Xylitol is produced from D-xylose as a metabolic intermediate in many xylose utilizing microorganisms in two ways: D-xylose is directly converted to xylitol by NADPH- dependent aldehyde reductase (EC 1.1.1.21), or D-xylose is first isomerized to D-

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xylulose by D-xylose isomerase (EC 5.3.1.5) and then reduced to xylitol by NADH — dependent xylitol dehydrogenase (EC 1.1.1.9) (Fig. 1) (<8). Many yeasts and mycelial fungi possess the enzyme xylose reductase which catalyzes the reduction of xylose to xylitol as a first step in xylose metabolism (9). Xylitol production is a relatively common feature among xylose-utilizing yeasts (10). In xylose fermenting yeasts, the initial reactions of xylose metabolism are the major limiting steps (77). This results in the accumulation of xylitol in culture medium, the degree varying with the culture conditions and the yeast strain used (72).

Onishi and Suzuki (73) examined 58 yeast strains belonging to the genera Saccharomyces, Debaryomyces, Pichia, Hansenula, Candida, Torulopsis, Kloeckera, Trichosporon, Cryptococcus, Rhodotorula, Monilia and Torula for polyalcohol production from pentose sugars such as D-xylose, L-arabinose and D-ribose. Candida polymorpha dissimilated aerobically these three pentoses and produced xylitol from xylose, L-arabitol from L-arabinose and ribitol from D-ribose at the yield of 30-40% of sugar consumed. Gong et al. (10) screened 20 strains of Candida belonging to 11 different species, 21 strains of Saccharomyces belonging to 8 species and 8 strains of Schizosaccharomyces pombe for their ability to convert xylose to xylitol. Significant quantities of xylitol were produced by all these yeast strains. Barbosa et al. (14) screened 44 yeasts from five genera (Candida, Hansenula, Kluyveromyces, Pichia and Pachysolen) for conversion of xylose to xylitol. All but two of the strains produced some xylitol with varying rates and yields. The best xylitol producers were localized largely in the species C. guilliermondii and C. tropicalis. Seven strains of C. guilliermondii from diverse isolation sources produced xylitol efficiently when grown in a simple medium containing 5.0% xylose within 24 h (75). However, xylitol essentially disappeared from all the cultures within 72 h. Sirisansaneeyakul et al. (16) selected C. mogii ATCC 18364 as an efficient xylitol producer (Yp/s = 0.62 g/g) from 11 strains of D-xylose utilizing yeasts. Debaryomyces hansenii was an efficient xylitol producer exhibiting a xylitol/ethanol ratio above 4 and a carbon conversion of 54% for xylitol (77). C. entomaea and Pichia guilliermondii produced 0.51 and 0.43 g xylitol/g xylose at pH 5.0 and pH 4.0, respectively and 34°C (18). Ambrosiozyma monospora NRRL Y-1484 produced about 22 g xylitol and 18 g ethanol from 100 g xylose per L when grown at 25°C under moderate aeration (79). A strain of C. tropicalis converted xylose to xylitol and did not produce ethanol (20). Significant quantity of xylitol was produced during ethanol fermentation by Pachysolen tannophilus (21t22) and Kluyveromyces cellobiovorus (23). Various thermo-tolerant yeasts have also been evaluated for the bioconversion of xylose into xylitol (24). Xylitol production ranged from 0.83 to 4.69 g from 10 g xylose.

A fungal strain of Petromyces albertensis produced xylitol when grown in a medium containing D-xylose (25). A large amount (36.8 g/L) of xylitol was obtained from a D-xylose (100 g/L) medium containing ammonium acetate and yeast extract at an initial pH of 7.0. The production of xylitol from xylose has been studied with bacteria such as Enterobacter liquefaciens (26, 27), Corynebacterium sp. (28, 29), and Mycobacterium smegmatis (30).

Onishi and Suzuki (37) screened 128 yeast strains for their ability to produce xylitol from glucose. They reported a sequential fermentation process of xylitol production from glucose (glucose^ D-arabitofcOD-xylulose^xylitol) without isolation

and purification of the intermediates, and the yield of xylitol was 11% from glucose. D. hansenii converted glucose to D-arabitol, Acetobacter suboxydans oxidized D-arabitol almost quantitatively to D-xylulose and C. guilliermondii var. soya reduced D-xylulose to xylitol. Table I summarizes production of xylitol from xylose by some Candida species.

Table L Production of xylitol from xylose by some Candida species

Yeast Fermentation Xylose Xylitol Xylitol

Time (g/L) (g/L) Yield

(h) (g/g)

Factors Affecting Xylitol Production

Medium Components. The conversion of xylose to xylitol by C. guilliermondii was affected by the nutrient source (14). Horitsu et al. (36) studied the influence of culture conditions on xylitol formation by C. tropicalis and optimized the volumetric xylitol production rate by the Box-Wilson method. In this respect, initial xylose concentration, yeast extract concentration and kLa were chosen as independent factors in 23-factorial design. Optimal product formation (г хуШ =2.67 g/L/h, C xytitol =110 g/L) was obtained at 172 g/L xylose, 21 g/L yeast extract and a kLa of 451.5 L/h.

Xylose Concentration. Initial xylose concentration is an important factor to obtain high xylitol production. Meyrial et al. (34) reported that an increase in the initial xylose concentration from 10 g/L to 300 g/L led to activation of xylitol production by C. guilliermondii. The xylitol yield increased gradually with substrate, the highest xylitol yield (0.75 g/g xylose) was obtained at a substrate concentration of 300 g/L. However, the growth of the yeast was gradually inhibited by an increase in initial xylose concentration in the medium. Both the yield and specific rate of cells production declined when xylose concentration initially present in the culture increased. Chen and Gong (32) reported a xylitol yield of 84.5% of theoretical and a maximum production rate of0.269 g/g/h from 249 g/L xylose by Candida sp. B-22. C. tropicalis HXP2 (37) and C. boidinii (33) produced the highest amounts of xylitol (144 g/L and 39 g/L, respectively) at respective values of substrate concentration of 200 g/L and 100 g/L. Dahiya (25) reported maximum xylitol production by P. albertensis was 36.8 g/L at the initial xylose concentration of 100 g/L. Xylitol production declined when the initial xylose concentration was increased to 150 g/L. This might be due to an osmotic effect on cells of P. albertensis or to substrate repression of xylose metabolizing enzymes.

When C. mogii was grown under oxygen-limited conditions in synthetic medium containing different concentrations of xylose (5-53 g/L), the xylitol formation rates showed a hyperbolic dependency on the initial substrate concentration (76).

Vandeska et al. (38) reported that an increase in initial xylose concentration induced xylitol production in C. boidinii but simultaneously acted as a growth inhibitory substrate leading to a long fermentation time. To overcome these problems, fed batch cultures were then used in which higher xylitol yields (0.57-0.68 g/g) and production rates (0.32-0.46 g/L/h) were obtained as compared with a batch process (39). A fed batch process with highest initial xylose concentration (100 g/L) and lowest level of aeration in the first phase, resulted in the highest yield of xylitol (75% of theoretical). A potentiometric biosensor for xylose to monitor fermentative conversion of xylose to xylitol was devised (40).

Presence of Other Sugars. Yahashi et al. (41) investigated the effect of glucose feeding on the production of xylitol from xylose by C. tropicalis. In the bench-scale fermenter (3 L scale) experiment, xylitol was produced at up to 104.5 g/L at 32 h cultivation and a yield of 0.82 (g/g xylose consumed) which is 1.3 times higher than that without glucose feeding. Meyrial et al. (34) evaluated the ability of C. guilliermondii to ferment non-xylose sugars such as glucose, mannose, galactose and L-arabinose commonly found in hemicellulose hydrolyzate. The strain did not convert glucose, mannose and galactose into their corresponding polyalcohol but only to ethanol and cell mass. Arabinose was converted to arabitol. Silva et al. (42) studied batch fermentation of xylose for xylitol production in stirred tank bioreactor. The efficiency of substrate conversion to xylitol was 66% in a medium containing xylose but decreased to 45% in a medium containing xylose and glucose. Vandeska et al. (39) investigated xylitol production by C. boidinii in fed batch fermentations with xylose (50, 100 g/L) and a mixture of glucose (25 g/L) and xylose (25 g/L). All fermentations were initially batch processes with high levels of aeration and rapid production of biomass. Faster growth occurred when a mixture of glucose and xylose was used instead of xylose. Glucose was assimilated first and maximal xylitol production was 39-41 g/L, compared with 46.5 and 59.3 g/L with xylose alone.

Nitrogen Sources and Organic Nutrients. Dahiya (25) studied the effect of 8 ammonium salts and 4 organic nitrogen sources on the production of xylitol from xylose by P. albertensis. Ammonium acetate was most effective for xylitol production. Yeast extract was the most suitable organic nutrient for enhancement of xylitol production. Lu et al. (35) investigated the effect of nitrogen sources [asparagine, casein hydrolyzate, glycine, Traders protein, yeast extract, urea, NaN03, NH 4N03, (NH4) 2S04, NH4C1, NH4H2P04 ] on xylitol production from xylose in shake flasks by an efficient xylitol producing yeast, Candida sp. L-102. Different nitrogen sources influenced xylitol production rate, average specific productivity, and xylitol yield. Maximum xylitol production (100 g/L of xylitol from 114 g/L of xylose) was obtained with urea (3 g/L) as the nitrogen source. Silva et al. (43) evaluated the xylose conversion into xylitol by C. guilliermondii in semi-synthetic media supplemented with different nitrogen sources [urea, NK^Cl, (NH4)2SOJ in a ratio C/N equal 25.6. The type of nitrogen source did not influence this bioconversion and the xylitol yield was around 80%. On the other hand,

Barbosa et al. {14) reported that the use of urea led to higher xylitol productivity by C. guilliermonctii than with ammonium sulfate, and supplementation of urea with casamino acids improved performance over urea alone only slightly. Yeast extract improved yields, but only slightly.

Magnesium and Biotin. Mahler and Guebel {44) studied the influence of Mg+2 concentration on growth, ethanol and xylitol production from xylose by Pichia stipitis NRRL Y-7124. Under constant oxygen uptake rate, biomass/xylose and biomass/Mg+2 yields increased with Mg+2 concentration with a maximum value at 4 mm. Ethanol was the main product formed. At low Mg+2 levels (1 mM), 49% of carbon flux to ethanol was redirected to xylitol production, and was correlated with intracellular accumulation ofNADH.

Lee et al. {45) reported that the relative amount of ethanol and xylitol accumulated in xylose fed aerobic batch cultures of P. tannophilus and C. guilliermondii depended on the limitation by biotin. In high biotin containing media (2 pg/L) P. tannophilus favored ethanol production over that of xylitol while C. guilliermondii favored xylitol formation.

Methanol Supplementation. Dahiya (25) reported that addition of 1% methanol to the medium with 100 g/L xylose increased the xylitol production from 36.8 g/L to 39.8 g/L by P. albertensis. No significant difference in fungal biomass and xylulose accumulation was observed and only 0.015% methanol was consumed. This could be due to the oxidation of methanol to yield NADH which would enhance the reduction of xylose and xylulose to xylitol. Vongsuvanlert and Tani {33) reported about 18 and 26% increase in xylitol production from xylose in presence of 1 and 2% methanol, respectively by C. boidinii. This is also the case with the production of sorbitol from glucose and iditol from L-sorbose by C. boidinii {46).

Initial Cell Density. Cao et al. {47) investigated the effect of cell density on the production of xylitol from xylose by Candida sp. B-22. The rate of xylitol production from xylose increased with increasing yeast cell density. At high initial yeast cell concentration of 26 mg/ml, 210 g/L of xylitol was produced from 260 g/L of xylose after 96 h of incubation with a yield of 81% of the theoretical value. Vandeska et al. {38) reported that high initial cell densities improved xylitol yields and specific production rates of xylitol by C. boidinii. The susceptibility of wood hydrolyzate to fermentation by D. hansenii NRRL Y-7426 was strongly dependent on the initial cell concentration {48).

Oxygen Supply. A variety of yeasts such as Candida, Hanensula, Klyveryomyces, and Pichia require oxygen for sugar uptake {49) and availability of oxygen has significant influence on xylose fermentation by these yeasts {10). However, oxygen limitation is the main factor stimulating the formation of xylitol {50). Roseiro et al (77) reported that xylitol production by D. hansenii required semianaerobic conditions. The presence of oxygen enhanced NADH oxidation and a high NAD+/NADH ratio led to xylitol oxidation to xylulose; therefore, less xylitol was accumulated. Thus the yield of xylitol depended strongly on the oxygen transfer rate (57). Horitsu et al. (56) reported that higher level of dissolved oxygen is required only at the earlier phase of cultivation and afterwards it should be decreased to the lower level of respiration by the yeast. Barbosa et al. {14) reported that increasing oxygen limitation led to increased xylitol productivity and decreased ethanol production with C. guilliermondii. Nolleau et al. (77) evaluated the ability of C. guilliermondii and C. parapsilosis to ferment xylose to xylitol under different oxygen transfer rates. In C. guilliermondii, a maximal xylitol yield of 0.66 g/g was obtained when oxygen transfer rate was 2.2 mmol/1* h. Optimal conditions to produce xylitol by C. parapsilosis (0.75 g/g) arose from cultures at pH 4.75 with 0.4 mmols of oxygen/1* h. The oxygen is not only an important factor to optimize the xylitol production but it is also an essential component for xylose assimilation. When aerobic batch cultures of C. guilliermondii and C. parapsilosis provided with xylose, were shifted to anaerobic conditions, the xylose concentration remained at a constant level and all metabolic activities stopped immediately. C. mogii produced xylitol from xylose under aerobic and oxygen-limiting conditions, but not without oxygen (76). Xylose conversion into xylitol by C. guilliermondii FTI20037 was investigated in a stirred tank bioreactor at different stirring rates {42). Maximal xylitol production (22.2 g/L) was obtained at 30°C, with an aeration rate of 0.46 wm using a stirring rate of 300 per min (lqa = 10.6 h’1). An increase of kLa caused an increase in the consumption of xylose in detriment to xylitol formation. Winkelhausen et al. (52) investigated xylitol formation by C. boidinni in oxygen limited chemostat culture. The production of xylitol by the yeast occurred under conditions of an oxygen limitation at specific oxygen uptake rates lower than 0.91 mmol/gh. The efffect of aeration on xylitol production from xylose by some yeasts is summarized in Table П.