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Polyols production during single and mixed substrate fermentations in Debaryomyces hansenii
Bioresource Technology 71 (2000) 245±251
Polyols production during single and mixed substrate fermentations in Debaryomyces hansenii F.M. Gõrio*, C. Amaro, H. Azinheira1, F. Pelica, M.T. Amaral-Collacßo INETI, IBQTA-Departamento de Biotecnologia, Unid. de Microbiologia Industrial e Bioprocessos, Estrada do Pacßo do Lumiar, 22, 1649-038 Lisboa, Portugal Received 30 December 1998; received in revised form 4 May 1999; accepted 15 May 1999
Abstract The kinetics of polyols production by Debaryomyces hansenii was studied both on single substrate and mixed substrate-containing media. From the single substrate experiments, polyols (xylitol and arabitol) and ethanol were produced from pentose sugars while ethanol was produced in signi®cant amounts only from glucose-grown D. hansenii. The maximal xylitol volumetric productivity (Qxylitol ), 0.28 g lÿ1 hÿ1 was obtained from D -xylose, whereas the maximal arabitol volumetric productivity (Qarabitol ), 0.04 g lÿ1 hÿ1 was observed with arabinose. When D. hansenii was cultivated with mixed substrates, a simultaneous sugar consumption pattern occurred both for glucose/arabinose and xylose/arabinose mixtures. The addition of low amounts of xylose to an arabinose medium led to a fourfold increase in the arabitol volumetric productivity: 0.17 g lÿ1 hÿ1 . Conversely, glucose addition had no eect on arabitol production. Xylitol was the main polyol produced for all tested cultivation conditions by D. hansenii. An enzymatic study of the ®rst two xylose-catabolic enzymes in glucose and xylose-grown D. hansenii revealed that both enzymes were induced by D -xylose. Glucose caused total inhibition of xylitol dehydrogenase, whereas xylose reductase was only partially repressed. Ó 1999 Elsevier Science Ltd. All rights reserved. Keywords: Mixed substrates; Pentoses; Polyols; Debaryomyces hansenii; Polyol-producing yeast
1. Introduction The obtaining of chemicals from lignocellulosic bioconversion processes predicates the total use of sugars obtained by cellulose and hemicellulose hydrolysis. Ethanol can be easily produced by yeasts during fermentation of glucose. This hexose is the major sugar obtained from cellulose hydrolysis of the lignocellulosic material. Hemicellulose breakdown by acid or enzymatic hydrolysis gives a complex mixture of sugars, which includes pentoses (xylose, arabinose), hexoses (glucose, mannose, galactose) and, additionally signi®cant amounts of uronic acids, acetate, furfural and other aromatic compounds (Ladisch and Svarczkopf, 1991). Depending on the raw material and on the hydrolysis conditions, pentoses (D -xylose and L -arabinose) are the
* Corresponding author. Tel.: +351 1 716 51 41; fax: +351 1 716 09 01; e-mail: [email protected] 1 Current address: Instituto de Investigacß~ ao Cientõ®ca Tropical, CEPTA, Tapada da Ajuda, 1301 Lisboa Codex, Portugal.
major components present in the hemicellulosic hydrolysates, particularly from hardwoods and cereal crops. Ethanol is not the sole chemical obtained from biological conversion of lignocellulosic materials, and other chemicals (e.g., xylitol and arabitol), which are currently manufactured through the chemical reduction of xylancontaining materials (Nigam and Singh, 1995), can be obtained from xylose metabolism by yeasts such as Candida guillermondii (Roberto et al., 1996), Candida parapsilosis (Furlan et al., 1991), Candida tropicalis (Horitsu et al., 1992), Candida boidinii (Vandeska et al., 1995) and Debaryomyces hansenii (Roseiro et al., 1991; Paraj o et al., 1996). The biological production of arabitol has been far less studied. The most promising arabitol-producing yeasts, Candida entomaea and Pichia guillermondii, yield up to 0.7 g lÿ1 of L -arabitol per gram of L -arabinose (Saha and Bothast, 1996). However, there is a lack of knowledge of how these yeasts respond to the presence of more than one sugar in the growth media for polyol production, which is a usual feature of media from hemicellulosic hydrolysates. This work reports comparative kinetic data obtained from batch cultivation with D. hansenii when
0960-8524/00/$ ± see front matter Ó 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 0 - 8 5 2 4 ( 9 9 ) 0 0 0 7 8 - 4
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hemicellulosic sugars were used either as single or mixed carbon and energy sources. The in¯uence of substrates on the pattern of polyols production by the yeast is also discussed. The data suggested that xylose and a xylose/arabinose mixture were the best substrates for xylitol and arabitol production, respectively.
2.5. Enzyme assays
2. Methods
Xylose reductase (XR, E.C. 1.1.1.21) and xylitol dehydrogenase (XDH, E.C. 1.1.1.9) enzyme activities, were assayed as described by Gõrio et al. (1989). Reaction rates were corrected for endogenous NAD(P)H consumption or production in crude extracts. Enzyme units (U) were de®ned as lmoles of NAD(P) reduced or NAD(P)H oxidized per min at room temperature. Speci®c activities were expressed as mU mgÿ1 of protein.
2.1. Organism
2.6. Analytical methods
D. hansenii was isolated by a batch enrichment technique from sugar cane and maintained as described before (Roseiro et al., 1991).
D -xylose, D -glucose, L -arabinose, xylitol and arabitol were determined by HPLC using a Sugar-Pak (Waters, Mildford, Mass., USA) column and a refractive index detector. The column temperature was 75°C and a ¯ow rate of 0.5 ml minÿ1 was used. Ethanol was measured by gas chromatography using a Carbowax 1540 column (2 m ´ 3 mm, 80/100 mesh) and a ¯ame ionization detector. Column, injector and detector temperatures were 50°C, 200°C and 250°C, respectively. Methanol (10 mm) was used as internal standard. Biomass was measured by gravimetry after sample ®ltration (0.45 lm), washing with distilled water and drying overnight to a constant weight at 105°C. Protein was determined by using the method of Lowry et al. (1951). Bovine serum albumin was used as standard protein.
2.2. Single and mixed-substrate fermentation Cultures were grown on a chemically-de®ned medium as described by Du Preez and van der Walt (1983), except that casamino acids were replaced by (NH4 )2 SO4 , (7.5 g lÿ1 ). The carbon source was supplied as single or mixed sugars which were sterilized separately. Yeast growth assays were carried out in 750 ml Erlenmeyer ¯asks containing 80 ml of the above medium and incubated in an orbital shaker set at 150 rpm and 30°C. The initial pH was 5.5. Inocula (5%, v/v) were grown in 500 ml Erlenmeyer ¯asks containing 80 ml of the de®ned medium and incubated under identical conditions for 16 h. All growth tests here presented were carried out in triplicate and the results shown are the average of at least two independent values. 2.3. Enzyme induction experiment Batch cultivations on the above chemically-de®ned medium containing glucose as single carbon source were run at 30°C in a Biola®tte (St. Germain en Laye, France) bioreactor of 3 l working volume and 1 vvm aeration rate. The pH was controlled at 5.5 with 1M NaOH and the agitation speed was 150 rpm. The inoculum (5%, v/v) was grown on 2% xylose for 16 h. A xylose pulse (90 g lÿ1 ) was carried out immediately after glucose depletion, and the growth and products accumulation were monitored for an additional 22 h. 2.4. Preparation of cell free-extracts Yeast samples from dierent stages of the bioreactor-grown cultures were harvested by centrifugation as described elsewhere (Gõrio et al., 1989). The cellfree extracts used for enzyme assays were prepared after cell disruption using the glass beads method (Ciriacy, 1975).
2.7. Fermentation parameter calculations The volumetric productivities were calculated by linear regression from the maximal slopes of the substrate (product) vs. time curves (Du Preez et al., 1989). The speci®c productivities were calculated by linear regression from the maximal slopes of dP (or dS)/X vs. time curves. 3. Results 3.1. Batch cultivation on single sugars with Debaryomyces hansenii Because a typical hemicellulosic hydrolyate contains a mixture of pentose and hexose sugars, a comparative study of growth pro®les of D. hansenii using mixed as well as single sugars was carried out. Fig. 1 shows the pro®les of sugar consumption rate, biomass and extracellular product formation rate during the batch growth of D. hansenii on dierent hemicellulosic sugars. As all shake-¯ask cultivations have a limited supply of oxygen, we use throughout this work the term ``semi-aerobic'' for the growth in such experimental conditions. For glucose-grown D. hansenii, only ethanol was produced, which indicates a mixed respiro-fermentative
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metabolism (Fig. 1(A)). Polyols were not detected for glucose-grown D. hansenii. When glucose concentration reached a value close to 3 g lÿ1 , ethanol was co-oxidised as second energy source. Conversely, arabinose-grown D. hansenii had a fully oxidative metabolism since ethanol was not detected (Fig. 1(B)). The maximal arabitol concentration (2.86 g lÿ1 ) was threefold higher than the maximal xylitol concentration (0.94 g lÿ1 ) with both maxima occurring at 120 h of cultivation time. Comparing the ethanol/hexose co-oxidation observed before (Fig. 1(A)), for arabinosegrown D. hansenii both polyols were consumed only after complete pentose utilisation in the cultivation medium. Xylose-grown D. hansenii utilised the carbon source mainly for growth purposes. Xylitol was the only polyol detected extracellularly (Fig. 1(C)). The maximal xylitol volumetric productivity was 0.28 g lÿ1 hÿ1 (Table 1). Again, xylitol was oxidized as carbon source when xylose concentration was below 3 g lÿ1 . Ethanol was produced in only residual amounts. Table 1 shows the fermentation parameters calculated for D. hansenii grown on single carbon sources. ÿ1 D -glucose supported the highest growth rate, 0.32 h . L -arabinose and D -xylose exhibited similar growth rates but of low magnitude compared to glucose. The maximal arabitol yield obtained for the L -arabinose medium was rather low, 0.10 g gÿ1 , compared to the maximal xylitol yield (0.59 g gÿ1 ) obtained for the xylose medium (Table 1). Under semi-aerobic conditions ethanol was produced in signi®cant amounts by D. hansenii only when glucose was the carbon and energy source. 3.2. Batch cultivation on a glucose/arabinose mixture of D. hansenii
Fig. 1. Batch cultivation pro®le of Debaryomyces hansenii grown semiaerobically in shake ¯asks on (A) D -glucose, (B) L -arabinose and (C) D -xylose. Glucose (s); Arabinose (n); Xylose (h); Ethanol (d); Arabitol (m); Xylitol (.); Biomass (n). The inocula were grown on glucose, arabinose and xylose, respectively.
When mixed sugars in a hexose/pentose ratio of 0.2 (w/w) were present in the initial culture medium, both sugars were simultaneously used as carbon and energy source for yeast growth, with no visible diauxic growth (Fig. 2). During the ®rst 10 h, glucose and arabinose were consumed simultaneously producing mainly biomass. However, arabitol started to accumulate before glucose depletion. After glucose exhaustion, xylitol was also formed from L -arabinose metabolism, although arabitol was the main metabolic product. The maximal volumetric arabitol and xylitol productivities were 0.03 and 0.02 g lÿ1 hÿ1 , respectively (Table 2). The arabitol yield (0.19 g gÿ1 ) was threefold higher than the xylitol yield (0.06 g gÿ1 ). Ethanol was not produced since all the glucose was rapidly consumed during the early stages of yeast growth where the oxygen limitation was still not drastic. When L -arabinose concentration had decreased to about 6 g lÿ1 , xylitol was co-metabolised with arabinose. Arabitol was also consumed, but at a slower rate (Fig. 2(B)).
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Table 1 Batch fermentation parameters by D. hansenii grown on glucose, arabinose and xylose Parameter ÿ1
l (h ) Yx=s (g gÿ1 ) YEtOH=s (g gÿ1 ) Yxylitol=s (g gÿ1 ) Yarabitol=s (g gÿ1 ) Qsubstrate (g lÿ1 hÿ1 ) qsubstrate (g gÿ1 hÿ1 ) Qxylitol (g lÿ1 hÿ1 ) Qarabitol (g lÿ1 hÿ1 ) Qethanol (g lÿ1 hÿ1 ) qxylitol (g gÿ1 hÿ1 ) qarabitol (g gÿ1 hÿ1 ) qethanol (g gÿ1 hÿ1 )
Carbon source Glucose
Arabinose
Xylose
0.32 0.55 0.23 ND ND 2.95 0.25 ND ND 1.04 ND ND 0.05
0.24 0.43 ND 0.03 0.10 0.39 0.06 0.01 0.04 ND 0.001 0.003 ND
0.25 0.31 0.01 0.59 ND 0.52 0.25 0.28 ND 0.01 0.06 ND 0.0002
ND ± not detected.
3.3. Batch cultivation on a xylose/arabinose mixture of D. hansenii The polyols produced by D. hansenii are derived only from pentoses, as shown in the previous single-substrate growth kinetics (Fig. 1). Therefore, we tried to increase the polyols production by using a initial mixture of xylose/arabinose (ratio of 0.2, w/w) in the growth medium composition (Fig. 3). Again, both sugars were simultaneously assimilated, reaching identical maximal volumetric consumption rates (Table 2). However, the arabinose volumetric consumption rate Qarabinose (0.09 g lÿ1 hÿ1 ) was only 47% of the Qxylose , 0.200 g lÿ1 hÿ1 during the ®rst 26 h of yeast growth. A similar arabinose consumption rate (0.20 g lÿ1 hÿ1 ) was obtained only after 60 h of cultivation time when xylose concentration became residual in the medium (<1 g lÿ1 ). Besides biomass, only polyols accumulated in xylose/arabinose D. hansenii cultures (Fig. 3). Arabitol was the main polyol produced and the maximal volumetric and speci®c productivities, 0.17 g lÿ1 hÿ1 and 0.013 g gÿ1 hÿ1 , respectively, were fourfold higher than the maximal volumetric and speci®c productivities obtained for the previous sugar mixture, 0.03 and 0.003 g gÿ1 hÿ1 , respectively (Table 2). The xylitol speci®c productivity was only 8% of that observed before for a xylose-grown D. hansenii culture (Fig. 1; Table 1). 3.4. Enzymatic study of the xylose-catabolic enzymes from D. hansenii In order to evaluate the inducible or constitutive type of the xylose catabolic enzymes, D. hansenii was grown in glucose medium for the ®rst 25 h then, just after glucose depletion, a pulse (90 g lÿ1 ) of xylose was added. The activities of the xylose-catabolic enzymes, NADPHlinked xylose reductase (XR) and NAD -linked xylitol
Fig. 2. Batch cultivation pro®le of D. hansenii grown semi-aerobically in shake ¯asks on a mixture of glucose and arabinose (ratio of 0.2, w/w). The inoculum was grown in L -arabinose. Symbols as in Fig. 1.
dehydrogenase (XDH), were measured during the exponential growth phase on glucose and immediately before the xylose pulse. After the pulse, additional enzyme activities were measured for the remainder of the growth phase (Table 3). Although a residual XR activity with D -xylose was detected when the carbon source was glucose (up to 59 mU/mg protein), 2 h after the xylose pulse the XR activity with D -xylose increased about sixfold. The increase of XR activity with arabinose was ®vefold in the same period. XDH enzyme was active only when xylose was present in the growth medium. The enzyme was speci®c for NAD and xylitol. When NADP and arabitol were used as substrates no activity
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Table 2 Parameters of mixed substrate fermentations by D. hansenii grown on glucose/arabinose and xylose/arabinose mixtures Carbon sources
l (hÿ1 ) Yx=s (g gÿ1 ) Yxylitol=s (g gÿ1 ) Yarabitol=s (g gÿ1 ) Qarabinose (g lÿ1 hÿ1 ) Qglucose (g lÿ1 hÿ1 ) Qxylose (g lÿ1 hÿ1 ) qarabinose (g gÿ1 hÿ1 ) qglucose (g gÿ1 hÿ1 ) qxylose (g gÿ1 hÿ1 ) Qxylitol (g lÿ1 hÿ1 ) Qarabitol (g lÿ1 hÿ1 ) qxylitol (g gÿ1 hÿ1 ) qarabitol (g gÿ1 hÿ1 )
Glucose/arabinose (ratio 0.2)
Xylose/arabinose (ratio 0.2)
0.17 0.24 a (0.64) 0.06 0.19 0.27 0.64 NA 0.03 0.02 NA 0.02 0.03 0.004 0.003
0.16 0.99 c 0.67 d (0.47) e 1.05 f 0.20 g (0.09) h NA 0.20 0.03 NA 0.07 0.03 0.17 0.005 0.013
b
a
Cell yield for glucose + arabinose; Cell yield for arabinose; c For consumed s xylose + arabinose; d For consumed s xylose; e For consumed s xylose + arabinose; f Assuming that all arabitol was produced from arabinose; g Calculated between 60 and 140 h of growth; h Calculated during the ®rst 26 h of growth; NA ± not applicable.
Fig. 3. Batch cultivation pro®le of D. hansenii grown semi-aerobically in shake ¯asks on a mixture of xylose and arabinose (ratio of 0.2, w/w). The inoculum was grown in L -arabinose. Symbols as in Fig. 1.
b
was detected (data not shown). This result closely agrees with the previously-reported substrate speci®city for the puri®ed XDH from D. hansenii (Gõrio et al., 1996). In the present experiments glucose produced total inactivation of pre-existing forms of XDH enzyme in D. hansenii, when the enzyme had been previously induced during the time course of a xylose-grown inoculum (Table 3). Moreover, after the xylose pulse, the XR and XDH activity measurements suggested that there was a dierent enzyme induction rate. XR was induced in a shorter time than to XDH, since 15 min after the xylose pulse (25.3 h) XR activity had increased by 29% and 40%, when assayed using xylose and arabinose, respectively, whereas XDH was still not detected (Table 3). 4. Discussion 4.1. Physiology of polyol synthesis by yeasts This paper presents the ®rst comprehensive study of the kinetics of xylitol and arabitol production by D. hansenii, a food-spoilage yeast. Polyol production by yeasts is generally regarded as undesirable, since polyols decrease the ethanol yield (Nunez et al., 1989). However, recent reports (Lu et al., 1995; Domõnguez et al., 1997) describing high yields for xylitol production by yeasts have turned interest to the study of the physiological and environmental parameters underlying the overpro-
Table 3 Crude extract enzyme activities from a batch fermenter cultivation of D. hansenii grown on D -glucose before and after a xylose pulse (90 g lÿ1 ). The pulse was added after 25 h of cultivation time Time (h) Enzyme activity (mU mgÿ1 protein)
9.5 24.8 25.3 27 47
Xylose reductase NADPH-linked with substrate
Xylitol dehydrogenase NAD -linked with substrate
D -xylose
L -arabinose
Xylitol
39 59 76 370 62
64 81 113 398 58
ND ND ND 468 ND
ND ± not detected.
duction of xylitol and other polyols by these organisms. Although the physiology of synthesis of polyols by yeasts is still not fully understood it appears to be related to the yeast strain, carbon source and oxygen availability (Ligthelm et al., 1988). Xylitol formation from xylose is believed to be due to a redox imbalance in cofactor production of the ®rst two oxidoreductive steps of xylose metabolism resulting in a metabolic NADH surplus under oxygen-limited growth conditions and leading to xylitol accumulation (PreziosiBelloy et al., 1997). The production of arabitol from arabinose is likely to be more complex, since at least four oxidoreductive enzymes (L -arabinose reductase, L -arabitol dehydrogenase, L -xylulose reductase and xylitol dehydrogenase) are required to convert this sugar into D -xylulose, in comparison to D -xylose metabolism where only two enzymes (xylose reductase and xylitol dehydrogenase) are required. After phosphorylation, D -xylulose is believed to enter into the pentose phosphate pathway in the same way as occurs for xylose
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metabolism by yeasts (Roseiro et al., 1991). Although D - and L -arabitol cannot be distinguished by HPLC analysis (Ruijter et al., 1997), the production of xylitol is a good indicator for the presence of L -arabitol in D. hansenii ®ltrates. 4.2. Kinetics of polyol production from single and mixed sugars in D. hansenii Xylitol was the main polyol produced at all tested growth conditions, exhibiting a maximum yield of 0.59 g gÿ1 of xylose from xylose-grown D. hansenii. Compared with other xylose-assimilating yeasts, D. hansenii polyol yield was higher than that obtained with Candida shehatae, 0.13 g gÿ1 (Du Preez et al., 1988) but lower than that obtained with Candida mogi, 0.70 g gÿ1 (Sirisansaneeyakul et al., 1995). Similar xylitol yields were reported for Candida tropicalis (0.55 g gÿ1 ; LohmeierVogel and Hahn-H agerdal, 1985) and for Candida guillermondii (0.59 g gÿ1 ; Meyrial et al., 1991). All reported literature data were taken from batch experiments with similar D -xylose concentration ranges (between 30±55 g lÿ1 ). Similar initial xylose concentrations are crucial for comparison among dierent literature data since the xylose concentration in¯uences the polyol yield (Roseiro et al., 1991). Higher xylitol yields have been reported for initial xylose concentrations above 100 g lÿ1 (Domõnguez et al., 1997). When the growth medium composition has a single hexose or pentose as sole carbon and energy source, the accumulation of polyols by D. hansenii was obtained only from pentoses, and not from hexoses (Fig. 1). Moreover, arabitol was produced only from arabinose, whereas xylitol was obtained from both xylose and arabinose catabolism. Similar results of arabitol formation from xylose had been reported before for Pachysolen tannophilus (Schneider et al., 1985) but the same was not observed for Pichia stipitis and C. tropicalis (McMillan and Boynton, 1994): these yeasts produced only arabitol from arabinose. The addition of small amounts of xylose to an arabinose medium led to arabitol overproduction by D. hansenii. The calculated arabitol yield on consumed arabinose was 1.05 which is equivalent to the theoretical maximal yield of 1.01 g arabitol produced/g arabinose consumed (Table 2). The stimulation of arabitol production for D. hansenii grown on the mixture xylose/ arabinose is still not well understood. Some authors have suggested the relatively innecient multistep redox assimilation pathway of L -arabinose (McMillan and Boynton, 1994). However, other metabolic dierences such as pathway regulation and sugar transport, might have an in¯uence. The slower arabinose consumption rate by D. hansenii when xylose concentration was not residual (Table 2) suggested the existence of a pentose catabolite
repression eect of xylose over arabinose. However, this behaviour occurred only at a low xylose/arabinose ratio. For a xylose/arabinose ratio of 4.0 (w/w), arabinose assimilation was fully repressed by xylose during the ®rst part of the growth phase, and arabinose started to be consumed at a slow rate only after the xylose level in the medium decreased from 50 to 20 g lÿ1 (Gõrio, Amaro and Amaral-Collacßo, unpublished). 4.3. Biochemical characteristics of xylose-catabolic enzymes in D. hansenii XR and XDH enzymes were induced by D -xylose in D. hansenii since both enzymes greatly increased their activities when the carbon source present was shifted from glucose to xylose (Table 3). Similar results have also been reported for P. tannophilus (Bolen and Detroy, 1985), P. stipitis (Bicho et al., 1988) and Aureobasidium pullulans (Machova, 1992). However, dierent degrees of response of D. hansenii cells upon the shift from glucose to xylose were observed. This may have been caused either by dierent permeabilities of sugars or dierences in enzyme induction-site speci®city (Sugai and Delgenes, 1995). In the yeast Pachysolen tannophilus XDH activity was also reported to be repressed to a much greater extent than XR activity in the presence of glucose (Bicho et al., 1988). XDH activity was not detected during yeast growth on glucose. A similar repression of XDH activity by glucose was reported in Pullularia pullulans, but the repression mechanism involved is not known (Lee, 1992). The higher XR activity for arabinose compared to xylose seems to be a general characteristic of xylose-assimilating yeasts (Bolen and Detroy, 1985; Ditzelm uller et al., 1984), with possibly one exception (Machova, 1992). Therefore, this enzyme should also belong to the aldose reductase family previously described in a wide range of yeasts, mammals and plants (Lee, 1998). Acknowledgements The authors are grateful to Ms. Amelia Marques for her excellent microbiological technical assistance. References Bicho, P.A., Runnals, P.L., Cunningham, J.D., Lee, H., 1988. Induction of xylose reductase and xylitol dehydrogenase activities in Pachysolen tannophilus and Pichia stipitis on mixed sugars. Appl. Environ. Microbiol. 54, 50±54. Bolen, P.L., Detroy, R.W., 1985. Induction of NADPH-linked D xylose reductase and NAD-linked xylitol dehydrogenase activities in Pachysolen tannophilus by D -xylose, L -arabinose, or D -galactose. Biotechnol. Bioeng. 27, 302±307. Ciriacy, M., 1975. Genetics of alcohol dehydrogenase in Saccharomyces cerevisiae. I. Isolation and genetic analysis of mutants. Mutat. Res. 29, 315±326.
F.M. Gõrio et al. / Bioresource Technology 71 (2000) 245±251 Ditzelm uller, G., Kubicek, C.P., W ohrer, W., R ohr, M., 1984. Xylose metabolism in Pachysolen tannophilus: puri®cation and properties of xylose reductase. Can. J. Microbiol. 30, 1330±1336. Domõnguez, J.M., Gong, C.S., Tsao, G.T., 1997. Prodution of xylitol from D -xylose by Debaryomyces hansenii. Appl. Biochem. Biotechnol. 63/65, 117±127. Du Preez, J.C., van Driessel, B., van prior, B.A., 1989. D -xylose fermentation by Candida shehatae and Pichia stipitis at low dissolved oxygen levels in fed-batch cultures. Biotechnol. Lett. 11, 131±136. Du Preez, J.C., van Driessel, B., Prior, B.A., 1988. The relation between redox potential and D -xylose fermentation by Candida shehatae and Pichia stipitis. Biotechnol. Lett. 10, 901±906. Du Preez, J.C., van der Walt, J.P., 1983. Fermentation of D -xylose to ethanol by a strain of Candida shehatae. Biotechnol. Lett. 5, 357± 362. Furlan, S.A., Bouilland, P., Strehaiano, P., Riba, J.P., 1991. Study of xylitol formation from xylose under oxygen limiting conditions. Biotechnol. Lett. 13, 203±206. Gõrio, F.M., Peito, M.A., Amaral-Collacßo, M.T., 1989. Enzymatic and physiological study of D -xylose metabolism by Candida shehatae. Appl. Microbiol. Biotechnol. 32, 199±204. Gõrio, F.M., Pelica, F., Amaral-Collacßo, M.T., 1996. Characterization of xylitol dehydrogenase from Debaryomyces hansenii. Appl. Biochem. Biotechnol. 56, 79±87. Horitsu, H., Yahashi, Y., Takamizawa, K., Kawai, K., Suzuki, T., Watanabe, N., 1992. Production of xylitol from D -xylose by Candida tropicalis: optimization of production rate. Biotechnol. Bioeng. 40, 1085±1091. Ladisch, M.R., Svarczkopf, J.A., 1991. Ethanol production and the cost of fermentable sugars from biomass. Biores. Technol. 36, 83± 95. Lee, H., 1992. Reversible inactivation of D -xylose utilization by D -glucose in the pentose-fermenting yeast Pachysolen tannophilus. FEMS Microbiol. Lett. 92, 1±4. Lee, H., 1998. The structure and function of yeast xylose (aldose) reductases. Yeast 14, 977±984. Ligthelm, M.E., Prior, B.A., du Preez, J.C., 1988. The oxygen requirements of yeasts for the fermentation of D -xylose and D -glucose to ethanol. Appl. Microbiol. Biotechnol. 28, 63±68. Lohmeier-Vogel, E., Hahn-H agerdal, B., 1985. The utilization of metabolic inhibitors for shifting product formation from xylitol to ethanol in pentose fermentations using Candida tropicalis. Appl. Microbiol. Biotechnol. 21, 167±172. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J., 1951. Protein measurement with folin phenol reagent. J. Biol. Chem. 193, 265±275. Lu, J., Tsai, L.B., Gong, C.S., Tsao, G.T., 1995. Eect of nitrogen sources on xylitol production from D -xylose by Candida sp. L-102. Biotechnol. Lett. 17, 167±170.
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Machov a, E., 1992. Induction of aldose reductase and polyol dehydrogenase activities in Aureobasidium pullulans by D -xylose, L -arabinose and D -glucose. . Appl. Microbiol. Biotechnol. 35, 374± 377. McMillan, J.D., Boynton, B.L., 1994. Arabinose utilization by xylosefermenting yeasts in fungi. Appl. Biochem. Biotechnol. 45/46, 569± 584. Meyrial, V., Delgenes, J.P., Moletta, R., Navarro, J.M., 1991. Xylitol production from D -xylose by Candida guillermondii: fermentation behaviour. Biotechnol. Lett. 13, 281±286. Nigam, P., Singh, D., 1995. Processes for fermentable production of xylitol ± a sugar substitute. Process Biochem. 30, 117±124. Nunez, M.J., Sanroman, A., Lopez, E., Vasquez, G., Lema, J.M., 1989. The D -xylose fermenting capacities of immobilized Pichia stipitis and Pachysolen tannophilus. Biotechnol. Lett. 11, 353±358. Paraj o, J.C., Domõnguez, H., Domõnguez, J.M., 1996. Production of xylitol from concentrated wood hydrolysates by Debaryomyces hansenii: eect of the initial cell concentration. Biotechnol. Lett. 18, 593±598. Preziosi-Belloy, L., Nolleau, V., Navarro, J.M., 1997. Fermentation of hemicellulosic sugars and sugar mixtures to xylitol by Candida parapsilosis. Enz. Microb. Technol. 21, 124±129. Roberto, I.C., Sato, S., Mancilla, I.M., 1996. Eect of inoculum level on xylitol production from rice straw hemicellulose hydrolysate by Candida guillermondii. J. Ind. Microbiol. 16, 348±350. Roseiro, J.C., Peito, M.A., Gõrio, F.M., Amaral-Collacßo, M.T., 1991. The eects of the oxygen transfer coecient and substrate concentration by Debaryomyces hansenii. Arch. Microbiol. 156, 484±490. Ruijter, G.J.G., Vanhanen, S.A., Gielkens, M.M.C., van de Vondervoort, P.J.I., Visser, J., 1997. Isolation of Aspergillus niger creA mutants and eects of the mutations on expression of arabinases and L -arabinose catabolic enzymes. Microbiol. 143, 2991±2998. Saha, B.C., Bothast, R.J., 1996. Production of L -arabitol from L -arabinose by Candida entomaea and Pichia guillermondii. Appl. Microbiol. Biotechnol. 45, 299±306. Schneider, H., Mahmourides, G., Mabelle, J.L., Lee, H., Maki, N., McNeil, H.J., 1985. Correlation between limitation of growth of Pachysolen tannophilus on D -xylose with the formation of ethanol and other products. Biotechnol. Lett. 7, 361±364. Sirisansaneeyakul, S., Staniszewski, M., Rizzi, M., 1995. Screening of yeasts for production of xylitol from D -xylose. J. Ferm. Bioeng. 80, 565±570. Sugai, J.K., Delgenes, J.P., 1995. Catabolite repression of induction of aldose reductase activity and utilization of mixed hemicellulosic sugars in Candida guillermondii. Curr. Microbiol. 31, 239±244. Vandeska, E., Amartey, S., Kuzmanova, S., Jeries, T., 1995. Eects of environmental conditions on production of xylitol by Candida boidinii. World J. Microbiol. Biotechnol. 11, 213±218.
Polyols production during single and mixed substrate fermentations in Debaryomyces hansenii F.M. Gõrio*, C. Amaro, H. Azinheira1, F. Pelica, M.T. Amaral-Collacßo INETI, IBQTA-Departamento de Biotecnologia, Unid. de Microbiologia Industrial e Bioprocessos, Estrada do Pacßo do Lumiar, 22, 1649-038 Lisboa, Portugal Received 30 December 1998; received in revised form 4 May 1999; accepted 15 May 1999
Abstract The kinetics of polyols production by Debaryomyces hansenii was studied both on single substrate and mixed substrate-containing media. From the single substrate experiments, polyols (xylitol and arabitol) and ethanol were produced from pentose sugars while ethanol was produced in signi®cant amounts only from glucose-grown D. hansenii. The maximal xylitol volumetric productivity (Qxylitol ), 0.28 g lÿ1 hÿ1 was obtained from D -xylose, whereas the maximal arabitol volumetric productivity (Qarabitol ), 0.04 g lÿ1 hÿ1 was observed with arabinose. When D. hansenii was cultivated with mixed substrates, a simultaneous sugar consumption pattern occurred both for glucose/arabinose and xylose/arabinose mixtures. The addition of low amounts of xylose to an arabinose medium led to a fourfold increase in the arabitol volumetric productivity: 0.17 g lÿ1 hÿ1 . Conversely, glucose addition had no eect on arabitol production. Xylitol was the main polyol produced for all tested cultivation conditions by D. hansenii. An enzymatic study of the ®rst two xylose-catabolic enzymes in glucose and xylose-grown D. hansenii revealed that both enzymes were induced by D -xylose. Glucose caused total inhibition of xylitol dehydrogenase, whereas xylose reductase was only partially repressed. Ó 1999 Elsevier Science Ltd. All rights reserved. Keywords: Mixed substrates; Pentoses; Polyols; Debaryomyces hansenii; Polyol-producing yeast
1. Introduction The obtaining of chemicals from lignocellulosic bioconversion processes predicates the total use of sugars obtained by cellulose and hemicellulose hydrolysis. Ethanol can be easily produced by yeasts during fermentation of glucose. This hexose is the major sugar obtained from cellulose hydrolysis of the lignocellulosic material. Hemicellulose breakdown by acid or enzymatic hydrolysis gives a complex mixture of sugars, which includes pentoses (xylose, arabinose), hexoses (glucose, mannose, galactose) and, additionally signi®cant amounts of uronic acids, acetate, furfural and other aromatic compounds (Ladisch and Svarczkopf, 1991). Depending on the raw material and on the hydrolysis conditions, pentoses (D -xylose and L -arabinose) are the
* Corresponding author. Tel.: +351 1 716 51 41; fax: +351 1 716 09 01; e-mail: [email protected] 1 Current address: Instituto de Investigacß~ ao Cientõ®ca Tropical, CEPTA, Tapada da Ajuda, 1301 Lisboa Codex, Portugal.
major components present in the hemicellulosic hydrolysates, particularly from hardwoods and cereal crops. Ethanol is not the sole chemical obtained from biological conversion of lignocellulosic materials, and other chemicals (e.g., xylitol and arabitol), which are currently manufactured through the chemical reduction of xylancontaining materials (Nigam and Singh, 1995), can be obtained from xylose metabolism by yeasts such as Candida guillermondii (Roberto et al., 1996), Candida parapsilosis (Furlan et al., 1991), Candida tropicalis (Horitsu et al., 1992), Candida boidinii (Vandeska et al., 1995) and Debaryomyces hansenii (Roseiro et al., 1991; Paraj o et al., 1996). The biological production of arabitol has been far less studied. The most promising arabitol-producing yeasts, Candida entomaea and Pichia guillermondii, yield up to 0.7 g lÿ1 of L -arabitol per gram of L -arabinose (Saha and Bothast, 1996). However, there is a lack of knowledge of how these yeasts respond to the presence of more than one sugar in the growth media for polyol production, which is a usual feature of media from hemicellulosic hydrolysates. This work reports comparative kinetic data obtained from batch cultivation with D. hansenii when
0960-8524/00/$ ± see front matter Ó 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 0 - 8 5 2 4 ( 9 9 ) 0 0 0 7 8 - 4
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hemicellulosic sugars were used either as single or mixed carbon and energy sources. The in¯uence of substrates on the pattern of polyols production by the yeast is also discussed. The data suggested that xylose and a xylose/arabinose mixture were the best substrates for xylitol and arabitol production, respectively.
2.5. Enzyme assays
2. Methods
Xylose reductase (XR, E.C. 1.1.1.21) and xylitol dehydrogenase (XDH, E.C. 1.1.1.9) enzyme activities, were assayed as described by Gõrio et al. (1989). Reaction rates were corrected for endogenous NAD(P)H consumption or production in crude extracts. Enzyme units (U) were de®ned as lmoles of NAD(P) reduced or NAD(P)H oxidized per min at room temperature. Speci®c activities were expressed as mU mgÿ1 of protein.
2.1. Organism
2.6. Analytical methods
D. hansenii was isolated by a batch enrichment technique from sugar cane and maintained as described before (Roseiro et al., 1991).
D -xylose, D -glucose, L -arabinose, xylitol and arabitol were determined by HPLC using a Sugar-Pak (Waters, Mildford, Mass., USA) column and a refractive index detector. The column temperature was 75°C and a ¯ow rate of 0.5 ml minÿ1 was used. Ethanol was measured by gas chromatography using a Carbowax 1540 column (2 m ´ 3 mm, 80/100 mesh) and a ¯ame ionization detector. Column, injector and detector temperatures were 50°C, 200°C and 250°C, respectively. Methanol (10 mm) was used as internal standard. Biomass was measured by gravimetry after sample ®ltration (0.45 lm), washing with distilled water and drying overnight to a constant weight at 105°C. Protein was determined by using the method of Lowry et al. (1951). Bovine serum albumin was used as standard protein.
2.2. Single and mixed-substrate fermentation Cultures were grown on a chemically-de®ned medium as described by Du Preez and van der Walt (1983), except that casamino acids were replaced by (NH4 )2 SO4 , (7.5 g lÿ1 ). The carbon source was supplied as single or mixed sugars which were sterilized separately. Yeast growth assays were carried out in 750 ml Erlenmeyer ¯asks containing 80 ml of the above medium and incubated in an orbital shaker set at 150 rpm and 30°C. The initial pH was 5.5. Inocula (5%, v/v) were grown in 500 ml Erlenmeyer ¯asks containing 80 ml of the de®ned medium and incubated under identical conditions for 16 h. All growth tests here presented were carried out in triplicate and the results shown are the average of at least two independent values. 2.3. Enzyme induction experiment Batch cultivations on the above chemically-de®ned medium containing glucose as single carbon source were run at 30°C in a Biola®tte (St. Germain en Laye, France) bioreactor of 3 l working volume and 1 vvm aeration rate. The pH was controlled at 5.5 with 1M NaOH and the agitation speed was 150 rpm. The inoculum (5%, v/v) was grown on 2% xylose for 16 h. A xylose pulse (90 g lÿ1 ) was carried out immediately after glucose depletion, and the growth and products accumulation were monitored for an additional 22 h. 2.4. Preparation of cell free-extracts Yeast samples from dierent stages of the bioreactor-grown cultures were harvested by centrifugation as described elsewhere (Gõrio et al., 1989). The cellfree extracts used for enzyme assays were prepared after cell disruption using the glass beads method (Ciriacy, 1975).
2.7. Fermentation parameter calculations The volumetric productivities were calculated by linear regression from the maximal slopes of the substrate (product) vs. time curves (Du Preez et al., 1989). The speci®c productivities were calculated by linear regression from the maximal slopes of dP (or dS)/X vs. time curves. 3. Results 3.1. Batch cultivation on single sugars with Debaryomyces hansenii Because a typical hemicellulosic hydrolyate contains a mixture of pentose and hexose sugars, a comparative study of growth pro®les of D. hansenii using mixed as well as single sugars was carried out. Fig. 1 shows the pro®les of sugar consumption rate, biomass and extracellular product formation rate during the batch growth of D. hansenii on dierent hemicellulosic sugars. As all shake-¯ask cultivations have a limited supply of oxygen, we use throughout this work the term ``semi-aerobic'' for the growth in such experimental conditions. For glucose-grown D. hansenii, only ethanol was produced, which indicates a mixed respiro-fermentative
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metabolism (Fig. 1(A)). Polyols were not detected for glucose-grown D. hansenii. When glucose concentration reached a value close to 3 g lÿ1 , ethanol was co-oxidised as second energy source. Conversely, arabinose-grown D. hansenii had a fully oxidative metabolism since ethanol was not detected (Fig. 1(B)). The maximal arabitol concentration (2.86 g lÿ1 ) was threefold higher than the maximal xylitol concentration (0.94 g lÿ1 ) with both maxima occurring at 120 h of cultivation time. Comparing the ethanol/hexose co-oxidation observed before (Fig. 1(A)), for arabinosegrown D. hansenii both polyols were consumed only after complete pentose utilisation in the cultivation medium. Xylose-grown D. hansenii utilised the carbon source mainly for growth purposes. Xylitol was the only polyol detected extracellularly (Fig. 1(C)). The maximal xylitol volumetric productivity was 0.28 g lÿ1 hÿ1 (Table 1). Again, xylitol was oxidized as carbon source when xylose concentration was below 3 g lÿ1 . Ethanol was produced in only residual amounts. Table 1 shows the fermentation parameters calculated for D. hansenii grown on single carbon sources. ÿ1 D -glucose supported the highest growth rate, 0.32 h . L -arabinose and D -xylose exhibited similar growth rates but of low magnitude compared to glucose. The maximal arabitol yield obtained for the L -arabinose medium was rather low, 0.10 g gÿ1 , compared to the maximal xylitol yield (0.59 g gÿ1 ) obtained for the xylose medium (Table 1). Under semi-aerobic conditions ethanol was produced in signi®cant amounts by D. hansenii only when glucose was the carbon and energy source. 3.2. Batch cultivation on a glucose/arabinose mixture of D. hansenii
Fig. 1. Batch cultivation pro®le of Debaryomyces hansenii grown semiaerobically in shake ¯asks on (A) D -glucose, (B) L -arabinose and (C) D -xylose. Glucose (s); Arabinose (n); Xylose (h); Ethanol (d); Arabitol (m); Xylitol (.); Biomass (n). The inocula were grown on glucose, arabinose and xylose, respectively.
When mixed sugars in a hexose/pentose ratio of 0.2 (w/w) were present in the initial culture medium, both sugars were simultaneously used as carbon and energy source for yeast growth, with no visible diauxic growth (Fig. 2). During the ®rst 10 h, glucose and arabinose were consumed simultaneously producing mainly biomass. However, arabitol started to accumulate before glucose depletion. After glucose exhaustion, xylitol was also formed from L -arabinose metabolism, although arabitol was the main metabolic product. The maximal volumetric arabitol and xylitol productivities were 0.03 and 0.02 g lÿ1 hÿ1 , respectively (Table 2). The arabitol yield (0.19 g gÿ1 ) was threefold higher than the xylitol yield (0.06 g gÿ1 ). Ethanol was not produced since all the glucose was rapidly consumed during the early stages of yeast growth where the oxygen limitation was still not drastic. When L -arabinose concentration had decreased to about 6 g lÿ1 , xylitol was co-metabolised with arabinose. Arabitol was also consumed, but at a slower rate (Fig. 2(B)).
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Table 1 Batch fermentation parameters by D. hansenii grown on glucose, arabinose and xylose Parameter ÿ1
l (h ) Yx=s (g gÿ1 ) YEtOH=s (g gÿ1 ) Yxylitol=s (g gÿ1 ) Yarabitol=s (g gÿ1 ) Qsubstrate (g lÿ1 hÿ1 ) qsubstrate (g gÿ1 hÿ1 ) Qxylitol (g lÿ1 hÿ1 ) Qarabitol (g lÿ1 hÿ1 ) Qethanol (g lÿ1 hÿ1 ) qxylitol (g gÿ1 hÿ1 ) qarabitol (g gÿ1 hÿ1 ) qethanol (g gÿ1 hÿ1 )
Carbon source Glucose
Arabinose
Xylose
0.32 0.55 0.23 ND ND 2.95 0.25 ND ND 1.04 ND ND 0.05
0.24 0.43 ND 0.03 0.10 0.39 0.06 0.01 0.04 ND 0.001 0.003 ND
0.25 0.31 0.01 0.59 ND 0.52 0.25 0.28 ND 0.01 0.06 ND 0.0002
ND ± not detected.
3.3. Batch cultivation on a xylose/arabinose mixture of D. hansenii The polyols produced by D. hansenii are derived only from pentoses, as shown in the previous single-substrate growth kinetics (Fig. 1). Therefore, we tried to increase the polyols production by using a initial mixture of xylose/arabinose (ratio of 0.2, w/w) in the growth medium composition (Fig. 3). Again, both sugars were simultaneously assimilated, reaching identical maximal volumetric consumption rates (Table 2). However, the arabinose volumetric consumption rate Qarabinose (0.09 g lÿ1 hÿ1 ) was only 47% of the Qxylose , 0.200 g lÿ1 hÿ1 during the ®rst 26 h of yeast growth. A similar arabinose consumption rate (0.20 g lÿ1 hÿ1 ) was obtained only after 60 h of cultivation time when xylose concentration became residual in the medium (<1 g lÿ1 ). Besides biomass, only polyols accumulated in xylose/arabinose D. hansenii cultures (Fig. 3). Arabitol was the main polyol produced and the maximal volumetric and speci®c productivities, 0.17 g lÿ1 hÿ1 and 0.013 g gÿ1 hÿ1 , respectively, were fourfold higher than the maximal volumetric and speci®c productivities obtained for the previous sugar mixture, 0.03 and 0.003 g gÿ1 hÿ1 , respectively (Table 2). The xylitol speci®c productivity was only 8% of that observed before for a xylose-grown D. hansenii culture (Fig. 1; Table 1). 3.4. Enzymatic study of the xylose-catabolic enzymes from D. hansenii In order to evaluate the inducible or constitutive type of the xylose catabolic enzymes, D. hansenii was grown in glucose medium for the ®rst 25 h then, just after glucose depletion, a pulse (90 g lÿ1 ) of xylose was added. The activities of the xylose-catabolic enzymes, NADPHlinked xylose reductase (XR) and NAD -linked xylitol
Fig. 2. Batch cultivation pro®le of D. hansenii grown semi-aerobically in shake ¯asks on a mixture of glucose and arabinose (ratio of 0.2, w/w). The inoculum was grown in L -arabinose. Symbols as in Fig. 1.
dehydrogenase (XDH), were measured during the exponential growth phase on glucose and immediately before the xylose pulse. After the pulse, additional enzyme activities were measured for the remainder of the growth phase (Table 3). Although a residual XR activity with D -xylose was detected when the carbon source was glucose (up to 59 mU/mg protein), 2 h after the xylose pulse the XR activity with D -xylose increased about sixfold. The increase of XR activity with arabinose was ®vefold in the same period. XDH enzyme was active only when xylose was present in the growth medium. The enzyme was speci®c for NAD and xylitol. When NADP and arabitol were used as substrates no activity
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Table 2 Parameters of mixed substrate fermentations by D. hansenii grown on glucose/arabinose and xylose/arabinose mixtures Carbon sources
l (hÿ1 ) Yx=s (g gÿ1 ) Yxylitol=s (g gÿ1 ) Yarabitol=s (g gÿ1 ) Qarabinose (g lÿ1 hÿ1 ) Qglucose (g lÿ1 hÿ1 ) Qxylose (g lÿ1 hÿ1 ) qarabinose (g gÿ1 hÿ1 ) qglucose (g gÿ1 hÿ1 ) qxylose (g gÿ1 hÿ1 ) Qxylitol (g lÿ1 hÿ1 ) Qarabitol (g lÿ1 hÿ1 ) qxylitol (g gÿ1 hÿ1 ) qarabitol (g gÿ1 hÿ1 )
Glucose/arabinose (ratio 0.2)
Xylose/arabinose (ratio 0.2)
0.17 0.24 a (0.64) 0.06 0.19 0.27 0.64 NA 0.03 0.02 NA 0.02 0.03 0.004 0.003
0.16 0.99 c 0.67 d (0.47) e 1.05 f 0.20 g (0.09) h NA 0.20 0.03 NA 0.07 0.03 0.17 0.005 0.013
b
a
Cell yield for glucose + arabinose; Cell yield for arabinose; c For consumed s xylose + arabinose; d For consumed s xylose; e For consumed s xylose + arabinose; f Assuming that all arabitol was produced from arabinose; g Calculated between 60 and 140 h of growth; h Calculated during the ®rst 26 h of growth; NA ± not applicable.
Fig. 3. Batch cultivation pro®le of D. hansenii grown semi-aerobically in shake ¯asks on a mixture of xylose and arabinose (ratio of 0.2, w/w). The inoculum was grown in L -arabinose. Symbols as in Fig. 1.
b
was detected (data not shown). This result closely agrees with the previously-reported substrate speci®city for the puri®ed XDH from D. hansenii (Gõrio et al., 1996). In the present experiments glucose produced total inactivation of pre-existing forms of XDH enzyme in D. hansenii, when the enzyme had been previously induced during the time course of a xylose-grown inoculum (Table 3). Moreover, after the xylose pulse, the XR and XDH activity measurements suggested that there was a dierent enzyme induction rate. XR was induced in a shorter time than to XDH, since 15 min after the xylose pulse (25.3 h) XR activity had increased by 29% and 40%, when assayed using xylose and arabinose, respectively, whereas XDH was still not detected (Table 3). 4. Discussion 4.1. Physiology of polyol synthesis by yeasts This paper presents the ®rst comprehensive study of the kinetics of xylitol and arabitol production by D. hansenii, a food-spoilage yeast. Polyol production by yeasts is generally regarded as undesirable, since polyols decrease the ethanol yield (Nunez et al., 1989). However, recent reports (Lu et al., 1995; Domõnguez et al., 1997) describing high yields for xylitol production by yeasts have turned interest to the study of the physiological and environmental parameters underlying the overpro-
Table 3 Crude extract enzyme activities from a batch fermenter cultivation of D. hansenii grown on D -glucose before and after a xylose pulse (90 g lÿ1 ). The pulse was added after 25 h of cultivation time Time (h) Enzyme activity (mU mgÿ1 protein)
9.5 24.8 25.3 27 47
Xylose reductase NADPH-linked with substrate
Xylitol dehydrogenase NAD -linked with substrate
D -xylose
L -arabinose
Xylitol
39 59 76 370 62
64 81 113 398 58
ND ND ND 468 ND
ND ± not detected.
duction of xylitol and other polyols by these organisms. Although the physiology of synthesis of polyols by yeasts is still not fully understood it appears to be related to the yeast strain, carbon source and oxygen availability (Ligthelm et al., 1988). Xylitol formation from xylose is believed to be due to a redox imbalance in cofactor production of the ®rst two oxidoreductive steps of xylose metabolism resulting in a metabolic NADH surplus under oxygen-limited growth conditions and leading to xylitol accumulation (PreziosiBelloy et al., 1997). The production of arabitol from arabinose is likely to be more complex, since at least four oxidoreductive enzymes (L -arabinose reductase, L -arabitol dehydrogenase, L -xylulose reductase and xylitol dehydrogenase) are required to convert this sugar into D -xylulose, in comparison to D -xylose metabolism where only two enzymes (xylose reductase and xylitol dehydrogenase) are required. After phosphorylation, D -xylulose is believed to enter into the pentose phosphate pathway in the same way as occurs for xylose
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metabolism by yeasts (Roseiro et al., 1991). Although D - and L -arabitol cannot be distinguished by HPLC analysis (Ruijter et al., 1997), the production of xylitol is a good indicator for the presence of L -arabitol in D. hansenii ®ltrates. 4.2. Kinetics of polyol production from single and mixed sugars in D. hansenii Xylitol was the main polyol produced at all tested growth conditions, exhibiting a maximum yield of 0.59 g gÿ1 of xylose from xylose-grown D. hansenii. Compared with other xylose-assimilating yeasts, D. hansenii polyol yield was higher than that obtained with Candida shehatae, 0.13 g gÿ1 (Du Preez et al., 1988) but lower than that obtained with Candida mogi, 0.70 g gÿ1 (Sirisansaneeyakul et al., 1995). Similar xylitol yields were reported for Candida tropicalis (0.55 g gÿ1 ; LohmeierVogel and Hahn-H agerdal, 1985) and for Candida guillermondii (0.59 g gÿ1 ; Meyrial et al., 1991). All reported literature data were taken from batch experiments with similar D -xylose concentration ranges (between 30±55 g lÿ1 ). Similar initial xylose concentrations are crucial for comparison among dierent literature data since the xylose concentration in¯uences the polyol yield (Roseiro et al., 1991). Higher xylitol yields have been reported for initial xylose concentrations above 100 g lÿ1 (Domõnguez et al., 1997). When the growth medium composition has a single hexose or pentose as sole carbon and energy source, the accumulation of polyols by D. hansenii was obtained only from pentoses, and not from hexoses (Fig. 1). Moreover, arabitol was produced only from arabinose, whereas xylitol was obtained from both xylose and arabinose catabolism. Similar results of arabitol formation from xylose had been reported before for Pachysolen tannophilus (Schneider et al., 1985) but the same was not observed for Pichia stipitis and C. tropicalis (McMillan and Boynton, 1994): these yeasts produced only arabitol from arabinose. The addition of small amounts of xylose to an arabinose medium led to arabitol overproduction by D. hansenii. The calculated arabitol yield on consumed arabinose was 1.05 which is equivalent to the theoretical maximal yield of 1.01 g arabitol produced/g arabinose consumed (Table 2). The stimulation of arabitol production for D. hansenii grown on the mixture xylose/ arabinose is still not well understood. Some authors have suggested the relatively innecient multistep redox assimilation pathway of L -arabinose (McMillan and Boynton, 1994). However, other metabolic dierences such as pathway regulation and sugar transport, might have an in¯uence. The slower arabinose consumption rate by D. hansenii when xylose concentration was not residual (Table 2) suggested the existence of a pentose catabolite
repression eect of xylose over arabinose. However, this behaviour occurred only at a low xylose/arabinose ratio. For a xylose/arabinose ratio of 4.0 (w/w), arabinose assimilation was fully repressed by xylose during the ®rst part of the growth phase, and arabinose started to be consumed at a slow rate only after the xylose level in the medium decreased from 50 to 20 g lÿ1 (Gõrio, Amaro and Amaral-Collacßo, unpublished). 4.3. Biochemical characteristics of xylose-catabolic enzymes in D. hansenii XR and XDH enzymes were induced by D -xylose in D. hansenii since both enzymes greatly increased their activities when the carbon source present was shifted from glucose to xylose (Table 3). Similar results have also been reported for P. tannophilus (Bolen and Detroy, 1985), P. stipitis (Bicho et al., 1988) and Aureobasidium pullulans (Machova, 1992). However, dierent degrees of response of D. hansenii cells upon the shift from glucose to xylose were observed. This may have been caused either by dierent permeabilities of sugars or dierences in enzyme induction-site speci®city (Sugai and Delgenes, 1995). In the yeast Pachysolen tannophilus XDH activity was also reported to be repressed to a much greater extent than XR activity in the presence of glucose (Bicho et al., 1988). XDH activity was not detected during yeast growth on glucose. A similar repression of XDH activity by glucose was reported in Pullularia pullulans, but the repression mechanism involved is not known (Lee, 1992). The higher XR activity for arabinose compared to xylose seems to be a general characteristic of xylose-assimilating yeasts (Bolen and Detroy, 1985; Ditzelm uller et al., 1984), with possibly one exception (Machova, 1992). Therefore, this enzyme should also belong to the aldose reductase family previously described in a wide range of yeasts, mammals and plants (Lee, 1998). Acknowledgements The authors are grateful to Ms. Amelia Marques for her excellent microbiological technical assistance. References Bicho, P.A., Runnals, P.L., Cunningham, J.D., Lee, H., 1988. Induction of xylose reductase and xylitol dehydrogenase activities in Pachysolen tannophilus and Pichia stipitis on mixed sugars. Appl. Environ. Microbiol. 54, 50±54. Bolen, P.L., Detroy, R.W., 1985. Induction of NADPH-linked D xylose reductase and NAD-linked xylitol dehydrogenase activities in Pachysolen tannophilus by D -xylose, L -arabinose, or D -galactose. Biotechnol. Bioeng. 27, 302±307. Ciriacy, M., 1975. Genetics of alcohol dehydrogenase in Saccharomyces cerevisiae. I. Isolation and genetic analysis of mutants. Mutat. Res. 29, 315±326.
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