jfpe_599

1..18

BIOETHANOL AND XYLITOL PRODUCTION FROM

DIFFERENT LIGNOCELLULOSIC HYDROLYSATES BY
SEQUENTIAL FERMENTATION
J. ARRIZON1, J.C. MATEOS1, G. SANDOVAL1, B. AGUILAR2, J. SOLIS2 and
M.G. AGUILAR3,4
1

Centro de Investigacin y Asistencia en Tecnologa y Diseo del Estado

de Jalisco A.C.
2

Universidad de Guadalajara
Guadalajara, Jalisco, Mxico
3

Instituto Tecnolgico de Veracruz/UNIDA

Av. Miguel A. de Quevedo 2779
Col. Formando Hogar,
CP. 91860, Veracruz, Veracruz Mxico
Accepted for Publication February 18, 2010

ABSTRACT
Sugarcane bagasse, Agave tequilana bagasse and coffee husks were
hydrolyzed by steam explosion followed by enzymatic treatment without
detoxifying process prior to fermentation. The hydrolysates underwent sequential fermentations using three different microorganisms to produce bioethanol
and xylitol. Sugarcane bagasse hydrolysate was found to be the best media for
bioethanol (0.44 g/g yield, 85.5% efciency) and xylitol (0.29 g/g yield) production and S. cerevisiae ITV01 performed efcient alcoholic fermentation in
the presence of a high acetic acid concentration (5.0 g/L, initial hydrolysate
concentration). For all hydrolysates, Candida tropicalis grew less; however,
it produced more xylitol than Candida magnolia (0.29 and 0.22 g/g, respectively). The effect of oxygen on xylitol production in Candida tropicalis was
also investigated, xylitol production improving as oxygen concentration level
increased (ask volume/medium volume ratios of 1.25, 3.3 and 6.6 yielded
0.143, 0.320 and 0.406 g/g, respectively). The maximum calculated efciency
for the conversion of consumed xylose to xylitol was 44.6%, with a maximum
xylitol production of 12.18 g/L.

Corresponding author. TEL: +52-229-9345701; FAX: +52-229-9345701; EMAIL: gaguilar@

itver.edu.mx

Journal of Food Process Engineering (2011) . All Rights Reserved.

2011 Wiley Periodicals, Inc.
DOI: 10.1111/j.1745-4530.2010.00599.x

J. ARRIZON ET AL.

PRACTICAL APPLICATIONS
Bioethanol and xylitol production by sequential fermentation process
using different lignocellulosic materials like sugarcane bagasse, Agave tequilana bagasse and coffee husks, is a new simple and interesting process because
it takes into account the total utilization of the principal component, the
lignocellulosic material (glucose and xylose) as well as the use of waste
material. One fermentation step produces two important products, bioethanol
and xylitol, which have various applications in different kinds of industry.

INTRODUCTION
According to the Food and Agriculture Organization of the United
Nations (FAO) (1999), more than 50 billion tons of agricultural products are
produced every year and 10% of them, mainly cellulose, followed by xylan,
the most abundant hemicellulose subcomponent, go to waste with no economic utilization whatsoever. In Mexico, sugarcane bagasse (SCB; MartnezJimnez et al. 2006) as well as Agave tequilana bagasse (ATB; Gschaedler
2004) and coffee husks (CHs; Regalado-Ortz 2006) are abundant industrial
wastes that constitute a big source of cellulose and hemicellulose; currently
little utilization of these materials is carried out, leading to environmental
problems. Different economically potential products can be obtained from
cellulose and hemicellulose by biotechnological processes.
Recently, the production of bioethanol from lignocellulosic wastes has
become an alternative for the production of sustainable energy sources. This
process requires delignication to liberate cellulose and hemicellulose from
their complex with lignin, subsequent depolymerization of the carbohydrate
polymers (cellulose and hemicellulose) to produce free sugars, and nally
fermentation of mixed hexose and pentose sugars to produce ethanol (Lee
1997). Cellulose can be hydrolyzed into glucose units by the action of cellulases. The classical cellulase system includes endoglucanase, exoglucanase,
and cellobiase (b-glucosidase). Endoglucanase attacks more or less randomly
at sites within (14)-b-D-glucan chains in amorphous regions of cellulose or at
the surface of microbrils; exoglucanase releases cellobiose from nonreducing ends of b-D-glucan chains and cellobiase hydrolyzes cellobiose and
water-soluble cellodextrins to glucose. Both exo-and endoglucanases are
inhibited by cellobiose, and this is often the rate-limiting step in cellulose
degradation (Lee 1997).
Xylan, the second most abundant component of lignocellulosic materials,
can be hydrolyzed by the action of xylanases to release xylose, which then
can be converted to xylitol by fermentation (Tran et al. 2004). Xylitol is a

BIOETHANOL AND XYLITOL PRODUCTION

ve-carbon sugar alcohol used in some interesting applications, such as to

prevent dental caries by inhibiting the metabolism of dental plaque formation
and the growth of bacteria that cause caries (Uhari et al. 1998) as well as to
provide an insulin-independent carbon source for diabetics (Van Eys et al.
1974). It is also used as a sweetener in various food products, such as chewing
gum, sweets, soft drinks and ice cream (Pepper and Olinger 1980). This has led
to a rapidly increasing demand for xylitol. Currently, most of the xylitol
production is manufactured from xylan by a chemical method. However, the
step to purify xylitol from other polyols and by-product sugars is relatively
complex (Winkelhausen and Kuzmanova 1998) and the strong chemical treatment is hazardous to the environment.
Xylitol production by the transformation of glucose and xylose has been
carried out with different fermentation systems (Granstrm et al. 2007). The
co-culture process (a mixture of two or more different kinds of cells grown
together) has been used in free or immobilized form, in order to utilize both
pentose and hexose sugars present in the enzymatic hydrolysates of lignocellulosic material (Hinfray et al. 1995). The main limiting factor of the
co-culture process is the low ethanol tolerance of pentose utilizing yeasts
(Laplace et al. 1993). The conversion of glucose and xylose to ethanol by
co-culture has been successfully carried out by Taniguchi et al. (1997) using a
respiratory decient mutant of S. cerevisiae and Pichia stipitis. However, the
other substitute for ethanol production can be the formation of xylitol from
lignocellulosic hydrolysates by yeasts such as Candida tropicalis and Candida
guillermondii (Barbosa et al. 1988; Horitsu et al. 1992). Normally the production of xylitol from lignocellulosic wastes by fermentation has been performed
with different fermentation systems using hydrolysates from different agricultural sources obtained by a thermal or chemical hydrolysis with a subsequent
detoxication process in order to eliminate fermentation inhibitors such
as acetic acid, furfural and phenolics (Paraj et al. 1996; Felipe et al. 1997;
Gurgel et al. 1998; Maciel de Mancilha and Karim 2003; Santos et al. 2003;
Carvalho et al. 2004, 2005; De Faveri et al. 2004; Mussatto and Roberto 2004;
Silva et al. 2006; Sreenivas-Rao et al. 2006). Therefore, the use of microorganisms, which can tolerate toxic compounds, such as acetic acid and furfural,
can decrease the cost of xylitol production, as the necessity of a detoxifying
process is avoided. In order to eliminate the production of toxic compounds
produced by chemical hydrolysis, some studies have performed enzymatic
hydrolysis of lignocellulosic wastes from beech wood and walnut shells for
xylitol production (Tran et al. 2004) and from corn cobs for bioethanol and
xylitol production (Latif and Rajoka 2001). Until now, there are no reports to
our knowledge dealing with the production of bioethanol and xylitol from ATB
and CH hydrolysates. In this study, a sequential fermentation process was
developed with acetic acid and furfural tolerant isolated yeast strains for the

J. ARRIZON ET AL.

simultaneous production of bioethanol and xylitol from CH, sugarcane and

ATB hydrolysates, without a detoxifying process. For the hydrolysis of these
agricultural wastes a combination of steam explosion followed by an enzymatic process was applied to release the fermentable sugars.
MATERIALS AND METHODS
Hydrolysis of Lignocellulosic Waste
The three industrial wastes: SCB, ATB and CHs were washed to remove
impurities, and then they were dried (at 60C, drying oven, Fisher Scientic,
Pittsburgh, PA) and milled (hammer mill, Pulvex 200, Mexico City, Distrito
Federal, Mexico) to obtain a particle size of 0.55 mm in sieving equipment
(Rotap RX-29 W.S. Tyler Inc., Mentor, OH). The dried industrial wastes were
then treated by the following thermal process. Steam explosion (170C, 7 kg/
cm2, for 5 min followed by rapid depressurization) was applied to 500 g of
each industrial waste (SCB, ATB and CH), which had been previously impregnated with 1% (w/w) H2SO4. Each treated industrial waste was then hydrolyzed with a commercial enzymatic cocktail (Celluzyme) in the following
manner: 9 mL CH3COOH buffer (0.05 mol/L, pH 5) was added per gram of
treated industrial waste, then 1 mL enzymatic solution was added (0.32 mg
celluzyme per mL CH3COOH buffer 0.05 mol/L, pH 5). The mix was incubated at 50C and 200 rpm for 348 h.
Bioethanol Fermentation on Lignocellulosic Hydrolysates
Medium Formulation. The three hydrolysate wastes were ltered and
the precipitates were removed, washed with distilled water and then mixed
with the supernatant until solutions with 30 g glucose per liter were achieved.
The following salts, KH2PO4 (5 g/L), Mg(NO3)26H2O (0.1530 g/L) and
CO(NH2)2 (3 g/L), were added to each of the solutions mentioned earlier in
order to formulate the medium for fermentation and subsequently adjusted to
pH 5.5. Using 250 mL Erlenmeyer asks containing 75 mL medium of each
formulated hydrolysate, sterilization was carried out (121C, 15 min) before
inoculation.
Fermentation. A Saccharomyces cerevisiae strain (JC-4, tolerant to
acetic acid, furfural and phenolics, ITV-01 collection, Veracruz, Veracruz,
Mexico), isolated from sugarcane molasses was propagated in a shaker incubator in the medium described earlier for fermentation (overnight culture,
30C and 200 rpm). 6 106 cells/mL were inoculated for each 75 mL of
sterilized medium and they were fermented for 24 h at room temperature

BIOETHANOL AND XYLITOL PRODUCTION

(3034C) without shaking. Samples were taken during fermentation at

0,6,12 and 24 h for cell count (microscope) and fermentation monitoring by
high-performance liquid chromatography (HPLC) analysis. It was necessary
to perform fermentations in four Erlenmeyer asks for each formulated
hydrolysate in order to have enough fermentation asks for replicates in the
subsequent xylitol fermentation.
Xylitol Fermentation on Lignocellulosic Hydrolysates
Medium Formulation. Once bioethanol fermentation was nished
(less than 24 h), the same fermented ask contents were formulated again with
the production medium consisting of yeast extract (1 g/L), KH2PO4 (5 g/L),
Mg (NO3)26H2O (0.1530 g/L) and CO(NH2)2 (3 g/L). The addition of 0.25,
1.65 and 1.8 g pure xylose to 75 mL SCB, ATB and CH hydrolysate medium,
respectively, was necessary in order to reach 30 g/L xylose as initial sugar
concentration for xylitol fermentation. The asks were adjusted to pH 5.5 and
sterilized at 121C for 15 min before inoculation.
Fermentation. A Candida tropicalis strain (IEC5 ITV collection, Veracruz, Veracruz, Mexico) and a Candida magnoliae strain (OFF1, CIATEJ
collection, Guadalajara, Mexico), which are also tolerant to acetic acid, furfural and phenolics, were isolated from SCB and mezcal fermentation, respectively. These strains were propagated in a shaker incubator in the medium
described earlier for xylitol fermentation (overnight culture, 30C and
200 rpm). C. tropicalis and C. magnoliae were inoculated (6 106 cells/mL)
in duplicate in Erlenmeyer asks (75 mL) containing the medium formulated
earlier, and fermented for 96 h at 30C in a shaking incubator (100 rpm).
Samples were taken during fermentation at 0, 6, 12, 24, 48, 72 and 96 h for cell
count (microscope) and fermentation monitoring by HPLC analysis.
Effect of Oxygenation on Xylitol Production
Once the comparison of xylitol production capacity between the two
Candida strains had been performed, the best xylitol producer (C. tropicalis)
and the best xylose source (SCB hydrolysate) were chosen to test the effect
of different oxygenation conditions on xylitol production. In the previous
fermentations, a 75-mL medium volume (mv) was introduced into a 250 mL
ask volume (fv) resulting in an fv/mv ratio of 3.3. The same 75 mL mv
was introduced into two different fv of 100 and 500 mL to produce lower
(fv/mv = 1.33) and higher (fv/mv = 6.6) oxygenation conditions, respectively.
These asks were prepared as described earlier and fermented under the same
conditions (fv/mv = 3.3; 6 106 cells/mL, 30C, 100 rpm, during 96 h).

J. ARRIZON ET AL.

TABLE 1.
HYDROLYSIS OF SUGARCANE BAGASSE, AGAVE TEQUILANA BAGASSE AND COFFEE
HUSKS WITH A COMBINATION OF STEAM EXPLOSION AND ENZYMATIC TREATMENT
Compound

Glucose
Xylose
Arabinose
Acetic acid

Agricultural waste g compound per g-1 agricultural waste

Sugarcane bagasse

Agave tequilana bagase

Coffe husk

0.1616 0.021
0.1462 0.015
0.0461 0.001
0.0239 0.005

0.0750 0.008
0.0210 0.005
0.0072 0.001
0.0160 0.003

0.0517 0.007
0.0103 0.009
0.0050 0.001
0.0321 0.004

HPLC Analysis Methods. Prior to HPLC measurement the samples

were pretreated in order to eliminate impurities as follows: 0.1 mL BaO
(0.15 mol/L) and 0.1 mL ZnSO4 (5% w/w) were added to 0.8 mL sample,
then it was centrifuged at 100 g for 10 min and nally the supernatant was
ltered with a 0.45 mm lter cellulose acetate. Glucose, xylose, arabinose,
ethanol, glycerol, acetic acid and xylitol concentrations were determined
by HPLC Waters 600 (TSP Spectra System, Waters, Milford, MA) using a
Biorad Aminex HPX-87H column (Bio-Rad Laboratories, Inc., Hercules,
CA) specic for the separation of alcohols, organic acids and sugars. The
column was maintained at 48C and the mobile phase was H2SO4
(0.005 mol/L) with a ow rate of 0.6 mL/min. Detection of the components
was performed by a differential refractometer Index Refraction detector
(Waters 2414, TSP Refracto Monitor V). Specialized software (DATA
APEX, Data Apex Company, Prague, Czech Republic) allowed the calculation of detected peaks areas. The maximum standard concentration was
20 g/L for the different components.

RESULTS
Hydrolysis of SCB, ATB and CH
From the three industrial wastes it can be observed that under the
hydrolysis conditions tested, SCB was the best sugar source (Table 1). The
glucose content in SCB was 2.15 and 3.12 times higher than ATB and CHs,
respectively (Table 1). The xylose content in SCB it was 6.9 and 14.2 times
higher than ATB and CH, respectively (Table 1). Arabinose content was also
higher in SCB, 6.4 and 9.22 times higher than ATB and CH, respectively
(Table 1). The content of acetic acid was higher in CH, followed by SCB and
ATB had the lowest content (Table 1).

BIOETHANOL AND XYLITOL PRODUCTION

Production of Bioethanol on Lignocellulosic Hydrolysates

by S. cerevisiae
In general the patterns of sugar consumption and ethanol production were
similar in the three hydrolysates; however, in ATB hydrolysate, slower sugar
consumption and ethanol production was observed (Fig. 1a,b). Glucose consumption was more than 95% for all hydrolysates (Table 2). In the case of cell
growth, great differences can be observed between ATB (23 106 cells/mL)
and SCB and CH (40 106 cells/mL) hydrolysates (Fig. 1c). Ethanol efciency (n) per amount of glucose (0.51 g/g experimental Yp/s bioethanol/theoretical
Yp/s bioethanol) was slightly higher in SCB and ATB (85.4 and 85.5%) than in
CH (83%) hydrolysates, as observed also in Yp/s bioethanol values (Table 2). The
glycerol produced by the end of fermentation was less than 1 g/L in the three
hydrolysates (Fig. 1d) and the respective Yp/s glycerol values for CH, SCB and
ATB were 0.0321, 0.026 and 0.0160 g/g (Table 2). As expected, the concentration of xylose and acetic acid remained constant during fermentation
(data not shown).

Xylitol Production in Lignocellulosic Hydrolysates to C. tropicalis

and C. magnoliae
In general, xylose consumption was higher and faster for C. tropicalis
yeast strain than for C. magnoliae, and in the case of SCB hydrolysate, xylose
consumption was slower than in ATB and CH hydrolysates for both Candida
strains (Fig. 2a,b). The percentage of total xylose consumed was higher for
C. tropicalis and C. magnoliae in SCB hydrolysate (87.8 and 87.7%, respectively) compared with the other hydrolysates (Table 3), whereas in ATB and
CH hydrolysates, the percentage of total xylose consumed was higher for
C. tropicalis (86.9 and 86.1%, respectively) than for C. magnoliae (66.0 and
71.6%, respectively) (Table 3). In Fig. 2c,d, it can be observed that in general,
xylitol production was higher for C. tropicalis than for C. magnoliae in the
three hydrolysates, and for both yeast strains the level of xylitol produced was
a function of the hydrolysate used, thus for SCB, ATB and CH hydrolysates
the maximum xylitol produced were 7.5, 5.2 and 3.0 g/L for C. tropicalis and
6.5. 4.2 and 1.2 g/L for C. magnoliae, respectively (Fig. 2c,d).
Xylitol efciency (n) per amount of xylose (0.917 g/g experimental
Yp/s xylitol/theoretical Yp/s xylitol) for SCB, ATB and CH hydrolysates were 31.6,
22.9 and 12.0 (%) for C. tropicalis and 26.1, 24.0 and 5.4 (%) for C. magnoliae, respectively (Table 3), and the respective Yp/s xylitol values for SCB,
ATB and CH were 0.29, 0.21 and 0.11 g/g for C. tropicalis and 0.24, 0.22
and 0.05 g/g for C. magnoliae (Table 3). Contrary to xylitol production,
cell growth was higher for C. magnoliae (6480 cells/106 mL) than for

J. ARRIZON ET AL.

FIG. 1. GLUCOSE CONSUMPTION (a) AND BIOETHANOL PRODUCTION (b), CELL

GROWTH (c) AND GLYCEROL PRODUCTION (d) WITH THE JC-4 SACCHAROMYCES
CEREVISIAE YEAST IN SCB (), ATB () AND CH ( ) HYDROLYSATES

BIOETHANOL AND XYLITOL PRODUCTION

TABLE 2.
BIOETHANOL YIELDS FOR S. CEREVISIAE IN SUGARCANE BAGASSE, AGAVE
TEQUILANA BAGASSE AND COFFEE HUSK HYDROLYSATES
Yield parameter

Efciency (%)
Consumed glucose (%)
Yp/s bioethanol
Yp/s glycerol

Agricultural waste
Sugarcane bagasse

Agave tequilana bagase

Coffe husk

85.4 2.35
98 1.07
0.435 0.0218
0.026 0.0015

85.5 2.42
95.5 2.31
0.436 0.066
0.016 0.003

83.5 3.90
100 0.05
0.426 0.015
0.032 0.001

C. tropicalis (4953 cells/106 mL) in the three hydrolysates and for both yeast
strains the growth was similar in all the hydrolysates (Fig. 2e,f).
Glycerol production was higher for C. magnoliae (2.24.8 g/L) than for
C. tropicalis (1.32.4 g/L) and for both yeast strains the production was the
lowest in CH hydrolysate (Fig. 2g,h). The corresponding Yp/s glycerol values for
SCB, ATB and CH hydrolysates were 0.04, 0.04 and 0.001 g/g for C. tropicalis
and 0.05, 0.18 and 0.04 g/g for C. magnoliae (Table 3).
At the beginning of fermentation 1.5, 0.93 and 0.73 g/L of acetic acid was
present in CH, SCB and ATB hydrolysates, respectively, and an average of
12 g/L of residual ethanol of the previous bioethanol fermentation was present
in all the hydrolysates. There was acetic acid consumption throughout fermentation for both Candida strains as well as additional ethanol production; the
calculated percentage of acetic acid consumed for SCB, ATB and CH hydrolysates were 50.9, 15.8 and 51.2% for C. tropicalis and 54.6, 44.4 and 53.5%
for C. magnoliae, respectively (Table 3). In all the hydrolysates the additional
ethanol produced was less than 4 g/L and the Yp/s ethanol values corresponding to
SCB, ATB and CH hydrolysates were 0.17, 0.08 and 0.15 g/g in C. tropicalis
and 0.16, 0.074 and 0.11 g/g in C. magnoliae, respectively (Table 3).
Effect of Oxygenation on Xylitol Production
Candida tropicalis and SCB hydrolysate were chosen to study the effect of
different oxygenation conditions on xylitol production, once the best xylitol
production was achieved and the best xylose source identied. In order to
increase and decrease the oxygenation conditions around the half value of
fv/mv = 3.3, the same volume of medium (75 mL) was introduced into asks
containing 100 and 500 mL, giving an fv/mv of 1.33 and 6.6, respectively.
Figure 3 shows that as fv/mv increased Yp/s xylitol also increased, the values being
0.143, 0.320 and 0.406 g/g for fv/mv values of 1.33, 3.3 and 6.6, respectively.
The maximum calculated efciency for the conversion of consumed xylose to
xylitol was 44.6%, with a maximum xylitol production of 12.18 g/L.

10

J. ARRIZON ET AL.

FIG. 2. XYLOSE CONSUMPTION [(a) IEC5 AND (b) OFF1] AND XYLITOL PRODUCTION
[(c) IEC5 AND (d) OFF1], CELL GROWTH [(e) IEC5 AND (f) OFF1] AND GLYCEROL
PRODUCTION [(g) IEC5 AND (h) OFF1] FOR C. TROPICALIS [(a, c, e, g) IEC5] AND
C. MAGNOLIAE [(b, d, f, h) OFF1], RESPECTIVELY, IN SCB (), ATB () AND
CH ( ) HYDROLYSATES

Efciency
Consumed xylose (%)
Consumed acetic acid (%)
Yp/s xylitol
Yp/s bioethanol
Yp/s glycerol

Yield parameter

26.8 1.5
87.7 3.6
54.6 2.1
0.24 0.012
0.16 0.004
0.05 0.003

32.1 1.3
87.8 4.5
50.9 2.3
0.29 0.051
0.17 0.003
0.04 0.002

23.0 1.5
86.9 4.1
15.8 1.8
0.21 0.032
0.08 0.005
0.04 0.001

C. tropicalis

C. magnoliae

C. tropicalis

24.2 0.9
66.0 2.7
44.4 2.7
0.22 0.023
0.074 0.003
0.18 0.007

C. magnoliae

Agave tequilana bagase

Sugarcane bagasse

Agricultural waste

12.8 1.7
86.1 3.7
51.2 1.9
0.11 0.007
0.15 0.004
0.001 0.0003

C. tropicalis

Coffe husk

5.8 0.10
71.6 3.2
53.5 2.7
0.05 0.003
0.11 0.003
0.04 0.003

C. magnoliae

TABLE 3.
XYLITOL YIELDS FOR C. TROPICALIS AND C.MAGNOLIA IN SUGARCANE BAGASSE, AGAVE TEQUILANA BAGASSE AND COFFEE
HUSK HYDROLYSATES

BIOETHANOL AND XYLITOL PRODUCTION

11

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J. ARRIZON ET AL.

1.33

3.3

6.6

FIG. 3. XYLITOL YIELD WITH C. TROPICALIS YEAST (IEC5) AT DIFFERENT fv/mv

RATIOS: 1.33, 3.3 AND 6.6

DISCUSSION
According with the results, the hydrolysis of lignocellulosic wastes by
the thermal enzymatic process applied was more efcient in SCB, followed
by ATB and less efcient for of CH. The acetic acid content was higher in
CH whereas in SCB and ATB was less and similar (Table 1). It has been
found that the three llignocellulosic wastes have different composition, SCB
contains 38.1% of glucose, 23.3% of xylose, 2.5% of arabinose and 1.1% of
galactose (Lee 1997), ATB is constituted of 43% of cellulose, 19% of hemicellulose, 15% of lignin and 1% of pectin (Gschaedler 2004) and in the case
of CH it contains 69.2% of crude ber and 6.77 of reducing sugars. On the
other hand, the cellulase and xylanase activity over the hydrolysis of lignocellulosic materials for sugars releasing depends on the degree of polymerization and crystallinity of the celluloses and its association with
hemicellulose and lignin (Dusterhoft et al. 2004). As the commercial enzymatic cocktail used in this study contains 50% of enzymatic activity over
carboxymethyl cellulose and 50% of enzymatic activity over beech wood
xylan, it could be possible that the differences observed in the released sugars
were dependent by the ability of the enzymes to attack the three different
lignocellulosic structures. It is well known that acetic acid and other fermentation inhibitors are produced by the breakdown of lignin from hemicelluloses by thermal-acid hydrolysis (Paraj et al. 1996; Gmez et al. 2004;
Mussatto and Roberto 2004), furthermore it has been shown that, as more
acetyl groups are present in the hemicelluloses more acetic acid is going to
be released by hydrolysis (Maloney et al. 1985), therefore it is possible that

BIOETHANOL AND XYLITOL PRODUCTION

13

CH contains more lignin that SCB and ATB. The bioethanol production, in
general an efcient conversion of the sugar consumed to the bioethanol produced was observed in the three hydrolysates even though the levels of acetic
acid present in the hydrolysates at the beginning of fermentation (8.2, 6.1 and
5.8 g/L for CH, SCB and ATB hydrolysates, respectively). It has been found
that acetic acid can be an inhibitor of microbial growth when present from 4
to 10 g/L (Ferrari et al. 1992; Lawford and Rosseau 1998) because it enters
to the cell membrane and decreases the intracellular pH, thus affecting the
metabolism of the microorganism (Maiorella et al. 1983; Van Zyl et al.
1991). Therefore, the S. cerevisiae ITV-01 strain employed for bioethanol
production from the three hydrolysates is tolerant to toxic compounds such as
acetic acid, which represents a technological advantage because it is not
necessary to remove these compounds and this contributes to the reduction of
costs for bioethanol production. It can be observed that in the case of ATB
hydrolysate sugar consumption and ethanol production was slower than SCB
and CH hydrolysates, thus it seems that it exist an inhibition during fermentation. This apparent inhibition was not caused by acetic acid concentration,
as the content was similar to SCB hydrolysate and lower than the acetic acid
content in CH hydrolysate. It has been found that A. tequilana contains
saponins (Cira et al. 2008) and these compounds are inhibitors of microorganisms (Miyakoshi et al. 2000), thus it could be possible that the presence
of these compounds in the hydrolysate could have an effect over the fermentation behavior in ATB hydrolysate. Nevertheless, the affect of acetic acid in
the conversion of xylose to xylitol as has been reported in other works
(Maciel de Mancilha and Karim 2003; Mussatto and Roberto 2004). In this
study, the inhibition effect by acetic acid content during xylitol fermentation
was not observed; on the contrary, this compound was consumed during
xylitol production, as cell growth was not affected by the presence of this
toxic compound in all the hydrolysates, it could be possible that acetic acid
was directly converted to biomass as reported by Augusto-Lima et al. (2004).
For the two yeast strains the percentage of xylose consumed and the level
of xylitol, glycerol and ethanol production in SCB hydrolysate were higher
than SCB and CH hydrolysates. In all the fermentations with C. tropicalis the
xylose consumption and xylitol production were higher than C. magnoliae,
whereas the cell growth and glycerol production were higher in C. magnoliae
than C. tropicalis. Ethanol production was similar for both strains in all
hydrolysates during glucose fermentation. It is well known that the redox
imbalance caused by a less oxygen availability resulted in low cell growth,
ethanol and glycerol production with a consequent increase in the conversion
of xylose to xylitol (Roseiro et al. 1991; Grio et al. 1994, 1999; Nobre et al.
1999; Grandstrm et al. 2000). Therefore, it can be concluded that C. tropicalis converted more efciently xylose to xylitol than C. magnoliae strain.

14

J. ARRIZON ET AL.

As the xylitol production efciency was higher forC. tropicalis strain in all
the hydrolysates and as the highest level of xylitol production was observed
in SCB hydrolysate. C. tropicalis and SCB hydrolysate were used in order
to see the effect of oxygenation conditions over xylitol production. In the
semi-aerobic conditions tested, as the oxygenation conditions increased
the yield of xylitol produced improved, this behavior has been observed in
other works (Kastner et al. 2003; Santos et al. 2003; De Faveri et al. 2004;
Sampaio et al. 2004). The maximum Yp/s xylitol obtained was 0.406 g/g corresponding to a calculated productivity of 0.15 g/L/h with 10.8 g/L of xylitol
produced in SCB hydrolysate and this is lower than the yields obtained
in sugarcane hydrolysates by Santos et al. (2003), Gurgel et al. (1998),
Sreenivas-Rao et al. (2006), Felipe et al. (1997), Carvalho et al. (2004, 2005)
with 0.54, 0.55, 0.72, 0.75, 0.81 and 0.81 g/g, respectively. It was also lower
than the yields obtained in rice straw hydrolysates by Silva et al. (2006),
Mussatto and Roberto (2004) and De Faveri et al. (2004) with 0.65, 0.72 and
0.73 g/g, respectively. In other lignocellulosic hydrolysates produced from
corn stover (Maciel de Mancilha and Karim 2003) and wood (Paraj et al.
1996), the yields were 0.41 and 0.520.67 g/g, respectively. Nevertheless,
the conversion efciency from xylose to xylitol in our study was lower than
in all the abovementioned studies, where a detoxication process was
applied in order to remove the inhibitory compounds. In our case, both
strains performed xylose fermentation to xylitol without detoxifying the
hydrolysates, which can reduce the cost of the global xylitol production
process. Tran et al. (2004) performed enzymatic hydrolysis of beech wood
and walnut shells, with a subsequent fermentation by C. tropicalis strain,
which yielded 0.22 g/g, lower than our results. Latif and Rajoka (2001)
performed a thermal pretreatment in corn cobs and then simultaneous
saccharication and fermentation with a thermoestable cellulases and
xylanases producer (C. thermophile) mixed with separate co-cultures of
S. cerevisiae and C. tropicalis for ethanol and xylitol production, respectively, the yields were 0.42 and 0.71 g/g for ethanol and xilytol, respectively,
which are lower (0.435 g/g) and higher (0.406 g/g) than our results, respectively. Therefore, the results of this study showed that the production of
bioethanol and xylitol by sequential fermentations without a detoxication
process of SCB hydrolysate is technically feasible with the inhibitors-tolerant
yeasts used. It must be pointed out that this is the rst study which evaluated
the use of wastes from tequila and coffee industries as sources for bioethanol
and xylitol production and according with levels of compounds produced,
ATB and CH hydrolysates can be better used for bioethanol than for xylitol
production. In order to optimize the xylitol production with C. tropicalis,
future studies have to be performed varying xylose concentration, oxygen
transfer and fermentation systems.

BIOETHANOL AND XYLITOL PRODUCTION

15

ACKNOWLEDGMENTS
We want to thanks to Fondo Sectorial de Investigacin SAGARPACONACYT project SAGARPA-2004-C01-139 and FOMIX-CONACYT
project 31346 for the nancial support of this research. We want to thanks also
to Elida Gastelum for technical support, Patricia Hayward for the critical
reading of the article.
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