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Enhanced Production of Bioxylitol from Corn Cobs by Candida magnoliae Kiyoshi Tada, Tohru Kanno, and Jun-ichi Horiuchi* Department of Biotechnology and Environmental Chemistry, Kitami Institute of Technology, Koen-cho 165, Kitami, Hokkaido, Japan ABSTRACT: An effective microbial process using Candida magnoliae was developed for xylitol production from xylose obtained from corn cob. The hydrolysate containing approximately 25 g-xylose/L and 3.0 g-glucose/L was obtained from 100 g-corn cobs/L by 1.0−3.0% sulfuric acid with 60 min of hydrolysis time. The corn cob hydrolysate was then treated by charcoal pellets which selectively removed more than 90% of inhibitors without affecting xylose concentration. Using the hydrolysate medium treated with the charcoal pellets, xylitol production using C. magnolia was successfully performed, resulting in a final production of approximately 18 g-xylitol/L from 25 g-xylose/L within 36 h. In total, 15−18 g xylitol could be produced from 100 g of corn cobs. From the batch experiments operated under strictly controlled oxygen supply conditions, it was found that the successful xylitol production occurred at the narrow range of the oxygen transfer rate (OTR) from 0.8 mmol-O2/L·h to 4.7 mmol-O2/L·h. A two-step fermentation consisting of the cell growth step using normal hydrolysate medium and the xylitol production step using the concentrated corn cob hydrolysate was effective to avoid serious inhibitory effect and to realize a high concentration of xylitol production (approximately 49 g/L).

1. INTRODUCTION Lignocellulosic biomass such as agricultural wastes, which contains cellulose, hemicellulose, and lignin, has been regarded as potential renewable feedstocks of biological processes for biofuels and fine chemicals production. Regarding the utilization of cellulose in biomass, there are many researches including bioethanol production; however, to enhance the successful utilization of lignocellulosic biomass, it is also important to develop a bioprocess utilizing pentose sugars such as xylose obtained from hemicelluloses which occupy more than 30% of agricultural wastes. One of the possible value-added products produced from pentose sugar is xylitol. Xylitol, a polyol that occurs widely in nature, has attracted much attention in the food and pharmaceutical industries because it has useful applications which include a sugar alternative for diabetics, the prevention of dental caries, and a natural sweetener.1,2 Xylitol is currently produced by a catalytic hydrogenation of D-xylose that is obtained from woody resources such as white birches by acid hydrolysis. However, the high production cost of this process and the necessity to minimize the environmental impact caused by excessive utilization of natural woody resources have led to extensive exploration of alternative processes for xylitol production. Microbial xylitol production using xylose obtained from renewable resources is a potential alternative candidate process because of its cheaper production cost and low environmental impact.3,4 Xylitol produced by fermentation instead of chemical hydrogenation is called bioxylitol. In this regard, we have reported the possibility of microbial xylitol production from corn cobs using a xylose utilizing yeast, Candida magnoliae5,6 which has an ability to convert xylose to xylitol under microaerbic conditions. Corn cobs were selected as a promising feedstock for xylitol production because they are renewable, low cost, rich in hemicellulose, and widespread in the world, but most of them are not utilized effectively.6 © 2012 American Chemical Society

In microbial xylitol production from corn cobs, the corn cobs are first hydrolyzed to release xylose from hemicellulose in the corn cobs by acid hydrolysis, and the corn cob hydrolysates containing xylose are then used as the medium for xylitol fermentation by xylose utilizing yeasts such as Candida sp. Therefore, we need to examine the hydrolysis conditions of the corn cobs and the culture conditions for maximizing xylitol production using the hydrolysates. Furthermore, it is known that furfural and its derivatives generated during the hydrolysis of corn cobs strongly inhibit the cell growth of yeasts. Therefore, it is also required to remove the inhibitors from the hydrolysate to prepare a medium suitable for xylitol production.7,8 In regards to the culture condition for microbial xylitol production, it has been pointed out that the supply of oxygen strongly influences the accumulation of xylitol.9−11 Therefore, it is important to evaluate the effect of the oxygen supply and to determine the optimal oxygen supply condition suitable for each microorganism employed for the production of xylitol. Furthermore, the increase in xylitol concentration by use of concentrated hydrolysates will contribute to the cost reduction of the purification step as well as the increase in the productivity. In this study, to establish total process of xylitol production from corn cobs, we investigated the hydrolysis conditions of corn cobs, an inhibitor removal method from the corn cob hydrolysates, and the effects of oxygen supply for the effective microbial xylitol production. Furthermore, to produce the high concentration of xylitol using concentrated hydrolysate with highly inhibitory effects, a two-step fermentation, which Special Issue: APCChE 2012 Received: Revised: Accepted: Published: 10008

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DO is usually employed as a parameter to control the aerobic environment in a fermentor; however, DO concentrations were almost zero throughout fermentation experiments because the oxygen supply is strictly limited in order to ensure a microaerobic environment. Therefore, we employed the volumetric oxygen transfer rate (OTR, mmol-O2/L/h) instead of DO as the index of oxygen supply condition. The OTR is defined under microaerobic condition (C ≃ 0) as follows.

increased cell concentration at the initial stage of fermentation followed by xylitol production using the concentrated hydrolysate, was investigated.

2. EXPERIMENTAL SECTION 2.1. Microorganisms and Culture Media. Candida magnoliae, (NIBH, Tsukuba, FERM P-16522) was used throughout this study. The strain has the ability to produce xylitol from xylose under microaerobic conditions as previously reported.5 The strain was grown on an agar slant containing 3 g of malt extract, 3 g of yeast extract, 5 g of peptone, 10 g of glucose, and 20 g of agar per liter and was maintained at 4 °C. One loop of cells was transferred to a 500-mL baffled flask containing 100 mL of medium consisting of 25 g of xylose, 1 g of casamino acid, 1.7 g of yeast nitrogen base without amino acid and ammonium sulfate, and 2.3 g of urea per liter. The culture was incubated at 30 °C for 24 h on a reciprocating shaker (110 stokes/min), and the flask culture broth was employed to seed a main culture. 2.2. Corn Cobs and Their Hydrolysis. Dried corn cobs, with a particle size smaller than 5 mm, which were locally purchased in Japan, were used. The average composition of the corn cobs was around 32% cellulose, 35% hemicellulose, 20% lignin, 4% ash and others. Sulfuric acid hydrolysis was employed for the hydrolysis of corn cobs. The 100 g of corn cobs was mixed with 1.0 L of diluted sulfuric acid with various concentrations, and these mixtures were hydrolyzed at 121 °C for 10 min, 60 min, and 120 min using an autoclave. After hydrolysis, the liquid fraction (corn cob hydrolysates) was filtered with a filter paper, and the pH was adjusted to 7.0 by the addition of 20% NaOH. To remove the inhibitors from the corn cob hydrolysates, the absorption experiments were performed using activated carbon with a specific surface area of 2000 m2/g (Kishida Chemicals, Japan) and charcoal pellets with a specific surface area of 200 m2/g (Okhotsk Carbonization Center, Hokkaido, Japan). The pore size distribution of the charcoal pellets and the activated carbon were analyzed by a porosimeter (Shimadzu 9400). The detailed characterization of the pellets was also reported previously.12 Prior to the adsorption treatment, the charcoal pellets were washed with tap water and dried in an oven at 110 °C overnight. The hydrolysates were mixed with the adsorbents at a ratio of 1: 10 (w/v) and placed on a shaker at 500 rpm at ambient temperature for 6 h. After the absorption treatment, the filtrate was recovered by filtration using a filter paper, and the corn cob hydrolysate medium was prepared by adding 1 g of casamino acid, 1.7 g of yeast nitrogen base without amino acids and ammonium sulfate, and 2.3 g of urea to 1 liter of the filtrate. The initial pH of the medium was set at 7.0. 2.3. Flask Culture. Batch experiments (in duplicate) were conducted by adding 2 mL of the preculture into a 500-mL baffled flask containing 100 mL of medium at 30 °C on a reciprocating shaker (110strokes/min). The initial pH of the corn cob hydrolysate medium was set at 7.0. 2.4. Batch Fermentation Using a Jar Fermentor. To examine the xylitol production under various oxygen supply conditions, a computer controlled 5-L jar fermentor with a 2-L working volume (D-type fermentor, Biott corporation, Japan) was used for batch experiments. A 50 mL portion of the preculture was added to 2.0 L of medium in the fermentor, and the temperature was automatically controlled at 30 °C. The culture pH was not controlled; however, the culture pH in the fermentor was almost constant at 7.0 during the experiments.

OTR =

dC = kLa(C* − C) ≅ kLaC* dt

Here, C* and C denote the saturated and current DO concentrations (mmol/L) in a fermentor respectively. kLa is a volumetric mass transfer coefficient. C is nearly zero during microaerobic fermentation because the supplied oxygen is completely consumed by the yeasts. Because kLa is a function of the agitation and aeration rates for a complete mixing jarfermentor, a desired OTR can be obtained by choosing the proper agitation rate and aeration rate. In this study, the relationship between OTR and the operating parameters including agitation and aeration rates was experimentally determined on the basis of the dynamic method in advance. Figure 1 shows the relationship between kLa and the aeration

Figure 1. Relationship between kLa and agitation at various aeration rates. Symbols for aeration rates: ●, 0.5 vvm; ▲, 0.25 vvm; ■, 0.1 vvm; ▼, 0.05 vvm.

rate under various agitation rates in the fermentor employed in this study. The results obtained were then used to realize various OTR conditions as required. Prior to an inoculation into the fermentor, the agitation and aeration rates were set so as to give the desired OTR and were maintained constant throughout a batch experiment. 2.5. Xylitol Yield and Productivity. The xylitol yield (Yx) and productivity (P, (g/L)·h) were defined as follows: Yx = P=

xylitol produced (g) xylose consumed (g) maximum xylitol concentration (g/L) fermentation time (h)

2.6. Analytical Methods. Xylose, xylitol, and acetic acid concentrations were determined by HPLC with a TSK-GEL SCX column (7.8 mm I.D. × 30 cm; TOSOH, Tokyo). Glucose was analyzed by an enzymatic method using glucose oxidase (Glucose analysis kit, GlucoseCII, Wako Pure Chemical Industries, Japan). Absorbance at 280 nm (A280) was employed as an index of the inhibitors including furfural and 510009

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concentrations in the acid hydrolysis of 100 g/L corn cobs. Figure 2a indicates that the highest xylose concentration of approximately 25 g-xylose/L was obtained under the condition of 1−5% sulfuric acid with 60 min of hydrolysis time. A 10 min hydrolysis was too short for successful xylose release from the corn cobs. At 120 min of hydrolysis time, the concentrations of xylose were low because their degradation was promoted under high sulfuric acid concentrations. Acetic acid, which might be a possible inhibitor for xylitol production, was not detected in the hydrolysates in all cases. Figure 2b shows that the glucose concentration in the hydrolysates was approximately 2−3 gglucose/L for all experiments, which suggests that little degradation of cellulose occurred in the dilute acid hydrolysis of corn cobs. Figure 2c indicates that the A280 value as an index of furfural and its derivatives increased as the concentration of sulfuric acid increased. This is because the high sulfuric acid concentration enhanced the generation of furfural from xylose released from corn cobs because furfural is readily generated by the dehydration of xylose during sulfuric acid hydrolysis. Apparently, the increase of the A280 value is associated with the decrease of xylose concentration. On the basis of these results, 1.0−3.0% sulfuric acid concentrations with 60 min of hydrolysis time were considered to be suitable for successful xylose recovery with relatively low inhibitor generation by the acid hydrolysis of corn cobs. Under these conditions, the A280 of hydrolysates ranged approximately from 100 to 300. The xylose yield over corn cobs was around 0.25 g-xylose/g-corn cobs. Because hemicellulose occupies around 35% of the dry corn cobs, the xylose yield over hemicellulose in the corn cobs becomes 0.71 g-xylose/ghemicellulose. Several flask cultures were then performed using the hydrolysates for xylitol production. However, the cell growth was strongly depressed due to the inhibitors generated during acid hydrolysis of the corn cobs, and the xylitol production did not occur at all for all experiments. 3.2. Selective Removal of Inhibitors from Hydrolysate by Charcoal Pellet Treatment. The removal of inhibitors was then examined by an absorption treatment using activated carbon and charcoal pellets. Figure 3 shows the time courses of A280 and xylitol concentration during the absorption treatment using activated carbon and charcoal pellets. As shown in Figure 3a, the inhibitors were immediately removed from the hydrolysates by the activated carbon treatment, while it took more than 4 h to successfully remove inhibitors by the charcoal pellet treatment. However, Figure 3b shows that the activated carbon also absorbed xylose, and its concentration decreased from 22.7 g/L to around 8.0 g/L. In contrast, xylose concentration did not change during the adsorption treatment with charcoal pellets. This indicates that charcoal pellets could selectively absorb only inhibitors from hydrolysates without affecting xylose concentration. To clarify the mechanism of selective removal of inhibitors by charcoal pellets, the pore size distribution of charcoal pellets and activated carbon was measured by a porosimeter. Figure 4 shows the results. As can be seen, the micropores around 1.0 nm which are effective to adsorb sugars including xylose were developed for the activated carbon; however, there were less micropores around 1.0 nm in the charcoal pellets because they were just carbonized and not activated. Therefore, it was considered that the difference in the volume of micropores around 1.0 nm resulted in the selectivity of inhibitor removal by the charcoal pellets. Compared with other treatments for inhibitor removal in

hydroxymethylfurfural (5-HMF) in the corn cob hydrolysates because the A280 value has been widely used as an index of phenolic compounds such as furfural which is known as the main inhibitory byproduct in chemically hydrolyzed biomass solution. In the preliminary analysis of the corn cob hydrolysate treated by 3.0% sulfuric acid, the concentrations of furfural and 5-HMF were 1130−1500 mg/L and 5.8−16.8 mg/L, respectively. Cell concentration was determined by measuring the optical density at 660 nm (OD660), and the OD660 was then converted into the dry cell concentration using a predetermined calibration curve.

3. RESULTS AND DISCUSSION 3.1. Effects of Sulfuric Acid Concentration and Hydrolysis Time on Corn Cob Hydrolysis. To maximize the xylose recovery from corn cobs, hydrolysis conditions were investigated at various sulfuric acid concentrations (0−20%) and hydrolysis times (10 min, 60 min, 120 min) at 121 °C. Figure 2 shows the xylose concentration (a), the glucose concentration (b), and the A280 (c) as a function of sulfuric acid

Figure 2. Hydrolysis of corn cobs at various sulfuric acid concentrations and hydrolysis times: (a) xylose concentration, (b) glucose concentration, and (c) absorbance at 280 nm (A280). Symbols for hydrolysis time: ●, 10 min; ■, 60 min, ▲120 min. 10010

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Figure 5. Effects of A280 of corn cob hydrolysate on growth and xylitol formation by Candida magnoliae. Symbols: ●, maximum xylitol concentration; ○, final dry cell concentration.

final xylitol and cell concentrations after 36 h of cultivation. The results indicate that the xylitol production became possible when the A280 in the hydrolysates was approximately less than 20. However, the A280 in the hydrolysates should be minimized as much as possible because, as Figure 5 shows, the final xylitol concentration increased as the A280 in the hydrolysates decreased. On the basis of these investigations, xylitol fermentation using corn hydrolysate treated with charcoal pellets was performed. As can be seen in Figure 6, xylose was gradually Figure 3. Time courses of A280 (a) and xylose concentration (b) during adsorption treatment by charcoal pellets and activated carbon. Symbols: ●, charcoal pellets; ▲, activated carbon.

Figure 6. Time courses of xylitol production using corn cob hyodrolystae by Candida magnoliae in a flask culture. Symbols: ●, dry cell concentration; ○ , xylose concentration; ▲ , xylitol concentration; △, ethanol concentration. Figure 4. Pore size distribution of charcoal pellets and activated carbon. Symbols: ○, charcoal pellets; ●, activated carbon.

consumed, followed by the growth of C. magnoliae. Xylitol production commenced after approximately 12 h of culture and steadily continued, resulting in a final production of approximately 18 g-xylitol/L from 25 g-xylose/L within 36 h. The xylitol yield over xylose consumed (Yx) was around 0.75 gxylitol/g-xylose consumed, and the xylitol productivity was 0.5 (g/L)/h. 3.4. Effects of Oxygen Supply on Xylitol Production. As described in the Introduction, it is known that the oxygen supply strongly influences the microbial xylitol production. Therefore, a proper oxygen supply condition for C. magnolia was investigated under strictly controlled oxygen supply conditions. Figure 7 and Table 1 show the effects of the oxygen transfer rate (OTR) on xylitol production by C. magnolia within the range of OTR from 0.0 to 16.5 mmol-O2/ L·h using a computer-controlled jar fermentor. Detailed

microbial medium preparation such as ion exchange, adsorption by moloecular sieve, and extraction,5 the charcoal pellet treatment would be useful in practical applications owing to the selectivity, the operational simplicity, and the low cost of the charcoal pellets. 3.3. Xylitol Production Using Corn Cob Hydrolysate Medium Treated with Charcoal Pellets. As previously mentioned, it is known that inhibitors including furfural and its derivatives generated during the acid hydrolysis of corn cobs inhibit the cell growth of yeast. To evaluate the effects of inhibitors on xylitol fermentation by C. magnoliae, flask cultures using corn cob hydrolysates with various inhibitor concentrations (A280 values) were carried out. Figure 5 summarizes the 10011

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Table 1. Xylitol Production from Corn Cob Hydrolysate Using Candida magnoliae under Various OTR Conditions OTR (mmolO2/ L·h)

initial xylose conc. (g/L)

residual xylose conc. (g/L)

final dry cell conc. (g/L)

final xylitol conc. (g/L)

Yx (gxylitol/ gxylose)

productivity (g/L·h)

0.0 0.4 0.8 1.6 4.7 16.5

23.7 25.8 31.1 22.4 18.3 23.4

20.9 21.8 0.0 0.5 1.4 0.0

0.60 0.80 1.82 1.85 3.41 9.31

0.0 0.0 18.8 13.1 8.4 0.0

0.0 0.0 0.60 0.60 0.50 0.0

0.0 0.0 0.157 0.182 0.175 0.0

production was not observed in this study. At the OTR of 0.8 and 1.6 mmol-O2/L·h, xylitol was successfully produced while cell growth was relatively low. At the OTR of 4.7 mmol-O2/ L·h, xylitol was produced but the yield was not very high, while the cell growth increased. Finally, at the OTR of 16.5 mmolO2/L·h, cell growth was extremely high; however, no xylitol production was observed. Table 1 summarizes the experimental results. Thus, it was found that the successful xylitol production by C. magnolia occurred within the narrow range of OTRs from 0.8 to 4.7 mmol-O2/L·h and that cell growth increased as the OTR increased; however, the highest xylitol yield (Y = 0.6) was obtained at an OTR of 0.8 and 1.6 mmol-O2/L·h. Vandeska et al.9 reported that an OTR of 14 mmol-O2/L·h was optimal for successful xylitol production with a YX of 0.48 and a P of 0.24 (g/L)·h from a synthetic medium containing 130 g/L using Candida boidinii. Their optimal OTR was rather large compared with our results. Figure 8 outlines the main metabolic pathway of xylitol biosynthesis in C. magnoliae. Xylose taken into the cell is first

Figure 7. Time courses of xylose concentration (a), xylitol concentration (b), and dry cell concentration (c) in microbial xylitol production using corn cob hydrolysate under various oxygen transfer rates (OTRs). Symbols for OTRs: ●, OTR = 0.4 mmol-O2/L·h (kLa = 1.67 h−1, agitation 100 rpm, and aeration 0.01 vvm); ▲, OTR = 1.6 mmol-O2/L·h (kLa = 6.51 h−1, agitation 100 rpm, and aeration 0.1 vvm); ■, OTR = 4.7 mmol-O2/L·h (kLa = 19.8 h−1, agitation 200 rpm, and aeration 0.5 vvm); ▼, OTR = 16.5 mmol-O2/L·h (kLa = 69.01 h−1, agitation 400 rpm, and aeration 0.5 vvm).

Figure 8. Metabolic pathways of xylitol biosynthesis in Candida magnoliae.

reduced to xylitol by xylose reductase (XR) associated with NADPH. When sufficient oxygen is supplied at the OTR of 16.5 mmol-O2/L·h, the synthesized xylitol is then converted to xyluose by xylitol dehydrogenase (XD) accompanied by the generation of NADH and utilized as a carbon source for energy metabolism and cell synthesis, resulting in high cell growth. However, if the oxygen supply is limited from 0.8 mmol-O2/L·h to 4.7 mmol-O2/L·h, the oxidation of NADH in the electron transport system does not work well; it then causes an imbalance between NAD+/NADH, leading to a decrease in the xylitol oxidation rate by XD. This causes an increase in the intracellular xylitol concentration, resulting in the successful excretion of xylitol. Furthermore, in the cases at OTRs of 0.0

experimental conditions for the OTRs are shown in the caption for Figure 7. At OTRs of 0.0 and 0.4, xylose uptake into cells was strongly inhibited due to the oxygen supply deficiency, resulting in no xylitol production and no cell growth of C. magnoliae. Though there are some reports showing ethanol production under anaerobic fermentation from xylose using Candida sp., ethanol 10012

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ratio is increased because the concentration of inhibitors is also increased. The results for a four times and six times concentration ratio also suggest that xylitol production using highly concentrated hydrolysate may become possible if cell growth was achieved at the initial stage of fermentation. Therefore, we considered a two-step batch fermentation; that is, the fermentation commenced with the use of a normal corn cob hydrolysate medium for successful cell growth, and then, the concentrated corn cob hydrolysate medium was added for high xylitol production. Figure 10 shows the results. The

and 0.4 where the oxygen supply is quite insufficient, the reduction of xylose to xylitol will not occur successfully because the generation of NADPH in the pentose−phosphate pathway, which is an essential substrate for xylose reduction, was limited. As such, the xylitol production by C. magnoliae was strongly influenced by the oxygen supply and need to keep the OTRs from 0.8 mmol-O2/L·h to 4.7 mmol-O2/L·h for successful xylitol production. 3.5. Xylitol Production of Concentrated Corn Cob Hydrolysates. It is important to increase xylitol concentration to achieve cost-effective xylitol production including the purification process of xylitol. However, as previously mentioned, the xylose concentration in the corn cob hydrolysate medium was around 20−25 g/L from 100 g/L corn cobs, which is then converted to 15−18 xylitol-g/L. These concentrations are low for the cost-effective xylitol production. Therefore, we then attempted the use of cob hydrolysate concentrated by evaporation to increase xylitol concentration. The corn cob hydrolysate treated with charcoal pellets (xylose concn = 21.2 g/L and A280 = 9.9) was concentrated twice (xylose concn = 42.3 g/L and A280 = 28.1), three times (xylose concn = 65.9 g/L and A280 = 53.2), four times (xylose concn = 77.3 g/L and A280 = 82.2) and six times (xylose concn = 114.3 g/L and A280 = 185). Xylitol fermentations using these concentrated hydrolysates were performed in order to evaluate the effects of the concentration ratio of hydrolysate on xylitol fermentation. Figure 9 shows the time courses of cell

Figure 10. Microbial xylitol production using concentrated corn cob hydrolysate by two-step fermentation. OTR = 0.84 mmol-O2/L·h (kLa = 3.51 h−1, agitation 100 rpm, and aeration 0.15 vvm). Arrow indicates the timing of concentrated hydrolysate addition. Symbols: ●, dry cell concentration; ○, xylose concentration; ▲, xylitol concentration.

fermentation was started using 2 L of normal corn cob hydrolysate medium with 31.9 g/L of xylose and cultivated for 36 h with the OTR of 0.84 mmol-O2/L·h. After the successful cell growth, 800 mL of concentrated corn cob hydrolysate medium (concentration ratio = 7, xylose concentration = 199 g/L, A280 = 280) was poured into the fermentor at 36 h. After the addition of the concentrated hydrolysate, the average concentration of xylose and A280 in the fermentor became 79.6 g/L and 111, respectively. As can be seen, xylose was steadily consumed after the addition of the concentrated corn cob hydrolysate medium, followed by the successful production of xylitol. The final xylose concentration, Yx, and P were 49.0 g/L, 0.62, and 0.314 (g/L)·h after 192 h cultivation, respectively. These results are sufficiently competitive compared with previous studies. The cell concentration was kept constant at a low concentration after 36 h, which indicates that the OTR was properly controlled so as to maximize xylitol production and to minimize unnecessary cell growth. The A280 of 111 in the mixed hydrolysate seemed too high for xylitol production according to Figure 4. However, despite this high A280 value, successful xylitol production was achieved because the cell growth was achieved at the initial stage of the fermentation. Thus, two-step fermentation, using a concentrated corn cob hydrolysate under the controlled OTR condition, was found to be effective for successful microbial xylitol production. Process Configuration for Microbial Xylitol Production. On the basis of the data obtained in this study, the process configuration showing material balance (dry weight base) for xylitol production is summarized in Figure11. Basically, three main unit operations are required for xylitol production from corn cobs: hydrolysis, adsorption, and fermentation. In this process, 100 kg of corn cobs was hydrolyzed to produce approximately 25 kg of xylose and the remainder became 53 kg of corn cob residues and 22 kg of soluble waste. Subsequently 25 kg of xylose was converted to

Figure 9. Time courses of cell growth and xylitol production using concentrated corn cob hydrolysate: (a) cell concentration, (b) xylitol concentration. Symbols for concentration ratio: ●, 1; □, 2; Δ, 3; □, 4; ○, 6.

concentration (a) and xylitol concentration (b) using concentrated hydrolysates. Cell growth was not affected until three times the concentration, but gradually decreased at four times the concentration, and finally no cell growth was observed at six times the concentration. This is due to the toxic effect by the concentrated inhibitors in the concentrated corn cob hydrolysate. Xylitol production was also achieved at four times the concentration; however, the lag phase for xylitol production was prolonged. Thus, xylitol production using concentrated hydrolysate was inhibited as the concentration 10013

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(7) Rivas, B.; Dominguez, J. M.; Dominguez, H.; Parajo, J. C. Bioconversion of Posthydrolysed Autohydrolysis Liquors: An Alternative for Xylitol Production from Corn Cobs. Enzyme Microb. Technol. 2002, 31, 431−438. (8) Tellez-Luis, S. J.; Ramirez, J. A.; Vazquez, M. Mathematical Modeling of Hemicellulosic Sugar Production from Sorghum Straw. J. Food Eng. 2002, 52, 285−291. (9) Vandeska, E.; Kuzmanova, S.; Jeffries, T. W. Xylitol Formation and Key Enzyme Activities in Candida boidinii under Different Oxygen Transfer Rates. J. Ferment. Bioeng. 1995, 80, 513−516. (10) Parajo, J. C.; Dominguez, H.; Dominguez, J. M. Biotechnological Production of Xylitol. Part 3: Operation in Culture Media Made from Lignocellulose Hydrolysates. Bioresour. Technol. 1998, 66, 25−40. (11) Winkelhausen E, Amartey S. A., Kuzmanova S 2004. Xylitol Production from D-xylose at Different Oxygen Transfer Coefficients in a Batch Bioreactor. Eng. Life Sci. 2004, 4, 150-154 (12) Horiuchi, J.; Tabata, K.; Kanno, T.; Kobayashi, M. Continuous Acetic Acid Production by a Packed Bed Bioreactor Employing Charcoal Pellets Derived from Waste Mushroom Medium. J. Biosci. Bioeng. 2000, 89, 126−130. (13) Agriculture & Livestock Industries Corporation, http://sugar. alic.go.jp/tisiki/ti_0503.htm (Accessed March 10, 2012).

Figure 11. Process configuration with material balance for microbial xylitol production from corn cobs (dry weight base).

15−18 kg of xylitol depending on culture conditions. According to the material balance in Figure 11, it should be noted that only 5 to 60 000 tons of agricultural wastes such as corn cobs have the potential to produce more than 7500−9000 tons of xylitol, which is enough to cover the overall xylitol demand in Japan.13 As can be seen in Figure 11, 53% of corn cobs still remain unutilized as corn cob residue which mainly contains cellulose. Further research is required to utilize them effectively.

5. CONCLUSIONS Enhanced process for microbial xylitol production from corn cob hydrolysates using Candida magnoliae was investigated. About 25 g-xylose/L solution was obtained from 100 g-corn cobs/L solution by acid hydrolysis and the xylitol production of approximately 18 g-xylitol/L using the corn cob hydrolysate was successfully carried out in combination with the removal of inhibitors by charcoal pellets. The xylitol production occurred only when the OTR ranged from 0.8 mmol-O2/L·h to 4.7 mmol-O2/L·h. It was demonstrated that a two-step fermentation using a concentrated corn cob hydrolysate under the control of OTR was effective for concentrated xylitol production.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +81-257-26-9385. Tel.: +81-157-26-9415. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by a grant-in-aid for scientific research by JSPS (No. 20560724) and JST (No. AS221Z02852E). REFERENCES

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