of *C. boidinni* NRRL Y17213 in a xylose model medium using shake flasks (50 g/L initial xylose concentration) and fermenter (130 g/L initial xylose concentration). The kLa values were varied between 0 and 46 1/h in shake flasks, and it was concluded that increasing kLa resulted in decreasing xylitol and increasing cell mass production. Interestingly, the highest specific xylitol yield (0.3 g/g) was achieved under anaerobic conditions (0 1/h kLa). As a comparison, Winkelhasuen et al. [50] achieved a specific xylitol yield of 0.16 g/g at 50 g/L initial xylose concentration and 16 1/h kLa after 96 h, which is lower than that of obtained in our study (0.43 g/g) at an initial xylose concentration of 55 g/L and kLa of 15 1/h (3.1 mmol O2/(L × h) OTR) after 72 h. The lower initial cell concentration (1.3 g/L) applied by Winkelhasuen et al. [50] compared to that of used in our study (5 g/L) could be one of the reasons for that. However, it is clear that xylitol producing capability can significantly differ with the subspecies of *Candida boidinii* also. Subspecies isolated from different environmental conditions might have XR and XDH enzymes with different characteristics, and different metabolic pathways for co-factors regeneration might have been activated in them, resulting in variable capability in xylitol fermentation under certain fermentation conditions. During the fermentations in a bioreactor using 130 g/L initial xylose and 5 g/L initial cell concentrations, Winkelhasuen et al. [50] achieved the highest specific xylitol yield (around 0.45 g/g) and xylitol volumetric productivity (around 0.26 g/(L × h)) at kLa of 47 1/h. It seemed that the increase in xylose and aeration together caused an increase in xylitol yield and productivity, however the increased initial cell mass could also have a positive effect on that. In our study, it was concluded that both initial xylose concentration and OTR had significant effects on the maximum xylitol yield and xylitol yield achieved after one day. However the interaction between xylose concentration and OTR had no significant effect within the investigated experimental range. It is worth it to note that the extent of their effects and their significant terms were different when the achievable maximum xylitol yield and the xylitol yield after a certain fermentation time (e.g., one day) was examined. In contrast, when the maximum xylitol volumetric productivity was evaluated, a clear interaction between OTR and initial xylose concentration was observed.

Xylitol fermentation on lignocellulosic hydrolysates by using *Candida boidinii* was previously tested by other studies. Santana et al. [51] investigated *Candida boidinii* XM02G (4 g/L initial cell mass) on cocoa pod husk hemicellulose hydrolysate detoxified by activated carbon, and a specific xylitol yield of 0.52 g/g was achieved after 372 h of fermentation. Fehér et al. [39] published a xylitol yield of 53% of theoretical and a xylitol volumetric productivity of 0.14 g/(L × h) reached after 72 h of fermentation by using *C. boidinii* NCAIM Y.01308 (5 g/L initial cell mass) on corn fibre hydrolysate detoxified by activated carbon. Lopez-Linares et al. [52] investigated the xylitol production of *C. boidinii* NCAIM Y.01308. (5 g/L initial mass) on exhausted olive pomace hydrolysate detoxified by ion-exchange resin and achieved 0.43 g/g specific xylitol yield and 0.07 g/(L × h) volumetric productivity after 96 h. In this study, the xylitol yield, the specific xylitol yield, and the volumetric productivity were 60%, 0.72 g/g, and 0.58 g/(L × h), respectively, on WB1/S after 24 h. Those results exceeded the ones mentioned before. It is also important to note that in the case of WB1/S, no detoxification step was required prior to the fermentation. In our previous study, xylitol fermentation was performed by using *Ogataea zsoltii* NCAIM Y.01540 on xylose-rich wheat bran hydrolysate [17]. Comparing the maximum xylitol yields and volumetric productivities achieved under the same conditions by using

*O. zsoltii* NCAIM Y.01540 (56% and 0.24 g/(L × h)) and *C. boidinii* NCAIM Y.01308 (60% and 0.58 g/(L × h)), xylitol production of *C. boidinii* NCAIM Y.01308 was found to be more advantageous. Based on these results, this study confirms that the xylose-rich hydrolysate of wheat bran is a suitable medium for xylitol fermentation without detoxification.

Mayerhoff et al. [53] investigated different yeast strains to ferment xylitol on sulfuric acid treated rice straw hydrolysate. In their work, the initial xylose concentration was 53.9 g/L and 50 mL medium was used in 125 mL-flasks at 200 rpm shaking. High specific xylitol yields (>0.6 g/g) were achieved after 75 h by several *Candida* strains such as *C. guilliermondii* FTI-20037, *C. mogii* NRRL Y-17032, *C. parapsilosis* IZ-1710, and *C. veronae* IZ-945. However, it was only 0.17 g/g by using *C. boidinii* NRRL Y-17213. Compared to that, a higher specific xylitol yield was achieved by *C. boidinii* NCAIM Y.01308 in our study (0.26 g/g after 72 h). Nitrogen source in the fermentation media is also an important factor influencing the xylitol production. Since rice straw hydrolysates contained very low amount of proteins, supplementation by ammonium-sulphate and peptone was tested in this study. Generally, organic nitrogen sources (e.g., yeast extract and urea) result in higher xylitol yield compared to the inorganic ones [54]. In accord with that, higher maximum xylitol yield was achieved on peptone-supplemented GRS/S (25%) compared to GRS/S (20%). Silvia and Roberto [55] also investigated the effect of the nutrient supplementation (2 g/L (NH4)2SO4, 0.1 g/L CaCl2\*2H2O and 10 g/L rice bran extract) of rice straw hydrolysate in order to improve the xylitol production of *C. guilliermondii* FTI 20037. They found that the nitrogen supplementation had no effect on the specific xylitol yield (0.36–0.37 g/g) achieved. In this study, a similar result was obtained, supplementation of GRS/S with peptone did not improve the specific xylitol yield (0.35 g/g after 24 h); however it increased the maximum xylitol yield from 20% to 25% of theoretical. Zeid et al. [56] investigated the effects of activated carbon treatment on xylitol production by *C. tropicalis* and *C. guilliermondii* using rice straw hydrolysate. After the activated carbon treatment, specific xylitol yields were increased from 0.25 g/g and 0.47 g/g to 0.61 g/g and 0.69 g/g in the cases of *C. tropicalis* and *C. guilliermondii*, respectively. Similarly, as a result of the activated carbon treatment of GRS/S, the maximum xylitol yield was increased from 25% to 30% in this study. Lopez Linares et al. [52] published a specific xylitol yield of 0.36 g/g achieved on exhausted olive pomace hydrolysate treated by activated carbon after 72 h of fermentation by *C. boidinii* NCAIM Y.01308 (5 g/L initial cell mass, 50 mL medium in 100 mL-flask, 150 rpm shaking). That is slightly lower than that obtained in our study (0.38 g/g) using peptone-supplemented GRS/S treated by activated carbon and the same yeast strain. One of the reasons for the low xylitol yields obtained in our study on rice straw hydrolysates is probably the presence of considerable amount of glucose (glucose/xylose ratio of 0.29 in GRS/S). A glucose/xylose ratio that is higher than 0.1 could negatively affect xylitol fermentation [52]. Moreover, the activated carbon treatment was not effective in removing the organic acids from GRS/S, which could also contribute to the insufficient bioconversion of xylose into xylitol. Bio-purification processes selectively removing organic acids and glucose from lignocellulosic hydrolysates [57,58] or appropriate genetic modifications of the xylose-fermenting microorganisms [59,60] are promising ways to overcome these kinds of obstacles.
