Separation and Purification Technology 78 (2011) 266–273

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Separation and Purification Technology

journal homepage: www.elsevier.com/locate/seppur

Comparative study on different strategies involved for xylitol purification from

culture media fermented by Candida tropicalis
Swati Misra, Pritesh Gupta, Shailendra Raghuwanshi, Kakoli Dutt, R.K. Saxena ∗
Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi 110021, India

a r t i c l e i n f o a b s t r a c t

Article history: Xylitol, a sugar substitute, is a high value product for pharmaceutical and food industries and its purifi-
Received 6 November 2010 cation being of commercial importance. In the present study, the purification of xylitol obtained through
Received in revised form 16 February 2011 Candida tropicalis by fermentation using synthetic xylose and corn cob hemicellulosic hydrolysate as sub-
Accepted 19 February 2011
strates were studied for liquid–liquid extraction (21.72 g/l xylitol extracted in 1:5 (v/v) of ethyl acetate)
and precipitation (67.44% xylitol recovery along with certain impurities). By this method xylitol recovery
Keywords:
is difficult and expensive for large scale processes. Therefore, activated charcoal treatment followed by
Xylitol
vaccum concentration and crystallization method for xylitol extraction was evaluated. The optimized
Fermentation broth
Activated charcoal treatment
conditions obtained for activated charcoal treatment followed by vaccum concentration and crystalliza-
Crystallization tion method were 15.0 g/l of charcoal concentration at 30 ◦ C for 1 h with 10 times super saturation of
Xylitol recovery initial concentration and crystallization temperature of −20 ◦ C for initiation and then at 8 ◦ C yielding
43.97%. After 4 cycles of crystallization, 76.20% and 68.06% xylitol crystallization yield was obtained in
50 ml and 5.0 l of the synthetic xylose fermentation broth by adapted strain of C. tropicalis respectively.
The effect of solvents on the crystalline structure of xylitol showed prismatic structure in the presence
of ethanol and orthorhombic needles in the presence of tetrahydrofuran. The purity of the xylitol was
characterized using 13 C and 1 H nuclear magnetic resonance, mass spectroscopy, and optical rotation,
confirming 98.99% purity in a pure crystallized form.
© 2011 Elsevier B.V. All rights reserved.

1. Introduction the complex composition of the fermentation broth [6]. In order

Industries producing polyol sweeteners have registered a grow-
ing demand for the consumption of sugar-free and low heat value
products. Among these, xylitol is an important sugar substitute
with certain interesting physical and chemical properties which
make it a high value compound for pharmaceutical, odontological
and food industries. At present, large scale commercial production
of xylitol is by an expensive catalytic hydrogenation of d-xylose
from acid hydrolysis of lignocellulosics [1]. Hence, it is worthwhile
to explore an alternative process for the effective production of xyl-
itol using micro-organisms which make use of the semi synthetic
media [2,3] or detoxified hemicellulosic hydrolysate [4] in order to
reduce the manufacturing costs with minimal environmental and
energy issues [5].
The recovery and purification of the product exists as a very
complicated step in several industrial fermentative processes,
which majorly depend on the nature of the product as well as on
Abbreviations: NMR, nuclear magnetic resonance; OR, optical rotation; MS, mass
spectroscopy; HPLC, high performance liquid chromatography.
∗ Corresponding author. Tel.: +91 11 24116559; fax: +91 11 24115270.
E-mail address: rksmicro@hotmail.com (R.K. Saxena).
to recover a product which requires higher purity for commercial-
ization [7], it is often implied that important steps characterized
by costs even higher than the production process are used. How-
ever, in literature on polyols, very little information is available
about xylitol recovery [8–10] and mainly reports are related to
the obtainment and treatment of the hemicellulosic hydrolysate,
its fermentation and metabolic bioconversion [11,12]. Until now,
on industrial scale, the xylitol obtained is separated and purified
by chromatographic methods [13,14]. Jandera and Churacek [15]
used cation exchange resin columns for xylitol separations followed
by crystallization at low temperatures of the xylitol-rich solutions.
Whereas, Gurgel et al. [8] used both anion and cation exchange
resins to purify xylitol from sugarcane bagasse hydrolysate fermen-
tation broth and observed a xylitol loss of about 46–57%. However,
such techniques tend to be expensive for industrial scale processes.
In order to overcome this hurdle, an efficient and econom-
ically competitive strategy for xylitol purification and recovery
from fermented broth was developed. The purification of solutions
by liquid–liquid extraction and precipitation is used in numerous
industrial processes in order to recover dissolved substances or to
remove undesirable impurities. However, the most efficient strat-
egy used for xylitol purification and its extraction is the activated
charcoal treatment followed by vaccum concentration and crystal-

1383-5866/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.seppur.2011.02.018
 S. Misra et al. / Separation and Purification Technology 78 (2011) 266–273
lization method [16]. In accordance with the literature reports, it
is well known that in the past few years, xylitol crystallization has
drawn more attention [17] and is believed to be the final step for
obtaining highly purified products [18,19]. The choice of the crys-
tallization method (cooling, evaporation, precipitation and salting
out) is dependent on the solubility and saturation slopes with the
temperature [20]. Mullin [21] explained the three zones involved
in the process of crystallization wherein he stated that at a very low
saturation concentration there was no possibility of nucleation or
crystal growth. The existing crystals dissolve in the medium. Sec-
ondly, in supersaturated metastable zone, there was an occurrence
of crystal growth but spontaneous nucleation does not take place.
It is believed to be the first stage in the crystallization process or
also known as primary nucleation wherein a series of bimolec-
ular collisions occurs and forms an aggregate of small number
of molecules of dissolved materials (embryos). Thirdly, there is a
labile zone where nuclei are formed spontaneously from a clear
solution. However, according to Martínez et al. [20], these three
zones are controlled not only by equilibrium, but also by process
parameters like agitation, temperature, solution purity and cooling
rate. Martínez et al. [4] also reported that during crystallization at
lower temperatures thermal degradation of compounds sensitive
to heat is minimized and also its unit operating costs exist low-
ered as compared to other recovery techniques due to high product
concentration.
The aim of the present investigation is to compare various
purification strategies for efficient xylitol purification and its
extraction from synthetic as well as detoxified corncob hemicellu-
losic hydrolysate fermented by a natural yeast isolate of C. tropicalis
which proved to be an efficient xylitol producer. The purification
strategy selected was optimized for maximum xylitol crystalliza-
tion yield and to evaluate its process economics.
2. Experimental
2.1. Chemicals used
The xylose used as substrate was obtained from corncob through
acid hydrolysis. The hydrolysate was detoxified using a combina-
tion of activated charcoal and pH adjustment followed by 2-fold
concentration in rotavapor. The initial concentration obtained
through this process is 40.16 g/l of xylose (details not given). The
synthetic xylose was purchased from Central Drug House (CDH,
Mumbai, India). All other medium components and chemicals used
were of analytical grade and were purchased locally (Himedia,
Qualigenes and Sisco Research Laboratories Ltd., India).
2.2. Media and fermentation conditions
The fermentation broth was prepared using synthetic xylose
(100.0 g/l for (unadapted) parent strain and 175.0 g/l for adapted
strain of Candida tropicalis) and corncob hemicellulosic hydrolysate
(obtained by acid hydrolysis) containing 40.16 g/l of xylose. The
medium was supplemented with (in g/l) yeast extract 5.0, KH2 PO4
2.0, and MgSO4 ·7H2 O 0.3. The appropriate medium was inoculated
with 5.0% of the seed inoculum and fermentation was carried out
at 30 ◦ C in 10 l fermentor with 5 l working volume (New Brunswick
Sci. Inc. Fermentor Bioflow IV, USA). The pH was controlled auto-
matically with 1 N NaOH/1 N HCl using a pH controller at pH 4.5.
Agitation and aeration rate were adjusted to 400 rpm with a con-
stant rate of 0.7 vvm (up to 24 h) and then shifted to 200 rpm and
0.3 vvm for rest of the fermentation run. Foaming was controlled
by adding silicon antifoam agent (50%, v/v, prepared in distilled
water). Samples were withdrawn periodically at intervals of 6 h and
analysed for xylitol production. The fermentation run was stopped
267
after 60 h (unadapted strain) and 42 h (adapted strain) in corncob
hemicellulosic hydrolysate medium and 84 h (unadapted strain)
and 96 h (adapted strain) of incubation in the presence of synthetic
xylose by this time more than 90–95% xylose was consumed. The
fermented broth was centrifuged at 10,000 rpm for 15 min in order
to separate the cells and solid particles (Sigma centrifuge 4X15,
Germany).
2.3. Extraction of xylitol from fermented broth
Three different methods were investigated, analysed and com-
pared for xylitol purification from culture broth.
2.3.1. Liquid–liquid extraction
The filtered fermented broth (50 ml) was extracted with ethyl
acetate, chloroform or dichloromethane in the ratio of 1:1 (v/v) at
30 ◦ C. The aqueous and organic fraction obtained with these sol-
vents was analysed for xylitol using HPLC.
2.3.2. Precipitation
The filtered fermented broth was mixed with each of ethanol,
acetone or tetrahydrofuran (THF) in 1:1 ratio (v/v), stirred and
allowed to stand for 60 min at 4 ◦ C. The precipitate was separated
by centrifugation (Sigma centrifuge 4X15, Germany) at 4000 rpm
for 10 min. The precipitate thus obtained was dissolved in water
and the left over solution was analysed for xylitol using HPLC.
2.3.3. Vaccum concentration and crystallization
2.3.3.1. Treatment with activated charcoal. After micro filtration
(GE Healthcare, USA), aliquots of the filtrate (50 ml) were trans-
ferred into 250 ml flasks for the treatment with 20 g/l of charcoal
concentration at 30 ◦ C. After magnetic agitation for 1 h, the mix-
ture was filtered using Whatmann filter paper No. 1. Samples of
the filtrate were analysed for the determinations of the initial con-
centrations of xylitol.
2.3.3.2. Crystallization tests. After treatment with activated char-
coal, fermented broth was concentrated in rotavapor (Buchi
Rotavapor R-210, Germany) at 55 ± 5 ◦ C up to the achievement of
the selected concentration. The aliquots of the concentrated solu-
tions were transferred to the glass petri plates and to that finely
ground commercial xylitol (1.0 g/l) was added in order to favor
nucleation of the crystals. The petriplates were kept at −20 ◦ C in
order to initiate crystal formation and once the process initiates
the petriplates were shifted at 8 ◦ C to increase the crystallization
yield.
2.4. Precipitated crystals estimation
After completion of crystallization, precipitated crystals were
removed from mother liquor through filtration using Whatmann
filter paper No. 1 and dried on the filter paper at room temperature.
Preweighted crystals were dissolved in water to determine their
contents of xylitol and its purity using HPLC.
2.5. Process optimization to enhance crystallization
Effect of various parameters such as treatment tempera-
ture (20–50 ◦ C), different concentrations of activated charcoal
(5.0–25.0 g/l), contact time (15–60 min.), xylitol saturation concen-
tration (5–15 times), crystallization temperature (−20 ◦ C, 8 ◦ C and
−20 ◦ C for 3–4 days and then transferred to 8 ◦ C), presence of xylose
in the concentrated broth were evaluated using one variable at a
time approach in order to enhance the crystallization yield and the
quality of crystals formed.
268 S. Misra et al. / Separation and Purification Technology 78 (2011) 266–273
Table 1a
Xylitol concentrations in aqueous phase and organic phase, after liquid–liquid extraction with chloroform and ethyl acetate.
Organic solvent Aqueous phase (g/l)
Chloroform (1:1) 51.62 ± 1.54
Ethyl acetate (1:1) 48.07 ± 1.44
Initial xylitol concentration: 66.78 g/l.
Table 1b
Various ratios of ethyl acetate were tried for the extraction of maximum xylitol concentration in organic phase.
Organic solvent Ratio (broth to organic solvent)
Ethyl acetate 1:1
Ethyl acetate 1:3
Ethyl acetate 1:5
Ethyl acetate 1:7
Initial xylitol concentration: 66.78 g/l.
2.6. Scale up of the crystallization process
After optimization of various parameters, the process was scaled
up to 5 l of the fermentation broth. The crystals were obtained
through repeated concentration of the mother liquor up to the
achievement of the selected concentration (supersaturation con-
centration) after each cycle of crystallization. The crystallization
was repeated number of cycles till a final yield was achieved.
Beyond this, it was not possible to further crystallize.
All the tests were performed in triplicates and the data of all
the experiments were statistically analysed and expressed as the
mean ± S.D. of three replicate experiments.
2.7. Analytical procedures
The concentrations of xylitol and xylose were determined at
37 ◦ C by high performance liquid chromatography (HPLC) using
a Hewlett Packard Series 1050 liquid chromatograph equipped
with a refractive index detector (RID) and a BIO-RAD Aminex HPX-
87H (300 mm × 7.8 mm) column. The optimized chromatographic
conditions were 5 mM H2 SO4 as mobile phase, with a flow rate
of 0.6 ml min−1 , temperature of 37 ◦ C and an injection volume of
20.0 ␮l. Samples were diluted when necessary prior to injection.
The aqueous and organic fraction obtained with the solvents
involved in the liquid–liquid extraction was analysed for xylitol
using HPLC.
The precipitate thus obtained during precipitation was dis-
solved in water and analysed for xylitol along with aqueous phase
using HPLC.
2.8. Analysis of xylitol for its purity
Evaluation of the purity of xylitol crystals obtained by activated
charcoal treatment followed by vaccum concentration and crystal-
lization method was performed using nuclear magnetic resonance
(NMR) (13 C NMR, 1 H NMR), mass spectroscopy (MS) and optical
rotation.
2.8.1. Nuclear magnetic resonance spectroscopy (NMR)
The 13 C NMR was performed using Bruker DPX 300 spectrome-
ter. The concentration of 20 mg/ml each of crystallized xylitol and
commercial xylitol was prepared separately in D2 O and spectra of
xylitol were analysed at 75.5 MHz. The 1 H nuclear magnetic res-
onance spectroscopy was performed using AV 300 spectrometer.
The concentration of 5 mg/ml each of crystallized xylitol and com-
mercial xylitol was prepared separately in D2 O and analysed at
300 MHz. Spectra were recorded and processed.
Organic phase (g/l) Distribution ratio (D)
14.04 ± 0.42 0.271 ± 0.00
17.01 ± 0.68 0.353 ± 0.01
Aqueous phase (g/l) Organic phase (g/l) Distribution ratio (D)
48.07 ± 1.92 17.01 ± 0.340 0.353 ± 0.00
46.52 ± 1.86 18.91 ± 0.567 0.406 ± 0.012
43.58 ± 1.30 21.72 ± 0.868 0.494 ± 0.019
43.74 ± 1.31 21.98 ± 0.879 0.502 ± 0.02
2.8.2. Mass spectroscopy (MS)
Mass spectrometry of xylitol was performed on an Esquire LC
electrospray injection (ESI) iontrap mass spectrometer equipped
with a nanospray source (Bruker Daltonics, Germany). Two hun-
dred microlitres of purified samples were evaporated and dissolved
in 1.5 ml 80:20% (v/v) acetonitrile:water. Aliquots of 50 ␮l were
acidified by addition of 3 ␮l 5% (v/v) formic acid prior to analysis.
Two microlitres of the samples were loaded into the nan-
otubes (Proxeon Biosystems, Odense, Denmark). Mass spectra were
recorded, each as an average of three scans from 100 to 200 m/z in
positive mode.
2.8.3. Optical rotation
The specific optical rotation of xylitol was analysed using a
polarimeter (Autopol® IV Polarimeter). The optimized polaro-
graphic conditions were 0.25% xylitol with a chamber temperature
of 29.4 ◦ C at 546 nm. The results of crystallized xylitol obtained
from fermented broth were compared with synthetic/commercial
xylitol.
3. Results and discussion
During fermentation in both synthetic xylose and corncob hemi-
cellulosic hydrolysate broth by unadapted (parent) and adapted
strains of C. tropicalis, complete consumption of xylose took place
with xylitol being the major end product (data not shown). How-
ever, the presence of certain other impurities or products in the
fermented broth necessitates the development of inexpensive
purification procedures for xylitol recovery. Of all the different
procedures given in literature, liquid–liquid extraction is used in
numerous industrial processes for xylitol extraction. The advan-
tages are that it is simple, clean and fast. Solvent recovery is also
easy owing to their low boiling points. Here, the extraction of the
undesirable impurities was carried out using ethyl acetate, chlo-
roform or dichloromethane. Maximum clarification of the broth
was obtained with ethyl acetate having 48.07 g/l xylitol in aqueous
phase and 17.01 g/l xylitol in organic phase (Table 1a). Differ-
ent permutations—combinations of fermentation broth and ethyl
acetate were evaluated for best ratio to clarify the broth. The
ratio of 1:5 (v/v) was selected resulting in 21.72 g/l xylitol in the
organic phase (Table 1b). Efficacy of ethyl acetate for removal of col-
ored substances from eucalyptus wood hydrolyzate has also been
reported by Parajó et al. [22]. Cruz et al. [23] and González et al.
[24] also reported the importance of using solvents for hydrolysate
purification through solvent extraction yielding a phenolic-rich
extract, which finally resulted in discoloration of the hydrolysate.
However, liquid–liquid extraction (ethyl acetate, chloroform and
dichloromethane) presents a tendency to form emulsion, slow-
ing the clarification process. Thus, another strategy for xylitol
 S. Misra et al. / Separation and Purification Technology 78 (2011) 266–273 269

Table 2
Extraction of xylitol through precipitation in aqueous phase with ethanol, acetone and tetrahydrofuran.

Organic solvent Aqueous phase (g/l) Organic phase (g/l) % Recovery of xylitol in aqueous phase % Recovery of xylitol in organic phase

Ethanol 38.62 ± 1.15 27.00 ± 0.81 57.83 ± 1.73 40.43 ± 1.21

Acetone 45.04 ± 1.80 20.44 ± 0.40 67.44 ± 2.69 30.60 ± 0.61
Tetrahydrofuran 33.69 ± 0.67 32.11 ± 1.28 50.44 ± 1.00 48.08 ± 1.92

Initial xylitol concentration: 66.78 g/l.

extraction is precipitation wherein acetone (organic solvent) was Table 3

able to clarify the broth with 67.44% xylitol recovery in aque-
ous phase along with certain impurities (medium components,
proteins and other metabolic intermediatory compounds during
fermentation process) (Table 2). The low selectivity of the solvent
was probably due to its low polarity that reduces the solubility of
xylitol in solution thereby causing its precipitation [25]. More than
60–70% impurities were removed from the solution as precipitate.
Solomons [26] also reported that acetone was the most selective
among all the organic solvents evaluated with only a 10.7% xylitol
loss.
Liquid–liquid extraction and precipitation involves the use
of solvents making xylitol recovery difficult and expensive for
large scale purification. Therefore, a more cost effective, efficient,
easy, less time consuming and environmental friendly procedure
is required. Thus, in the present investigation, another strat-
egy for xylitol extraction was developed using activated charcoal
treatment followed by vaccum concentration and crystallization
method. In this method, before recovery, the initial xylitol concen-
tration in the synthetic xylose fermented broth [unadapted (parent)
strain of C. tropicalis] was 66.78 g/l which after recovery by activated
charcoal treatment resulted in 63.31 g/l of xylitol. Thus, there was
a loss of only 3.47 g/l of xylitol, which was adsorbed to charcoal.
This filtered fermented broth was concentrated ten times through
rotavapor and commercial xylitol was added (1 g/l) for nucleation.
Incubation was at −20 ◦ C for 3–4 days to initiate xylitol formation
and then transferred at 8 ◦ C. Yield of 39.33% xylitol was achieved
when 1.306 g of xylitol crystals were obtained from 50 ml of the fer-
mented broth (theoretical yield of 3.32 g xylitol from 50 ml). During
crystallization, the xylitol crystals and the mother liquor contain-
ing uncrystallized xylitol were analysed followed by recycling of
mother liquor for crystallization. Since, crystallization is an impor-
tant stage of xylitol purification [6,10,16,27], various parameters
were optimized for amelioration of the yield and quality of xylitol
crystals.
3.1. Optimization of treatment temperature
The treatment temperature was optimized in the range from
20 to 50 ◦ C with optimal temperature of 30 ◦ C yielding 39.33% xyl-
itol crystallization yield (Table 3). Contrary to our results, Gurgel
et al. [8] reported 80 ◦ C as optimal treatment temperature for the
process of crystallization. How and Morr [28] explained that an
increase in treatment temperature, led to absorption of impurities
and colored compounds from fermented broth due to molecular
Effect of different treatment temperatures on xylitol crystallization yield in the pres-
ence of 20.0 g/l of charcoal in the activated charcoal treatment followed by vaccum
concentration and crystallization method.
Tt WC (g) % YC (WC /YT ) × 100
◦
20 C 1.276 ± 0.04 38.43 ± 1.15
30 ◦ C 1.306 ± 0.05 39.33 ± 1.57
37 ◦ C 1.214 ± 0.02 36.56 ± 0.73
50 ◦ C 0.992 ± 0.01 29.87 ± 0.29
Tt : treatment temperature; WC : weight of the crystals; YT : theoretical yield
(3.32 g/50.0 ml); % YC : % xylitol crystallization yield.
vibration intensification and also has a positive effect on xylitol
crystallization yield.
3.2. Optimization of charcoal concentration
Optimal xylitol yield of 43.31% of xylitol crystallization yield
(1.438 g of xylitol crystals per 50 ml) was obtained with charcoal
concentration of 15.0 g/l at 30 ◦ C (Table 4). Contrary to our results,
Sampaio et al. [16] reported a higher charcoal concentration of
20.0 g/l as the optimal for clarifying the fermented broth. How
and Morr [28] also noted that at higher charcoal concentrations,
the effect of contact time is reduced. This is due to higher adsor-
bent/adsorbate ratio and lower adsorbate saturation concentration
resulting in better yield.
3.3. Optimization of xylitol saturation concentration
The supersaturation of xylitol concentration was optimized in
the range from 5.0 to 15.0 times by concentration in rotavapor
at 55 ± 5 ◦ C (supersaturation temperature). The final concentra-
tion of 637.08 g/l of xylitol was selected for crystallization (i.e.
when the solution containing xylitol was concentrated 10 times
to its original concentration). This concentration falls under labile
supersaturated zone wherein supersaturated and rapid nucleation
occurs. Results show that 43.37% of xylitol crystallization yield
(1.44 g of xylitol crystals per 50 ml) was achieved at charcoal con-
centration of 15.0 g/l and 30 ◦ C (Table 5). The reports of de Faveri
et al. [9] support our findings as they have noted that increasing
xylitol concentration and decreasing temperature improves xyl-
itol recovery through crystallization. Later in 2004, they further
reported the optimal xylitol supersaturation value as 728 g/l and
that a cooling temperature of −6 ◦ C leads to 54.0% of xylitol crys-
tallization yield [6]. Wei et al. [17] and Martínez et al. [29] also

Table 4
Effect of the concentration of the charcoal on xylitol crystallization yield in the presence of 30 ◦ C as the optimized treatment temperature in the activated charcoal treatment
followed by vaccum concentration and crystallization method.

CC (g/l) WC (g) (WTot C − WF ) % YC (WC /YT ) × 100 AC

5.0 1.324 ± 0.01 5.78 ± 0.06 Small; less in amount

10.0 2.018 ± 0.08 26.92 ± 1.07 Fair in amount
15.0 2.578 ± 0.10 43.31 ± 1.73 Clear, large crystals
20.0 2.416 ± 0.07 38.85 ± 1.16 Clear crystals, slightly black
25.0 1.925 ± 0.04 23.79 ± 0.47 Lesser in amount

CC : concentration of charcoal; WF : weight of filter paper (g); WTot C : total weight (g); WC : weight of the crystals (g); % YC : % of xylitol crystallization yield; AC : appearance of
crystals; YT : theoretical yield (3.32 g/50.0 ml).
270 S. Misra et al. / Separation and Purification Technology 78 (2011) 266–273

Table 5 Table 7
Effect of different xylitol saturation concentrations on xylitol crystallization yield Effect of different charcoal concentrations on the number of cycles of crystallization
in the activated charcoal treatment followed by vaccum concentration and crystal- and on the xylitol crystallization yield in the presence of 30 ◦ C as the optimized
lization method. treatment temperature in the activated charcoal treatment followed by vaccum
concentration and crystallization method from synthetic xylose fermented broth.
n Cxy (g/l) WC (g) (WTot C − WF ) % YC (WC /YT ) × 100
CC (g/l) N WTot C (g) (WF + WC ) % YC (WC /YT ) × 100
5n 361.31 ± 7.22 0.285 ± 0.005 8.58 ± 0.17
10n 637.08 ± 19.11 1.44 ± 0.043 43.37 ± 1.30 5.0 2.0 0.325 ± 0.006 9.70 ± 0.19
15n 909.41 ± 36.37 1.398 ± 0.041 42.41 ± 1.27 10.0 4.0 1.651 ± 0.049 49.7 ± 1.49
15.0 4.0 2.530 ± 0.10 76.2 ± 3.04
n: number of times fermented broth concentrated; Cxy : concentration of xylitol; WC :
20.0 4.0 2.087 ± 0.083 62.6 ± 2.50
weight of crystals; % YC : % of xylitol crystallization yield.
25.0 3.0 1.156 ± 0.034 34.8 ± 1.04

CC : charcoal concentration; N: no. of cycles; WTot C : total weight; % YC : % of xylitol

Table 6 crystallization yield.
Effect of different crystallization temperatures on xylitol crystallization yield
obtained from fermented broth containing 637 ± 19.11 g/l xylitol in the activated
charcoal treatment followed by vaccum concentration and crystallization method. Table 8
◦
Effect of different charcoal concentrations on the number of cycles of crystallization
Temperature ( C) WC (g) (WTot C − WF ) % YC (WC /YT ) × 100
and on the xylitol crystallization yield in the presence of 30 ◦ C as the optimized
−20 ◦ C 1.319 ± 0.026 39.72 ± 0.79 treatment temperature in the activated charcoal treatment followed by vaccum
−20 + 8 ◦ C 1.46 ± 0.043 43.97 ± 1.31 concentration and crystallization method from fermented broth of corncob hemi-
8 ◦C 1.427 ± 0.042 42.98 ± 1.28 cellulosic hydrolysate.

WC : weight of crystals; % YC : % of xylitol crystallization yield. CC (g/l) N WTot C (g) (WF + WC ) % YC (WC /YT ) × 100

5.0 1.0 1.888 ± 0.02 3.20 ± 0.03

reported an increased xylitol crystallization yield with an opti-
mal xylitol saturation concentration of 750 g/l and 745.3 g/l for
the process of crystallization. Earlier, Amiard [30] explained the
phenomenon involved behind the addition of xylitol crystals in
the metastable zone (secondary nucleation) during the process
of crystallization which helps in preferential crystallization and is
widely used as in many industrial processes. Similarly, the impor-
tance of adding xylitol has also been reported by Canilha et al. [27]
wherein they explained that the inhibition to crystallize xylitol in
a fermented wheat straw hemicellulosic hydrolysate medium was
overcome by the addition of commercialized xylitol crystals to the
medium.
3.4. Optimization of crystallization temperature
The effect of initial crystallization temperature at −20 ◦ C, 8 ◦ C
and at 20 ◦ C for 3–4 days with subsequent transfer at 8 ◦ C resulted
in more or less similar yields (Table 6). But on further evaluation
the two phase crystallization temperature (−20 ◦ C and 8 ◦ C) was
more beneficial. At lower temperature (−20 ◦ C) crystal initiation is
in a shorter time with higher degree of purity and an increase in the
crystallization yield takes place when further incubation is carried
out at 8 ◦ C. On the other hand, crystallization at 8 ◦ C, not only delays
initiation of crystallization up to six days but also results in lower
yield. Also the crystals formed at 8 ◦ C were brittle and smaller in size
as compared to two phase crystallization temperature. Similarly,
Sampaio et al. [16] have reported that lowering the crystallization
temperature, results in effective crystallization in terms of crystal-
lization yield, while completely opposite behavior is observed for
degree of crystal purity.
3.5. Effect of the presence of xylose on crystallization
During fermentation, more than 95–98% xylose was consumed
with very little xylose left unutilized which has no effect on the
crystallization yield and on the degree of purity of the xylitol crys-
tals. On the contrary, Sampaio et al. [16] reported that the presence
of xylose ensures 1.6-fold increases in the crystallization yield
(42.0%) and enhances the rate constant of crystal growth. On the
other hand, Wei et al. [17] reported a negative effect on crystalliza-
tion in the presence of residual sugars (xylose).
The activated charcoal treatment followed by vaccum con-
centration and crystallization method resulted in a xylitol
crystallization yield of 43.97% which after 4 cycles resulted in net
10.0 2.0 1.691 ± 0.05 34.08 ± 1.02
15.0 3.0 2.019 ± 0.08 53.43 ± 2.13
20.0 3.0 1.819 ± 0.05 42.12 ± 1.26
25.0 2.0 1.471 ± 0.03 20.56 ± 0.41
CC : charcoal concentration; N: no. of cycles; WTot C : total weight; % YC : % of xylitol
crystallization yield.
76.20% of the xylitol crystallization yield at the temperature of 30 ◦ C
with 15.0 g/l of charcoal concentration (2.53 g/3.32 g) (Table 7).
Earlier, Sampaio et al. [16] have also reported that activated
charcoal treatment followed by vaccum concentration and crystal-
lization method is an efficient method to clarify fermented broth
with the optimized conditions of 20 g/l of charcoal at 25 ◦ C for 1 h
with a xylitol crystallization yield of 42.0%. On the other hand,
Gurgel et al. [8] reported that the optimal conditions for the treat-
ment is 25 g/l of charcoal in 100 ml of the fermented broth at pH 6.0,
80 ◦ C for 1 h. However they were not able to separate xylitol crystals
from the broth due to higher viscosity and colored solution.
3.6. Xylitol recovery and its purification obtained from corn cob
hemicellulosic hydrolysate
The activated charcoal treatment followed by vaccum concen-
tration and crystallization method proved to be beneficial for xylitol
extraction from synthetic xylose solution (Faveri et al. [6]; Sam-
paio et al. [16] and Martínez et al. [29]). The optimized conditions
obtained were used to enhance xylitol crystallization in the fer-
mented corncob hemicellulosic hydrolysate. It has been reported
that xylitol crystallization from corncob hemicellulosic hydrolysate
is difficult due to the presence of several impurities resulting
from acid hydrolysis of lignocelluloses [8,9]. Experimental studies
have shown that the impurities can be removed by detoxifica-
tion of the hydrolysate through pH adjustment (initially at pH
10.0 then shifts to pH 4.5). Combining this process with acti-
vated charcoal treatment (data not shown) reduces the cost of
xylitol recovery thereby making the process more feasible. Also
the use of untreated hydrolysate hinders the purification pro-
cess of xylitol from hydrolysate. Finally, on using the optimized
conditions obtained previously for extracting xylitol crystals from
the hemicellulosic hydrolysate resulted in crystallization yield of
24.40% (0.226 g/0.93 g) at 30 ◦ C and 15.0 g/l of charcoal concentra-
tion which after 3 cycles of crystallization resulted in net 53.43% of
xylitol crystallization yield (0.496 g/0.93 g) by unadapted (parent)
strain of C. tropicalis (Table 8).
 S. Misra et al. / Separation and Purification Technology 78 (2011) 266–273
Fig. 1. Photograph of purified and crystalline xylitol obtained after activated char-
coal treatment followed by vaccum concentration and crystallization method.
3.7. Process scaled up for xylitol crystallization
The volume of the fermentation broth containing synthetic
xylose was scaled up to 5 l through unadapted (parent) strain of
C. tropicalis using the initial xylitol concentration of 66.78 g/l. The
theoretical yield was estimated to be 333.90 g of xylitol in 5 l of
broth. Experimentally, however, 230.79 g of xylitol crystals with
69.12% xylitol crystallization yield was obtained after 4 cycles of
crystallization.
Using adapted strain of C. tropicalis, a theoretical yield of
525.95 g of xylitol in 5 l of broth was estimated. Experimentally,
68.06% yield (357.99 g of xylitol crystals) was obtained after 4 cycles
of crystallization (Fig. 1). This is the first report of an economically
viable purification and xylitol extraction through crystallization
strategy using activated charcoal treatment followed by vaccum
concentration and crystallization method. On the contrary, de
Faveri et al. [9] showed that through optimal crystallization con-
ditions obtained using response surface methodology, a xylitol
crystallization yield of 54.0% was obtained in synthetic xylose fer-
mented medium. Whereas, Wei et al. [17] reported a xylitol purity
of 95.0% and enhanced xylitol crystallization yield of 60.20% in their
xylitol purification studies.
Fig. 2. Photograph showing crystalline structure of xylitol in different solvents as analysed through electron microscopy: (a) prismatic structure of xylitol in ethanol and (b)
orthorhombic needles of xylitol in tetrahydrofuran.
271
The volume of the fermentation broth obtained from the corn-
cob hemicellulosic hydrolysate was treated [using a combination of
activated charcoal and pH adjustment (initially adjusted to pH 10.0
then shift at pH 4.5)] and concentrated in rotavapor. The process
was scaled up to 5 l through unadapted (parent) strain of C. tropi-
calis using the initial xylitol concentration of 18.62 g/l (theoretical
yield is 93.10 g in 5 l of broth). Xylitol crystallization yield of 46.49%
(43.19 g of xylitol) was achieved after 3 cycles of crystallization.
On the other hand, using adapted strain of C. tropicalis, resulted
in an initial xylitol concentration of 27.13 g/l (theoretical yield
of 135.65 g of the xylitol in 5 l of broth). Crystallization resulted
in 64.96 g of xylitol; yield of 47.89% obtained after 3 cycles of
crystallization. Rivas et al. [31] similarly reported that 47.0% crystal-
lization yield was obtained from fermented corncob hemicellulosic
hydrolysate broth. Martínez et al. [10] reported xylitol crystalliza-
tion from fermented sugarcane hemicellulosic hydrolysate with
92–94% purity of xylitol crystals. Further studies showed that xyl-
itol yield was improved by processing of mother liquors through
successive cycles of crystallization. Similar has been reported by
Heikkilae et al. [32].
The morphology of the purified and extracted xylitol crys-
tal obtained through large scale purification from the synthetic
xylose and corncob hemicellulosic hydrolysate fermented broth
was studied. The xylitol crystal formed in the synthetic xylose con-
taining fermented broth is white granular crystal while, the crystals
obtained from hemicellulosic hydrolysate were pale yellow and
sticky, owing to larger viscosity of the residual sugars and also due
to the presence of pigments and inorganic salts. The existence of
the residual sugar in the fermented hydrolysate affects the degree
of purity of the crystals and its crystallization yield. Wei et al. [17]
also reported similar results when they studied the influence of the
residual sugars on the purity and xylitol crystallization yield.
3.8. Effect of solvent on the crystalline structure of xylitol
Xylitol showed change in the shape of the crystal in the pres-
ence of different solvents. Through electron microscopic studies,
it was observed that xylitol showed regular tetrahedral structure
when dissolved in water (Fig. S1). In organic solvents, a prismatic
structure was observed in ethanol (Fig. 2a) and orthorhombic nee-
dles in tetrahydrofuran (Fig. 2b). Hao et al. [33] also studied and
272 S. Misra et al. / Separation and Purification Technology 78 (2011) 266–273
Fig. 3. Nuclear magnetic resonance (13 C) of purified and crystallized xylitol obtained
from fermented broth through activated charcoal treatment followed by vaccum
concentration and crystallization method.
Fig. 4. Nuclear magnetic resonance (1 H) of purified and crystallized xylitol obtained
from fermented broth through activated charcoal treatment followed by vaccum
concentration and crystallization method.
showed a strong influence of solvents on the crystalline behav-
ior of xylitol. Similarly, Mullin [21] and Randolph and Larson [34]
also reported the influence of solvents on the solubility and the
crystalline structure of the crystalline materials in solutions.
3.9. Analysis of xylitol for its purity
The purity of the xylitol was characterized through nuclear
magnetic resonance (NMR) (13 C NMR, 1 H NMR). The xylitol
obtained using activated charcoal treatment followed by vaccum
concentration and crystallization strategy was white, crystalline
in appearance having a m.p. (93.5 ◦ C) 13 C NMR (75 MHz, D2 O):
70.67, 69.52, and 61.37; 1 H NMR (300 MHz, D2 O); ı3.72–3.54
(m, 7H) showed similar spectra when compared to the standard
xylitol. White crystalline m.p. (94.0 ◦ C) 13 C NMR (75 MHz, D2 O):
71.88, 70.73, 62.58; 1 H NMR (300 MHz, D2O); ı3.74–3.59 (m, 7H)
(Figs. 3 and 4, Figs. S2 and S3), mass spectroscopy having a mass of
152.14 showed 175.29 due to the addition of sodium (23.14) dur-
ing analysis (Fig. 5 and Fig. S4), optical rotation of xylitol is 0.016
(average) (Fig. 6 and Fig. S5). Through all these techniques, it was
confirmed that the xylitol crystal purified by charcoal treatment
followed by vaccum concentration and crystallization method was
98.99% pure. Martínez et al. [29] in their study reported 98.5–99.2%
purity of xylitol crystals through a process of batch cooling crystal-
Fig. 5. Mass spectroscopy of purified and crystallized xylitol obtained from
fermented broth through activated charcoal treatment followed by vaccum con-
centration and crystallization method.
Fig. 6. Optical rotation of purified and crystallized xylitol obtained from fermented
broth through activated charcoal treatment followed by vaccum concentration and
crystallization method.
lization. Whereas, Sampaio et al. [16] reported the purity of xylitol
crystals in the range from 96.0 to 97.8%.
4. Conclusions
This work reveals the feasibility of using activated charcoal
treatment followed by vaccum concentration and crystallization
strategy over other two purification strategies. Since, activated
charcoal treatment followed by vaccum concentration and crystal-
lization method is cost effective, easy and environmental friendly
route for purification and crystallization of xylitol through micro-
bial fermentation of synthetic xylose and corn cob hemicellulosic
hydrolysate. The experimental procedure and data provide a sound
basis for large scale industrial production and purification of this
high valued sugar alcohol in order to enhance the yield and quality
of xylitol crystals formed through crystallization.
Acknowledgments
This study was supported by Council of Scientific and Industrial
Research (CSIR, India) in the form of a grant. S.M. is the recipient of
the CSIR-SRF (Grant no. 9/45(939)/2010-EMR-I).
 S. Misra et al. / Separation and Purification Technology 78 (2011) 266–273
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.seppur.2011.02.018.
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