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Bioresource Technology 100 (2009) 1238–1245

Comparative hydrolysis and fermentation of sugarcane

and agave bagasse
J.M. Hernández-Salas, M.S. Villa-Ramı́rez, J.S. Veloz-Rendón, K.N. Rivera-Hernández,
R.A. González-César, M.A. Plascencia-Espinosa, S.R. Trejo-Estrada *
Centro de Investigación en Biotecnologı́a Aplicada del Instituto Politécnico Nacional (CIBA-IPN), Exhacienda San Juan Molino, Km 1.5 Carretera Estatal
Tecuexcomac-Tepetitla, Tepetitla de Lardizábal, Tlaxcala, Mexico

Received 2 June 2003; received in revised form 12 August 2006; accepted 12 September 2006

Abstract

Sugarcane and agave bagasse samples were hydrolyzed with either mineral acids (HCl), commercial glucanases or a combined treat-
ment consisting of alkaline delignification followed by enzymatic hydrolysis. Acid hydrolysis of sugar cane bagasse yielded a higher level of
reducing sugars (37.21% for depithed bagasse and 35.37% for pith bagasse), when compared to metzal or metzontete (agave pinecone and
leaves, 5.02% and 9.91%, respectively). An optimized enzyme formulation was used to process sugar cane bagasse, which contained Cel-
luclast, Novozyme and Viscozyme L. From alkaline–enzymatic hydrolysis of sugarcane bagasse samples, a reduced level of reducing sugar
yield was obtained (11–20%) compared to agave bagasse (12–58%). Selected hydrolyzates were fermented with a non-recombinant strain
of Saccharomyces cerevisiae. Maximum alcohol yield by fermentation (32.6%) was obtained from the hydrolyzate of sugarcane depithed
bagasse. Hydrolyzed agave waste residues provide an increased glucose decreased xylose product useful for biotechnological conversion.
 2007 Elsevier Ltd. All rights reserved.

Keywords: Sugarcane; Agave; Bagasse; Fermentation; Hydrolysis

1. Introduction all factory power from burning the residual bagasse

There is an increased interest in producing bioethanol as
an octane booster or as a liquid fuel. Lignocellulosic mate-
rials from different crop residues have been used for con-
version to ethanol (Lynd et al., 1991; Wiselogel et al.,
1996; Rabinovich, 2006).
One of the most extensively used agricultural residues is
sugarcane bagasse. In Latin America, sugarcane is widely
produced and provides the main source of fermentable
carbohydrates for alcohol production. In 1997–98 Brazil
produced more than 15,000,000 m3 of ethanol obtained
from alcoholic fermentation of sugarcane juice, providing
*
Corresponding author. Address: Bogota No. 5, Colonia America
Norte, Puebla, CP 72340, Mexico. Tel.: +52 555 7296000x87804; fax: +52
248 4870762.
E-mail addresses: sr_tres@hotmail.com, sertre@hotmail.com (S.R.
Trejo-Estrada).
(almost 100 million tons), (Boddey, 1993; CFC–ISO–
GEPLACEA, 1999).
Using simultaneous saccharification and fermentation
(SSF), conversion of lignocellulosic residues such as
bagasse to ethanol is technically and economically feasible
(Philippidis et al., 1992; Hinman et al., 1992).
In Mexico, sugar cane (47 million tons produced in
1997) is used entirely for sugar production. The byproduct
blackstrap molasses (1.8 million tons produced in 1997) is
fermented and distilled to produce alcohol. Molasses is also
used as a feed supplement for cattle production or sold on
to the international markets as a fermentation raw mate-
rial. Bagasse, an important residue from sugarcane pro-
cessing (13.6 million tons per year), could become an
important biomass source for saccharification and fermen-
tation to produce bioethanol, but only 3% is processed
in Mexico’s pulp and paper industry (González-César,
2002).

0960-8524/$ - see front matter  2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2006.09.062
 J.M. Hernández-Salas et al. / Bioresource Technology 100 (2009) 1238–1245
Four million vehicles in Mexico City have an estimated
daily consumption of 17.2 million liters of gasoline. Assum-
ing that an alcohol gasoline blend (containing 8% anhy-
drous ethanol in the mixture) was used to alleviate air
pollution, then all the molasses produced in the country
would need to be fermented to anhydrous alcohol. How-
ever, distilleries in Mexico produce only 53 million liters
of alcohol per year, a volume equivalent to the potential
biofuel consumption of 5 weeks in Mexico City. In Mexico
there are five major cities that will require improved motor
vehicle emissions (González-César, 2002).
Considerable attention has been given to crop residues
derived from sugarcane, corn, wheat, rice and other cereals,
as well as residues from the pulp and paper industry (Wis-
elogel et al., 1996). Other sources have been considered for
converting lignocellulose to fermentable sugars, such as
legume shrub (Sericea lespedeza), sorghum (Sorghum bico-
lor) (Wiselogel et al., 1996), horse gram (Dolichos biflorus)
(Reddy and Reddy, 2005), switchgrass (Panicum virgatum)
(Pimentel and Patzek, 2005), and peat moss (Forsberg
et al., 1986).
The use of sugarcane bagasse in chemistry and biotech-
nology has been recently reviewed (Pandey et al., 2000).
Sugarcane bagasse has a complex structure, and is primar-
ily composed of 25% lignin, 25% hemicellulose and 40–50%
cellulose (Pandey et al., 2000; Neureiter et al., 2002). Con-
version of sugarcane bagasse into fermentable sugars is
possible through thermal, chemical, or enzymatic hydroly-
sis (Beguin and Aubert, 1994; Neureiter et al., 2002; Martin
et al., 2002; Laser et al., 2002).
Treating lignocellulosic agricultural residues with min-
eral acids can be used to release fermentable sugars. The
level and composition of the sugars released depends on
the type of acid and its concentration in the hydrolysis mix-
ture (Israilides et al., 1978). The hemicellulose fraction can
be easily extracted and hydrolyzed by dilute acid treatment.
Using an HCl solution, 85% of the xylose was recovered in
the hydrolyzate (Du Toit et al., 1984), whereas treatment
with dilute sulfuric acid released mainly xylose, which
could in turn be converted to ethanol by fermentation with
a selected strain of Pichia stipitis (Roberto et al., 1991).
Variations of hydrolysis conditions provided a method
for acid hydrolysis of sugarcane bagasse, which proved to
be useful to produce 23 g of xylose per 100 g of bagasse,
a near theoretical yield (Neureiter et al., 2002).
Alkaline treatment of sugarcane bagasse digests of the
lignin matrix and makes cellulose and hemicellulose avail-
able to enzyme degradation (Pandey et al., 2000; Rodri-
guez-Vazquez and Diaz-Cervantes, 1994; Aiello et al.,
1996). Similar treatment of sugarcane leaves enhanced sub-
sequent hydrolysis by a cellulolytic enzyme complex
(Krishna et al., 1998; Bhat, 2000). Alternatively, biological
delignification of bagasse is possible using selected strains
of Panus tigrinus, a white rot fungus (Costa et al., 2002; Gon-
calves et al., 2002).
Commercial enzyme preparations have been used to
convert sugarcane bagasse to fermentable sugars. Krishna
1239
et al. (1998) used Trichoderma reesei cellulase and cellobi-
ase to hydrolyze sugarcane leaves after alkaline delignifica-
tion. More recently, Martin et al. (2002) used a mixture of
endo-glucanases and cellobiases to saccharify steam pre-
treated sugarcane bagasse. The resultant hydrolyzate had
a sugar composition similar to that reported from chemi-
cally treated bagasse, when analyzed by a modified TAPPI
standard method (Neureiter et al., 2002).
To provide a more diversified group of locally produced
raw materials for bioethanol production, we initiated a
study to understand the conversion of lignocellulosic mate-
rials derived from sugarcane and agave bagasse.
Vast areas of Mexico have agave species, wild perennial
succulent xerophytes that grow in dry habitats. Agave
plants are used for fiber (henequén) production in the
Yucatan peninsula (Sánchez-Marroquı́n, 1963); for tequila
and mezcal production in southern, west-central and wes-
tern Mexican states; and also for producing a traditional
fermented beverage called pulque, in the highlands of Mex-
ico’s central plateau, including agricultural areas within the
limits of Mexico City (Sánchez-Marroquı́n, 1963; Idarraga
et al., 1999). The agave residues examined in the present
study are by-products from pulque manufacturing and
are derived from Agave atrovirens (which is called
‘‘maguey’’ in Mexico), a species widely distributed in the
Mexico’s central states: Tlaxcala, Puebla and Hidalgo
(Sánchez-Marroquı́n, 1963).
Pulque production starts by extracting sap or ‘‘agua-
miel’’, the fermentation substrate, which is fermented by
mixed populations of yeast, fermentative and lactic acid bac-
teria. When the sap extraction is done, a cellulosic residue
called metzal is obtained. ‘‘Metzontete’’ is the whole agave
plant as it ends its productive life, providing an excellent cel-
lulosic material for bioconversion to fermentable sugars.
Although the composition of A. atrovirens bagasse has
not been reported, fiber from other agave species has been
recently studied. Cedeño-Cruz and Alvares-Jacobs (1999)
reported that the composition of bagasse from Agave
tequilana (var. Weber, from the tequila industry) is 43% cel-
lulose; 19% hemicellulose and 15% lignin. Its potential use
for pulp and paper production and for animal feeding has
been recently demonstrated (Idarraga et al., 1999; Iñiguez-
Covarrubias et al., 2001). It is possible that the increased
glucan and decreased lignin content of agave fiber may pro-
vide a good source of fermenting sugars produced by chem-
ical and enzymatic hydrolysis and saccharification.
Fermentation of hydrolyzate from lignocellulosic resi-
dues is necessary to produce alcohol (Wyman, 1996). For
that purpose, both pentose-utilizing yeast strains (P. stipitis;
Roberto et al., 1991), and non-pentose-utilizing yeast strains
(Saccharomyces cerevisiae; Krishna et al., 1998) have been
used. Recombinant strains of Escherichia coli, Zymomonas
mobilis and S. cerevisiae capable of hexose and pentose
catabolism and high ethanol production have also been con-
structed. Their use has made the conversion of lignocellulose
to ethanol economically feasible (Mohagheghi et al., 2002;
Moniruzzaman and Ingram, 1998; Martin et al., 2002).
1240 J.M. Hernández-Salas et al. / Bioresource Technology 100 (2009) 1238–1245
The aim of the present study was to examine the produc-
tion of free sugars through chemical or enzymatic hydroly-
sis of agave residues obtained from the pulque industry,
and to compare that process to the better understood pro-
cess of saccharification of sugarcane bagasse. Evidence is
presented that supports the potential use of fermentable
sugars thus obtained for ethanol production by
fermentation.
2. Methods
2.1. Sources of lignocellulose
Sugarcane bagasse samples were kindly provided by
Ingenio Central Motzorongo, a sugar factory based in
Motzorongo, Veracruz, Mexico.
Agricultural agave residues (from A. atrovirens), metzal
and metzontete were kindly provided by Rancho San Isidro,
a pulque producer from Nanacamilpa Tlaxcala, Mexico (see
Table 1).
Mezontete was thoroughly washed with distilled water,
and dried in an oven at 70 C for 72 h. Metzal was only
dried, using the same procedure. Agave and sugarcane res-
idues were ground separately in a small disc mill (Bauer
Bros. Co. Springfield, Ohio, USA. SS mod. 148-2, 3540
RPM, 300 mm ID). The cellulosic materials were then
sieved through stainless steel sieves. The fractions between
0.149 and 1.68 mm were collected for further processing.
2.2. Steam pretreatment
A steam treatment was applied to agave and sugarcane
bagasse. Two grams of either metzal, metzontete or sugar-
cane bagasses were mixed with distilled water. The mix-
tures were autoclaved at 121 C and 1.1 kg/cm2 for 4 h.
2.3. Acid hydrolysis
Lignocellulosic materials were also hydrolyzed with
dilute acid and steam treatment. HCl (1.2% v/v) was added
to bagasse samples in a 5:1, 10:1 or 15:1 ratio (mL of solu-
tion/g of bagasse by weight) which accounted for concen-
trations equivalent to 30, 60 and 90 mg HCl/g of dry
matter. Each suspension was autoclaved at 121 C and
1.1 kg/cm2 for 4 h. After hydrolysis 0.1 N NaOH was
Table 1
Lignocellulosic materials from sugarcane and agave
Substrates
Metzal (Mz): The cellulosic material obtained from scrapping agave
pinecone before and during ‘‘aguamiel’’ production
Mezontete (Mt): The whole agave biomass (leaves and pinecone) left
after ‘‘aguamiel’’ production has ceased
Pith bagasse (B): Short fibers from the vascular bundle of sugarcane
stalk. Low density, porous material
Depithed bagasse (Bc): Long fibers from the cortex of sugarcane stalk.
High density
added and the pH adjusted to 5.0. The mixtures were then
diluted with distilled water to a final dilution of 30:1
(30 mL of liquid phase/g of bagasse). The homogenized
mixture was then filtered and the liquid saved for analysis.
2.4. Alkaline and enzymatic hydrolysis
Bagasse samples (2 g) from agave and sugarcane were
delignified with alkali before enzymatic hydrolysis. This
treatment was carried by the use of a dilute NaOH solution
(2% w/v) applied at a ratio of 5 mL of solution/g of
bagasse. Final concentration of NaOH was 50 mg/g of
bagasse. The mixture was then autoclaved at 121 C and
1.1 kg/cm2 for 4 h.
After this treatment, the pH was adjusted with NaOH
(0.25 M), to 5.0–7.5, depending on the optimum pH value
reported by the enzyme manufacturer. The mixture was
then blended with the corresponding enzyme preparation
and diluted with distilled water to a final dilution of 15:1
(mL of liquid phase/g of bagasse). For enzymatic treat-
ment, delignified bagasse was treated with 0.40 g of the cor-
responding enzyme. Final concentrations were 6% bagasse
and 1.33% of enzyme (20% (w/w) of enzyme per bagasse
dry weight). When several enzymes were used, each one
was added at the same concentration.
Enzymatic hydrolysis was performed in a water bath set
at 55 C for 4 h. All the enzyme preparations were kindly
provided by Novozymes (Mexico). The preparations used
were Viscozyme, Celluclast, Novozyme, Cellubrix and
Pulpzyme.
Pulpzyme HC: Is a liquid preparation produced by sub-
merged fermentation of modified genetically strain of
Bacillus sp. with an activity of 1000 AXU/g (xylanase units
per gram).
Cellubrix L: Is a liquid preparation of cellulase and cel-
lobiase produced by separated fermentations of a strain of
Thichoderma longibrachiatum and a strain of Aspergillus
niger.
Novozyme: Is a liquid preparation produced by sub-
merged fermentation of monocomponent endo-glucanase
by modified genetically strain of Aspergillus spp. with an
activity of 5000 ECU/g (endo-cellulase units per gram).
Celluclast: Is a liquid preparation of cellulase produced
by submerged fermentation of strain of T. reesei with an
activity of 1.5 l Celluclast 700 EGU/g (endo-glucanase
units per gram).
Viscozyme: Is a liquid multienzyme complex preparation
of arabinase, b-glucanase, hemicellulase, cellulase and
xylanase produced by a strain of Aspergillus aculeatus with
an activity of 100 FBG/g (fungal beta-glucanase, units per
gram).
2.5. Reducing sugars
The reducing sugar concentration was determined by the
3,5-dinitro salicylic acid (DNS) method (Miller, 1959), with
a spectrophotometer (Hewlet Packard, Palo Alto, CA)
 J.M. Hernández-Salas et al. / Bioresource Technology 100 (2009) 1238–1245
Model 8453. Hydrolysis by either chemical (acid or alkali)
or enzymatic methods, was expressed as the yield for sugars
by the quantification of the reducing sugars concentration
in the filtrate, as determined by the DNS method. Yields
for sugars were calculated using the formula referred by
Neureiter et al. (2002) as described below.
Y ¼ ðCV =W Þ  100
C is the concentration of reducing sugars (g/L), V is total
volume of the liquid phase (L) and W is dry weight of
the corresponding lignocellulosic material (g). Y is the yield
of sugars expressed as percent of reducing sugars in dry
weight.
2.6. Fermentation
The hydrolyzates were adjusted to pH 5 and amended
with mineral nutrients (NH4)2SO4 (6 g/L), KH2PO4
(7.5 g/L), K2HPO4 (2.4 g/L), MgSO4  7H2 O (0.6 g/L),
CaCl2 (0.001 g/L), CuSO4  5ðH2 OÞ (0.001 g/L), MnCl
(0.001 g/L), ZnSO4 (0.001 g/L), CoCl2 (0.001 g/L) and
sodium molybdate (0.001 g/L). The liquid media was ster-
ilized and distributed in Erlenmeyer flasks (450 mL in 1 L
flasks), inoculated with a commercial strain of S. cerevisiae
(SAFMEX, Mexico 1 · 107 cfu/mL) and incubated at
30 C for 48 h. The flasks were sampled (50 mL) to deter-
mine total reducing sugars and alcohol concentration.
3. Analytical methods
Samples were analyzed for sugars by HPLC. One milli-
liters of each sample was diluted 15-fold and 10 ml of this
mix was passed through 3 cm3 Sep-Pak C-18 cartridges
(Waters, Milford, MA); (preequilibrated with 5 mL metha-
nol and 10 mL of deionized water), in order to eliminate
hydrophobic substances. The clarified samples were then
filtered through 0.22-lm Acrodisc membrane filters
(Waters, Milford, MA); (Wright, 1995). The filtered sam-
ples were injected (5 lL) into a Shodex SC1011 (Shoko
Co., Tokyo, Japan) column and eluted with water at a flow
rate of 1.0 mL/min and a constant temperature of 80 C.
Refractive index detection was used (Hewlett–Packard,
D112), for HPLC analysis (Hewlett–Packard series 1100,
USA), to determine monosaccharide composition. Individ-
ual solutions of true glucose and xylose (Sigma–Aldrich,
USA) were used to prepare calibration curves.
3.1. Ethanol analysis
Fermentation musts were distilled and the distillate ana-
lyzed by gas chromatography using an HP6890 chromato-
graph (Hewlett–Packard, USA) with an FID detector. The
inlet temperature was 200 C, the mode was split flow
(40:1) and the carrier gas was nitrogen. The HP Innowax
column was 30.0 m long with a diameter of 340 lm, with
a film thickness of 0.15 lm. The oven was warmed to an
initial temperature of 40 C, held for 3 min, and then raised
1241
to 70 C at a rate of 3 C/min. The oven temperature was
then raised to 180 C at a rate of 10 C/min.
4. Results and discussion
4.1. Acid hydrolysis
Acid hydrolysis of bagasse was performed using HCl.
The greatest post-treatment sugar concentration was
detected in ryegrass straw using this acid, when compared
to other mineral acids (Israilides et al., 1978).
Fig. 1 shows the results of acid hydrolysis of agave waste
residues. Both metzal and metzontete were treated with
HCl at a concentration of 1.2% (v/v). An increase in the
amount of acid solution from 5 to 15 (volume of acid solu-
tion (mL)/weight of metzal (g)) correlated with an
increased saccharification. When compared to a control
treated only with water and autoclaved (1.2% hydrolysis),
the concentration of 15 mL of acid solution per gram of
metzal yielded 10% hydrolysis. In contrast, only 4.8% by
weigh of metzontete was converted to reducing sugars
using hydrochloric acid (Fig. 1).
Sugarcane waste by-products, namely pith bagasse and
depithed bagasses, were also hydrolyzed by dilute acid
(Fig. 2). Even from the lowest (5:1) ratio of acid solution
to bagasse, more than 30% by weight was converted to
reducing sugars, and at the highest ratio (15:1), a sacchar-
ification level of more than 35% was obtained.
Acid hydrolysis of sugarcane bagasse yielded a higher
level of reducing sugars (37.21% for sugarcane depithed
bagasse and 35.37% for sugarcane pith bagasse), when
compared to either metzal or metzontete (5.02% and
9.91%, respectively).
The analysis of variance was performed using the
Student t test. When the control (no treatment) was com-
pared to the acid treated bagasse samples (90 mg HCl per
gram of bagasse), statistically significant differences were
found (F = 6.15 > F0.10 = 5.54) for every kind of bagasse
14
12
Reducing Sugars (% w/w dry matter)
10
8 Metzal Metzontete
6
4
2
0
Control 30 60 90 control 30 60 90
(mg of HCl/g of dry matter)
Fig. 1. Acid hydrolysis of agave bagasse. Effect of HCl (mg of HCl/g of
bagasse), on the hydrolysis of agave bagasse (yield of reducing sugars).
1242 J.M. Hernández-Salas et al. / Bioresource Technology 100 (2009) 1238–1245

45 80
Depithed Bagasse Pith Bagasse

Reducing Sugars (% w/w dry matter)

40 70 Agave Bagasse(Metzal) Agave Bagasse(Metzontete)
Reducing Sugars (% w/w dry matter)

60
35
50
30
40
25
30

20 20

15 10

0
10

viscozyme

viscozyme
novozyme
novozyme
pulpzyme

pulpzyme

celluclast
celluclast

cellubrix
cellubrix
Control

Control
5

0
Control 30 60 90 Control 30 60 90 Fig. 3. Effect of different enzyme preparations on the release of reducing
(mg of HCl/g of dry matter) sugars from hydrolysis of alkaline-treated agave bagasse.

Fig. 2. Effect of acid concentration (mg HCl/g of dry matter) on the

release of reducing sugars from the hydrolysis of sugarcane bagasse (yield The differences observed in saccharification of alkaline
of reducing sugars). treated bagasse samples were clearly due to the specific
enzyme preparation used. Statistically significant differ-
ences were obtained by ANOVA analysis using the t test
tested. But when the different bagasse materials were com- (F0 = 2.99 > F0.10 = 2.27).
pared with respect to their hydrolysis yield under dilute From alkaline–enzymatic hydrolysis of sugarcane
HCl treatment, no significant differences were found bagasse samples (Fig. 4), the reducing sugar yield was
(F0 = 0.95 < F0.10 = 5.39). The four kinds of bagasse lower compared to agave bagasse. For depithed bagasse,
tested, despite their different origins, responded similarly 13–18% reducing sugars were generated, whereas 11–20%
to the dilute acid treatment. saccharification was obtained from pith bagasse. In this
case, Cellubrix and Celluclast resulted in the highest hydro-
4.2. Enzymatic hydrolysis lytic activity of all enzyme preparations tested.
Statistically significant differences were determined by
Agave and sugarcane bagasse samples were also treated the ANOVA analysis when the alkaline–enzymatic sacchar-
with commercial enzyme preparations after autoclave pre- ification (the reducing sugar yield), was studied as an effect
treatment. At optimal pH and appropriate concentrations, of the kind of bagasse used (F0 = 12.33 > F0.10 = 2.40).
only from treatments with Novozyme and Viscozyme L, The yield of saccharification products from the alkaline–
5–7% hydrolysis was obtained (data not shown). enzymatic treatment of agave bagasse almost doubles the
An additional step of delignification using dilute NaOH yield from sugarcane bagasse. This sharp difference can
was then tested. This process renders cellulose more avail- be explained by the different lignin content of the corre-
able for enzymatic hydrolysis (Ghosh and Singh, 1993; sponding tissues. Paturau (1969) and other authors (Pandey
Rodriguez-Vazquez and Diaz-Cervantes, 1994; Aiello et al., 2000; Neureiter et al., 2002) report an average lignin
et al., 1996; Pandey et al., 2000). content of 20–25% for both pith and depithed sugarcane
Alkaline delignification was performed using a dilute bagasse, whereas Cedeño-Cruz and Alvares-Jacobs (1999)
NaOH solution (2% w/v) mixed with 5 mL of solution
per gram of bagasse by weight and autoclaved for 4 h.
After the pH was adjusted to 7, the partially delignified Pith bagasse
Depithed bagasse
materials were treated with enzyme preparations, either
Reducing Sugars (% w/w dry matter)

20
alone or in combinations.
In contrast with results obtained from the acid treat-
15
ment, the use of an alkaline–enzymatic process on agave
bagasse (metzal and metzontete) resulted in a higher con-
10
centration of reducing sugars (Fig. 3). From the treatment
of metzal and metzontete with dilute alkali and autoclave
5
(controls in Fig. 3), only 5% of these materials were trans-
formed to reducing sugars. Subsequent enzymatic hydroly-
0
sis of metzal generated from 22% to 58% saccharification,
viscozyme
viscozyme

novozyme
novozyme
pulpzyme

pulpzyme
celluclast

celluclast
cellubrix

cellubrix
Control
Control

depending on the enzyme preparation, whereas the met-

zontete values ranged from 12% to 36%. In both cases,
treating of delignified samples with Viscozyme L and Cel- Fig. 4. Effect of different enzyme preparations on the release of reducing
luclast showed the highest hydrolysis rates. sugars from hydrolysis of alkaline-treated sugarcane bagasse.
 J.M. Hernández-Salas et al. / Bioresource Technology 100 (2009) 1238–1245 1243

report 15% for tequila agave, a similar level described for 70 Mz Alkaline-enzymatic hydrolysis
other agave species (Idarraga et al., 1999). Acid hydrolysis Mt
B
An optimized enzyme formulation was developed for 60
Bc
pith and depithed bagasse which contained Celluclast, 50
Mz
Mt
Novozyme and Viscozyme L in equal proportions. After

Glucose (g/l)
B
the alkaline treatment described previously, each enzyme 40 Bc

concentrate was added at a rate of 0.19 mL per gram of

30
bagasse. The same formulation was used for all four sub-
strates, and the sugars released by alkaline and enzymatic 20

treatment were characterized by HPLC.

10
Interestingly, the combination of enzymes used for full
hydrolysis did not include a pure cellobiase preparation 0

like Novozyme 188, which was previously used for sugar-

Fig. 6. Glucose content of sugarcane and agave hydrolyzates determined
cane hydrolysis (Martin et al., 2002). It is possible that cel- by HPLC-IR. Released by optimized enzyme preparation.
lobiase is present in some of the commercial enzyme
preparations tested. Another explanation comes from the
difference between steam-enzymatic treatments reported
in other studies (Martin et al., 2002) and the alkaline–enzy- 40 Acid hydrolysis Enzymatic hydrolysis
matic approach presented here. Alkaline treatment deligni- Mz
35 Mt
fies more efficiently than regular steam explosion B
(Rodriguez-Vazquez and Diaz-Cervantes, 1994). 30
Bc
Mz
Fig. 5 shows the results of bagasse saccharification by Mt
Xylose (g/l)

alkaline–enzymatic treatment for each class of lignocellu- 25 B

Bc
lose material. HPLC analysis was performed for acid 20
hydrolyzed samples (data not shown). The only carbohy-
drates analyzed quantitatively in these samples were glu- 15

cose and xylose. 10

Other non-characterized monosaccharides (mostly gal-
actose and diverse pentose sugars) were separated and 5

detected but not quantified. Figs. 6 and 7 refer to the 0

amount of glucose and xylose released by acid or alka-
line–enzymatic treatments from sugarcane or agave
bagasse, measured by HPLC-IR. Glucose concentration
was clearly higher in hydrolyzates derived from the alka-
line–enzymatic treatment for each bagasse sample
(F0 = 26.57 > F0.10 = 4.06) As expected, the amount of glu-
cose liberated from the hydrolysis of sugarcane pith and
Agave bagasse (Metzal)
Agave bagasse (Metzontete)
Sugar Cane bagasse (Depithed)
Sugar Cane bagasse (Pith)
70
Reducing Sugars (%w/w dry matter)
0 2 4 6 8 10
Fig. 7. Xylose from samples of sugarcane and agave bagasse hydrolyzates
determined by HPLC-IR. Released by optimized enzyme preparation.
partially dephited bagasse was three times the amount of
xylose liberated. When saccharification is compared within
either the acid or the alkaline–enzymatic treated samples,
no statistically significant differences can be attributed to
the kind of bagasse used (F0 = 0.4 < F0.10 = 3.62).
This results are consistent with the data reported by
Paturau (1969), Wiselogel et al. (1996), Neureiter et al.
(2002) and Martin et al. (2002).
Agave waste residues showed different hydrolysis prod-

60
ucts depending on the type of tissue. Metzal, the residue
50
from pinecone extraction, released more sugars compared
40 to the whole agave residue (metzontete). The concentration
of glucose released from metzal is 2.5 times higher than the
30
concentration of xylose released by the alkaline–enzymatic
20 treatment.
Acid hydrolysis released less glucose compared to xylose
10
from sugarcane tissues and metzontete. This trend was pre-
0 viously reported from acid hydrolysis (Du Toit et al., 1984)
and uncatalyzed conversion (Jacobsen and Wyman, 2002)
Fig. 5. Reducing sugars released from hydrolysis of alkaline-treated
sugarcane and agave bagasse. An optimized mixture was used, containing of sugarcane bagasse. In contrast, acid hydrolysis of metzal
three different enzyme preparations. Celluclast, Novozyme and Viscozyme rendered twice as much glucose as xylose, and almost half
at a concentration of 0.57 g of enzyme/g dry bagasse. of the amount obtained from alkaline–enzymatic hydroly-
1244 J.M. Hernández-Salas et al. / Bioresource Technology 100 (2009) 1238–1245
Table 2
Enzyme preparations used in hydrolysis of sugarcane and agave bagasse
Enzymes from Novo Nordisk
Pulpzyme HC: Is a liquid preparation produced by submerged
fermentation of modified genetically strain of Bacillus sp. With an
activity of 1000 AXU/g (Xilanase units per gram)
Cellubrix L: Is a liquid preparation of cellulase and cellobiase produced
by separated fermentations of a strain of Thichoderma longibrachiatum
and a strain of Aspergillus niger
Novozyme: Is a liquid preparation produced by submerged fermentation
of mono-component endo-glucanase by modified genetically strain of
Aspergillus spp. With an activity of 5000 ECU/g (endo-cellulase units
per gram)
Celluclast: Is a liquid preparation of cellulase produced by submerged
fermentation of strain of Thichoderma reesei: With an activity of 1.5 l
Celluclast 700 EGU/g (endo-glucanase units per gram)
Viscozyme: Is a liquid multi-enzyme complex preparation of arabinase,
b-glucanase, hemicellulase, cellulase and xylanase produced by a strain
of Aspergillus aculeatus. With an activity of 100 FBG/g (fungal beta-
glucanase, units per gram)
sis with the same material. A higher glucan content or the
type of hemicellulose present (Type A or B, Du Toit et al.,
1984) may explain the difference of monosaccharides
released from metzal, a material present only in agave pine-
cone (see Table 2).
The origin of metzal is structurally similar to the
A. tequilana bagasse, but metzal does not undergo a ther-
mal treatment during processing. Hemicellulose should still
be present in the fiber, but the hydrolyzate should have a
lower concentration of toxic products (like hydroxy methyl
furfural), when compared to bagasse from the tequila
industry (Idarraga et al., 1999; Martin et al., 2002).
5. Fermentation
Fermentation of sugarcane and agave bagasse hydrolyz-
ates was performed with a non-recombinant distillers strain
of S. cerevisiae. Table 3 shows data on the content of reduc-
ing sugars in the hydrolyzates and the ethanol yield after
fermentation. The ethanol yield is much lower than that
obtained by SSF as reported by Philippidis et al. (1992).
As reported for sugarcane leaves (Krishna et al., 1998),
the fermentation of hydrolyzates yielded an ethanol concen-
tration that correlates with the bioconversion of glucose.
No glucose, only xylose was detected by HPLC analysis
of hydrolyzates after fermentation ceased (data not shown).
Table 3
Sugars and alcohol yield of the fermentation of hydrolyzates from sugarcane and agave bagasse
Raw material and treatment Sugar yield
Raw material Hydrolysis Reducing sugars (RS), g/L
Agave metzal (Mz) Acid 24.82
Agave mezontete (Mt) Acid 19.45
Sugarcane depithed (B) Acid 35.43
Sugarcane pith (Bc) Acid 29.9
Agave metzal (Mz) Alkaline–enzymatic 56.37
Agave metzontete (Mt) Alkaline–enzymatic 28.44
Sugarcane depithed (B) Alkaline–enzymatic 38.38
Sugarcane pith (Bc) Alkaline–enzymatic 50.08
6. Conclusions
The data presented here supports the use of agave
bagasse for saccharification processes to obtain valuable
fermentation products. In particular the use of metzal and
pinecone waste residues from the pulque industry would
provide an excellent source of a cellulosic material easily
hydrolyzed to a glucose rich, xylose sparse product, compa-
rable to other biotechnologically important bioresources,
like corn straw, or sugarcane bagasse. In the present study,
experimental data demonstrates a yield of 56% of sugars
from the conversion of agave metzal, by a combined alka-
line–enzymatic treatment. A similar treatment for sugar-
cane depithed bagasse allowed a similar sugar yield, but a
much higher alcohol yield by fermentation (32.6% alcohol
by weight of sugars present in the hydrolyzate).
Both agave and sugarcane are important raw materials
for manufacturing sugar and fermented beverages in Méx-
ico. The distribution of the corresponding cultivars is geo-
graphically distinctive, but their combined distribution
covers an important proportion of Mexican land. Lignocel-
lulosic by-products derived from the industrial processing
of those bioresources may provide raw materials for pro-
ducing bioethanol, using chemical and biotechnological
processes. Bioethanol, a renewable source of liquid fuel,
has proved to be an octane booster and an excellent alter-
native to decrease air pollution. Bioethanol may serve as an
alternative suitable for cities such as Mexico City and
Guadalajara, the most densely populated areas in Mexico.
Acknowledgements
The authors thank M.Sc. Adrián González Romo from
Colegio de Posgraduados Campus Puebla, Dr. Sonia Silva
Gómez and Dr. Ricardo Pérez Avilés from Posgrado de
Ciencias Ambientales, Universidad Autónoma de Puebla
for their assistance. Thanks to Dr. Don L. Crawford, Uni-
versity of Idaho, for helpful review of the manuscript. This
work was mainly supported by Sistema Regional Zaragoza
(SIZA) from Consejo Nacional de Ciencia y Tecnologı́a
(CONACYT-México) grant no. 980502014 and Fundacion
PRODUCE Puebla. This work was partially funded by
PIFI, COFAA and Programa de Becas Institucionales
(SIP) from Instituto Politécnico Nacional (IPN-México).
Ethanol yield
g/L (48 h) Yield (% w/w initial RS)
7.4 29.81
6.5 33.42
5.0 14.11
4.7 15.72
6.6 11.71
6.3 22.23
12.5 32.57
12.9 25.76
 J.M. Hernández-Salas et al. / Bioresource Technology 100 (2009) 1238–1245
References
Aiello, C., Ferrer, A., Ledesma, A., 1996. Effect of alkaline treatments at
various temperatures on cellulase and biomass production using
submerged sugarcane bagasse fermentation with Trichoderma reesei
QM 9414. Bioresource Technology 57 (1), 13–18.
Beguin, P., Aubert, J.P., 1994. The biological degradation of cellulose.
FEMS Microbiology Reviews 13, 25–58.
Bhat, M.K., 2000. Cellulases and related enzymes in biotechnology.
Biotechnology Advances 18, 355–383.
Boddey, R., 1993. Green energy from sugarcane. Chemistry and Industry,
355–358.
Cedeño-Cruz, M., Alvares-Jacobs, J., 1999. Production of tequila from
agave. In: Murtagh, J. (Ed.), The Alcohol Textbook, second ed.
Nottingham University Press, UK.
CFC–ISO–GEPLACEA, 1999. Project for promoting carburant alcohol.
Final report. Edited by: Common Fund for Commodities, Interna-
tional Sugar Organization, Group of Latin America and Caribbean
sugar exporting countries, Sao Paulo, Brazil.
Costa, S.M., Goncalves, A., Esposito, E., 2002. Action of White-Rot
Fungus Panus tigrinus on Sugarcane Bagasse: evaluation of selectivity.
Applied Biochemistry and Biotechnology 98/100 (1/3), 357–364.
Du Toit, P., Oliver, S., Van Bijon, P., 1984. Sugarcane bagasse as a
possible source of fermentable carbohydrates. I. Characterization of
bagasse with regard to monosaccharide, hemicellulose, and amino acid
composition. Biotechnology and Bioengineering 26, 1071–1078.
Forsberg, C.W., Schellhorn, H., Gibbins, L., Maine, F., Mason, E., 1986.
The release of fermentable carbohydrate from peat by steam explosion
and its use in the microbial production of solvents. Journal of
Biotechnology and Bioengineering 28, 176–184.
Ghosh, P., Singh, A., 1993. Physicochemical and biological treatment for
enzymatic/microbial conversion of lignocellulosic biomass. Advances
in Applied Microbiology 39, 295–333.
Goncalves, A., Costa, S., Esposito, E., 2002. Panus tigrinus strains used in
delignification of sugarcane bagasse prior to kraft pulping. Applied
Biochemistry and Biotechnology 98/100 (1/3), 373–382.
González-César, R., 2002. Sustentabilidad de la producción e intro-
ducción de alcohol como carburante para uso en automotores en la
Ciudad de México. Masters thesis. Benemérita Universidad Autónoma
de Puebla (BUAP), México.
Hinman, N., Schel, D., Riley, C., Bergeron, P., Walter, P., 1992.
Preliminary estimate of the cost of ethanol production for SSF
Technology. Applied Biochemistry and Biotechnology 34–35, 639–649.
Idarraga, G., Ramos, J., Zúñiga, V., Sahin, T., Young, R.A., 1999. Pulp
and paper from blue agave waste from tequila agave. Journal of
Agricultural and Food Chemistry 47, 4450–4455.
Iñiguez-Covarrubias, G., Lange, S., Rowell, R., 2001. Utilization of
byproducts from the tequila industry. Part 1: Agave bagasse as a raw
material for animal feeding and fiberboard production. Bioresource
Technology 77 (ER1), 25–32.
Israilides, C., Grant, G., Han, Y., 1978. Sugar level, fermentability, and
acceptability of straw treated with different acids. Applied and
Environmental Microbiology 36, 43–46.
Jacobsen, S., Wyman, C., 2002. Xylose monomer and oligomer yields for
uncatalyzed hydrolysis of sugarcane bagasse hemicellulose at varying
solids concentration. Industrial and Engineering Chemistry Research
41 (6), 1454–1461.
Krishna, S.H., Prasanthi, K., Chowdary, G.V., Ayyanna, C., 1998.
Simultaneous saccharification and fermentation of pretreated sugar-
cane leaves to ethanol. Process Biochemistry 33, 825–830.
Laser, M., Schulman, D., Allen, S., Lichwa, J., Antal, M., Lynd, L., 2002.
A comparison of liquid hot water and steam pretreatment of sugarcane
1245
bagasse for bioconversion to ethanol. Bioresource Technology 81
(ER1), 33–44.
Lynd, L., Cushman, J., Nichols, R., Wyman, C., 1991. Fuel ethanol from
cellulosic biomass. Science 51, 1318–1323.
Martin, C., Galbe, M., Wahlbom, C., Hahn-Hagerdal, B., Jonsson, L.,
2002. Ethanol production from enzymatic hydrolysates of sugarcane
bagasse using recombinant xylose-utilizing Saccharomyces cerevisiae.
Enzyme and Microbial Technology 31 (3), 274–282.
Miller, G., 1959. Use of dinitrisalicylic acid reagent for determination of
reducing sugars. Analytical Chemistry 31, 426–429.
Mohagheghi, A., Evans, K., Chou, Y., Zhang, M., 2002. Cofermentation
of glucose, xylose, and arabinose by genomic dna-integrated xylose/
arabinose fermenting strain of Zymomonas mobilis AX101. Applied
Biochemistry and Biotechnology 98 (1–3), 885–898.
Moniruzzaman, M., Ingram, L., 1998. Ethanol production from dilute
acid hydrolysate of rice hulls using genetically engineered Escherichia
coli. Biotechnology Letters 20 (10), 943–947.
Neureiter, M., Danner, H., Thomasser, C., Saidi, B., Braun, R., 2002.
Dilute-acid hydrolysis of sugarcane bagasse at varying conditions.
Applied Biochemistry and Biotechnology 98/100 (1/3), 49–58.
Pandey, A., Soccol, C., Soccol, V., 2000. Biotechnological potential of
agro-industrial residues. I: Sugarcane bagasse. Bioresource Technol-
ogy 74 (2), 69.
Paturau, J., 1969. By-Products of the Cane Sugar Industry, An
Introduction to their Industrial Utilization. Elsevier Publishing
Company, pp. 01–115.
Philippidis, G., Smith, T., Wyman, C., 1992. Study of the enzymatic
hydrolysis of cellulose for production of fuel ethanol by the simulta-
neous saccharification and fermentation process. Biotechnology and
Bioengineering 41, 846–853.
Pimentel, D., Patzek, T., 2005. Ethanol production using corn, switch-
grass, and wood; biodiesel production using soybean and sunflower.
Natural Resources Research 14 (1), 65–76.
Rabinovich, M., 2006. Ethanol production from materials containing
cellulose: the potential of Russian research and development. Applied
Biochemistry and Microbiology 42 (1), 1–26.
Reddy, L., Reddy, O., 2005. Improvement of ethanol production in
very high gravity fermentation by horse gram (Dolichos biflorus)
flour supplementation. Letters in Applied Microbiology 41 (5),
440–444.
Roberto, I., Lacis, L., Barbosa, M., De Mancilha, I., 1991. Utilization of
sugarcane bagasse hemicellulosisc hydrolysate by Pichia stipitis for the
production of ethanol. Process Biochemistry 26, 15–21.
Rodriguez-Vazquez, R., Diaz-Cervantes, D., 1994. Effect of chemical
solutions sprayed on sugarcane bagasse pith to produce single cell
protein: physical and chemical analysis of pith. Bioresource Technol-
ogy 47 (2), 159.
Sánchez-Marroquı́n, A., 1963. Industrialización del Agave. Revista de la
Sociedad Quı́mica. Universidad Nacional Autonoma de México
(UNAM), Mexico.
Wiselogel, A., Tyson, S., Johnson, D., 1996. Biomass feedstock resources
and composition. In: Wyman, C.E. (Ed.), Handbook of Bioethanol:
Production and Utilization. Taylor & Francis, Washington, DC, USA,
ISBN 1-56032-553-4 (Chapter 6).
Wright, S.A., 1995. Distillery quality control. In: Kelsall, D.R., Lyons,
T.P., Murtagh, J.E. (Eds.), The Alcohol Textbook: Ethanol Produc-
tion by Fermentation and Distillation. Nottingham University Press,
UK (Chapter 19).
Wyman, C.E., 1996. Ethanol production from lignocellulosic biomass:
overview. In: Wyman, C.E. (Ed.), Handbook of Bioethanol: Produc-
tion and Utilization. Taylor & Francis, Washington, DC, USA, ISBN
1-56032-553-4 (Chapter 1).