Short communication

Influence of the toxic compounds present in brewer’s spent

grain hemicellulosic hydrolysate on xylose-to-xylitol bioconversion

by Candida guilliermondii

Solange I. Mussatto *, Giuliano Dragone, Ines C. Roberto

Departamento de Biotecnologia, Faculdade de Engenharia Quımica de Lorena, Rodovia Itajuba-Lorena,

km 74.5, Cx. Postal 116, Cep: 12600-970, Lorena, SP, Brazil

Received 29 April 2005; received in revised form 24 June 2005; accepted 30 June 2005

Abstract

Fermentation media containing different concentrations of toxic compounds were prepared from brewer’s spent grain (BSG) hemi-

cellulosic hydrolysate, and used for xylose-to-xylitol bioconversion by Candida guilliermondii. Such fermentation media were composed of

the hydrolysate in the following ways: raw (RH); concentrated four-fold (CH); concentrated and treated with activated charcoal (TCH); raw

supplemented with sugars until a concentration four-fold higher (SRH); concentrated and subsequently diluted but supplemented with sugars

until a concentration four-fold higher (SDCH). All media presented an initial xylose concentration of 85 g/l, except RH, which contained 23 g/

l xylose. Fermentation results revealed that the sugars supplementation to raw hydrolysate favored the xylitol production. Nevertheless, xylitol

production from CH was negatively affected due to the high concentration of toxic compounds present in the medium. The hydrolysate

treatment with activated charcoal partially removed the toxic compounds, and the xylitol production was higher than in CH, but not so efficient

as in SRH. It was thus concluded that to obtain an efficient xylose-to-xylitol bioconversion from BSG hydrolysate, the sugars concentration

must be increased, but the toxic compounds concentration must be reduced to the same level present in the raw hydrolysate.

# 2005 Elsevier Ltd. All rights reserved.

Keywords: Brewer’s spent grain; Hemicellulosic hydrolysate; Xylose; Toxic compounds; Xylitol; Candida guilliermondii

www.elsevier.com/locate/procbio

Process Biochemistry 40 (2005) 3801–3806

1. Introduction

Xylitol is a natural food edulcorant with sweetener power

similar to sucrose, but that can substitute this sugar with

advantages because it presents important clinical properties

and considerable pharmaceutical applications [1]. For these

reasons, the xylitol use in the food and pharmaceutical

industries has increased in the last years. On an industrial

scale, xylitol is produced by the catalytic hydrogenation of

D-xylose from hemicellulosic hydrolysates, a high-cost

process that requires high pressure (up to 50 atm) and

temperature (80–140 8C), use of an expensive catalyst

(Raney-Nickel), use of extensive xylose purification steps,

and yields 50–60% xylitol [2,3]. Nevertheless, xylitol

can also be produced by microorganisms (fermentative

* Corresponding author. Tel.: +55 12 3159 5027; fax: +55 12 3153 3165.

E-mail address: solange@debiq.faenquil.br (S.I. Mussatto).

1359-5113/$ – see front matter # 2005 Elsevier Ltd. All rights reserved.

doi:10.1016/j.procbio.2005.06.024

pathway), and this process appears to be more economical

than the chemical industrially used.

Various xylose-rich hemicellulosic materials, such as rice

straw, sugarcane bagasse, eucalyptus wood, corn stover and

brewer’s spent grain, can serve as abundant and cheap

feedstocks for xylitol production by fermentation [4–8].

These materials are normally hydrolysed with dilute acid to

liberate xylose, the sugar that is used as substrate for xylitol

production. However, several compounds that are toxic to

the microorganisms are also liberated in the sugar liquor

during the acid hydrolysis of these materials. Such

compounds include hydroxymethylfurfural and furfural

(resulted from the degradation of hexoses and pentoses,

respectively), acetic acid (released from the hemicellulose

acetyl groups) and phenolics (resulted from the partial lignin

degradation) [9,10]. These toxic compounds can inhibit the

microbial metabolism during the hydrolysates fermentation,

and the maximum allowable concentration for each one of

S.I. Mussatto et al. / Process Biochemistry 40 (2005) 3801–38063802

them in a fermentation medium cannot be established, since

it strongly depends on many factors, such as the degree of

adaptation of the microorganism to the medium, the

fermentation process employed, and the simultaneous

presence of several other inhibitors, which can have

synergistic effects [9].

The present work evaluated the influence of the toxic

compounds present in BSG hydrolysate on xylose-to-xylitol

bioconversion by the yeast Candida guilliermondii FTI

20037. Assays were carried out with different hydrolysate-

based media, which were prepared by concentration of the

hydrolysate, its detoxification or not with activated charcoal,

and its supplementation or not with sugars. The hydrolysate

media thus prepared contained around 85 g/l xylose, but they

differed in the concentration of toxic compounds. Fermen-

tation of these hydrolysates made possible to verify the

effect of the toxic compounds on xylitol production as well

as on the yeast productive capacity.

2. Materials and methods

2.1. Raw material and hydrolysate preparation

BSG originated from a process employing 100% malt

(without addition of other cereal adjuncts), was supplied by

the microbrewery of the Faculty of Chemical Engineering of

Fig. 1. Schematic representation of the procedures used for preparation

Lorena. As soon as obtained, the material was washed with

water until neutral pH and dried at 50 � 5 8C to 10%

moisture content. The hemicellulosic hydrolysate was

prepared by acid hydrolysis in 1.5-l stainless-steel batch

reactors, as optimized by Mussatto and Roberto [4]. After

hydrolysis, the resulting solid material was separated by

centrifugation and the filtrate (raw hydrolysate) was stored

at 4 8C. Part of the raw hydrolysate was concentrated under

vacuum in a 4-l evaporator at 70 � 5 8C, to obtain a xylose

content of approximately 85 g/l.

2.2. Microorganism cultivation

Cells of the yeast C. guilliermondii FTI 20037 (ATCC

201935), were maintained at 4 8C on malt extract agar

slants. The inocula were prepared by transferring a slant of

cells to test tubes containing about 5 ml of sterilized water.

Aliquots of 2 ml of this suspension were then transferred to

250 ml Erlenmeyer flasks containing 100 ml of the

medium consisted of 20 g/l xylose, 3.0 g/l (NH4)2SO4,

0.1 g/l CaCl2�2H2O and 20% (v/v) rice bran extract. The

culture was incubated at 200 rpm, 30 8C for 24 h. Subse-

quently, the cells were recovered by centrifugation

(1100 � g, 20 min), washed, and resuspended in the

fermentation medium.

Solutions of all the components were prepared as

described by Mussatto and Roberto [4].

of the fermentation media from BSG hemicellulosic hydrolysate.

S.I. Mussatto et al. / Process Biochemistry 40 (2005) 3801–3806 3803

Table 1

Composition of the brewer’s spent grain hydrolysates used as fermentation

medium for xylitol production

Compound Concentration in the hydrolysate (g/l)

RH SRH CH TCH SDCH

Glucose 1.51 6.00 6.08 6.02 5.93

Xylose 22.97 84.21 88.62 86.94 86.52

Arabinose 10.24 38.75 40.70 39.96 39.77

Furfural 0.66 0.61 0.01 0.00 0.00

Hydroxymethylfurfural 0.10 0.08 0.10 0.01 0.00

Acetic acid 1.25 1.22 3.83 1.53 1.05

Total phenolics 4.01 3.93 10.38 5.38 3.34

2.3. Media and fermentation conditions

The procedures used for preparation of the fermentation

media from raw and concentrated hydrolysates are presented

in Fig. 1.

The pH of the hydrolysates was adjusted by addition of

NaOH (pellets) and the formed precipitate was removed by

centrifugation (1100 � g, 20 min). The hydrolysate treat-

ment with activated charcoal was based on the addition of

2.5 g charcoal per 100 g of hydrolysate, at pH 2.0,

150 rpm, 45 8C for 60 min. All the five obtained

fermentation media were analysed by chromatography

in order to determine the sugars and toxic compounds

concentration.

Fermentation assays were performed in 250 ml Erlen-

meyer flasks containing 100 ml of medium inoculated with

an initial cell concentration of 1 g/l. The flasks were agitated

in a rotatory shaker at 200 rpm, 30 8C, for 30 h (RH) or 96 h

(SRH, CH, TCH and SDCH). The fermentation runs were

monitored through periodic sampling in order to determine

cell growth, glucose and xylose consumption, and xylitol

production.

2.4. Analytical methods

Sugars and toxic compounds concentrations were

determined by high-performance liquid chromatography

(HPLC) [11]. Vanillin, syringaldehyde, ferulic acid and

syringic acid concentrations were determined under the

same conditions employed for hydroxymethylfurfural and

furfural determination. The total phenolic compounds

concentration was estimated by ultraviolet spectroscopy

at 280 nm, according to the methodology described by

Mussatto and Roberto [11]. Cell concentration was

determined in a spectrophotometer at 600 nm, by means

of a calibration curve (dry weight versus optical density

[OD]) obtained from cells grown in hydrolysate medium, at

200 rpm, 30 8C, for 24 h.

3. Results and discussion

3.1. Composition of the BSG hydrolysate

The main components of the raw BSG hydrolysate (RH)

are shown in Table 1. It can be noted in this table that under

the employed hydrolysis conditions, hemicellulose sugars

were the main monosaccharides liberated in the liquor, being

the xylose + arabinose concentration 22-fold higher than the

glucose concentration. These results demonstrate that the

hydrolysis process was highly selective to liberate hemi-

cellulose sugars. Besides, the low glucose concentration

favors the hydrolysate use as fermentation medium for the

xylose-to-xylitol bioconversion, since high concentrations

of this hexose inhibit the xylose metabolism by yeasts [12–

14].

Furan derivatives compounds were also found in RH, and

among these, furfural was present in a higher concentration

than hydroxymethylfurfural. This is justifiable, since

pentoses sugars are present in large quantity in the

hydrolysate, and furfural is formed by their decomposition;

while hydroxymethylfurfural is formed by the decomposi-

tion of glucose, a hexose present in small amount in the

hydrolysate. Notwithstanding, the low concentration of

furfural and hydroxymethylfurfural in RH suggests that,

under the employed hydrolysis conditions, pentose and

hexose sugars were few susceptible to degradation. Acetic

acid and phenolic compounds were also present in RH, as

previously reported by Mussatto and Roberto [4]. Phenolics

were the toxic compounds present in the highest concentra-

tion in RH, followed by acetic acid in a concentration 3.2

times lower.

When the raw hydrolysate was concentrated, the sugars

content increased proportionally to the employed factor

(around four-fold), showing that there was no sugar

degradation during the concentration process (Table 1).

On the other hand, the toxic compounds concentration did

not increase proportionally to the employed factor. The

furfural concentration, for example, was drastically reduced

during this process because under vacuum furfural presents a

boiling point at 54–55 8C [15]. The hydroxymethylfurfural

concentration was maintained constant, suggesting that this

compound was partially volatilized or degraded during the

concentration step. The acetic acid concentration increased

three-fold. According to Rodrigues et al. [16] the low pH of

the hydrolysate (�1.0) favors the partial volatilization of this

acid during vacuum concentration process, since under this

condition, the acetic acid is in the undissociated form

(pKa = 4.75). The total phenolics content increased 2.6-fold,

suggesting that some of these compounds are also volatile at

70 8C under vacuum.

The hydrolysate treatment with activated charcoal

reduced the concentration of the toxic compounds and

practically did not interfere in the sugars concentration.

Furfural and hydroxymethylfurfural were practically elimi-

nated from the hydrolysate by this treatment, while the

acetic acid and phenolic compounds concentrations were

reduced in 60 and 48%, respectively. A still lower

concentration of these compounds was obtained when the

S.I. Mussatto et al. / Process Biochemistry 40 (2005) 3801–38063804

Table 2

Fermentative parameters and final cell concentration obtained during the

xylitol production from BSG hydrolysates

Hydrolysate YP/Sa QP YX/S

a YP/Xa h X

RH 0.65 0.38 0.20 2.34 70.88 4.91

SRH 0.79 0.86 0.06 10.00 86.15 6.23

CH 0.37 0.13 0.10 2.28 40.35 5.28

TCH 0.55 0.18 0.13 2.83 59.98 6.02

SDCH 0.67 0.78 0.07 7.60 73.06 7.37

YP/S = xylitol yield coefficient for xylose consumed (g/g); QP = xylitol

volumetric productivity (g/l h); YX/S = cell mass yield coefficient for sub-

strate (xylose + glucose) consumed (g/g); YP/X = xylitol yield coefficient

with respect to cell mass (g/g); h = xylitol production efficiency (% of the

maximum theoretical value = 0.917 g/g); X = total cell concentration at the

fermentation end (g/l).a Values calculated by linear regression of the obtained results.

concentrated hydrolysate was diluted and subsequently

supplemented with sugars (SDCH). In SDCH, the acetic acid

and phenolic concentrations were 72 and 68% lower than in

concentrated hydrolysate, being similar to the concentra-

tions present in the diluted hydrolysates (RH and SRH).

Nevertheless, the furfural and hydroxymethylfurfural pre-

sence in the diluted hydrolysates constitutes the main

difference between these and the SDCH.

3.2. Fermentation of BSG hydrolysates

The initial xylose concentration in the fermentation

medium is one of the most important factors that interfere on

xylitol production. For concentrations of this pentose lower

than 50 g/l, the kinetic behavior of the yeast is deviated from

xylitol production to the cell mass formation [17,18]. This

behavior was also observed in the present work, since the

highest YX/S value (cell mass yield coefficient for consumed

substrate) was obtained from RH (Table 2). In fact, RH

proportioned a final cell concentration similar to that

obtained from CH, but in a fermentation time three times

lower. For this reason, the hydrolysates must be initially

concentrated to be efficiently used as fermentation medium

Fig. 2. Xylose consumption (A) and xylitol production (B) from BSG

for xylitol production. Nevertheless, when the raw BSG

hydrolysate was concentrated, the concentration of some

toxic compounds (acetic acid and phenolics) was also

increased, and consequently interfered in the xylose-to-

xylitol bioconversion. It can be noted in Table 2 that the

fermentative parameters YP/S, QP and YX/S were negatively

affected when the hydrolysate was concentrated. However,

the yeast productive capacity (YP/X) was similar to that

obtained in RH. This demonstrates that the increase in the

toxic compounds concentration interfered mainly in the

xylose consumption by the yeast, since the consumed xylose

was converted into xylitol with the same efficiency as in RH.

The glucose consumption was not affected by the increase in

the toxic compounds concentration, since this sugar was

totally consumed by the yeast at the beginning of all the

fermentations. On the other hand, the arabinose was not

consumed in any medium during all the considered

fermentation time.

Fig. 2 shows the xylose consumption (A) and xylitol

production (B) by C. guilliermondii from BSG hydrolysates

containing high sugars concentration. It can be noted that the

yeast performance was strongly affected by the concentra-

tion of toxic compounds present in the hydrolysate. The

xylose consumption (Fig. 2A) was similar to the hydro-

lysates CH and TCH, suggesting that, although the high

removal of total toxic compounds (around 50%), the

hydrolysate treatment with activated charcoal did not favor

the consumption of this pentose. However, the xylitol

production from treated hydrolysate (Fig. 2B) was slightly

improved, and this reflected in an increase in all the

fermentative parameters values (Table 2). These results

suggest that the xylitol production from BSG hydrolysate is

favored when the toxic compounds concentration in the

medium is decreased. Nevertheless, it must be detached that

the conditions employed in this study for hydrolysate

treatment with activated charcoal were previously optimized

for rice straw hydrolysate [19]. BSG hydrolysate treated

under these conditions did not promote a significant

improvement on xylitol production as observed for rice

hydrolysates: SRH (&), CH (*), TCH (~) and SDCH (!).

S.I. Mussatto et al. / Process Biochemistry 40 (2005) 3801–3806 3805

Table 3

Concentration of some phenolic compounds found in the BSG hydrolysates

Compound Concentration in the hydrolysate (g/l)

RH SRH CH TCH SDCH

Vanillin 0.0302 0.0291 0.0719 0.0359 0.0218

Syringaldehyde 0.1776 0.1753 0.6127 0.3163 0.1932

Ferulic acid 0.0598 0.0595 0.2246 0.0898 0.0709

Syringic acid nd nd 0.0435 0.0165 0.0122

nd = not detected.

straw hydrolysate. This permits to conclude that the

hydrolysate treatment conditions must be optimized for

each raw material used.

The xylose consumption from SRH and SDCH was faster

than from CH or TCH (Fig. 2A). Besides, the SRH and

SDCH also proportioned a higher xylitol concentration at

the end of the process (Fig. 2B). Among these, the maximum

xylitol production occurred in SRH (62.3 g/l in 72 h),

corresponding to a yield coefficient of 0.79 g/g and a

volumetric productivity of 0.86 g/l h. Although both

hydrolysates presented similar cell yield for consumed

substrate (YX/S), the values of xylitol yield coefficient with

respect to cell mass (YP/X) confirmed that the yeast

productive capacity was higher in SRH than in SDCH

(Table 2). Analysing the composition of these two

hydrolysates (Table 1) it can be noted that the main

difference among them is in terms of furfural, compound

that was present in SRH and absent in SDCH. This fact

suggests that besides the presence of furfural in the

hydrolysate (0.61 g/l) has not inhibited the fermentative

process, it favored the xylitol production. Ojamo et al. [20]

reported a beneficial effect of low furfural concentrations

(0.6 g/l) on xylitol production. Besides, several authors

reported that furfural is only toxic to the microorganisms

when present in concentrations higher than 1 g/l [21–23].

Another possible explanation for the difference observed

during the fermentations of the SRH and SDCH (Table 2),

would be in function of the individual concentration of some

phenolic compounds. Total phenolic compounds consist of a

number of phenolic compounds (vanillin, syringaldehyde,

ferulic and syringic acids, among others) precedents from

the partial lignin degradation during the acid hydrolysis

process. Such compounds present different toxicity degrees

for the microorganisms. For example, Cortez [24] observed

that vanillin concentrations up to 2 g/l favored the xylitol

production byC. guilliermondii, while the syringaldehyde in

the same concentration, was strongly toxic to this

microorganism. In the present work, vanillin and syringal-

dehyde were found in the BSG hydrolysates composition, as

well as ferulic and syringic acids (Table 3). When the

hydrolysate was concentrated, the syringaldehyde concen-

tration increased 3.4-fold, while the concentration of

vanillin, which is not considered toxic to this microorgan-

ism, increased 2.4-fold. It is possible thus that during the

hydrolysate concentration, phenolic compounds of highest

toxicity for the microorganism had their concentration

increased in larger proportion than the phenolic compounds

of lower toxicity. This could explain the highest toxicity of

the SDCH when compared to the SRH, in spite of both

present similar contents of total phenolic compounds.

Comparing the TCH and SRH fermentations, it can be

noted a large difference in the fermentative process results,

which mainly reflected in an increase of 4.8 times in QP

(from 0.18 to 0.86 g/l h, respectively), and 3.5 times in YP/X(from 2.83 to 10.0 g/g, respectively). When analysing the

composition of these two hydrolysates (Table 1) the large

difference observed in their fermentations can be attributed

to the higher concentration of total phenolic compounds in

TCH. In fact, among the toxic compounds present in BSG

hydrolysate, the phenolics appears to be the most inhibitory

to the microorganism, because their concentration was the

highest among all toxic compounds. On the other hand, as

the acetic acid is strongly toxic for C. guilliermondiiwhen in

concentrations higher than 3.0 g/l [25], it is probable that

this acid has also influenced in the high toxicity observed for

the medium formulated from concentrated BSG hydrolysate

(CH). It must be also considered that, although the acetic

acid was present in the media TCH, SRH and SDCH in

concentrations considered as not inhibitory to the micro-

organism, the presence of the phenolic compounds in these

media can potentialize the acetic acid toxicity, due to the

synergistic effect.

4. Conclusions

The xylitol production by C. guilliermondii yeast from

BSG hydrolysate was strongly influenced by the concentra-

tion of toxic compounds present in the medium, the lowest

fermentative parameters values being attained from con-

centrated hydrolysate medium, which contained the highest

inhibitors concentration (3.83 g/l acetic acid and 10.38 g/l

total phenolics). As the xylitol production depends on the

initial xylose concentration in the medium, it was thus

concluded that to increase the efficiency of this bioprocess

from BSG hydrolysate containing 85 g/l xylose, the toxic

compounds concentration (mainly the phenolics) must be

reduced to the levels present in the raw hydrolysate, which

did not interfere in the microbial metabolism.

Acknowledgements

The authors gratefully acknowledge CAPES, CNPq and

FAPESP, Brazil.

References

[1] Mussatto SI, Roberto IC. Xylitol: an edulcorant with benefits for

human health. Brazil J Pharmaceut Sci 2002;38:401–13.

[2] Parajo JC, Domınguez H, Domınguez JM. Biotechnological produc-

tion of xylitol. Part 1. Interest of xylitol and fundamentals of its

biosynthesis. Bioresour Technol 1998;65:191–201.

S.I. Mussatto et al. / Process Biochemistry 40 (2005) 3801–38063806

[3] Saha BC. Hemicellulose bioconversion. J Ind Microbiol Biotechnol

2003;30:279–91.

[4] Mussatto SI, Roberto IC. Acid hydrolysis and fermentation of brewer’s

spent grain to produce xylitol. J Sci Food Agric, in press.

[5] Santos JC, Converti A, Carvalho W, Mussatto SI, Silva SS. Influence

of aeration rate and carrier concentration on xylitol production from

sugarcane bagasse hydrolyzate in immobilized-cell fluidized bed

reactor. Process Biochem 2005;40:113–8.

[6] Mussatto SI, Roberto IC. Kinetic behaviour of Candida guilliermondii

yeast during xylitol production from highly concentrated hydrolysate.

Process Biochem 2004;39:1433–9.

[7] Mancilha IM, Karim MN. Evaluation of ion exchange resins for

removal of inhibitory compounds from corn stover hydrolyzate for

xylitol fermentation. Biotechnol Prog 2003;19:1837–41.

[8] Canilha L, Almeida e Silva JB, Solenzal AIN. Eucalyptus hydrolysate

detoxification with activated charcoal adsorption or ion-exchange

resins for xylitol production. Process Biochem 2004;39:1909–12.

[9] Mussatto SI, Roberto IC. Alternatives for detoxification of diluted-

acid lignocellulosic hydrolyzates for use in fermentative processes—a

review. Bioresour Technol 2004;93:1–10.

[10] Palmqvist E, Hahn-Hagerdal B. Fermentation of lignocellulosic

hydrolysates. I. Inhibition and detoxification. Bioresour Technol

2000;74:17–24.

[11] Mussatto SI, Roberto IC. Chemical characterization and liberation of

pentose sugars from brewer’s spent grain. J Chem Technol Biotechnol,

in press.

[12] Tavares JM, Duarte LC, Amaral-Collaco MT, Gırio FM. The influence

of hexoses addition on the fermentation of D-xylose in Debaryomyces

hansenii under continuous cultivation. Enzyme Microb Technol

2000;26:743–7.

[13] Walther T, Hensirisak P, Agblevor FA. The influence of aeration and

hemicellulosic sugars on xylitol production by Candida tropicalis.

Bioresour Technol 2001;76:213–20.

[14] Preziosi-Belloy L, Nolleau V, Navarro JM. Fermentation of hemi-

cellulosic sugars and sugar mixtures to xylitol by Candida parapsi-

losis. Enzyme Microb Technol 1997;21:124–9.

[15] Perry RH, Green DW. Perry’s chemical engineering handbook, 7th

ed., New York: McGraw-Hill, 1997.

[16] Rodrigues RCLB, Felipe MGA, Almeida e Silva JB, Vitolo M, Gomez

PV. The influence of pH, temperature and hydrolyzate concentration

on the removal of volatile and nonvolatile compounds from sugarcane

bagasse hemicellulosic hydrolyzate treated with activated charcoal

before or after vacuum evaporation. Brazil J Chem Eng 2001;18:299–

311.

[17] Parajo JC, Dominguez H, Dominguez JM. Production of xylitol from

raw wood hydrolysates by Debaryomyces hansenii NRRL Y-7426.

Bioprocess Eng 1995;13:125–31.

[18] Felipe MGA, Mancilha IM, Vitolo M, Roberto IC, Silva SS, Rosa SA.

Preparation of xylitol by fermentation of a hydrolysate of hemicellu-

lose obtained from sugarcane bagasse. Brazil Arch Biol Technol

1993;36:103–14.

[19] Mussatto SI, Roberto IC. Optimal experimental condition for hemi-

cellulosic hydrolyzate treatment with activated charcoal for xylitol

production. Biotechnol Prog 2004;20:134–9.

[20] Ojamo H, Ylinen L, Linko M. Process for the preparation of xylitol

from xylose by cultivating Candida guilliermondii. US Patent WO 88/

05467; 28 julho 1988.

[21] Martinez A, Rodriguez ME, York SW, Preston JF, Ingram LO. Effects

of Ca(OH)2 treatments (‘‘overliming’’) on the composition and toxi-

city of bagasse hemicellulose hydrolysates. Biotechnol Bioeng

2000;69:526–36.

[22] Parajo JC, Dominguez H, Dominguez JM. Improved xylitol

production with Debaryomyces hansenii Y-7426 from raw or detox-

ified wood hydrolysates. Enzyme Microb Technol 1997;21:

18–24.

[23] Roberto IC, Lacis LC, Barbosa MFS, Mancilha IM. Utilization

of sugarcane bagasse hemicellulosic hydrolysate by Pichia stipitis,

for the production of ethanol. Process Biochem 1991;26:

15–21.

[24] Cortez DV. Influencia dos produtos de degradacao da lignina na

bioconversao de xilose em xilitol por Candida guilliermondii. Master

Dissertation. Faculdade de Engenharia Quımica de Lorena, Faenquil,

Lorena, Brazil; 2005.

[25] FelipeMGA, Veira MV, Vitolo M,Mancilha IM, Roberto IC, Silva SS.

Effect of acetic acid on xylose fermentation to xylitol by Candida

guilliermondii. J Basic Microbiol 1995;35:171–7.