Journal Identification = PRBI Article Identification = 9237 Date: June 27, 2011 Time: 8:19 am

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Process Biochemistry 46 (2011) 1619–1626

Contents lists available at ScienceDirect

Process Biochemistry

jo u rn al hom epage: www.elsev ier .com/ locate /procbio

n environmentally friendly and efficient method for xylitol bioconversion withigh-temperature-steaming corncob hydrolysate by adapted Candida tropicalis

e Wang1, Ming Yang1, Xiaoguang Fan, Xintao Zhu, Tao Xu, Qipeng Yuan ∗

tate Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology, Beijing 100029, China

r t i c l e i n f o

rticle history:eceived 25 November 2010eceived in revised form 4 May 2011ccepted 5 May 2011

eywords:orncob hemicellulose hydrolysate

a b s t r a c t

This study reports a new process to prepare corncob hemicellulose hydrolysate through high tempera-ture steaming (HTS) for xylitol bioconversion by adapted Candida tropicalis. Under the optimal corncobhydrolysis conditions of 160 ◦C and 120 min, the maximum xylose yield was more than 20%. The optimalfermentative parameters from the HTS hydrolysate is as follows: initial xylose concentration of 140 g l−1,initial pH 6.0, initial cell concentration of 1.2 g l−1 and 30 ◦C using a two-step dissolved oxygen processwith a rotary shaker speed at 200 rpm for the first 24–26 h and then at 150 rpm until 48 h of fermenta-

−1 −1

ermentationigh temperature steamingylitolandida tropicalis

tion. The highest xylitol yield (71.4%) and volumetric productivity (2.12 g l h ) were obtained from theHTS hydrolysate, which were 158% and 149%, respectively, higher than the results obtained from the acidhydrolysate. Additionally, the amount of inhibitors produced by the HTS hydrolysis and the burden of ionexchange purification after fermentation with HTS hydrolysate were much lower compared with the acidhydrolysate. Therefore, fermentation with corncob HTS hydrolysate is an environmentally friendly andefficient method to produce xylitol, demonstrating a wide potential application in xylitol bioconversion.

. Introduction

As a five-carbon sugar alcohol, xylitol has been widely used as natural sweetener because it is beneficial for nutrition [1] andealth (the prevention of caries) [2] and is useful in low-sugar foodreparation for diabetic patients [3]. A major industrial method forylitol production is chemical synthesis from xylose, which is com-only obtained from the acid hydrolysis of plant hemicellulosicaterial using a nickel catalyst. Corncob is an abundant residual

gricultural by-product that contains about 30% hemicellulose [4].orncob may be used as a raw material for xylitol synthesis becauseur previous studies have shown that its hemicellulose fractionould be utilized to produce xylose or xylo-oligosaccharides (XO)5]. The xylitol chemical synthesis process includes high pressurend temperature (7 MPa, 130 ◦C) [6] as well as expensive sep-ration and purification steps [7]. Compared with this process,

n alternative process for xylitol fermentation from hemicellu-ose hydrolysate utilizing microorganisms represents a renewablerocess with moderate reaction conditions and low energy require-

∗ Corresponding author at: West Room 309, Science and Technology Building,ailbox No. 75, College of Life Science and Technology, Beijing University of Chem-

cal Technology, No. 15 Beisanhuan East Road, Chaoyang District, Beijing 100029,hina. Tel.: +86 10 64437610; fax: +86 10 64437610; mobile: +86 13811402874.

E-mail address: yuanqp@mail.buct.edu.cn (Q. Yuan).1 These authors contributed equally to this work.

359-5113/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.oi:10.1016/j.procbio.2011.05.004

© 2011 Elsevier Ltd. All rights reserved.

ments, which ensures high product selectivity, low cost and safety[8]. It has been shown that Candida tropicalis (C. tropicalis) is adesirable microorganism for xylitol production with high yield andvolumetric productivity from xylose-containing hydrolysate [9].

Environmental pollution and equipment corrosion caused byindustrial hemicellulose hydrolysate preparation using sulfuricacid is significant [10]. Specifically, sulfuric acid left in the acidhydrolysate and corncob residue may damage the environmentand equipment. Furthermore, a number of degradation productsthat are generated during the acid hydrolysis process are rec-ognized fermentation inhibitors, such as acetic acid, formic acid,furfural, hydroxymethylfurfural (HMF) and phenolic compounds[11], necessitating complicated detoxification and purificationtreatments before and after xylitol fermentation [12]. By com-parison, lignocellulosic material hydrolysis by high temperaturesteaming (HTS) has a lower environmental impact, requires lowercapital investment and less hazardous process chemicals, and gen-erates the recycled and sustainable corncob residue, much lowerconductivity in the HTS hydrolysate and less inhibition to xyli-tol fermentation later [10,13]. Therefore, HTS hydrolysis may bean alternative process for hemicellulose hydrolysate preparationthat is more useful for xylitol bioconversion, broth purification andcrystallization [14].

HTS hydrolysis of corncob, but not acid hydrolysis, has alreadybeen applied in xylo-oligosaccharides production [5,15]. However,there are no reports on the use of corncob hydrolysis by HTS to pre-pare the xylose-abundant hydrolysate for xylitol bioconversion by

Journal Identification = PRBI Article Identification = 9237 Date: June 27, 2011 Time: 8:19 am

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icroorganisms. Although the corncob HTS hydrolysis process wasmployed by Rivas [14], there was little xylose produced; the mainydrolytic products in the HTS hydrolysate were XO. To yield xylose

rom these oligomers, a further hydrolysis step with sulfuric acidould be necessary. Moreover, HTS was used to recover xylose from

he bagasse by Boussarsar [10], but the xylose yield was much lowerhen compared with the acid hydrolysate. The enzymatic hydrol-

sis of oligomers was necessary to increase the recovery of xyloseonomer. Consequently, to overcome the shortcomings observed

n previous studies, we developed a new process to prepare corn-ob HTS hemicellulose hydrolysate for use in an environmentallyriendly and efficient method of xylitol bioconversion by adapted. tropicalis.

. Materials and methods

.1. Raw materials and pretreatment

Corncob (Southern suburb of Beijing, China) was locally collected after harvest-ng. It was air-dried in the sun, milled into 10 mesh particles and stored until use.o make the material fragile to facilitate hydrolysis by HTS, and decrease the pro-uction of inhibitors [5], 100 g of corncob (dry matter) was pretreated by soaking in00 ml of 0.1% (v v−1) sulfuric acid at room temperature for 24 h. Then, the suspen-ion was centrifuged, and the supernatant was recycled. The collected solids wereashed with distilled water until the washing solution reached pH 5.5–6.0.

.2. Preparation of corncob hemicellulose hydrolysate by HTS or sulfuric acid

After the pretreatment, the corncob was hydrolyzed in a 5 L reactor with oneurbine with a four-plane blade and which was capable of operating at high pressuresnd temperatures. The actual working volumes used for hydrolysis by HTS weredjusted, as required, by adding deionized water [16]. A range of temperatures (140,50, 160, 170, 180, 190 or 200 ◦C), pressures (0.36–1.56 MPa) and residence times60, 90, 120, 150 or 180 min) were investigated in preparing the HTS hydrolysate.he optimal condition of sulfuric acid hydrolysis at 100 ◦C with 2.0% (v v−1) sulfuriccid in deionized water with a final volume of 2–4 L for 120 min was used to preparehe sulfuric acid hydrolysate [17].

The raw HTS or sulfuric acid hydrolysates were obtained by cooling theydrolysates to 40 ◦C and filtering to remove the insoluble compounds. Therocessed HTS or sulfuric acid hydrolysates were obtained by maintaining theydrolysate at 100 ◦C for 15 min in the opened reactor to remove some of the volatile

nhibitors to fermentation and concentrate the sugar content. After cooling downo 40 ◦C, the hydrolysates were neutralized to pH 7.0 with 1 M NaOH and filteredo remove the insoluble compounds to obtain the processed HTS or sulfuric acidydrolysates.

The raw and processed HTS or sulfuric acid hydrolysates were concentrated by factor of 2–10 times by vacuum evaporation at 55 ◦C for storage and fermentation.

.3. Microorganism and xylitol fermentation experiments

.3.1. Microorganism screening and cultivation adaptationC. tropicalis As 2.1776 was purchased from China General Microbiological

ulture Collection Center (Beijing, China). After UV-mutagenesis, as previouslyescribed [18], C. tropicalis JA 309, one of the mutated strains, was used in thistudy because it demonstrated the highest xylitol yield and volumetric productivitymong all the mutants. The strain was cultured on an agar slant (pH 6.0) containing

g l−1 yeast extract, 4 g l−1 glucose, 10 g l−1 xylose and 20 g l−1 agar at 30 ◦C for 48 hnd stored at 4 ◦C.

The adaptation culture medium (initial pH 5.0–6.0) contained an increasing gra-ient of concentration of processed HTS hydrolysate from 40 to 140 g l−1 xylose andhe following components: 10 g l−1 glucose, 10 g l−1 yeast extract, 3 g l−1 KH2PO4,

g l−1 (NH4)2HPO4 and 0.1 g l−1 MgSO4·7H2O. The total xylose concentration in theedium was fixed at 140 g l−1 by adding some xylose solid. With increasing amounts

f the concentrated HTS hydrolysate, the adaptability of C. tropicalis JA 309 grad-ally increased. Finally, the adapted cell was able to tolerate the processed HTSydrolysate with an initial xylose concentration = 140 g l−1 in the medium. More-ver, after each batch of adaptation cultivation, the adapted cell was checked in theested fermentation culture (the processed HTS hydrolysate with an initial xyloseoncentration = 140 g l−1, 12 g l−1 yeast extract, 3 g l−1 KH2PO4, 2 g l−1 (NH4)2HPO4

nd 0.1 g l−1 MgSO4·7H2O) for their ability to produce xylitol. The volume of mediumas 50 ml in 250 ml flasks during both the microorganism screening and adaptation

ultivation.

.3.2. Preparation of cell inoculumCells from the slant were used to aseptically inoculate 50 ml of inoculum

edium in 250 ml flasks at 30 ◦C for 24 h with shaking at 200 rpm. The inoculumedium (initial pH 5.0–6.0) includes 20 g l−1 xylose, 10 g l−1 glucose, 10 g l−1 yeast

stry 46 (2011) 1619–1626

extract, 3 g l−1 KH2PO4 and 2 g l−1 (NH4)2HPO4. The cell inoculum prepared withoutcentrifugation was directly inoculated into the fermentation medium with variousinitial cell concentrations.

2.3.3. Xylitol bioconversion conditionsThe various fermentative conditions of initial hydrolysate concentration, initial

cell concentration, initial pH value and fermentation temperature were investigatedin 250 ml Erlenmeyer flasks containing 50 ml of the fermentation medium (the rawand processed HTS or sulfuric acid hydrolysates of different initial xylose concen-tration with 12 g l−1 yeast extract, 3 g l−1 KH2PO4, 2 g l−1 (NH4)2HPO4 and 0.1 g l−1

MgSO4·7H2O). The two-step dissolved oxygen (DO) process was adopted using therotary shaker speed of 200 rpm from the beginning of the process to 24–26 h and150 rpm to the end of fermentation.

Xylitol fermentation was carried out in a 20 L bioreactor (B. Braun Biotech Inter-national, Germany) provided with temperature, stirring, aeration and pH valuecontrollers, a DO value controller and two turbines with six-plane blades. The vol-ume of medium was fixed at 14 L with stirring at a rate of 200 rpm. DO conditionsby various ventilations of air through one volume of broth per minute (v v−1 min−1)were examined in the bioreactor.

The initial pH of the medium was adjusted by the addition of 1 M NaOH or 1 MHCl, and the pH value in the broth altered naturally throughout the cultivationsin all the experiments. Fermentation was monitored through periodic sampling todetermine cell growth, arabinose and xylose consumption and xylitol yield, andfermentation was stopped when the xylitol concentration no longer increased inthe broth.

2.4. Analysis methods

The composition of the dried raw material and the solid residue after hydrol-ysis was analyzed by following methods previously published in the literature[15,19,20]. The cell optical density (OD) was determined at 550 nm using a UV spec-trophotometer (Shimadzu UV-1700, Jiangsu, China) and converted to cell dry weightby an appropriate calibration curve. Phenolic compounds were measured by theFolin–Ciocalteu (FC) method as described by Wang [21]. The absorbance was mea-sured at 765 nm on the UV spectrophotometer described above. A standard curvewith serially diluted phenol solutions was used for calibration. The reducing sugarand total sugar in the samples was directly determined using our previously pub-lished method [5]. A standard curve with serial xylose solutions was used for sugarcalibration. The conductivities of the hydrolysates were measured by a DDS-12Adigital conductivity meter (Lida Instrument Factory, Shanghai, China).

All samples were filtered through 0.22 �m filters and diluted prior to high-performance liquid-chromatography (HPLC) analysis. The analysis was performedusing a Hitachi HPLC system (Hitachi, Tokyo, Japan) and the N2000 software (EjerTechnol. Co. Ltd., Zhejiang, China). Xylitol and individual sugars in the hydrolysatesand broths were measured on a Sugar-pak1 column (Waters, Milford, MA, USA)at 80 ◦C. As the mobile phase, ultra-pure water was supplied at a flow rate of0.5 ml min−1. Aliphatic acids such as acetic acid and formic acid were measuredon an Aminex HPX-87H column (Bio-Rad, Hercules, CA, USA) at 45 ◦C. As the mobilephase, 5 mM H2SO4 was supplied at a flow rate of 0.6 ml min−1. Detections were per-formed using a refractive index detector (Hitachi, Tokyo, Japan). Xylitol yield wasdetermined by the ratio of final xylitol concentration to initial xylose concentra-tion. Furfural and HMF were measured on a BioSil C18 column (Bio-Rad, Hercules,CA, USA) at room temperature. The mobile phase consisted of 40% (v v−1) aque-ous methanol, adjusted to pH 3 by the addition of 1 M HCl and supplied at a flowrate of 0.6 ml min−1. A UV-detector (Hitachi, Tokyo, Japan) at 230 nm was used fordetection.

Results represent the average of three independent experiments conducted withthree replicates for each condition. Standard deviations are below 3.0%.

3. Results and discussion

3.1. Composition and characteristics of the HTS and acidhydrolysates

The corncob raw material contains 40–44% cellulose, 31–33%hemicellulose, 16–18% lignin and 3–5% ash. Xylose, the basic rawmaterial for xylitol fermentation from the hemicellulosic fraction,was the most abundant monosaccharide in the HTS hydrolysate(Table 1). Xylose, glucose, arabinose, mannose and galactose werepresent in the two hydrolysates, with the smaller amounts of themonosaccharides and more XO yields found in the HTS hydrolysatecompared to the acid hydrolysate. The amounts of monosaccha-

rides in the processed HTS and sulfuric acid hydrolysates were24.3% and 31.1%, respectively. The yields of XO in the processedHTS and sulfuric acid hydrolysates were 5.24% and 0.31%, respec-tively (Table 1). However, the total sugar in the HTS hydrolysate

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Table 1Composition of the hydrolysates with HTS (160 ◦C, 120 min) or 2.0% sulfuric acid (100 ◦C, 120 min) treatments.a

Composition of the hydrolysates (drymatter of corncob (%))

Treatments

HTS 2% Sulfuric acid

Rawb Processedb Rawb Processedb

(mean ± SD) (mean ± SD) (mean ± SD) (mean ± SD)

Xylose 20.1 ± 0.40 18.3 ± 0.30 26.2 ± 0.50 23.9 ± 0.40Glucose 2.97 ± 0.05 2.64 ± 0.04 3.56 ± 0.06 3.18 ± 0.07Mannose 0.85 ± 0.02 0.74 ± 0.03 1.13 ± 0.03 0.95 ± 0.04Galactose 0.87 ± 0.03 0.75 ± 0.02 1.02 ± 0.03 0.90 ± 0.02Arabinose 1.98 ± 0.07 1.83 ± 0.11 2.33 ± 0.14 2.16 ± 0.13Xylo-oligosaccharides 5.85 ± 0.14 5.24 ± 0.13 0.42 ± 0.03 0.31 ± 0.02Xylan 1.08 ± 0.04 0.97 ± 0.03 –c –c

Acetic acid 0.72 ± 0.02 0.28 ± 0.02 1.19 ± 0.03 0.42 ± 0.02Formic acid 0.28 ± 0.01 0.14 ± 0.01 0.36 ± 0.02 0.20 ± 0.01Others (furfural and HMF) 0.60 ± 0.02 0.12 ± 0.01 0.74 ± 0.03 0.17 ± 0.01Phenolic compounds 0.82 ± 0.03 0.45 ± 0.02 0.92 ± 0.03 0.52 ± 0.02pH 4.02 ± 0.03 6.81 ± 0.04 0.72 ± 0.03 6.73 ± 0.05Conductivity (�s cm−1) 938 ± 7.0 455 ± 5.0 18170 ± 20.0 3275 ± 10.0

a After hydrolysis of the corncob, the solid residue contains the most cellulose (which is practically not affected by the hydrolysis), lignin (less than 10% of lignin isdepredated by hydrolysis to phenolic compounds and so on) and residual hemicelluloses (less than 5% of hemicelluloses are residual in the waste stream).

b For the production methods for the raw and processed samples; please refer to Section 2.2.

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nd sulfuric acid hydrolysate were similar (values in the processedTS and sulfuric acid hydrolysates were 30.6% and 31.4%, respec-

ively), demonstrating that HTS hydrolysis was a good choice forxtracting sugars from corncob. The amounts of furfural, HMF,cetic acid, formic acid and phenolic compounds, which are allnown inhibitors of microbial fermentation [22], were determinedn both the HTS and acid hydrolysates. Severe inhibition to the fer-

entation has been found with high levels of a cocktail of thesenhibitors [23], although other researchers have discovered that. tropicalis has the capacity to metabolize phenolic compounds,hich were translated into the formation of biomass [24,25]. The

nhibitors listed above were observed in the HTS hydrolysate at lowevels (Table 1). These results indicate that the degree of hydrol-sis by HTS was milder, with some XO remaining in the HTSydrolysate. With respect to lignocellulose from corncob, even theydrolyzed products were broken down more significantly withreater inhibitor production by the sulfuric acid hydrolysis [10].igh levels of the inhibitors in the broth were not only harmful

o the yeast growth and xylitol bioconversion [26], but also to therocesses of xylitol purification and crystallization [11]. This inhi-ition in the acid hydrolysis was limited after performing with aomplicated detoxification before fermentation [23]. At the veryeast, a method such as the activated charcoal treatment knowns Carvalheiro’s method [27] had to be used to decrease inhibitorevels in the acid hydrolysate, which may increase the cost. By con-rast, because there were fewer inhibitors in the HTS hydrolysateTable 1), xylitol fermentation could be performed directly withouthe detoxification [28].

Table 1 shows that the final pH value of the HTS hydrolysate,hich decreased with elevated hydrolysis temperature, was muchigher than that of the sulfuric acid hydrolysate. Furthermore, theonductivities of the raw and processed HTS hydrolysates wereuch lower than those of the raw and processed sulfuric acid

ydrolysates (938 �s cm−1 vs. 18170 �s cm−1 and 455 �s cm−1 vs.275 �s cm−1). In other words, the conductivity increments by HTSydrolysis were only one-fifth of those by sulfuric acid hydrolysis

or 1 g xylose production, suggesting that much fewer ions were

roduced in the hydrolysate by HTS. Higher ion concentrations

n hydrolysates mean a greater burden for ion removal using ionxchange resin in the subsequent processing, with more associatedost and environmental pollution.

Xylose was the main product from our final HTS hydrolysatewith a small quantity of XO also present (xylose yield of 20.1%, XOyield of 5.8%). In contrast, XO that was not thoroughly hydrolyzedto xylose was the main product in the final hydrolysate with HTSobtained by Boussarsar (xylose yield of 10.2%, XO yield of 11.8%)[10]. It was suggested that a secondary hydrolysis using enzymesshould be used to increase the recovery of xylose monomer from XOin Boussarsar’s research. Moreover, XO was also the primary prod-uct in the HTS hydrolysate obtained by Rivas [14] (xylose yield of2.4%, XO yield of 20.3%). Consequently, this HTS hydrolysis had to befollowed by a secondary hydrolysis with 0.5% sulfuric acid at 125 ◦Cfor 165 min to obtain more xylose, resulting in the conductivity ofRivas’s final hydrolysate being much higher than that of our finalhydrolysate (conductivities: 6430 �s cm−1 vs. 938 �s cm−1). There-fore, it is more suitable to obtain the xylose-abundant hydrolysateby the HTS hydrolysis method described by our research, withoutsecondary hydrolysis with either acid or enzymes, providing anenvironmentally friendly and efficient method to generate xylosefor use in xylitol bioconversion.

3.2. Optimization of HTS hydrolysis

Fig. 1A shows that the highest reducing sugar yield (24.3%)and xylose yield (20.1%) in the processed HTS hydrolysate wereobtained at 170 ◦C and 160 ◦C, respectively, with a residence timeof 120 min. The reducing sugar yield increased steadily with anincreasing temperature below 170 ◦C. When the temperature wasgreater than 160 ◦C, the xylose yield increased slightly by furtherXO hydrolysis, and the amount of XO and glucose increased slightlyby the hydrolysis of xylan and cellulose, respectively, resulting ina slight increase of reducing sugar [5]. However, the xylose con-centration was reduced during the HTS hydrolysis over 160 ◦C for120 min because the consumption of xylose by degradation and theMaillard reaction was more than the xylose produced by hydrolysis,resulting in more fermentative inhibitors (furfural, acid, phenoliccompounds and melanoidins) produced in the hydrolysate [22].Therefore, 160 ◦C is the optimum temperature for xylose produc-

tion by HTS hydrolysis.

As shown in Fig. 1B, increases in reducing sugar and xyloseyields were not apparent when the hydrolysis time was maintainedfor longer than 120 min at 160 ◦C. On the contrary, the amount of

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1622 L. Wang et al. / Process Biochemistry 46 (2011) 1619–1626

Fig. 1. Effects of temperature, time and ratio of water to corncob mass onHTS hydrolysis. (A) Yields of reducing sugar and xylose in the processed HTScorncob hemicellulose hydrolysate with different heating temperatures (heatingtime = 120 min, the ratio of water to corncob mass is 20:1). (B) Yields of reducingsugar and xylose in the processed HTS corncob hemicellulose hydrolysate for differ-ent lengths of heating time (temperature = 160 ◦C, the ratio of water to corncob massis 20:1). (C) Yields of reducing sugar and xylose in the processed HTS corncob hemi-cellulose hydrolysate with different ratio of water to corncob mass (dry weight ratioof water to corncob mass at 4 ◦C; temperature = 160 ◦C and heating time = 120 min).Ycc

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Fig. 2. Results of xylitol fermentation tests by each batch of the adapted C. tropicalisfrom the processed HTS hydrolysate with the following initial conditions: xyloseconcentration = 140 g l−1, pH 6.0 and cell concentration = 1.2 g l−1. The fermentationwas performed at 30 ◦C using a two-step DO process with the rotary shaker speed of

ields of reducing sugar and xylose (%): the percent of the final reducing sugar con-entration (g l−1) or xylose concentration (g l−1) divided by the initial corncob massoncentration (g l−1).

nhibitors increased rapidly under this high temperature for such long time (data not shown), which is consistent with the pub-ished data [20]. Considering the cost savings inherent in shorterncubations and the decreasing inhibitor production, the optimumydrolysis time was 120 min, with favorable yields of reducingugar and xylose (24.9% and 20.1%, respectively) obtained in therocessed HTS hydrolysate.

Fig. 1C shows that the yields of reducing sugar and xylosencreased with an increasing ratio of water to corncob mass. Whenhe ratio was between 10 and 120, with HTS hydrolysis performedt 160 ◦C for 120 min, the yields of reducing sugar and xylose in the

200 rpm for the first 24–26 h and 150 rpm to the end of fermentation at 48 h. Xylitolyield (%): the percent of the final xylitol concentration (g l−1) in the broth dividedby the initial xylose concentration (g l−1) in the medium.

processed hydrolysate increased from 18.4% to 28.3% and 16.6% to21.4%, respectively. However, because corncob is an abundant andcheap natural resource, it was more suitable to reduce the costsof energy consumption and the amount of concentration requiredlater by reducing the ratio of water to corncob mass to 20:1 in theHTS hydrolysis. When the ratio of water to corncob mass was 20:1,the optimal yields of reducing sugar and xylose (24.2% and 19.5%,respectively) obtained in the processed HTS hydrolysate (Fig. 1C)accounted for approx. 75% of the reducing sugar and xylose yieldsin the processed acid hydrolysate.

3.3. The effect of HTS hydrolysate concentration on xylitolfermentation

As shown in Table 1, to decrease the inhibitor concentration,the processed HTS and sulfuric acid hydrolysates were employedas described in Section 2.2 before fermentation, resulting in a lossof sugar less than 6% and the removal of some volatile compounds(almost all furfural and HMF, 45–65% of formic acid and aceticacid, as well as 40–50% of the phenolic compounds). However, theresidual inhibitors, particularly accompanying the processed con-centrated hydrolysates with a high initial sugar concentration, hadnegative effects on cell growth and xylitol yield, which is in agree-ment with the previous literature [1]. Consequently, cell adaptationcultivation using batches was adopted to improve xylitol biocon-version. The inoculum of the first batch used for strain adaptationwas the inoculum preparation described in Section 2.3.2. The cellsuspension from the first batch adaptation broth was divided intotwo parts. One part was used as the inoculum for a second batchwith an initial cell concentration of 1.5 g l−1, and the other part wasused as the inoculum for xylitol fermentation to test the results ofthe cell adaptation (Fig. 2). The results of the fermentation were alltested in the same manner. It was found that with an increase ofthe adaptation batch times, the tolerance capacity of C. tropicalisto the concentrated HTS hydrolysate gradually increased (Fig. 2).There were gradually increasing yields of xylitol and cell mass, anddecreasing amounts of residual xylose. A xylitol yield of 0.71 g g−1,a cell dry weight of 11 g l−1 and a residual xylose yield of less than

5 g l−1 was obtained after 20 batches of cell adaptation cultivation(each batch lasted about 48 h), and a negligible increase of xyli-tol was observed after more than 21 batches (Fig. 2). By contrast,the tolerance capacity of C. tropicalis to the concentrated sulfu-

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L. Wang et al. / Process Biochemi

Fig. 3. The effect of the initial pH value on the xylitol bioconversion process from theprocessed HTS hydrolysate with initial xylose concentration of 140 g l−1 and initialcell concentration of 1.2 g l−1 at 30 ◦C and a two-step DO process at the rotary shakerseo

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peed of 200 rpm from the beginning of the process to 24–26 h and 150 rpm to thend of fermentation at 48 h. Adjustment of the various initial pH values were carriedut by the addition of 1 M NaOH or 1 M HCl.

ic acid hydrolysate increased insignificantly, with a resulting lowylitol yield and cell mass as well as much more residual xyloseemaining in the broth. Consequently, without complicated detox-fication before fermentation, the HTS hydrolysate could be directlyermented to xylitol by the adapted C. tropicalis.

.4. The effect of the initial cell concentration on xylitolermentation

With an increase of initial cell concentration from 0.42 g l−1

o 1.25 g l−1, the xylitol yield and xylitol volumetric productiv-ty increased from 88.7 g l−1 to 100.8 g l−1 and 1.56 g l−1 h−1 to.10 g l−1 h−1, respectively (Table 2). Meanwhile, the fermentationeriod was decreased from 56.6 h to 45.4 h (Table 2). When the

nitial cell concentration was higher than 1.62 g l−1, the increase innitial cell concentration over the limit tolerated by the culture mayead to a decrease in the DO availability in the medium, resulting in

ore xylose residue in the broth. In addition, more produced xyli-ol and supplied nutrient were consumed by large numbers of cellsith a decrease in xylitol yield and volumetric productivity. More-

ver, the medium was significantly diluted by the large amountf inoculum, increasing the cost of fermentation, which is consis-ent with published data [9]. When the initial cell concentrationas lower than 0.81 g l−1, the xylitol yield decreased dramaticallyith a decrease in initial cell concentration. This finding is also in

greement with the previous literature [29], suggesting that xylitolield and volumetric productivity increase, and both the negativeffects of inhibitors and cell death from assimilation or degradationre limited when the initial cell concentration is suitable. Therefore,he optimum initial cell concentration for xylitol fermentation fromhe HTS hydrolysate was 1.25 g l−1.

.5. The effects of initial pH values and fermentationemperatures on xylitol fermentation

If the initial pH value was in the range of 5.75–6.25, optimum fer-entation results for xylitol yield, xylose consumption and biomass

ormation were obtained (Fig. 3). This may be because inhibition ofylitol fermentation by the undissociated form of acetic acid, which

ncreased with the increase in hydrolysate concentration, primarilyepends on the alteration of the initial pH value in the medium [30].

t is consistent with the conclusion of the previous literatures thathe negative effect on xylitol bioconversion from the hydrolysate is

stry 46 (2011) 1619–1626 1623

mainly attributable to the undissociated form of acetic acid in themedium [11,31]. Therefore, an adjustment of the initial pH value ofthe medium was adopted (optimum initial pH 6.0) to decrease theinhibition of xylitol fermentation by undissociated acetic acid.

When the fermentation temperature was maintained at25–35 ◦C, a xylitol yield of 0.58–0.71 g g−1 and a xylitol volumet-ric production of 1.36–2.12 g l−1 h−1 were obtained (Table 3). Thexylitol volumetric production was highest and the biomass wasthe largest when the fermentation temperature was 35 ◦C because35 ◦C is suitable for cell growth and proliferation, with an enhance-ment on fermentation efficiency [1]. However, excessive biomassformation consumes more xylose and xylitol in the broth as carbonsources [7], resulting in a decrease in the amount of xylose availableas the substrate for xylitol bioconversion. Moreover, the residualxylose concentration was higher in the broth maintained at 35 ◦Ccompared with 30 ◦C (3.8 g l−1 vs. 3.1 g l−1) because of the higheractivity of xylose reductase at 30 ◦C. This finding is in agreementwith previously published literature [28]. Therefore, the optimalfermentation temperature was determined to be 30 ◦C, resulting inthe highest xylitol yield, the lowest residual xylose concentrationand a moderate cell dry weight.

3.6. Fermentation process in the bioreactor

During xylitol fermentation, the DO condition is a key factor,not only in cell growth by adjusting carbon flow, but also in xylitolbioconversion by regulating enzyme activity [32]. Some inhibitorswere present in the fermentation medium, and from Fig. 4, it canbe observed that the xylitol volumetric productivity was largerand the negative effects on cell growth by the inhibitors wereless when higher aeration was supplied by method A (aerobiccondition) rather than the lower aeration provided by method C(oxygen-limited condition). This may be due to the presence ofhydrolysis-derived inhibitors [12]. However, oxygen-limited con-ditions are the optimum DO condition for the bioconversion ofcommodity xylose liquor to xylitol because a great deal of NADH isaccumulated and the activity of xylitol dehydrogenase is restrained[33]. Therefore, it is necessary to find a suitable method to con-trol the DO condition for xylitol bioconversion directly from theHTS hydrolysate. Fig. 4 shows that the two-step DO adjustment(method B) was the optimum DO condition for xylitol bioconver-sion directly from the HTS hydrolysate, resulting in the maximumxylitol production (100.8 g l−1), xylitol yield (71%) and xylitol volu-metric productivity (2.12 g l−1 h−1) with the lowest residual xylose(6.8 g l−1) and arabinose (2.1 g l−1) as well as moderate cell dryweight (10.8 g l−1) after fermentation for 48 h. This may be due tothe higher aeration (1.0 v v−1 min−1) used to obtain aerobic condi-tions in the first step from the beginning of fermentation to 26 hthat was adopted for achieving not only a higher consumption ofinhibitors (e.g. acetic acid) by the yeast but also decreased fermen-tation time and increased cell growth and xylitol yield, which isconsistent with previously published results [31,34]. Because theconcentration of inhibitors was low and the cell concentration wassuitable at 26 h after the start of fermentation, a lower aerationrate (0.4 v v−1 min−1) was used to obtain oxygen-limiting condi-tions in the second stage between 26 h and 48 h of fermentation.The lower aeration rate in the second step maintained moderate cellgrowth and decreased the cellular consumption of xylose and xyli-tol and restrained the activity of xylitol dehydrogenase to increasethe xylitol accumulation, consistent with the previous literature[29].

During the fermentation, it was found that glucose in the HTS

hydrolysate blocked the production of the enzymes responsiblefor the metabolism of xylose, and a sequential consumption of thetwo main sugars is observed. In addition, galactose, mannose andarabinose began to be consumed after the glucose was depleted,

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1624 L. Wang et al. / Process Biochemistry 46 (2011) 1619–1626

Table 2Effects of the initial cell concentration on xylitol fermentation from the HTS hydrolysate by C. tropicalis.a

Mean ± SD

X0 (g l−1)b S0 (g l−1)c t (h)d S (g l−1)e P (g l−1)f X (g l−1)g QP (g l−1 h−1)h YP/S0(g g−1)i

0.42 ± 0.01 138.5 ± 2.0 56.6 ± 0.3 2.82 ± 0.04 88.7 ± 1.2 6.7 ± 0.12 1.56 ± 0.03 0.63 ± 0.020.81 ± 0.02 140.3 ± 2.1 49.2 ± 0.2 3.05 ± 0.05 97.4 ± 1.6 8.9 ± 0.14 1.94 ± 0.03 0.70 ± 0.021.25 ± 0.02 141.1 ± 2.4 45.4 ± 0.3 3.51 ± 0.04 100.8 ± 2.0 11.1 ± 0.16 2.10 ± 0.05 0.71 ± 0.011.62 ± 0.03 141.5 ± 2.2 50.7 ± 0.3 4.29 ± 0.05 95.6 ± 2.3 11.5 ± 0.21 1.89 ± 0.04 0.66 ± 0.012.06 ± 0.03 139.6 ± 2.1 57.9 ± 0.3 3.06 ± 0.05 88.5 ± 2.1 12.6 ± 0.22 1.53 ± 0.03 0.62 ± 0.02

a Results of xylitol bioconversion from the processed HTS hydrolysate by C. tropicalis at different initial cell concentration with an initial pH 6.0, at 30 ◦C and with a two-stepDO process at the rotary shaker speed of 200 rpm from the beginning until 24–26 h and 150 rpm to the end of fermentation.

b Initial cell concentration (g l−1).c Initial xylose concentration (g l−1).d Fermentation time (h).e Residual xylose concentration (g l−1).f Final xylitol concentration (g l−1).g Final cell concentration (g l−1).h Volumetric productivity of xylitol (g l−1 h−1).i Xylitol yield (g g−1), the ratio of the final xylitol concentration (g l−1) and the initial xylose concentration (g l−1).

Table 3Effects of fermentation temperature on xylitol fermentation from the HTS hydrolysate by C. tropicalis.a

Mean ± SD

T (◦C)b t (h) S (g l−1) P (g l−1) YP/S0(g g−1) QP (g l−1 h−1) X (g l−1)

10 ± 0.2 150 ± 0.3 116.4 ± 0.2 10.2 ± 0.2 0.06 ± 0.01 0.06 ± 0.01 6.12 ± 0.115 ± 0.2 133 ± 0.3 95.6 ± 0.2 18.4 ± 0.3 0.11 ± 0.01 0.12 ± 0.01 8.08 ± 0.220 ± 0.2 79 ± 0.3 75.8 ± 0.2 45.1 ± 0.4 0.30 ± 0.02 0.53 ± 0.02 10.17 ± 0.325 ± 0.2 60 ± 0.2 37.2 ± 0.2 83.7 ± 0.5 0.58 ± 0.01 1.36 ± 0.03 11.22 ± 0.330 ± 0.2 46 ± 0.2 3.1 ± 0.2 102.1 ± 0.5 0.71 ± 0.01 2.05 ± 0.03 11.67 ± 0.335 ± 0.2 43 ± 0.2 3.8 ± 0.2 96.6 ± 0.6 0.67 ± 0.02 2.12 ± 0.03 12.34 ± 0.340 ± 0.2 36 ± 0.2 37.9 ± 0.2 41.4 ± 0.4 0.27 ± 0.01 1.07 ± 0.02 11.45 ± 0.345 ± 0.2 38 ± 0.2 111.1 ± 0.2 12.7 ± 0.2 0.07 ± 0.01 0.39 ± 0.01 8.33 ± 0.2

a Results of xylitol bioconversion from the processed HTS hydrolysate by C. tropicalis at the different fermentation temperatures (initial xylose concentration = 140 g l−1,i tary sf

wacsa4pwo

TC

ct

nitial cell concentration = 1.2 g l−1, initial pH 6.0 and two-step DO process at the roermentation).

b Temperature (◦C).

hich is consistent with previous observations [26,28,32]. Sugarsnd alcohols, except for xylitol, xylose, arabinose, XO and xylan,ould not be detected in the final broth, indicating that most of theugars were utilized by C. tropicalis as carbon sources, which is ingreement with previous studies [17]. In total, 15.2 g l−1 of XO and

−1

.1 g l of xylan were left in the broth (Table 4). However, as inap-ropriate carbon sources for microbial metabolism, XO and xylanere hardly decomposed or biotransformed by C. tropicalis with-

ut influencing xylitol bioconversion and could be removed from

able 4ompositions of the xylitol fermentation broths using C. tropicalis from the HTS and sulfu

Compositions of fermentation broths (g l−1) Treatments

HTS

Rawb

(mean ± SD)

Residual xylose 6.33 ± 0.16

Xylitol 85.7 ± 1.2

Xylitol yield (%) 61.4 ± 1.0

Xylitol volumetric productivity (g l−1 h−1) 1.82 ± 0.04

Arabinose 3.1 ± 0.05

Xylo-oligosaccharides 18.8 ± 0.31

Xylan 5.7 ± 0.12

Other sugars 1.21 ± 0.03

Final cell concentration 11.0 ± 0.18

Conductivity (�s cm−1) 1815 ± 10.0

a Results of xylitol bioconversion from the HTS and sulfuric acid corncob hemicellulose

oncentration = 140 g l−1, initial pH 6.0, initial cell concentration = 1.2 g l−1, 30 ◦C for 47 h ao 24–26 h and 150 rpm to the end of fermentation).

b For the production methods for the raw and processed samples; please refer to Sectioc Cannot be detected.

haker speed of 200 rpm from the beginning to 24–26 h and 150 rpm to the end of

the broth by the subsequent separation process to become valuableby-products.

Table 4 shows that the maximum xylitol yields and xyli-tol volumetric productivities, fermented by C. tropicalis directlyfrom the raw and processed HTS hydrolysate, were 61.8% and

−1 −1 −1 −1

1.81 g l h and 71.4% and 2.12 g l h , respectively. These val-ues were 1.74- and 1.70-fold and 1.58- and 1.49-fold of the xylitolyields and xylitol volumetric productivities, respectively, fer-mented from the sulfuric acid hydrolysate (35.6% and 1.07 g l−1 h−1

ric acid hydrolysates by HPLC analysis.a

2% sulfuric acid

Processedb Rawb Processedb

(mean ± SD) (mean ± SD) (mean ± SD)

2.42 ± 0.05 70.27 ± 1.4 44.35 ± 0.0598.1 ± 1.4 49.8 ± 1.1 67.9 ± 0.0570.1 ± 1.1 35.6 ± 1.0 48.5 ± 0.052.08 ± 0.05 1.06 ± 0.02 1.41 ± 0.05

2.4 ± 0.06 4.3 ± 0.08 2.8 ± 0.0516.2 ± 0.27 1.4 ± 0.04 1.0 ± 0.03

4.6 ± 0.11 –c –c

1.03 ± 0.03 1.61 ± 0.04 1.32 ± 0.0311.7 ± 0.15 8.5 ± 0.02 10.6 ± 0.25

1380 ± 10.0 19080 ± 30.0 4185 ± 15.0

hydrolysate by C. tropicalis under the proper fermentation conditions (initial xylosend a two-step DO process at the rotary shaker speed of 200 rpm from the beginning

n 2.2.

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Fig. 4. The effect of the DO conditions on the xylitol bioconversion process from theprocessed HTS hydrolysate by C. tropicalis fermentation of 14 L of medium in a 20 Lbioreactor with an initial xylose concentration of 140 g l−1, an initial pH 6.0, and aninitial cell concentration of 1.2 g l−1, at 30 ◦C for 48 h. (A) Results of xylose and arabi-nose consumption, xylitol yields and cell dry-mass with DO conditions of aeration of1.0 v v−1 min−1 (aerobic condition) by the A method. (B) Results of xylose and arabi-nose consumptions, xylitol yields and cell dry-mass with DO conditions of aerationof 1.0 v v−1 min−1 from 0 to 26 h (aerobic condition) and 0.4 v v−1 min−1 after 26 h(oxygen-limited condition) of fermentation by the B method. (C) Results of xyloseand arabinose consumptions, xylitol yields and cell dry-mass with DO conditions ofaeration of 0.4 v v−1 min−1 (oxygen-limited condition) by the C method.

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and 45.2% and 1.42 g l−1 h−1, respectively). Moreover, to obtain 1 gof xylitol, the total conductivity increments after fermentation fromthe raw and processed HTS hydrolysate were about only one-fourthand one-sixth of the conductivity increments, respectively, afterfermentation from the raw and processed sulfuric acid hydrolysate.In addition, it has also been reported that inhibition on xylitol fer-mentation from the acid hydrolysate that was only neutralizedwas severe [17,35]. Therefore, xylitol bioconversion by C. tropi-calis directly from the HTS hydrolysate, rather than a sulfuric acidhydrolysate, has a wide range of potential applications.

Acknowledgments

This work was supported by the National High-tech Researchand Development Program (2009AA02Z202 to Qipeng Yuan), theNatural Science Foundation of China (20976009 to Qipeng Yuan)and Beijing Nova Program (2010B013 to Ming Yang).

References

[1] Sreenivas-Rao R, Pavana-Jyothi C, Prakasham RS, Sarma PN, Venkateswar-RaoL. Xylitol production from corn fiber and sugarcane bagasse hydrolysates byCandida tropicalis. Bioresour Technol 2006;97:1974–8.

[2] Emidi A. Xylitol its properties and food application. Food Technol1978;32:20–32.

[3] Pepper T, Olinger PM. Xylitol in sugar-free confections. Food Technol1988;42:98–106.

[4] Santos JC, Converti A, Carvalho W, Mussatto SI, Silva SS. Influence of aer-ation rate and carrier concentration on xylitol production from sugarcanebagasse hydrolyzate in immobilized-cell fluidized bed reactor. Process Biochem2005;40:113–8.

[5] Yuan QP, Zhang H, Qian ZM, Yang XJ. Pilot-plant production of xylo-oligosaccharides from corncob by steaming, enzymatic hydrolysis andnanofiltration. J Chem Technol Biotechnol 2004;79:1073–9.

[6] Mikkola JP, Vainio H, Salmi T, Sjöholm R, Ollonqvist T, Väyrynen J. Deactivationkinetics of Mo-supported Raney Ni catalyst in the hydrogenation of xylose toxylitol. Appl Catal A 2000;196:143–55.

[7] Liaw WC, Chen CS, Chang WS, Chen KP. Xylitol production from rice strawhemicellulose hydrolysate by polyacrylic hydrogel thin films with immobilizedCandida subtropicalis WF79. J Biosci Bioeng 2008;105:97–105.

[8] Tada K, Horiuchi JI, Kanno T, Kobayashi M. Microbial xylitol production fromcorn cobs using Candida magnoliae. J Biosci Bioeng 2004;98:228–30.

[9] Converti A, Perego P, Sordi A, Torre P. Effect of starting xylose concentration onthe microaerobic metabolim of Debaryomyces hansenii. Appl Biochem Biotech-nol 2002;101:15–29.

10] Boussarsar H, Rogé B, Mathlouthi M. Optimization of sugarcane bagasse conver-sion by hydrothermal treatment for the recovery of xylose. Bioresour Technol2009;100:6537–42.

11] Larsson S, Palmqvist E, Hahn-Hägerdal B, Tengborg C, Stenberg K, Zacchi G,et al. The generation of fermentation inhibitors during dilute acid hydrolysis ofsoftwood. Enzyme Microb Technol 1999;24:151–9.

12] Parajó JC, Domínguez H, Domíhguez JM. Improved xylitol production withDebaryomyces hansenii Y-7426 from raw or detoxified wood hydrolysates.Enzyme Microb Technol 1997;21:18–24.

13] Ballesteros I, Oliva JM, Negro MJ, Manzanares P, Ballesteros M. Enzymic hydrol-ysis of steam exploded herbaceous agricultural waste (Brassica carinata) atdifferent particule sizes. Process Biochem 2002;38:187–92.

14] Rivas B, Domínguez JM, Domínguez H, Parajó JC. Bioconversion of posthydrol-ysed autohydrolysis liquors: an alternative for xylitol production from corncobs. Enzyme Microb Technol 2002;31:431–8.

15] Garrote G, Domínguez H, Parajó JC. Autohydrolysis of corncob: study ofnon-isothermal operation for xylooligosaccharide production. J Food Eng2002;52:211–8.

16] Negro MJ, Manzanares P, Ballesteros I, Oliva JM, Cabanas A, Ballesteros M.Hydrothermal pretreatment conditions to enhance ethanol production frompoplar biomass. Appl Biochem Biotechnol 2003;108:87–100.

17] Domínguez JM, Cao NJ, Gong CS, Tsao GT. Dilute acid hemicellulosehydrolysates from corn cobs for xylitol production by yeast. Bioresour Technol1997;61:85–90.

18] Cassier-Chauvat C, Fabre F. A similar defect in UV-induced mutagenesis con-ferred by the rad6 and rad18 mutations of Saccharomyces cerevisiae. Mutat Res1991;254:247–53.

19] Cheng KK, Zhang JA, Ling HZ, Ping WX, Huang W, Ge JP, et al. Optimization

of pH and acetic acid concentration for bioconversion of hemicellulose fromcorncobs to xylitol by Candida tropicalis. Biochem Eng J 2009;43:203–7.

20] Ruiz E, Cara C, Manzanares P, Ballesteros M, Castro E. Evaluation of steamexplosion pre-treatment for enzymatic hydrolysis of sunflower stalks. EnzymeMicrob Technol 2008;42:160–6.

Journal Identification = PRBI Article Identification = 9237 Date: June 27, 2011 Time: 8:19 am

1 chemi

[

[

[

[

[

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[

[

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626 L. Wang et al. / Process Bio

21] Wang T, Jónsdóttir R, Ólafsdóttir G. Total phenolic compounds, radical scav-enging and metal chelation of extracts from Icelandic seaweeds. Food Chem2009;116:240–8.

22] Helle S, Cameron D, Lam J, White B, Duff S. Effect of inhibitory compoundsfound in biomass hydrolysates on growth and xylose fermentation by a genet-ically engineered strain of S. cerevisiae. Enzyme Microb Technol 2003;33:786–92.

23] Martín C, Jonssön LJ. Comparison of the resistance of industrial and labora-tory strains of Saccharomyces and Zygosaccharomyces to lignocellulosic-derivedfermentation inhibitors. Enzyme Microb Technol 2003;32:386–95.

24] Yan J, Wen JP, Li HM, Yang SL, Hu ZD. The biodegradation of phenol at highinitial concentration by the yeast Candida tropicalis. Biochem Eng J 2005;24:243–7.

25] Weng XY, Sun JY. Biodegradation of free gossypol by a new strain of Candidatropicalis under solid state fermentation: effects of fermentation parameters.Process Biochem 2006;41:1663–8.

26] García Martín JF, Cuevas M, Bravo V, Sánchez S. Ethanol production from olive

prunings by autohydrolysis and fermentation with Candida tropicalis. RenewEnergy 2010;35:1602–8.

27] Carvalheiro F, Duarte LC, Lopes S, Parajó JC, Pereira H, Gírio FM. Evaluation ofthe detoxification of brewery’s spent grain hydrolysate for xylitol productionby Debaryomyces hansenii CCMI 941. Process Biochem 2005;40:1215–23.

[

stry 46 (2011) 1619–1626

28] Parajó JC, Domínguez H, Domínguez JM. Biotechnological production of xyli-tol. Part 1. Interest of xylitol and fundamentals of its biosynthesis. BioresourTechnol 1998;65:191–201.

29] Felipe MGA, Alves LA, Silva SS, Roberto IC, Mancilha IM, Almeidae-Silva JB.Fermentation of Eucalyptus hemicellulosic hydrolysate to xylitol by Candidaguilliermondii. Bioresour Technol 1996;56:281–3.

30] Morita TA, Silva SS, Felipe MGA. Effects of initial pH on biological synthesisof xylitol using xylose-rich hydrolysate. Appl Biochem Biotechnol 2000;84-86:751–9.

31] Lima LHA, Felipe MGA, Vitolo M, Torres FAG. Effect of acetic acid present inbagasse hydrolysate on the activities of xylose reductase and xylitol dehydro-genase in C. guilliermondii. Appl Microb Biotechnol 2004;65:734–8.

32] Winkelhausen E, Kuzmanova S. Microbial conversion of d-xylose to xylitol. JFerment Bioeng 1998;86:1–14.

33] Nigam P, Singh D. Production of xylitol—a sugar substitute. Process Biochem1995;30:117–24.

34] Perego P, Converti A, Palazzi E, Del Borghi M, Ferraiolo G. Fermentation of hard-

wood hemicellulose hydrolysate by Pachysolen tannophilus, Candida shehataeand Pichia stipitis. J Indus Microb Biotechnol 1990;6:157–64.

35] Roberto IC, Felipe MGA, Lacis LS, Silva SS, Mancilha IM. Utilization of sugarcane bagasse hemicellulosic hydrolysate by Candida guilliermondii for xylitolproduction. Bioresour Technol 1991;36:271–5.