Xylitol fermentation on semidefined medium

Preliminary studies showed that Candida boidiniican convert xylose into xylitol by using semidefined fermentation medium, however, the achievable xylitol yield was strongly affected by the aeration condition. In order to simulate different aeration conditions in shake flasks, the fermentations were performed at three levels of filling ratio (0.35, 0.5 and 0.65) each of them at two levels of rotation speed (125 and 220 rpm) using semidefined medium. Filling ratio is defined as the ratio of the medium volume to the flask volume. Oxygen transfer rate of the different settings of filling ratio and rotation speed was determined. The investigation of the effect of OTR to the xylitol yield achievable was performed with 1 g/L initial cell concentration and 30 g/L initial xylose concentration.

The higher filling ratio resulted in the lower OTR, while the higher rotation speed increased the OTR, during shake flask fermentations (Figure 21). At the rotation speed of 220 rpm, the OTR value decreased from 6.6 mmol/(L×h) to 5.2 mmol/(L×h) by increasing the filling ratio from 0.35 to 0.65. At the rotation speed of 125 rpm, the OTR value decreased from 4.2 mmol/(L×h) to 1.9 mmol/(L×h) by increasing the filling ratio from 0.35 to 0.65. The xylitol yield continuously increased by decreasing the OTR value until 2.8 mmol/(L×h), where a xylitol yield of 44% was obtained (Figure 21). That condition of aeration can be referred to as microaerobic condition (Walther et al., 2001), and was set for the subsequent experiments. Further decrease in the OTR resulted in significant decrease in the xylitol yield (Figure 21). The xylitol fermentation performed at 1 g/L initial cell concentration and 30 g/L initial xylose concentration at 2.8 mmol/(L×h) OTR is referred to as base case in the following sections.

Figure 20: Arabinose biopurification on semidefined medium using Candida boidinii NCAIM Y.01308

Standard deviations are calculated from duplicates.

6.2.2. Xylitol fermentation on semidefined medium

Preliminary studies showed that Candida boidiniican convert xylose into xylitol by using semidefined fermentation medium, however, the achievable xylitol yield was strongly affected by the aeration condition. In order to simulate different aeration conditions in shake flasks, the fermentations were performed at three levels of filling ratio (0.35, 0.5 and 0.65) each of them at two levels of rotation speed (125 and 220 rpm) using semidefined medium. Filling ratio is defined as the ratio of the medium volume to the flask volume. Oxygen transfer rate of the different settings of filling ratio and rotation speed was determined. The investigation of the effect of OTR to the xylitol yield achievable was performed with 1 g/L initial cell concentration and 30 g/L initial xylose concentration.

The higher filling ratio resulted in the lower OTR, while the higher rotation speed increased the OTR, during shake flask fermentations (Figure 21). At the rotation speed of 220 rpm, the OTR value decreased from 6.6 mmol/(L×h) to 5.2 mmol/(L×h) by increasing the filling ratio from 0.35 to 0.65. At the rotation speed of 125 rpm, the OTR value decreased from 4.2 mmol/(L×h) to 1.9 mmol/(L×h) by increasing the filling ratio from 0.35 to 0.65. The xylitol yield continuously increased by decreasing the OTR value until 2.8 mmol/(L×h), where a xylitol yield of 44% was obtained (Figure 21). That condition of aeration can be referred to as microaerobic condition (Walther et al., 2001), and was set for the subsequent experiments. Further decrease in the OTR resulted in significant decrease in the xylitol yield (Figure 21). The xylitol fermentation performed at 1 g/L initial cell concentration and 30 g/L initial xylose concentration at 2.8 mmol/(L×h) OTR is referred to as base case in the following sections.

Figure 21: Xylitol yields achieved on semidefined medium using Candida boidinii NCAIM Y.01308 at different aeration conditions

Filling ratios and rotation speeds applied are indicated under the xylitol yields. Standard deviations are calculated from triplicates.

Xylitol fermentations of the same initial conditions (1 g/L initial cell concentration and 30 g/L initial xylose concentration) using C. boidinii followed the same trends, which is demonstrated in Figure 22.

Figure 22: Fermentation profile of xylitol production on semidefined medium under microaerobic condition (2.8 mmol/(L×h) OTR) at 1 g/L initial cell concentration and 30 g/L initial xylose concentration by using Candida boidiniiNCAIM Y.01308 (base case)

Standard deviations are calculated from triplicates.

64

Xylitol concentration keeps increasing as long as xylose is available in the fermentation broth. After xylose is depleted, C. boidinii starts to consume xylitol, hence xylitol concentration has a maximum value during the fermentation. Small amount of ethanol is always produced simultaneously with xylitol, however, after xylose depletion the ethanol is also consumed. The cell mass continuously increases, which indicates that xylitol and ethanol are used to form cell mass after xylose depletion.

The effects of initial cell concentration, initial xylose concentration and methanol addition to the xylitol yield were investigated. The effect of high initial xylose concentration for the fermentative capacity of C. boidiniiwas also investigated using 70 g/L initial xylose concentration (Table 12). The high initial xylose concentration resulted in a xylitol yield of 40%, which is slightly lower compared to the xylitol yield of the base case (44%) (Table 12). The xylitol concentration continuously increased until the end of the fermentation resulting in a volumetric productivity of 0.3 g/(L×h), which is similar to that obtained in the base case (0.28 g/(L×h)).

The effect of high cell density on the fermentation process was investigated using 5 g/L initial cell concentration. High initial cell density resulted in significantly higher xylitol yield within shorter fermentation time, compared to the base case, as 58% xylitol yield was achieved in one day (Table 12). The volumetric productivity of the fermentation was 0.73 g/(L×h).

Addition of methanol as a co-substrate did not result in significant increase of xylitol yield, as a xylitol yield of 60% was obtained when 12 g/L methanol was added to the fermentation broth containing 30 g/L initial xylose and 5 g/L initial cell concentrations (Table 12). On the other hand, longer time was needed (2 days) to reach the highest xylitol concentration compared to that of without methanol addition (1 day), which resulted in a volumetric productivity of 0.38 g/(L×h).

Table 12: Fermentation conditions, maximal xylitol yields achieved on semidefined media and times required to achieve the maximal yield

OTR (mmol/Lh)

Initial cell concentration

(g/L)

Initial xylose concentration

(g/L)

Co-substrate Xylitol yield (% of theoretical) Time (day)

2.8 1 30 - 44 (4) 2

2.8 1 70 - 40 (1) 4

2.8 5 30 - 58 (5) 1

2.8 5 30 12 g/L

MeOH 60 (5) 2

Standard deviations are calculated from triplicates and indicated in parenthesis.

According to our investigation of xylitol production on semidefined media using C.

boidinii, aeration and initial cell density have the greatest effects on xylitol yield. Xylitol production is favoured under microaerobic condition (2.8 mmol/(L×h) OTR) using increased (5 g/L) initial cell concentration.

Xylitol concentration keeps increasing as long as xylose is available in the fermentation broth. After xylose is depleted, C. boidinii starts to consume xylitol, hence xylitol concentration has a maximum value during the fermentation. Small amount of ethanol is always produced simultaneously with xylitol, however, after xylose depletion the ethanol is also consumed. The cell mass continuously increases, which indicates that xylitol and ethanol are used to form cell mass after xylose depletion.

The effects of initial cell concentration, initial xylose concentration and methanol addition to the xylitol yield were investigated. The effect of high initial xylose concentration for the fermentative capacity of C. boidinii was also investigated using 70 g/L initial xylose concentration (Table 12). The high initial xylose concentration resulted in a xylitol yield of 40%, which is slightly lower compared to the xylitol yield of the base case (44%) (Table 12). The xylitol concentration continuously increased until the end of the fermentation resulting in a volumetric productivity of 0.3 g/(L×h), which is similar to that obtained in the base case (0.28 g/(L×h)).

The effect of high cell density on the fermentation process was investigated using 5 g/L initial cell concentration. High initial cell density resulted in significantly higher xylitol yield within shorter fermentation time, compared to the base case, as 58% xylitol yield was achieved in one day (Table 12). The volumetric productivity of the fermentation was 0.73 g/(L×h).

Addition of methanol as a co-substrate did not result in significant increase of xylitol yield, as a xylitol yield of 60% was obtained when 12 g/L methanol was added to the fermentation broth containing 30 g/L initial xylose and 5 g/L initial cell concentrations (Table 12). On the other hand, longer time was needed (2 days) to reach the highest xylitol concentration compared to that of without methanol addition (1 day), which resulted in a volumetric productivity of 0.38 g/(L×h).

Table 12: Fermentation conditions, maximal xylitol yields achieved on semidefined media and times required to achieve the maximal yield

OTR (mmol/Lh)

Initial cell concentration

(g/L)

Initial xylose concentration

(g/L)

Co-substrate Xylitol yield (% of theoretical) Time

(day)

2.8 1 30 - 44 (4) 2

2.8 1 70 - 40 (1) 4

2.8 5 30 - 58 (5) 1

2.8 5 30 12 g/L

MeOH 60 (5) 2

Standard deviations are calculated from triplicates and indicated in parenthesis.

According to our investigation of xylitol production on semidefined media using C.

boidinii, aeration and initial cell density have the greatest effects on xylitol yield. Xylitol production is favoured under microaerobic condition (2.8 mmol/(L×h) OTR) using increased (5 g/L) initial cell concentration.

6.2.3. Integration of arabinose biopurification and xylitol fermentation using corn