Biotechnol. Prog.

2005, 21, 1639−1643 1639

Variables That Affect Xylitol Production from Sugarcane Bagasse

Hydrolysate in a Zeolite Fluidized Bed Reactor
Júlio C. Santos, Solange I. Mussatto,* Mário A. A. Cunha, and Silvio S. Silva
Departamento de Biotecnologia, Faculdade de Engenharia Quı́mica de Lorena, Rodovia Itajubá-Lorena, km 74,5,
12600-970, Lorena, SP, Brazil

The operational conditions for xylitol production by fermentation of sugarcane bagasse

hydrolysate in a fluidized bed reactor with cells immobilized on zeolite were evaluated.
Fermentations were carried out under different conditions of air flowrate (0.0125-
0.0375 vvm), zeolite mass (100-200 g), initial pH (4-6), and xylose concentration (40-
60 g/L), according to a 24 full factorial design. The air flowrate increase resulted in a
metabolic deviation from product to biomass formation. On the other hand, the pH
increase favored both the xylitol yield (YP/S) and volumetric productivity (QP), and the
xylose concentration increase positively influenced the xylitol concentration. The best
operational conditions evaluated were based on the use of an air flowrate of 0.0125
vvm, 100 g of zeolite, pH 6, and xylose concentration of 60 g/L. Under these conditions,
38.5 g/L of xylitol were obtained, with a YP/S of 0.72 g/g, QP of 0.32 g/L‚h, and cell
retention of 25.9%.

Introduction operational conditions, and recently, the use of im-

Fluidized bed reactors (FBR) have been used in many
studies of microbial processes for high-value products
obtainment and wastewater treatment. The interest in
this kind of reactor is due to the several advantages that
it presents, which include high stability in long run tests,
facility of aeration control, good mass transfer charac-
teristics, and lower shearing stress for the biocatalyst
than in the stirred tank reactors. In general, the pro-
cesses performed in these bioreactors require immobiliza-
tion of the microorganisms on solid particles that are
immersed in a cultivation medium. Consequently, the
kind and quantity of particles employed as carrier for the
microorganism are important variables that can influence
the FBR operation, affecting the biomass growth and/or
the product formation. Some examples of cell carriers
used in FBR include diatomite, sand, calcium alginate
with alumina, and polyurethane. In previous works, FBR
was employed for xylitol production in a system with cells
of Candida guilliermondii FTI 20037 immobilized on
porous glass beads (1-3).
Xylitol is a polyalcohol with sweetener power similar
to that of sucrose and interesting properties that permit
its application in the food, odontological, and pharma-
ceutical industries (4). Currently, xylitol is produced by
a high-cost chemical process, but it can also be produced
by microorganisms (fermentative pathway). In fact, the
xylitol production by fermentation has received important
attention by researchers because this process appears to
be more economical than the chemical route, since it
requires mild conditions of pressure and temperature and
the hydrolysate can be used without extensive xylose
purification (5, 6). Many studies about xylitol production
by fermentation of hemicellulosic hydrolysates have been
thus performed aiming to establish the best process
* To whom correspondence should be addressed. Fax: +55 12
3153 3165. E-mail:solange@debiq.faenquil.br.
10.1021/bp050219n CCC: $30.25 © 2005 American Chemical Society and American Institute of Chemical Engineers
Published on Web 09/14/2005
mobilized cell systems has been evaluated in different
bioreactors (1-3, 7, 8).
In a previous work (7) zeolite was employed as im-
mobilization carrier for xylitol production in Erlenmeyer
flasks, and the YP/S and QP results were lower than those
obtained in a system using free cells. In that work, the
low performance of the zeolite system was attributed to
the direct exposure of the immobilized cells to the
mechanical stress caused by friction among the beads in
the flasks. As a better attrition control in the FBR com-
pared to the Erlenmeyer flasks is expected, the present
work deals with the determination of the operational
conditions for xylitol production in a FBR, using cells of
C. guilliermondii FTI 20037 immobilized by adsorption
on NaX-zeolite. The evaluated operational conditions
were air flowrate, zeolite mass, xylose concentration, and
initial pH. The employed substrate was the hemicellulosic
hydrolysate of sugarcane bagasse, an abundant byprod-
uct of sucro-alcoholic industries in Brazil.
Material and Methods
Hydrolysate Preparation. The employed sugarcane
bagasse was supplied by the Usina Guarany (Olı́mpia/
SP, Brazil). The hemicellulosic hydrolysate was prepared
by acid hydrolysis in a 250 L stainless steel reactor, which
was loaded with the sugarcane bagasse and a sulfuric
acid solution (100 mg acid/g dry weight of bagasse) in a
solid/liquid ratio of 1:10 (w/w). The hydrolysis was
performed at 120 °C for 10 min, and the composition of
the obtained hemicellulosic hydrolysate was (g/L) xylose
17.49, glucose 0.99, arabinose 1.02, and acetic acid 2.05.
The produced hydrolysate was subsequently concen-
trated by vacuum evaporation under 70 ( 5 °C in order
to reach a xylose concentration of 40-60 g/L. Afterward,
it was treated with activated charcoal in order to remove
compounds that are toxic to the yeast metabolism, as
described by Alves et al. (9). Finally, the hydrolysate was
autoclaved at 112 °C for 15 min.
1640
Inoculum Preparation and Fermentation Media.
Cells of C. guilliermondii FTI 20037 were maintained at
4 °C on malt extract agar slants. A loopful of a slant
culture was transferred to 125-mL Erlenmeyer flasks
containing 50 mL of the growth medium, which consisted
of 30 g/L xylose, 3.0 g/L (NH4)2SO4, 0.10 g/L CaCl2‚2H2O,
and 10% v/v rice bran extract. The flasks were main-
tained at 30 °C, 200 rpm for 24 h. Subsequently, the cells
were collected by centrifugation at 2000g for 20 min,
rinsed twice with sterile water, and utilized as inoculum.
Fermentation media were composed of the treated
hydrolysate supplemented with the following nutrients:
(NH4)2SO4 3.0 g/L; CaCl2‚2H2O 0.1 g/L and rice bran
extract 10% v/v.
Fluidized Bed Reactor (FBR) Operation and Cell
Immobilization. The FBR used in this work has been
described elsewhere (2). The bed was composed of NaX-
type zeolite UOP WE 894, purchased from Plury Quı́mica
S.A. (Diadema, SP, Brazil), whose physicochemical charac-
teristics were presented in a previous work (6). To be used
in the experiments, the zeolite beads were first treated
with a 0.5% HCl solution at 200 rpm for 4 h, washed with
distilled water for 24 h, and dried at 150 °C.
The FBR was loaded with 100-200 g of pretreated
carrier, and steam at 100 °C was passed during 30 min.
The accumulated water was removed and the bioreactor
was loaded with 1.6 L of fermentation medium and an
inoculum suspension containing 1.6 g dry-weight of free
cells. At the beginning of the fermentation, the yeast was
immobilized in situ by adsorption onto the zeolite surface.
Batch fermentations were carried out at 30 °C and were
monitored by sampling 5 mL of medium for each 24 h to
determine the xylitol, xylose, and free cell concentrations.
At the end of fermentation (when at least 90% xylose was
consumed) samples of zeolite beads were collected from
the reactor and employed in the analysis methodology
for immobilized cell concentration determination.
The initial pH of the media was adjusted with 6 N
NaOH/H2SO4, and the aeration rate was adjusted as
indicated in the experimental design. The bed fluidization
was promoted by fluid recirculation (2) and the ascendant
liquid velocity was 6.08 cm/s, corresponding to a bed
porosity of about 80%.
Experimental Design. A 24 full factorial design with
three replicates in the central point was utilized. The
levels of the independent variables selected for this study
were (coded values in parentheses): aeration rate of
0.0125 (-1) and 0.0375 vvm (+1); zeolite mass of 100 (-1)
and 200 g (+1); xylose concentration of 40 (-1) and 60
g/L (+1), and initial pH of 4.0 (-1) and 6.0 (+1). The
xylitol volumetric productivity (QP), xylitol yield (YP/S),
xylitol concentration (P), total cell concentration (X), and
cell retention (ηi), all at the end of fermentation, were
taken as dependent variables or responses. The effects
of the independent variables on these responses were
analyzed through the Statistica 5.1 software (StatSoft,
Tulsa/OK).
Analytical Methods and Response Variables Cal-
culation. Xylose and xylitol concentrations were mea-
sured by HPLC using a Shimadzu chromatograph, model
LC-10-AD (Tokyo, Japan), equipped with a Bio-Rad (Her-
cules, CA) Aminex HPX-87H (300 mm × 7.8 mm) column
and a refractive index RID 6A detector. Samples were pre-
viously filtered through a Sep Pak C18 filter and injected
in the chromatograph under the following conditions: 45
°C column temperature, 0.01 N H2SO4 as the mobile
phase, 0.6 mL/min flow rate, and 20 µL injection volume.
Methylene blue test for staining dead cells was em-
ployed to verify cell viability (10). Free cell concentration
Biotechnol. Prog., 2005, Vol. 21, No. 6
was determined by optical density measurements at 600
nm using a spectrophotometer (Beckman DU 640B,
California). The dry mass of cells immobilized in the
carrier was determined by drying the samples at 105 °C
until constant mass and subsequently burning all organic
substances at 950 °C until calcination. The difference
between carrier masses measured at 950 and 105 °C was
assumed to represent the dry mass of immobilized cells
in the sample and was employed to calculate the im-
mobilized cell concentration. The ash content of cells,
determined by a similar procedure, was taken into
consideration for calculations. Adsorbing materials be-
sides cells were previously quantified in blank experi-
ments without cells and taken into account when calcu-
lating the mass of immobilized cells.
Xylitol yield (YP/S, in g/g) was considered as the ratio
between xylitol production (g/L) and xylose consumption
(g/L), while the xylitol volumetric productivity (QP, in g/L‚
h) was the ratio between xylitol production (g/L) and
fermentation time (h). Cell retention (ηi) was assumed
to be the ratio between the immobilized and the total cell
concentration. The efficiency of xylose-to-xylitol biocon-
version was calculated as the ratio between experimental
YP/S value (g/g) and the theoretical YP/S value (0.917 g/g)
proposed by Barbosa et al. (11).
Results and Discussion
Table 1 shows the fermentation results obtained ac-
cording to the employed 24 full factorial design. It can be
noted in this table that the highest xylitol volumetric
productivities (QP) were obtained in experiments 2 and
10 and in the central points. The highest xylitol concen-
trations (P) were obtained at the minimum level of
aeration rate and maximum level of xylose concentration.
The highest efficiency of xylose-to-xylitol bioconversion
was 78% (corresponding to a YP/S value of 0.72 g/g) and
was obtained by using the minimum levels of aeration
rate and zeolite mass and the maximum levels of initial
pH and xylose concentration (experiment 10).
A statistical analysis of the estimated effects of the
variables for the responses related to the xylitol produc-
tion (YP/S, QP, and P), and total cell concentration and
retention (X and ηi) was thus performed and can be
visualized in Table 2. Such analysis showed that the
variables initial pH and air flowrate had a significant
influence on YP/S (p < 0.10 and p < 0.05 for the pH and
air flowrate, respectively). When the pH value was
increased from 4.0 to 6.0, an average increase of 0.13 g/g
was observed on YP/S. Otherwise, the air flowrate increase
from 0.0125 to 0.0375 vvm decreased the xylitol yield in
0.17 g/g.
The pH effect is strongly related to the acetic acid
presence in hemicellulosic hydrolysates (12, 13). When
the fermentations are carried out under low pH values,
the acetic acid is present in the medium mainly in the
nondissociated form, which can cross the plasmatic
membrane and go into the cell. When this acid gets into
the cytoplasm, it dissociates and reduces the intracellular
pH. According to the uncoupling theory, explained by
Palmqvist and Hahn-Hägerdal (13), the drop in the
intracellular pH, resulting from inflow of weak acids, is
neutralized by the action of the plasma membrane
ATPase, which pumps protons out of the cell at the
expense of ATP hydrolysis. As a consequence, a fraction
of the energy obtained by the xylose metabolism would
be used in this “proton pump”, decreasing the product
formation. The negative effect of the air flowrate on YP/S
is due to a deviation of the cellular metabolism from
Biotechnol. Prog., 2005, Vol. 21, No. 6 1641

Table 1. Experimental Matrix of the 24 Full Factorial Design for Evaluation of Xylitol Production in a FBR with Zeolite
Bed, under Different Operational Conditions
real values of the variables response variables
expt zeolite mass (g) initial pH xylose concn (g/L) air flowrate (vvm) YP/S (g/g) QP (g/L‚h) P (g/L) X (g/L) ηi (%)
1 200 6 60 0.0375 0.33 0.27 19.56 11.26 3.81
2 100 6 60 0.0375 0.45 0.37 26.47 11.95 9.12
3 200 4 60 0.0375 0.49 0.15 22.18 16.46 1.94
4 100 4 60 0.0375 0.09 0.02 5.34 16.62 9.51
5 200 6 40 0.0375 0.46 0.27 19.19 15.46 7.18
6 100 6 40 0.0375 0.50 0.29 20.81 8.71 0.46
7 200 4 40 0.0375 0.46 0.26 18.39 14.94 0.94
8 100 4 40 0.0375 0.08 0.01 3.08 14.70 7.07
9 200 6 60 0.0125 0.58 0.21 30.64 11.00 13.00
10 100 6 60 0.0125 0.72 0.32 38.49 6.37 25.90
11 200 4 60 0.0125 0.56 0.20 33.50 11.16 29.84
12 100 4 60 0.0125 0.58 0.22 36.38 11.06 14.10
13 200 6 40 0.0125 0.60 0.24 21.18 6.94 9.22
14 100 6 40 0.0125 0.44 0.23 16.00 5.21 18.62
15 200 4 40 0.0125 0.30 0.07 11.26 14.60 25.34
16 100 4 40 0.0125 0.46 0.11 19.09 7.03 16.79
17 150 5 50 0.0250 0.55 0.39 28.11 9.93 20.04
18 150 5 50 0.0250 0.56 0.35 24.81 9.60 20.31
19 150 5 50 0.0250 0.53 0.29 27.99 12.29 16.52

Table 2. Effects, Standard Errors (SE), and p-values for the Response Variables, According to the 24 Full Factorial
Design
xylitol yield xylitol xylitol volumetric total cell cell
YP/S concentration P productivity QP concentration X retention ηi
variablesa effects SE p effects SE p effects SE p effects SE p effects SE p
mean 0.46 (0.03 22.24 (1.37 0.23 (0.02 11.33 (0.48 13.14 (1.66
A -0.17 (0.07 0.03b -8.94 (2.99 0.02b 0.01 (0.05 0.92 4.59 (1.04 0.00b -14.10 (3.61 0.00b
B 0.06 (0.07 0.36 10.45 (2.99 0.01b 0.04 (0.05 0.50 1.04 (1.04 0.35 2.70 (3.61 0.48
C 0.13 (0.07 0.08c 5.39 (2.99 0.11 0.15 (0.05 0.02b -3.71 (1.04 0.01b -2.28 (3.61 0.55
D 0.06 (0.07 0.40 1.28 (2.99 0.68 0.01 (0.05 0.81 2.52 (1.04 0.04b -1.29 (3.61 0.73
A*B -0.10 (0.07 0.17 -7.43 (2.99 0.04b -0.04 (0.05 0.45 -0.42 (1.04 0.70 -0.52 (3.61 0.89
A*C 0.02 (0.07 0.74 3.87 (2.99 0.23 0.05 (0.05 0.39 -0.13 (1.04 0.91 2.56 (3.61 0.50
A*D 0.10 (0.07 0.17 4.63 (2.99 0.16 0.05 (0.05 0.32 -0.99 (1.04 0.37 -1.79 (3.61 0.63
B*C -0.04 (0.07 0.53 -0.95 (2.99 0.76 0.00 (0.05 1.00 0.03 (1.04 0.98 1.39 (3.61 0.71
B*D -0.03 (0.07 0.68 -1.48 (2.99 0.63 -0.04 (0.05 0.47 -1.55 (1.04 0.18 -1.22 (3.61 0.74
C*D -0.09 (0.07 0.19 -4.08 (2.99 0.21 -0.07 (0.05 0.21 0.58 (1.04 0.59 -3.94 (3.61 0.31
a A, air flowrate; B, xylose concentration; C, pH; D, zeolite mass. b Significant at 95% confidence level. c Significant at 90% confidence

level.

product to biomass formation under high aeration rates.
In fact, the air flowrate had a positive influence on the
total cell concentration (Table 2), resulting in an average
increase of 4.59 g/L in this response value. The deviation
of the yeast metabolism from xylitol production to bio-
mass formation has been reported by some authors (8,
14-16) and is related to coenzyme regeneration (4, 6).
Regarding the xylitol concentration (P), the statistical
analysis (Table 2) revealed that the variables xylose
concentration and air flowrate, as well as their interac-
tion were significant for this response (p < 0.05). The
xylose concentration increase resulted in an average
increase of about 10 g/L in the xylitol concentration, and
the aeration increase resulted in an average decrease of
about 9 g/L for this response. The interaction between
xylose concentration and air flowrate had a negative
effect on xylitol concentration. The effect of the air
flowrate has a similar explanation to that previously
given for the effect of the aeration on YP/S. The negative
effect of the interaction between the variables xylose
concentration and air flowrate on xylitol concentration
suggests that besides the individual effect of these
variables on xylitol production, the simultaneous increase
in the xylose concentration and decrease in the air
flowrate potentialize their individual effects on xylitol
production.
The statistical analysis of the data for the response QP
(Table 2) showed that the pH was the only variable with
influence on this response, presenting a main and posi-
tive effect (p < 0.05). Such effect is also related to the
acetic acid presence in hydrolysate, as previously explain-
ed. In fact, the initial pH was a variable of great influence
on xylitol production since both responses, xylitol yield
and productivity, were favored when the initial pH was
increased from 4.0 to 6.0. On the other hand, the pH
increase resulted in a low total cell concentration (nega-
tive effect, Table 2) at the end of fermentations. This fact
suggests that at the highest pH level, the yeast metabo-
lism was directed to product formation.
Additionally, Table 2 shows the effect of the studied
variables on the cell retention (ηi), defined as the fraction
of cells that were adsorbed onto the zeolite surface at the
end of the process. As can be observed, only the air
flowrate had a significant influence (p < 0.05) on this
response, and on average when the aeration was in-
creased from 0.0125 to 0.0375 vvm, the cell retention
decreased about 14%. This effect can be attributed to the
strong agitation caused by the high aeration employed,
which could have promoted a cell detachment from the
zeolite surfaces. Another possible explanation would be
the positive effect of the air flowrate on the cell concen-
tration (Table 2) that would promote an increase mainly
in the free cells concentration in the medium, causing
as consequence a decrease in the cell retention value.
Another important observation that can be stated
about the obtained results is that, at the end of fermen-
1642
Figure 1. Xylose consumption and xylitol production under the conditions that promoted the highest xylitol concentration and YP/S
value (air flowrate of 0.0125 vvm; initial pH of 6.0; xylose concentration of 60 g/L, and mass of carrier of 100 g (experiment 10)).
tations, the fraction corresponding to adsorbed cells was
lower than 30% of the total yeast population (Table 1),
showing a mixed system with free and immobilized cells.
The low cell retention can be attributed to the low area
of the employed carrier available for cell immobilization
(7). However, at the highest obtained ηi, the quantity of
yeast adsorbed onto the zeolite surface would be enough
to be used as inoculum in a new fermentation cycle in a
repeated batch system. Moreover, the cell viability analy-
ses showed that both free and immobilized cells were
viable almost in their totality.
On the whole, the results in FBR attained in the
present work were better than the former obtained in
Erlenmeyer flasks (7), and the high YP/S value (0.72 g/g,
experiment 10, Table 1) reached seems to confirm the
hypothesis of better attrition control in the FBR com-
pared to the flasks. In fact, by using FBR, the cells were
exposed to a minor mechanical stress compared to the
previous work employing flasks, which could explain the
higher xylitol concentration and YP/S value obtained.
However, the QP value was still low and perhaps operat-
ing the FBR under less turbulent conditions could
increase it.
Figure 1 shows the profile of substrate consumption
and product formation at the conditions that promoted
the highest YP/S and P values (experiment 10, Table 1).
As can be observed, the highest xylitol concentration was
obtained at 120 h of fermentation, when the consumption
of xylose was about 90%. After this time, the xylitol
concentration stayed constant for some hours and, near
144 h of fermentation, started to decrease. This decrease
suggests the xylitol consumption by the yeast when the
xylose was exhausted from the medium.
Conclusions
The xylitol production from sugarcane bagasse by
fermentation in a fluidized bed reactor with cells im-
mobilized onto a zeolite bed was influenced by the
employed operational conditions. Under the best opera-
tional conditions (initial pH of 6.0, air flowrate of 0.0125
vvm, xylose concentration of 60 g/L, and 100 g of zeolite),
a high YP/S value (0.72 g/g) was obtained and the xylitol
concentration attained 38.49 g/L. In addition, a total cell
concentration of 6.37 g/L was obtained with about 26%
of the total yeast population being adsorbed onto the
Biotechnol. Prog., 2005, Vol. 21, No. 6
zeolite surface. Regarding the independent variables, the
pH increase from 4.0 to 6.0 directed the cell metabolism
to xylitol production, whereas a more aerated system
resulted in a biomass formation increase, affecting the
xylitol production. The increase in xylose concentration
favored xylitol production, and the increase in zeolite
mass resulted in a higher total cell concentration.
Acknowledgment
The authors gratefully thank the FAPESP, CAPES,
and CNPq for financial support.
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Accepted for publication August 10, 2005.
BP050219N