Journal of Food Engineering 61 (2004) 407–412

www.elsevier.com/locate/jfoodeng

Optimization of xylitol recovery by crystallization from synthetic

solutions using response surface methodology
D. De Faveri, P. Torre, P. Perego, A. Converti *

Department of Chemical and Process Engineering, ‘‘G.B. Bonino’’, Genoa University, Via Opera Pia 15, 16145 Genoa, Italy
Received 21 December 2002; accepted 27 April 2003

Abstract
The combined effects of initial xylitol supersaturation value (Xyts ) and cooling temperature (Tc ) on xylitol crystallization from
synthetic solutions were investigated using response surface methodology. A 32 full-factorial design was employed for experimental
design. Synthetic solutions were subjected to crystallization, aimed at contributing to the development of a complete biotechno-
logical process, including preliminary xylose-to-xylitol bioconversion in hemicellulose hydrolyzate and final xylitol recovery by
crystallization. The crystallization methodology consisted of concentration of synthetic solutions up to supersaturation, cooling of
the supersaturated solutions, separation of crystals by centrifugation, and final filtration. By means of response surface analysis, the
statistical model identified the operating conditions (Xyts ¼ 728 g/l and Tc ¼
6:0
C) under which purity degree (0.97) and xylitol
crystallization yield (0.54) were simultaneously optimized. On the other hand, a purity degree close to 1.0 to meet industrial
standards is expected at Xyts ¼ 583 g/l and Tc ¼
2:4
C, but the crystallization yield would be unsatisfactory (0.25).
 2003 Elsevier Ltd. All rights reserved.

Keywords: Xylitol; Xylose-to-xylitol bioconversion; Crystallization; Response surface methodology

1. Introduction (Paraj
o et al., 1998a). Such processes consist of fer-

Xylitol is a natural carbon polyalcohol that has at-
tracted attention in recent years for its interesting
properties, which make it an important product for the
food and pharmaceutical industry. It is a compound
with a sweetness similar to that of sucrose, is non-
cariogenic, tolerated by diabetics, recommended for
obese people and, because of its negative heat of disso-
lution, used as a part of the coating of pharmaceutical
products (Paraj
o, Dom
ınguez, & Dom
ınguez, 1998a;
Ylikahri, 1979).
Xylitol is commercially produced by a chemical pro-
cess consisting of catalytic hydrogenation of the xylose
present in lignocellulosic hydrolyzates, but the solutions
produced by this process require expensive purification
and separation steps to obtain pure xylitol (Winkel-
hausen & Kuzmanova, 1998). Alternatively, it can be
produced by biotechnological methods, based on the use
of microorganisms, in particular yeasts, and/or enzymes
*
Corresponding author. Tel.: +39-10-3532593; fax: +39-10-
3532586.
E-mail address: converti@unige.it (A. Converti).
0260-8774/$ - see front matter  2003 Elsevier Ltd. All rights reserved.
doi:10.1016/S0260-8774(03)00148-1
mentation of agro-industrial residues, which could po-
tentially compete with the traditional chemical method.
Pachysolen tannophilus, Candida guillermondii and,
above all, Debaryomyces hansenii were widely employed
as xylitol producers in previous studies (Converti &
Dom
ınguez, 2001; Converti, Perego, & Dom
ınguez,
1999b; Cruz, Dom
ınguez, Dom
ınguez, & Paraj
o, 2000;
Felipe, Vitolo, Mancilha, & Silva, 1997; G
ırio, Amaro,
Azinheira, Pelica, & Amaral-Collacßo, 2000; G
ırio,
Roseiro, S
a-Machado, Duarte-Reis, & Amaral-Collacßo,
1994; Paraj
o, Dom
ınguez, & Dom
ınguez, 1997;
Roberto, Sato, & Mancilha, 1996).
Apart from the nature and composition of hydrolyz-
ates, which vary widely, xylitol production by these
yeasts depends on oxygen level, pH, temperature, start-
ing xylose concentration, inoculum level and presence of
inhibiting compounds (furfural, acetic acid, phenolic
compounds and metal cations) (Converti, Perego, &
Dom
ınguez, 1999a; Converti, Perego, Dom
ınguez, &
Silva, 2001; Converti, Perego, Torre, & Silva, 2000;
Paraj
o, Dom
ınguez, & Dom
ınguez, 1998b,c). The bio-
technological production of xylitol uses renewable re-
sources, such as agro-industrial residues, which represent
408 D. De Faveri et al. / Journal of Food Engineering 61 (2004) 407–412
an inexpensive source of raw materials and helps
to minimize environmental and energetic problems
(Roberto et al., 1995). However, xylitol recovery is the
hardest step of the whole fermentation process because
of the low product concentration as well as the complex
composition of the fermentation broth. The literature on
polyol recovery from fermented broths is poor because
of the innovative characteristics of the process (Gurgel,
Mancilha, Pecßanha, & Siqueira, 1995). The aim of the
present study is to complete an experimental protocol for
optimization of xylitol crystallization conditions, some
results of which have recently been presented (De Faveri,
Perego, Converti, & Del Borghi, 2002). In particular,
supersaturation xylitol value and cooling temperature
have been identified in that work as the major factors
affecting the polyol crystallization, because of their in-
fluence on crystallization kinetics.
Response surface methodology (RSM) is a collection
of mathematical and statistical techniques widely used
to determine the effects of several variables, which was
already used to optimize different bioconversion pro-
cesses (Dasu & Panda, 2000; Rao, Kim, & Rhee, 2000).
In addition, this method was successfully applied to the
optimization of medium composition (Roseiro, Esgalh-
ado, Amaral-Collacßo, & Emery, 1992), conditions of
enzymatic hydrolysis (Ma & Ooraikul, 1986), parame-
ters of food preservation (King, 1993) and fermentation
(Rosi, Costamagna, & Bertuccioli, 1987). Many appli-
cations deal with the study of the effects of two or more
factors. In general, factorial designs are more efficient
for this purpose than the classical method of studying
one variable at a time. Besides, they are necessary to
avoid misleading conclusions and allow the effects of a
factor being estimated at several levels of the other
factor, yielding results that are valid over a wide range
of experimental conditions (Montgomery, 2001).
In this study, a 32 full-factorial design combined with
RSM has been used to determine the influence of su-
persaturation level and cooling temperature on xylitol
crystallization procedure as well as to identify the opti-
mum crystallization conditions.
2. Materials and methods
2.1. Crystallization procedure and synthetic solutions
Experiments were performed in a bench-scale system
described in detail in previous work (De Faveri et al.,
2002). It consisted of a rotavapor (B€
uchi, R-114, Flawil,
Switzerland) used as low pressure-concentration unit
(P ¼ 1:6  104 Pa, T ¼ 30–50
C), a crystallization unit
working at atmospheric pressure, a centrifuge equipped
with a process parameter control system (ALC Inter-
national, ALC4237R, Milan, Italy), and a vacuum fil-
tration system. The crystallization unit consisted of
25 ml glass test tubes submerged in an ethylene glycol
bath (Julabo, F25, Seelbach, Germany) and gently stir-
red by means of a mechanical system. As soon as the
crystallization temperature was reached, finely ground
commercial xylitol was added, up to 1.0 g/l, to favour
the nucleation of xylitol crystals. After crystallization,
the solutions were subjected to centrifugation at 5000
rpm for 15 min to separate xylitol crystals. Finally, the
precipitated crystals were separated by vacuum filtra-
tion, through filters with 0.45-lm pore diameter, and re-
dissolved in water to be analyzed. Synthetic solutions
contained both xylitol and xylose, with a 3:1 ratio, to
simulate the actual composition of fermented hemi-
cellulose hydrolyzates. Some tests taken from a previous
work (De Faveri et al., 2002) were integrated with a set
of new experiments to permit the statistical study.
2.2. Analytical methods
Xylose and xylitol concentrations were determined by
HPLC (Bio-Rad, HRLC 800, Richmond, VI) using an
ion-exchange AMINEX column (Bio-Rad, HPX-87C,
Richmond, VI) and a refractometer (Bio-Rad, 1770,
Richmond, VI). A 30% acetonitrile-water solution was
used as the mobile phase. Calibration curves were ob-
tained by injecting standard solutions with concentra-
tions of xylose ranging from 3.0 to 15 g/l and xylitol
from 9.0 to 45 g/l. The analyses were carried out at 70
C
and a flow rate of 0.6 ml/min.
2.3. Experimental design
A 32 full-factorial experimental design was employed
to collect the data necessary for optimization of the
crystallization procedure. One central point was added
to estimate the experimental error as well as to investi-
gate the suitability of the proposed model. Coding of the
independent variables was done according to the fol-
lowing equation:
ð Xi
Xo Þ
xi ¼ i ¼ 1; 2; 3; . . . ; k ð1Þ
DXi
where xi and Xi are the dimensionless and the actual
values of the independent variable i, Xo the actual value
of the independent variable i at the central point, and
DXi the step change of Xi corresponding to a unit vari-
ation of the dimensionless value.
The initial xylitol supersaturation value and cooling
temperature were selected as the independent variables
due to their influence on the process kinetics, as previ-
ously described (De Faveri et al., 2002). The range and
the levels of both coded and actual values of the inde-
pendent variables tested in this work are listed in Table 1.
The behavior of the system can be described by the
following second-order polynomial equation:
 D. De Faveri et al. / Journal of Food Engineering 61 (2004) 407–412 409

Table 1 Table 2
Experimental range and levels of both coded and actual values of the Experimental design of xylitol crystallization and corresponding
independent variables: Xyts ¼ xylitol supersaturation value; Tc ¼ cool- experimental results
ing temperature
Runs Independent variables Responses
Independent Variable Levels
x1 x2 Yxyt PD
variable symbol
)1 0 +1
1 )1 )1 0.38 0.92
X1 Xyts (g/l) 582 656 730 2 )1 +1 0.20 0.98
X2 Tc (
C) )10 )52 0 3 +1 )1 0.60 0.89
4 +1 +1 0.45 0.93
5 0 +1 0.34 0.97
6 +1 0 0.56 1.0
X X X 7 )1 0 0.31 1.0
Y ¼ bo þ bi xi þ bii x2i þ bij xi xj ð2Þ 8 0 )1 0.44 0.95
9 0 0 0.38 1.0
where Y is the predicted response, bo is the intercept 10 0 0 0.39 0.99
coefficient, bi are the linear terms, bii are the squared x1 ¼ coded value of xylitol supersaturation, x2 ¼ coded value of cooling
terms, bij are the interaction terms, and xi and xj rep- temperature, Yxyt ¼ xylitol crystallization yield, PD ¼ purity degree.
resent the coded levels of the independent variables. The
‘‘Statistica’’ software (trial version 6.0, StatSoft, Tulsa,
OK) was used for the regression analysis, while the 3.1. Response surface for xylitol crystallization yield
‘‘Design Expert’’ software (trial version 6.07, Stat-Ease,
Minneapolis, MN) was used for graphical optimization. Neglecting the statistically insignificant terms, the
model of response surface for xylitol crystallization yield
is represented by the equation:
Y ¼ 0:405 þ 0:120x1
0:0720x2 ð3Þ
3. Results and discussion
The results of the regression analysis, listed in Table 3,
The dependent variables selected in the present study demonstrate, for the crystallization yield, that the qua-
for the statistical analysis were the xylitol crystallization dratic terms of cooling temperature and xylitol super-
yield (Yxyt ), expressed as the mass ratio of recovered saturation value as well as the interaction term are not
xylitol to starting xylitol, and the purity degree (PD), statistically significant. The linear regression coefficients
calculated as the mass ratio of recovered xylitol to total show the positive effect of xylitol supersaturation and
precipitate. The supersaturation ratio (r), defined as the the negative one of cooling temperature on xylitol
ratio of starting xylitol supersaturation value (Xyts ) to crystallization yield. That is in good agreement with
final concentration of xylitol in the saturated solution at previous observations (De Faveri et al., 2002), which
the same temperature and purity (Xytf ), was also cal- suggested increasing xylitol concentration and decreas-
culated from the experimental data to better identify ing temperature to improve xylitol recovery by crystal-
the optimum conditions. The experimental design and lization. The regression coefficients were estimated with
the corresponding experimental results are shown in a satisfactory determination coefficient (r2 ¼ 0:9604).
Table 2. Statistical evaluation of the model was by the FisherÕs

Table 3
Results of the regression analysis for xylitol crystallization yield (Yxyt ) and purity degree (PD)
Term Yxyt PD
Coefficient Standard error t-value p-value Coefficient Standard error t-value p-value
Mean 0.3943 0.0138 – – 1.0050 0.0119 – –
(0.4050)a (0.0083) (1.0050) (0.0110)
x1 0.1200 0.0094 12.7173 0.0002 )0.0133 0.0081 )1.642 0.1760
(0.1200) (0.0107) (11.1862) (<0.0001) ()0.0133) (0.0075) ()1.780) (0.1352)
x2 )0.0717 0.0094 )7.5950 0.0016 0.0200 0.0081 2.4623 0.0695
()0.0717) (0.0107) ()6.6807) (0.0003) (0.0200) (0.0075) (2.6700) (0.0443)
x21 0.0314 0.0151 2.0771 0.1064 )0.0150 0.0130 )1.1517 0.3136
()0.0150) (0.0120) ()1.2488) (0.2670)
x22 )0.0136 0.0151 )0.8969 0.4205 )0.0550 0.0130 )4.2228 0.0134
()0.0550) (0.0120) ()4.5788) (0.0059)
x1 x 2 0.0075 0.0117 0.6490 0.5517 )0.0050 0.0100 )0.5026 0.6416
a
Values between brackets were calculated without taking into account the terms that the regression analysis revealed to be not significant.
410 D. De Faveri et al. / Journal of Food Engineering 61 (2004) 407–412

Table 4
Results of ANOVA for xylitol crystallization yield
Source Sum of squares Degrees of freedom Mean square F -value p-value
Model 0.12 2 0.059 84.88 <0.0001
Residual 4.833 · 10
3 7 6.905 · 10
4
Lack of fit 4.783 · 10
3 6 7.972 · 10
4 15.94 0.1894
Pure error 5.000 · 10
5 1 5.000 · 10
5
Total 0.12 9

theless, also the linear and quadratic terms of xylitol

supersaturation have been kept in the equation in order
to diminish the errors.
The results of the ANOVA for purity degree, listed in
Table 5, demonstrate that the statistical model is sig-
nificant at the 5% probability level (p < 0:05). The above
equation fitted the experimental data with an acceptable
determination coefficient (r2 ¼ 0:8763). The response
surface in Fig. 2 identifies a maximum (1.0 PD) cor-
responding to a cooling temperature close to )5
C,
while, as suggested by the statistically significance of the
model terms, xylitol supersaturation does not seem to
affect PD.

3.3. Graphical optimization

Fig. 1. Response surface plot of xylitol crystallization yield as a
function of xylitol supersaturation level (Xyts ) and cooling tempera-
ture (Tc ).
test for analysis of variance (ANOVA) whose results are
shown in Table 4. The model F -value of 84.88 means
that the regression is statistically significant at a 99%
confidence level (p < 0:0001). Fig. 1 shows the three-
dimensional graph representing the response surface for
xylitol crystallization yield.
3.2. Response surface for purity degree
The response surface for purity degree is described by
the equation:
Y ¼ 1:005
0:013x1 þ 0:020x2
0:015x21
0:055x22 ð4Þ
In this case, only the linear (at a 10% probability level)
and quadratic (at a 5% probability level) terms of cool-
ing temperature were shown to be significant; never-
It was possible, by means of Eqs. (3) and (4), to ap-
proach a graphical optimization of the system using
the ‘‘Design Expert’’ software. In this way, ranges were
identified where the two independent variables ensured
maximum values of both xylitol crystallization yield and
purity degree. The curves of the two models were
overlaid and minimum threshold values of 0.50 and 0.95
were imposed for the above dependent variables, re-
spectively.
The results of the graphical optimization are shown
in Fig. 3, where the white area represents the region
where both imposed conditions are satisfied, while the
flag in the same figure shows the values of the inde-
pendent variables as well as of the responses of a rep-
resentative point inside of this area. The optimal
operating conditions identified by this point correspond
to a 728 g/l xylitol supersaturation value and a cooling
temperature of )6.0
C, which should lead to 0.97 purity
degree and 0.54 xylitol crystallization yield.
The statistical results obtained in this study suggest
on the whole that, to attain a crystallization product as

Table 5
Results of ANOVA for purity degree
Source Sum of squares Degrees of freedom Mean square F -value p-value

3
Model 0.012 4 2.982 · 10 8.86 0.0172
Residual 1.683 · 10
3 5 3.367 · 10
4
Lack of fit 1.633 · 10
3 4 4.083 · 10
4 8.17 0.2560
Pure error 5.000 · 10
5 1 5.000 · 10
5
Total 0.014 9
 D. De Faveri et al. / Journal of Food Engineering 61 (2004) 407–412
Fig. 2. Response surface plot of purity degree as a function of xylitol
supersaturation level (Xyts ) and cooling temperature (Tc ).
Fig. 3. Optimum region identified by the overlaid plots of the two
responses (xylitol crystallization yield and purity degree).
pure as possible, it would be better to operate at a high
cooling temperature but not too high a supersaturation
level. In fact the response surface for crystallization yield
is described by a linear model, predicting improvements
of this parameter either by increasing xylitol super-
saturation value or decreasing cooling temperature. On
the other hand, the most significant term of the purity
degree model is the cooling temperature quadratic term,
which exhibits a parabolic trend. However, since its
correlation coefficient is negative, a decrease in cooling
temperature is expected to greatly reduce purity degree.
Besides, a not-excessive supersaturation level should
make the control of the crystallization procedure easier,
as suggested by the use of 1:12 < r < 1:40 in the
industrial crystallization practice (Semlali Aouragh
411
Fig. 4. Optimum region identifying complete purity of crystals at
typical industrial conditions for crystallization procedure (Yxyt < 0:25
corresponding to r < 1:34).
Hassani, Saidi, & Bounahmidi, 2001). So, as illustrated
in Fig. 4, if 0.25 would be selected as the highest crys-
tallization yield, corresponding to a supersaturation
ratio of 1.34, the purity is expected to be nearly complete
at a 583 g/l xylitol supersaturation value and a cooling
temperature of )2.4
C.
4. Conclusions
Batch crystallization tests were carried out using
xylitol–xylose synthetic solutions to investigate the
combined effects of xylitol supersaturation level (Xyts )
and cooling temperature (Tc ) on crystallization proce-
dure. Statistical optimization method predicted best
operating conditions (Xyts ¼ 728 g/l and Tc ¼
6:0
C),
under which both purity degree (0.97) and xylitol crys-
tallization yield (0.54) were simultaneously optimized.
This response was in good agreement with previous
observations, which suggested operating either at high
supersaturation level to improve xylitol crystallization
yield, or at a mean cooling temperature to improve
purity degree. Besides, the model showed that it is
possible to increase the purity degree by lowering the
crystallization yield, in order to make the crystallization
procedure slower, the process control easier and to
achieve near total purity of crystals. This paper con-
tributes to the development of a complete biotechno-
logical process, including preliminary xylose-to-xylitol
bioconversion in hemicellulose hydrolyzate and final
xylitol recovery by crystallization, which could compete
with the traditional chemical method. Further tests of
crystallization under optimal operative conditions, using
fermented hemicellulose hydrolyzate solutions, are
412 D. De Faveri et al. / Journal of Food Engineering 61 (2004) 407–412
needed to confirm these results as well as to definitively
demonstrate the possible influence of other compounds
present in fermented broths.
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