Enzyme and Microbial Technology 31 (2002) 431–438

Bioconversion of posthydrolysed autohydrolysis liquors:

an alternative for xylitol production from corn cobs
B. Rivas, J.M. Domı́nguez, H. Domı́nguez, J.C. Parajó∗
Department of Chemical Engineering, Faculty of Science, University of Vigo (Campus Ourense), Polytechnical Building, As Lagoas, 32004 Ourense, Spain
Received 4 December 2001; received in revised form 25 February 2002; accepted 11 March 2002

Abstract
A readily fermentable hydrolysate was prepared by treating corn cobs in sequential steps of autohydrolysis (in aqueous media) and
posthydrolysis (in the presence of sulphuric acid). The fermentation of the liquors obtained with this processing scheme by Debaryomyces
hansenii resulted in 18% increase in productivity and 25% increase in yield in comparison with the results obtained in fermentation media
made by direct prehydrolysis of corn cobs. Two D. hansenii strains were compared for their ability to produce xylitol in media made with
autohydrolysis–posthydrolysis liquors. The best strain was adapted to raw and to detoxified, concentrated hydrolysates. Using membrane
sterilisation to avoid the thermal degradation of the culture media, productivities up to 1.49 g l−1 h−1 and product yields up to 0.73 g g−1
were obtained in batch cultures carried out at the optimal concentrations of xylose and biomass.
© 2002 Elsevier Science Inc. All rights reserved.
Keywords: Corn cobs; Autohydrolysis; Posthydrolysis; Debaryomyces hansenii; Xylitol

1. Introduction as well as the neutralising agent), the handling of neutral-

Corn cobs are an agricultural byproduct of lignocellu-
losic nature. As other lignocellulosic materials (LCMs), corn
cobs can be fractionated using a sequence of chemical treat-
ments allowing the separation of cellulose, hemicellulose
and lignin (untouched or as degradation products) in sepa-
rate streams. With this approach, the various fractions com-
ing from the process can be utilised for different product
applications.
The first stage of an LCM multi-step fractionation pro-
cess can be an acid hydrolysis, which leads to a solid
residue made up of cellulose and lignin useful for further
processing and to a liquid phase containing a variety of
hemicellulose-degradation products (including monomeric
sugars such as xylose, glucose, mannose, galactose, ara-
binose and rhamnose), sugar degradation products as fur-
fural and hydroxymethylfurfural (HMF), extractive-derived
compounds, phenolics coming from lignin degradation
(acid-soluble lignin), acetic acid and uronic acids [1,2]. The
hydrolysis liquors can be used to make fermentation media
suitable for a variety of purposes. The main drawbacks of
prehydrolysis are the equipment corrosion caused by the
mineral acid, the cost of reagents (the acid used as a catalyst
∗Corresponding author. Tel.: +34-98883887033; fax: +34-988387033.
E-mail address: jcparajo@uvigo.es (J.C. Parajó).
0141-0229/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved.
PII: S 0 1 4 1 - 0 2 2 9 ( 0 2 ) 0 0 0 9 8 - 4
isation sludges, the possibility that the lignin remaining
in solid phase undergoes repolymerisation reactions (lim-
iting its potential for further chemical utilisation) and the
generation of compounds acting as fermentation inhibitors.
In comparison with prehydrolysis, the autohydrolysis
treatments show several advantages for lignocellulose frac-
tionation. In this technology, water and LCM are the only
reagents, and a selective hemicellulose depolymerisation is
reached by the catalytic action of hydronium ions coming
from both water and in situ generated compounds (such as
acetic acid, uronic acid and phenolic acid) to give sugar
oligomers, monosaccharides and acetic acid as main reaction
products. The mildly acidic conditions used in the chemical
processing result in liquors with limited inhibitor concentra-
tion, particularly those coming from sugar decomposition
and acid-soluble lignin. The solid phase from autohydroly-
sis contains cellulose (which is practically not affected by
autohydrolysis), lignin and residual hemicelluloses.
Since the sugar oligomers produced in autohydrolysis
cannot be assimilated by yeasts, a posthydrolysis stage
(catalysed by acids or enzymes) is needed to produce the
corresponding sugars. The lower cost, simplicity and faster
kinetics of the acid postydrolysis makes it preferable over
enzymatic hydrolysis. Under selected conditions, the acid
posthydrolysis of oligomers can be carried out with minimal
decomposition into furfural [3,4]. Acetic acid (generated
432 B. Rivas et al. / Enzyme and Microbial Technology 31 (2002) 431–438
from acetylated oligomers) and other sugars (such as arabi-
nose and glucose) are posthydrolysis byproducts, and their
presence may affect the fermentability of liquors.
The fermentation of culture media obtained from direct
treatment of LCM can be hindered (or even prevented) by
inhibitors. Because of this, physico-chemical [5–8] and/or
biological detoxification [9,10] are necessary for a satisfac-
tory fermentation of hydrolysates. Since higher severity of
the chemical treatments results in increased inhibitory po-
tential of hydrolysates [11–14], mild operational conditions
are desirable.
Xylitol, a pentitol with high sweetening power, anticari-
ogenic properties and suitable for diabetics, has technolog-
ical and biological properties fostering its utilisation in the
food industry [15,16]. Even though xylitol can be produced
by a variety of micro-organisms, yeasts have been the most
widely studied ones. In this field, several Candida sp. and
D. hansenii strains have been employed in reported studies
[5,6,17–22].
This work deals with the fermentation of xylose-
containing liquors obtained from either autohydrolysis–
posthydrolysis or prehydrolysis of corn cobs, a raw material
rich in xylan (a hemicellulosic polymer made up of xylose
units). The effects caused by key factors affecting the fea-
sibility of the studied process (including the adaptation of
micro-organisms to both raw and detoxified hydrolysates
and the utilisation of increased initial xylose concentrations)
are assessed.
2. Materials and methods
2.1. Raw material
Corn cobs were collected locally, milled to a particle
size smaller than 1 mm and stored until use. Their average
composition, determined according to standard methodol-
ogy [23], was 31.7% of cellulose, 34.7% of hemicellulose,
20.3% of lignin and 3.4% of acetyl groups (oven-dry basis).
2.2. Preparation of hydrolysates
2.2.1. Autohydrolysis
Corn cobs were subjected to nonisothermal autohydrol-
ysis in a Parr reactor to reach 202 ◦ C with 8 g water/g dry
solid [23]. The solid and liquid phases were separated by
filtration and the composition of autohydrolysis liquors was
as follows: xylooligosaccharides (as xylose equivalent),
25.4 g l−1 ; degree of xylooligosaccharide acetylation, 0.224
acetyl groups per xylose unit; xylose, 3.05 g l−1 ; arabinose,
1.75 g l−1 ; glucose, 0.75 g l−1 ; furfural, 0.55 g l−1 and acetic
acid, 1.75 g l−1 .
2.2.2. Acid posthydrolysis of autohydrolysis liquors
Acid posthydrolysis of autohydrolysis liquors was carried
out to cleave the xylooligosaccharides into the correspond-
ing sugars at 125 ◦ C with 0.5% H2 SO4 for 165 min. [3].
The liquid phase from posthydrolysis was neutralised with
CaCO3 to a final pH of 6.5 and the precipitated CaSO4 was
separated from the supernatant by filtration. The composi-
tion of the liquors after posthydrolysis was 28.2 g xylose l−1 ,
2.1 g glucose l−1 , 3.2 g arabinose l−1 , 3.9 g acetic acid l−1 ,
1.2 g furfural l−1 and less than 0.05 g HMF l−1 .
2.2.3. Prehydrolysis
Acid prehydrolysis of corn cobs was carried out in
autoclave treatments at 130 ◦ C with 2% sulphuric acid
for 15 min using a liquid/wood ratio = 8 g g−1 [24].
The liquors contained 35.3 g xylose l−1 , 3.2 g glucose l−1 ,
4.6 g arabinose l−1 , 3.7 g acetic acid l−1 , 0.3 g furfural l−1
and less than 0.1 g HMF l−1 .
2.2.4. Concentration of hydrolysates
In selected experiments, the xylose concentration of
liquors was increased to the desired level by vacuum evap-
oration at temperatures below 40 ◦ C.
2.2.5. Charcoal adsorption
Powdered charcoal (Probus, Madrid, Spain) was activated
with hot water and dried at room temperature. Charcoal
detoxification of hydrolysates was carried out by contact-
ing hydrolysate and charcoal (mass ratio: 10 g g−1 ) at room
temperature under stirring for 1 h. The liquid phase was re-
covered by filtration and used for making culture media.
2.2.6. Membrane sterilisation
Concentrated, detoxified hydrolysates were filtered
through a 0.2 ␮m membranes (Nalgene) before fermenta-
tion.
2.3. Fermentation methods
2.3.1. Micro-organisms
D. hansenii NRRL Y-7426 was kindly provided by the
“National Center for Agricultural Utilisation Research”
(Peoria, Illinois, USA). D. hansenii CCMI 942 was kindly
provided by Dr. Francisco Gı́rio (Instituto Nacional de
Engenheria e Tecnologia Industrial, Lisbon, Portugal).
2.3.2. Solid culture and fermentation media
Freeze-dried cells were grown in a culture medium con-
taining (per liter) 10 g commercial xylose, 3 g yeast extract,
3 g malt extract and 5 g peptone. Micro-organisms were
maintained in agar slant tubes containing a medium formu-
lated with the same components and concentrations as the
previous one plus 20 g agar.
The hydrolysates were supplemented per liter with 3 g
yeast extract, 3 g malt extract and 5 g peptone. Fermenta-
tion was performed at 30 ◦ C under microaerophilic condi-
tions using 250 ml Erlenmeyer flasks (containing 50 ml of
culture media) placed in an orbital shaker (New Brunswick)
at 200 rpm. The initial pH of media was set at 6.5.
 B. Rivas et al. / Enzyme and Microbial Technology 31 (2002) 431–438
2.4. Analytical methods
A Hewlett Packard high-performance liquid chromato-
graphic system was used to analyse glucose, xylose, arabi-
nose, acetic acid, ethanol, xylitol, furfural and HMF. The
system consisted of an HP-1050 Intelligent Auto Sampler,
an HP-1047A Refractive Index detector, an HP-1050 Diode
Array UV detector and an HP-1050 pump. Separation was
achieved at 50 ◦ C using an Interaction ION-300 column
eluted with 0.4 ml min−1 of 0.01 M sulphuric acid.
Biomass concentration was measured by filtration
(0.45 ␮m membrane filters), washing and oven-drying to
constant weight at 90 ◦ C.
3. Results and discussion
3.1. Fermentation of raw hydrolysates with two yeast
strains
In a preliminary set of experiments, two D. hansenii
strains (NRRL Y-7426 and CCMI 942) were compared for
their ability to produce xylitol in media made from raw,
corn cob-derived, autohydrolysis–posthydrolysis liquors.
First of all, the yeasts were adapted to the fermentation
medium. For this purpose, inocula obtained from experi-
ments carried out with diluted hydrolysates were used to start
successive batch cultures carried out with increasing xylose
concentrations, using inocula obtained from the previous ex-
periment. This operational mode allowed to adapt the cells to
the inhibitors present in the hydrolysates, shortening the lag
periods and improving the reaction kinetics. In spite of that,
the performance of both yeasts was poor, showing relatively
prolonged lag phases. Table 1 lists data concerning batch
fermentations performed in Erlenmeyer flasks. D. hansenii
NRRL Y-7426 showed a better performance with a maxi-
mum xylitol concentration (P) of 12.2 g l−1 (volumetric pro-
ductivity QP = 0.51 g l−1 h−1 , product yield YP/S = 0.54 g
g−1 ) in comparison with 3.8 g xylitol l−1 (QP = 0.08 g
l−1 h−1 , YP/S = 0.31 g g−1 ) obtained with D. hansenii
CCMI 942. Because of this, the first strain was selected for
further experiments.
3.2. Fermentability of media obtained by prehydrolysis
and by autohydrolysis–posthydrolysis
Considering the qualitative advantages described upwards
for the media obtained by the sequence autohydrolysis–
Table 1
Xylitol production in autohydrolysis–posthydrolysis media by: (a) D. hansenii CCMI 942 and (b) D. hansenii NRRL Y-7426
S0 (g l−1 ) P0 (g l−1 ) X0 (g l−1 ) t (h) S (g l−1 )
(a) 21.7 1.0 2.1 47 12.8
(b) 23.1 0.5 1.8 24 1.5
Results are the average of two independent experiments conducted with two replicates for each one. Standard deviations were in the range of 2–3% of
the mean.
433
posthydrolysis, these liquors were assayed for possible im-
provements in fermentability. A set of batch tests was car-
ried out with the yeast D. hansenii NRRL Y-7426 in order
to assess the effects of inhibitors present in media derived
from prehydrolysis or from autohydrolysis–posthydrolysis.
Starting with similar initial xylose concentration (S0 about
24 g l−1 ), the maximum xylitol concentration, volumetric
productivity and xylitol yield were higher for fermenta-
tions using media made by autohydrolysis–posthydrolysis
(12.5 g l−1 , 0.53 g l−1 h−1 and 0.55 g g−1 , respectively)
than for fermentations using media made by prehydrol-
ysis (11.0 g l−1 , 0.45 g l−1 h−1 and 0.44 g g−1 , respecti-
vely). Because of this, all the fermentation media used
in further experiments were performed with autohydrolysis–
posthydrolysis media.
3.3. Fermentation of detoxified, concentrated hydrolysates
In order to improve both productivity and yield, the
liquors from autohydrolysis–posthydrolysis were vacuum
evaporated. During this stage, the volatile compounds
present in the reaction media (such as acetic acid or furfural)
were partially removed, whereas the concentrations of xy-
lose and nonvolatile byproducts (namely the lignin-derived
fraction, which can act as a fermentation inhibitor) increased
proportionally.
In a set of experiments carried out with initial xylose
concentrations twice higher than the original hydrolysates
(data not shown), the xylose was scarcely consumed at the
end of the fermentation owing to the effect of the inhibitors
present in the media. Because of this, charcoal detoxifica-
tion (a technology already applied to wood hydrolysates, see
[5,6,20]) was employed to improve the fermentability of the
media.
3.3.1. Adaptation of yeasts to detoxified media
Poor performance was observed using unadapted
micro-organisms in charcoal-detoxified media containing
up to 45 g xylose l−1 (see Fig. 1). The fermentation showed
long fermentation times (72 h), limited product yield
(YP/S = 0.22 g g−1 ) and slow reaction kinetics, leading to
low productivity (QP = 0.09 g l−1 h−1 ).
In order to limit the inhibition effects, the yeasts were
adapted to hydrolysates by performing successive batch cul-
tures, each of them carried out with cells coming from
the previous assay. Fig. 1 shows that just two sequential
fermentations were required to fully adapt the yeasts to
the charcoal-detoxified hydrolysates, because no significant
P (g l−1 ) X (g l−1 ) EtOH (g l−1 ) QP (g l−1 h−1 ) YP/S (g g−1 )
3.8 5.3 0.9 0.08 0.31
12.2 4.7 0.8 0.51 0.54
434 B. Rivas et al. / Enzyme and Microbial Technology 31 (2002) 431–438

Fig. 1. Effect of D. hansenii NRRL Y-7426 adaptation on the bioconversion of charcoal-treated hydrolystes obtained by autohydrolysis–psthydrolysis
of corn cobs. Results represent the average of two independent experiments conducted with two replicates for each condition. Standard deviations were
below 2.5% of the mean. S, final xylose concentration (g l−1 ); P, final xylitol concentration (g l−1 ); X, final dry weight biomass concentration (g l−1 );
EtOH, final ethanol concentration (g l−1 ); QP , volumetric productivity (g l−1 h−1 ); YP/S , product yield (g g−1 ).

improvement was observed when additional adaptation steps
were carried out.
3.3.2. Fermentation of membrane-sterilised media
When concentrated fermentation media were sterilised
by autoclaving at 121 ◦ C, significant losses of xylose were
observed (from 75–175 to 53–150 g xylose l−1 ). Literature
data dealing with the harmful effect of hydrolysate expo-
sure to high temperatures evidenced or assumed the for-
mation of toxic compounds. In this field, improvements
in the fermentation of oak wood hydrolysates (prepared
by steam explosion), when the sterilisation at 121 ◦ C was
replaced by a thermal treatment at 60 ◦ C, have been re-
ported [25,26]. In this work, media sterilised in autoclave at
121 ◦ C were effectively converted into xylitol when the ini-
tial xylose concentration was 53 g l−1 or lower (see Table 2),
but the fermentation did not proceed in media containing
64 g xylose l−1 .
In order to avoid the detrimental effect caused by auto-
claving, the possible benefits derived from the utilisation
of membrane sterilisation was tested in a new set of ex-
periments. The results of Table 2 show comparative ad-
vantages for membrane-sterilised media: for example, xy-
lose was rapidly consumed in media containing initially
66 g xylose l−1 , leading to a maximum xylitol concentration
of 41 g l−1 (QP = 1.08 g l−1 h−1 , YP/S = 0.62 g g−1 ).
3.3.3. Influence of the initial xylose concentration
Based on the above findings, adapted yeasts were em-
ployed for the bioconversion of concentrated, detoxified,
membrane-sterilised hydrolysates containing xylose concen-
trations in the range 37–155 g l−1 . Fig. 2 shows the time
course of representative fermentation assays. Productivities
above 1 g l−1 h−1 were obtained for initial xylose concentra-
tions in the range 66–100 g l−1 . The best volumetric produc-
tivity (QP = 1.22 g l−1 h−1 ) corresponded to the experiment
carried out with 97 g xylose l−1 , in which the xylitol con-
centration reached 68.8 g l−1 (YP/S = 0.71 g g−1 ). The high-
est xylitol concentrations (74 and 81 g l−1 ) were obtained
for experiments starting with xylose concentrations of 116
and 130 g l−1 , respectively; but in both assays the volumet-
ric productivity and the xylitol yield decreased with respect
to the previous case. The unfavourable balance between the
positive effect caused by increased substrate concentration
and the detrimental effect of increased inhibitor concentra-
tion led to decreased productivity and yield in the experiment
carried out with the maximum substrate concentration tested
(155 g l−1 ). This finding is in agreement with literature data.
For example, reported studies found that the fermentative
xylitol production from charcoal-treated sugarcane bagasse
hydrolysates improved when the liquors were concentrated
up to 60 g of xylose l−1 , but higher concentration degrees
resulted in decreased xylose consumption even if additional

Table 2
Influence of the sterilisation technique on the bioconversion of charcoal-treated, autohydrolysis–posthydrolysis hydrolysates into xylitol using the yeast
D. hansenii NRRL Y-7426
S0 (g l−1 ) P0 (g l−1 ) X0 (g l−1 ) t (h) S (g l−1 ) P (g l−1 ) X (g −1 ) EtOH (g l−1 ) QP (g l−1 h−1 ) YP/S (g g−1 )

(a) 40.5 2.1 3.0 31 3.4 19.1 5.8 7.4 0.55 0.46
(b) 53.5 1.9 1.8 48 1.6 31.6 4.8 9.3 0.62 0.57
(c) 64.4 4.8 2.3 – – – – – – –
(d) 37.4 0.9 0.8 24 1.7 21.0 5 3.5 0.84 0.56
(e) 48.2 1.8 3.4 24 2.4 28.5 10.8 4.5 1.11 0.58
(f) 65.6 2.0 4.5 36 2.1 41.1 10.8 6.0 1.08 0.62
Here (a), (b) and (c): autoclave-sterilised media; (d), (e) and (f): membrane-sterilised media. Results are the average of two independent experiments
conducted with two replicates each. Standard deviations were in the range of 1–3% of the mean.
 B. Rivas et al. / Enzyme and Microbial Technology 31 (2002) 431–438 435

Fig. 2. Effect of the initial xylose concentration on the D. hansenii NRRL Y-7426 fermentative behaviour for xylitol production using concentrated,
charcoal-detoxified, membrane-sterilised media made from autohydrolysis–posthydrolysis liquors. (a) S0 = 37 g xylose l−1 ; (b) S0 = 66 g xylose l−1 ;
(c) S0 = 85 g xylose l−1 ; (d) S0 = 97 g xylose l−1 ; (e) S0 = 117 g xylose l−1 ; (f) S0 = 133 g xylose l−1 ; (g) S0 = 155 g xylose l−1 . The symbols (䉬)
xylitol concentration; (䊉) xylose concentration; (䉱) ethanol concentration; (∗) biomass concentration. Results represent the average of two independent
experiments conducted with two replicates for each condition. Standard deviations were below 3% of the mean.

detoxification treatments (like cation-exchange resins) were
applied [27]. In the same way, the xylitol production by
Candida guilliermondii in media also made from sugarcane
bagasse hydrolysates improved (40% in cell concentration,
20% in xylitol yield and 44% in xylitol productivity) when
the initial xylose concentration was increased from 37.6 to
54.5 g l−1 , but detrimental effects were observed for xylose
concentrations beyond this limit [28].
3.3.4. Effect of the initial biomass concentration
The effect of the initial cell biomass was considered in
concentrated hydrolysates containing around 97 g xylose l−1
436 B. Rivas et al. / Enzyme and Microbial Technology 31 (2002) 431–438

Table 3
Fermentation parameters showing the influence of the initial cell concentration on the bioconversion of membrane-sterilised, charcoal-treated,
autohydrolysis–posthydrolysis media
X0 (g l−1 ) S0 (g l−1 ) P0 (g l−1 ) t (h) S (g l−1 ) P (g l−1 ) X (g l−1 ) EtOH (g l−1 ) QP (g l−1 h−1 ) YP/S (g g−1 )

3 97.2 2.2 54.8 3.9 68.8 10.2 7.1 1.22 0.71

12.5 106.1 3.0 46.0 5.6 71.1 20.8 7.0 1.48 0.68
17.4 92.6 4.0 39.0 12.8 62.0 24.6 6.7 1.49 0.73
20.3 92.0 1.9 36.5 11.0 53.7 24.0 6.0 1.42 0.64
25.1 89.7 1.1 45.0 4.5 55.2 29.8 6.7 1.21 0.63
54.6 95.3 4.8 48.8 2.4 67.8 56.2 9.4 1.24 0.71
Results are the average of two independent experiments conducted with two replicates each. Standard deviations were in the range of 2–4% of the mean.

(the optimum substrate concentration determined previ-
ously). According to literature data, high cell density en-
hances the volumetric productivity and the product yield,
limiting both the toxic effects of some inhibitors and the
cell death caused by their assimilation or degradation. For
example, the inhibitory effect of acetic acid present in Eu-
calyptus wood hydrolysates was overcame by increasing
the C. guilliermondi cell concentration up to just 3 g l−1
[29], whereas other studies used 10 g of cells l−1 [30], 50 g
of cells l−1 [5] or up to 75 g l−1 [31] for related purposes.
The wide range of initial cell concentrations leading to
optimal bioconversion determined in different studies can
be ascribed to the interrelationship between the different
operational variables affecting the bioconversion: for exam-
ple, increases in cell concentration above a given threshold
may lead to a reduction in the oxygen availability in the
medium which can be responsible for decreased yields and
productivities.
Table 3 shows the fermentation parameters determined in
experiments with different initial cell concentrations. The
best results were achieved when the initial cell concentra-
tion was kept in the range of 12.5–17.4 g l−1 . Although the
xylitol concentration was in the range of 62–71 g l−1 , in-
creased cell concentrations reduced the fermentation time
from 55 to 39 h and improved the volumetric productivity
up to 1.49 g l−1 h−1 . In these cases, the product yields were
0.68 and 0.73 g g−1 .
The results achieved in this work compare favourably
with the data reported for the batch production of xylitol
in synthetic media containing mixtures of sugars simulat-
ing the composition of hemicellulosic hydrolysates with
initial xylose concentrations in the range of 50–80 g l−1
[19,32,33]. More sophisticated fermentation strategies (such
as the utilisation of two fermentation stages with two dif-
ferent fermentation substrates [34] or the utilisation of
immobilised, genetically modified yeasts [35]) were needed
to achieve higher productivities. The approach described
in this work allows better results than the ones achieved
with hydrolysates from other hardwoods [6,32,36] or agri-
cultural residues as rice straw [37–39]. The low severity
of the chemical processing steps used in this work is a
possible explanation of improvements observed with re-
spect to the studies dealing with the acid hydrolysis of the
same raw material [40,41]. To our knowledge, in litera-
ture studies dealing with hydrolysate-derived media, the
maximal productivity reported in this work has been only
improved using a more elaborated fermentation technol-
ogy (high cell density cultures with membrane bioreactors)
[42].
4. Conclusions
The sequence autohydrolysis–posthydrolysis was suc-
cessfully assayed for the production of xylose-containing
fermentation media prepared from corn cob hydrolysates. In
comparison with the conventional acid hydrolysis (prehy-
drolysis), the autohydrolysis–posthydrolysis increased the
productivity by 18% and the product yield by 25%. In this
kind of media, D. hansenii NRRL Y-7426 presented a su-
perior performance for xylitol production than D. hansenii
CCMI 942.
When the xylose-containing liquors were concentrated
by vacuum evaporation to obtain solutions with xylose
concentrations above 54 g l−1 , the fermentation proceeded
only when the media was sterilised by membrane filtra-
tion. Using membrane sterilisation, media containing up
to 155 g xylose l−1 could be fermented, even though the
best fermentation conditions corresponded to about 97 g
initial xylose per litre. Operating at the optimum initial
cell concentration, up to 62 g of xylitol l−1 were obtained
from media containing 93 g of xylose l−1 . The correspond-
ing volumetric productivity (1.49 g l−1 h−1 ) and xylitol
yield (0.73 g g−1 ) compare well with data reported for
the fermentative production of xylitol from synthetic or
hydrolysate-derived fermentation media.
Acknowledgments
Authors are grateful to the Commission of the European
Communities and to the Spanish Ministry of Education for
the financial support of this work (projects FAIR-CT98-3811
and ALI98-1625-CE), as well as to Ms. Aida Nespereira
and Ms. Antonia Rodrı́guez Jardón for their technical
assistance.
 B. Rivas et al. / Enzyme and Microbial Technology 31 (2002) 431–438
References
[1] Maloney MT, Chapman TW, Baker AJ. Dilute acid hydrolysis of
paper birch: kinetic study of xylan and acetyl-group hydrolysis.
Biotechnol Bioeng 1985;27:355–61.
[2] Frazer FR, McCaskey TA. Wood hydrolysate treatments for improved
fermentation of wood sugars to 2,3-butanediol. Biomass 1989;18:
31–42.
[3] Garrote G, Domı́nguez H, Parajó JC. Generation of xylose solutions
from Eucalyptus globulus wood by autohydrolysis–posthydro-
lysis: posthydrolysis kinetics. Biores Technol 2001;79(2):155–
64.
[4] Garrote G, Domı́nguez H, Parajó JC. Manufacture of
xylose-based fermentation media from corn cobs by hydrolysis
of autohydrolysis liquors. Appl Biochem Biotechnol 2001;95(3):
195–207.
[5] Parajó JC, Domı́nguez H, Domı́nguez JM. Production of xylitol
from concentrated wood hydrolysates by Debaryomyces hansenii:
effect of the initial cell concentration. Biotechnol Lett 1996;18:
593–8.
[6] Parajó JC, Domı́nguez H, Domı́nguez JM. Improved xylitol
production with Debaryomyces hansenii Y-7426 from raw or
detoxified wood hydrolysates. Enzyme Microb Technol 1997;21:
18–24.
[7] Cruz JM, Domı́nguez JM, Domı́nguez H, Parajó JC. Solvent
extraction of hemicellulosic wood hydrolysates: a procedure
useful for obtaining both detoxified fermentation media and
polyphenols with antioxidant activity. Food Chem 1999;67:
147–53.
[8] Larsson S, Reimann A, Nilvebrant NO, Jönsson LF. Comparison of
different methods for the detoxification of lignocellulose hydrolysates
of spruce. Appl Biochem Biotechnol 1999;77–79:91–103.
[9] Palmqvist E, Hahn-Hägerdal B, Szengyel Z, Zachhi G, Rèczey K.
Simultaneous detoxification and enzyme production of hemicellulose
hydrolysates obtained after steam pretreatment. Enzyme Microb
Technol 1997;20:286–93.
[10] Jönsson LJ, Palmqvist E, Nilvebrandt NO, Hahn-Hägeldal B.
Detoxification of wood hydrolysates with laccase and peroxidase
from the white-rot fungus Trametes versicolor. Appl Microbiol
Biotechnol 1998;49:691–7.
[11] Boussaid A, Robinson J, Cai Y, Gregg DJ, Saddler JN. Fermentability
of the hemicellulose-derived sugars from steam-exploded softwood
(Douglas Fir). Biotechnol Bioeng 1999;64:284–9.
[12] Palmqvist E, Hahn-Hagerdal B. Fermentation of lignocellulosic
hydrolysates. I. Inhibition and detoxification. Bioresour Technol
2000;74(1):17–24.
[13] Palmqvist E, Hahn-Hagerdal B. Fermentation of lignocellulosic
hydrolysates. II. Inhibitors and mechanisms of inhibition. Bioresour
Technol 2000;74(1):25–33.
[14] Larsson S, Quintana-Sáinz A, Reimann AN, Nilvebrant NO, Jonson
LJ. Influence of lignocellulose-derived aromatic compounds on
oxygen-limited growth and ethanolic fermentation by Saccharomyces
cerevisiae. Appl Biochem Biotechnol 2000;84–86:617–32.
[15] Emodi A. Xylitol. Its properties and food applications. Food Technol
1978;1:28–32.
[16] Pepper T, Olinger PM. Xylitol in sugar-free confections. Food
Technol 1988;42:98–106.
[17] Gı́rio FM, Peito MA, Amaral-Collaço MT. In: Grassi G, Gosse G,
dos Santos G, editors. Biomass for energy and industry, vol. 2.
Amsterdam: Elsevier, 1990.
[18] Gı́rio FM, Roseiro JC, Sá-Machado P, Duarte-Reis AR,
Amaral-Collaço MT. Effect of oxygen transfer rate on levels of key
enzymes of xylose metabolism in Debaryomyces hansenii. Enzyme
Microb Technol 1994;16:1074–8.
[19] Gı́rio FM, Amaro C, Azinheira H, Pelica F, Amaral-Collaço
MT. Polyols production during single and mixed substrate
fermentation in Debaryomyces hansenii. Bioresour Technol 2000;71:
245–51.
437
[20] Parajó JC, Domı́nguez H, Domı́nguez JM. Xylitol production from
Eucalyptus wood hydrolysates extracted with organic solvents.
Process Biochem 1997;7:599–604.
[21] Domı́nguez JM, Gong CS, Tsao GT. Production of xylitol from
d-xylose by Debaryomyces hansenii. Appl Biochem Biotechnol
1997;63–65:117–27.
[22] Granstrom TB, Aristidou AA, Jokela J, Leisola M. Growth
characteristics and metabolic flux analysis of Candida milleri.
Biotechnol Bioeng 2000;70:197–207.
[23] Garrote G, Domı́nguez H, Parajó JC. Autohydrolysis of corn cob:
study of nonisothermal operation for xylooligosacharide production,
J. Food Eng. 2002;52:211–8.
[24] Cruz JM, Domı́nguez JM, Domı́nguez H, Parajó JC. Preparation of
fermentation media from agricultural wastes and their bioconversion
into xylitol. Food Biotechnol 2000;14:79–97.
[25] Lee WG, Lee JS, Shin CS, Park SC, Chang HN, Chang YK.
Ethanol production using concentrated oak wood hydrolysates
and methods to detoxify. Appl Biochem Biotechnol 1999;77–79:
547–59.
[26] Lee WG, Park BG, Chang YK, Chang HN, Lee JS, Park
SC. Continuous ethanol production from concentrated wood
hydrolysates in an internal membrane-filtration reactor. Biotechnol
Prog 2000;16:302–4.
[27] Domı́nguez JM, Gong CS, Tsao GT. Pretreatment of sugar cane
bagasse hemicellulose hydrolysate for xylitol production by yeasts.
Appl Biochem Biotechnol 1996;57–58:49–56.
[28] Felipe MGA, Vitolo M, Mancilha IM, Silva SS. Environmental
parameters affecting xylitol production from sugar cane bagasse
hemicellulosic hydrolyzate by Candida guilliermondii. J Ind
Microbiol Biotechnol 1997;18:251–4.
[29] Felipe MGA, Alves LA, Silva SS, Roberto IC, Mancilha IM, Almeida
e Silva JB. Fermentation of Eucalyptus hemicellulosic hydrolysate
to xylitol by Candida guilliermondii. Bioresour Technol 1996;56:
281–3.
[30] Palmqvist E, Hahn-Hägerdal B, Galbe M, Zachhi G. The effect of
water-soluble inhibitors from steam-pretreated willow on enzymatic
hydrolysis and fermentation. Enzyme Microb Technol 1996;19:
470–6.
[31] Olsson L, Hahn-Hägerdal B. Fermentation of lignocellulosic
hydrolysates for ethanol production. Enzyme Microb Technol
1986;18:312–31.
[32] Preziosi-Belloy L, Nolleau V, Navarro JM. Xylitol production from
aspenwood hemicellulose hydrolyzate by Candida guilliermondii.
Biotechnol Lett 2000;22:239–43.
[33] Walthers T, Hensirisak P, Agblevor FA. Model compound studies:
influence of aeration and hemicellulosic sugars on xylitol production
by Candida tropicalis. Appl Biochem Biotechnol 2001;91–93:
423–35.
[34] Kim JH, Ryu YW, Seo JH. Analysis and optimisation of a
two-substrate fermentation for xylitol production using Candida
tropicalis. J Ind Microbiol Biotechnol 1999;22:181–6.
[35] Roca E, Meinander N, Hahn-Hägerdal B. Xylitol production
by immobilised recombinant Saccharomyces cerevisiae in a
continuous packed-bed bioreactor. Biotechnol Bioeng 1996;51:
317–26.
[36] Converti A, Perego P, Domı́nguez JM. Microaerophilic metabolism
of Pachysolen tannophilusat different pH. Biotechnol Lett 1999;21:
719–23.
[37] Mayerhoff ZDVL, Roberto IC, Silva SS. Production of xylitol by
Candida mogii from rice straw hydrolyzate: Study of environmental
effects using statistical design. Appl Biochem Biotechnol 1998;70–
72:149–59.
[38] Rodrigues DCGA, Silva SS, Prata AMR, Felipe MDGA.
Biotechnological production of xylitol from agroindustrial residues:
evaluation of bioprocesses. Appl Biochem Biotechnol 1998;70–
72:869–75.
438 B. Rivas et al. / Enzyme and Microbial Technology 31 (2002) 431–438

[39] Acosta E, Silva SS, Felipe MDGA. Aspect of xylitol formation
in sugarcane bagasse hydrolysate by Candida guilliermondii in the
presence of tetracycline. Appl Biochem Biotechnol 1999;77–79:
347–54.
[40] Kim SY, Oh DK, Kim JH. Evaluation of xylitol production from corn
cob hemicellulose hydrolysate by Candida parapsilosis. Biotechnol
Lett 1999;21:891–5.
[41] Leathers TD, Dien BS. Xylitol production from corn fiber
hydrolysates by a two-stage fermentation process. Process Biochem
2000;35:765–9.
[42] Cruz JM, Domı́nguez JM, Domı́nguez H, Parajó JC. Xylitol
production from barley bran hydrolysates by continuous
fermentation with Debaryomyces hansenii. Biotechnol Lett 2000;22:
1895–8.