Bioresource Technology 101 (2010) 47544766

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Bioresource Technology
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Production of bioethanol from sugarcane bagasse: Status and perspectives

C.A. Cardona *, J.A. Quintero, I.C. Paz
Departamento de Ingeniera Qumica, Universidad Nacional de Colombia Sede Manizales, Cra. 27 No. 64-60, Manizales, Colombia

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

Article history: Lignocellulosic biomass is considered as the future feedstock for ethanol production because of its low
Received 15 August 2009 cost and its huge availability. One of the major lignocellulosic materials found in great quantities to be
Received in revised form 22 October 2009 considered, especially in tropical countries, is sugarcane bagasse (SCB). This work deals with its current
Accepted 23 October 2009
and potential transformation to sugars and ethanol, considering pretreatment technologies, detoxica-
Available online 28 November 2009
tion methods and biological transformation. Some modeling aspects are exposed briey. Finally stability
is discussed for considering the high nonlinear phenomena such as multiplicity and oscillations, which
Keywords:
make more complex the control as a result of the inhibition problems during fermentation when furfural
Sugarcane bagasse
Ethanol
and formic acid from SCB hydrolysis are not absent.
Pretreatment 2009 Elsevier Ltd. All rights reserved.
Lignocellulosic
Stability

1. Introduction (MSW). Numerous studies for developing large-scale production

For large-scale biological production of fuel ethanol, it is desir-
able to use cheaper and more abundant substrates. When produc-
ing ethanol from maize (made up from starch chains) or sugarcane
(in the form of either cane juice or molasses) the raw material con-
stitutes about 4070% of the production cost (Sendelius, 2005;
Quintero et al., 2008). By using waste products from forestry, agri-
culture and industry, the costs of the feedstocks may be reduced.
Lignocellulose (complex polymer made up from three carbohy-
drates: cellulose hemicelluloses and lignin) is considered as an
attractive feedstock for the production of fuel ethanol, because of
its availability in large quantities at low cost (Cardona and Snchez,
2007; Cheng et al., 2008) and for reducing competition with food
but not necessarily with feed. Today the production cost of ethanol
from lignocellulose is still too high, which is the major reason why
ethanol from this feedstock has not made its breakthrough yet.
Many lignocellulosic materials have been tested for bioethanol
production as was reviewed by Snchez and Cardona (2008). In
general, prospective lignocellulosic materials for fuel ethanol pro-
duction can be divided into six main groups: crop residues (cane
bagasse, corn stover, wheat straw, rice straw, rice hulls, barley
straw, sweet sorghum bagasse, olive stones and pulp), hardwood
(aspen and poplar), softwood (pine and spruce), cellulose wastes
(newsprint, waste ofce paper and recycled paper sludge), herba-
ceous biomass (alfalfa hay, switchgrass, reed canary grass, coastal
Bermudagrass and thimothy grass), and municipal solid wastes
* Corresponding author. Tel.: +57 6 8879300x50417; fax: +57 6 8879300x50452.
E-mail address: ccardonaal@unal.edu.co (C.A. Cardona).
of ethanol from lignocellulosics have been carried out in the world.
However, the main limiting factor is the higher degree of complex-
ity inherent to the processing of this feedstock. This is related to
the nature and composition of lignocellulosic biomass (which con-
tain up to 75% of cellulose and hemicelluloses). Cellulose and
hemicelluloses should be broken down into fermentable sugars
in order to be converted into ethanol or other valuable products
(xylans, xylitol, hydrogen and enzymes). But this degradation pro-
cess is complicated, energy-consuming and non-completely devel-
oped (Snchez and Cardona, 2008). With the advent of modern
genetics and other tools the cost of producing sugars from these re-
calcitrant fractions and converting them into products like ethanol
can be signicantly reduced in the future.
Several reviews have been published on the theme of fuel eth-
anol production especially from lignocellulosic biomass (Lin and
Tanaka, 2006; Cardona and Snchez, 2007; Snchez and Cardona,
2008). Lignocellulosic materials from different crop residues have
been used for conversion to ethanol. One of the major lignocellu-
losic materials found in great quantities to be considered, espe-
cially in tropical countries, is sugarcane bagasse (SCB), the
brous residue obtained after extracting the juice from sugar cane
(Saccharum ofcinarum) in the sugar production process (Martn
et al., 2007a).
SCB is produced in large quantities by the sugar and alcohol
industries in Brazil (Martnez et al., 2003; Hernndez-Salas et al.,
2009), India (Martnez et al., 2003; Chandel et al., 2007), Cuba
(Martnez et al., 2003), China (Martnez et al., 2003; Cheng et al.,
2008), Mxico (Hernndez-Salas et al., 2009), Indonesia (Restuti
and Michaelowa, 2007) and Colombia (Quintero et al., 2008). In

0960-8524/$ - see front matter 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.biortech.2009.10.097
 C.A. Cardona et al. / Bioresource Technology 101 (2010) 47544766
general, 1 ton of sugarcane generates 280 kg of bagasse, and
5.4  108 dry tons of sugarcane are processed annually throughout
the world (Cerqueira et al., 2007). About 50% of this residue is used
in distillery plants as a source of energy (Pandey et al., 2000); the
remainder is stockpiled. Therefore, because of the importance of
SCB as an industrial waste, there is great interest in developing
methods for the biological production of fuel and chemicals that
offer economic, environmental, and strategic advantages (Adsul
et al., 2004).
In the approximately 80 sugarcane producing countries there is
a potential to make better use of the SCB. Subjected to improved
energy efciency, sugar producers could supply energy either as
co-generated electricity, or as fuel ethanol through cellulose
hydrolysis followed by fermentation (Botha and Blottnitz, 2006).
The most common use for SCB is the energy production by com-
bustion (Ramjeawon, 2008). In addition, SCB can be used also to
produce chemical compounds such as furfural or hydroxymethyl-
furfural (Almazn et al., 2001), paper paste (Pattra et al., 2008) or
ethanol (Laser et al., 2002). The use of SCB in chemistry and bio-
technology has been reviewed elsewhere (e.g. Pandey et al., 2000).
As raw material, SCB should be analyzed from composition,
structure and surface properties. SCB is primarily composed of lig-
nin (2030%), cellulose (4045%) and hemicelluloses (3035%)
(Peng et al., 2009). Because of its lower ash content, 1.9% (Li et
al., 2002), bagasse offers numerous advantages compared with
other agro-based residues such as paddy straw, 16% (Goh et al.,
2009), rice straw, 14.5% (Guo et al., 2009) and wheat straw, 9.2%
(Zhao and Bai, 2009). Work on structure and surface characteriza-
tion of SCB has not been done extensively, but some works can be
found (Zhao et al., 2007; Quintero and Cardona, 2009). In a previ-
ous work (Quintero and Cardona, 2009) SCB was obtained from a
small sugarcane juice factory and milled for its structural analysis.
Obtained bers had smooth surface layers and characteristic elon-
gations with lengths over 200 lm (this was obtained from SEM
micrographs with in a JEOL JSM-5910LV microscope). XRD analysis
(Rigaku MiniFlex II unit with CuKa used at 30 kV and 15 mA, dif-
fraction angle ranged from 35 to 2 with a scan speed of 5/min)
showed that crust and marrow bagasse exhibit different structures
and crystallinity. Crust bagasse presents two diffraction peaks at 2h
values of 18.04 and 21.9, while marrow bagasse presents only a
peak at 21.86, characteristic of the cellulose structures. It is
important to note, that most of the developments in SCB transfor-
mation to sugars and ethanol have the common scientic basis
with other lignocellulosic materials, due to the fact that there are
not considerable qualitative differences in composition and
structure.
Overall fuel ethanol production from SCB includes ve main
steps: biomass pretreatment, cellulose hydrolysis, fermentation
of hexoses, separation and efuent treatment (see Fig. 1). Further-
more, detoxication and fermentation of pentoses released during
the pretreatment step can be carried out. Solid fraction from pre-
treatment contains the cellulose which is later hydrolisated, and
liquid fraction contains the hemicellulose hydrolysate. Once cellu-
lose hydrolysis is completed, the resulting hydrolysate is fer-
mented and converted into ethanol. This process is called
separate hydrolysis and fermentation (SHF). SHF is one of the
congurations that have been tested more extensively. Pentose
fermentation, when it is carried out, is accomplished in an inde-
pendent unit. The need of separate fermentations is due to the
fact that pentose utilizing microorganisms ferment pentoses and
hexoses slower than microorganisms that only assimilate hex-
oses. Moreover, these microorganisms are more sensitive to the
inhibitors and to the produced ethanol. For this reason, the hemi-
cellulose hydrolysate resulting from pretreatment should be
detoxied. If the fermentation of the hemicelluloses and cellulose
hydrolysates is carried out in a separate way, less liquid volumes
4755
of hydrolysate have to be detoxied. The ideal organism for the
production of ethanol would be the one which can utilize pentose
and hexose sugars generated by lignocellulose hydrolysis (Chan-
del et al., 2007).
Present paper deals with the uses, pretreatment and biologi-
cal transformation of SCB into added value products, emphasiz-
ing on fuel ethanol production. Potential uses of lignocellulosic
biomass depend on its composition and in some extend of its
availability. Moreover, the required pretreatment is a function
of the structure complexity. Main pretreatment methods for
SCB are presented. Potential applications of bagasse hydrolysate
and the detoxication methods are discussed. Finally, some
modeling and stability aspects are considered. Separation and
purication, and efuent treatment technologies are not dis-
cussed in this paper, because, these technologies are well estab-
lished for other types of raw material. Additionally efuent
product and wastes are similar, despite the highly variations in
raw material composition.
2. Pretreatment methods
Lignocellulosic materials do not contain monosaccharides
readily available for bioconversion. Instead they contain polysac-
charides, such as cellulose and hemicelluloses, which have to be
hydrolyzed, by means of acids or enzymes, to fermentable sug-
ars. Enzymatic hydrolysis is a promising way for obtaining sug-
ars from lignocellulosic materials, but the low enzymatic
accessibility of the native cellulose is a key problem for bio-
mass-to-ethanol processes. Cellulose in plants is closely associ-
ated with hemicelluloses and lignin. The lignin is partly
covalently associated with hemicelluloses, thus preventing the
access of hydrolytic agents to cellulose. In addition, the crystal-
line structure of cellulose itself represents an extra obstacle to
hydrolysis. A pretreatment is required for removing lignin and
hemicelluloses, reducing cellulose crystallinity and increasing
the porosity of the material (Keller et al., 2003). This enhances
the enzymatic susceptibility of cellulose. An effective pretreat-
ment must preserve the utility of the hemicelluloses and avoid
the formation of inhibitors (Laser et al., 2002). An economical
pretreatment should use inexpensive chemicals and require sim-
ple equipment and procedures (Martn et al., 2007a).
Several pretreatment methods have been investigated for dif-
ferent lignocellulosic materials (reviewed by Sun and Cheng
(2002); Cardona and Snchez (2007); Snchez and Cardona
(2008)), steam explosion, solvent extraction, and thermal pretreat-
ment using acids or bases (Mosier et al., 2005); along with biolog-
ical pretreatments with white rot fungi (Itoh et al., 2003). Among
all these methods, acid pretreatment is still the method of choice
in several model processes. Pretreatment methods already investi-
gated for bagasse include acid pretreatment with different acids
(Gmez et al., 2004, 2006; Rodriguez-Chong et al., 2004; Chandel
et al., 2007; Cheng et al., 2008; Pattra et al., 2008; Hernndez-Salas
et al., 2009), steam explosion (Martn et al., 2002a; Sendelius,
2005; Hernndez-Salas et al., 2009), alkaline treatment (Hernn-
dez-Salas et al., 2009, alkaline dewaxed (Peng et al., 2009), biolog-
ical treatment (Li et al., 2002; Camassola and Dillon, 2009), wet
oxidation (Martn et al., 2007a), organosolv pretreatment (Pasquini
et al., 2005a, b; Pereira et al., 2007; Tu et al., 2008), liquid hot water
pretreatment (Laser et al., 2002) and pretreatments with peracetic
acid (Teixeira et al., 1999) or with ammonia water (Kurakake et al.,
2001). Table 1 shows some of the most pretreatment methods used
for bagasse exploitation with their respective operation conditions
and in some cases sugar yields. Higher yields are presented with
acid hydrolysis. Little information is presented for alkaline pre-
treatment because delignication is its main objective.
4756 C.A. Cardona et al. / Bioresource Technology 101 (2010) 47544766
Fig. 1. Processes scheme of fuel ethanol production from sugarcane bagasse. Possibilities for reactionreaction integration are shown inside the shaded boxes: CF, co-
fermentation; SSF, simultaneous saccharication and fermentation; SSCF, simultaneous saccharication and co-fermentation.
2.1. Acid pretreatment
The hydrolysis with dilute acids (sulphuric, hydrochloric or ace-
tic acid are habitual, typically 110% weight) is usually called acid
hydrolysis or prehydrolysis and consists in the hydrolysis of the
hemicellulosic fraction at moderate temperature (in the range
100150 C). The hemicellulose fraction of SCB represents up to
35% of the total carbohydrates that can be readily hydrolyzed to
monomeric sugars by dilute acid. However, the concentration of
reducing sugar in the hydrolysate is relatively low due to high li-
quid/solid ratio during the acid hydrolysis. So the hydrolysate
should be concentrated before fermentation (Cheng et al., 2008).
The acid medium attacks the polysaccharides, especially hemi-
celluloses that are easier to be hydrolyzed than cellulose. There-
fore, the cellulose and lignin fractions remain almost unaltered in
the solid phase and can be further processed, being considered
suitable for SCB pretreatment as shown by Gmez et al. (2004,
2006). Depending on the operational conditions, the liquid phase
of the hydrolysates will be constituted by sugar (xylose, glucose
and arabinose), products of decomposition of the hemicelluloses
(such as oligomers from the polymers and acetic acid generated
from the hydrolysis of acetyl groups linked to sugars) and/or the
decomposition products from monosaccharides (such as furfural,
product of dehydration of pentoses, and 5-hydroxymethylfurfural
(HMF), product of dehydration of hexoses) (Gmez et al., 2006).
These products are growth inhibitors of microorganisms. There-
fore, the hydrolysates can be used as fermentation media if the
concentration of inhibitors remains low (Gmez et al., 2004). The
most used acid is H2SO4, among other acids that can be used such
as HCl or HNO3.
2.1.1. Acid pretreatment using sulphuric acid (H2SO4)
Pattra et al. (2008) has evaluated the hydrolysis of SCB using
H2SO4 at various concentrations (0.257.0% volume) and reaction
times (15240 min) at 121 C, 1.5 kg/cm2 in autoclave. Optimal
conditions obtained were 0.5% H2SO4 and 60 min, which yielded
24.5 g/L of total sugar. At these conditions the highest glucose con-
centration was obtained: 11 g glucose/L; 11.29 g xylose/L; 2.22 g
arabinose/L; 2.48 g acetic acid/L and 0.12 g furfural/L were ob-
tained. An increase from 0.5% to 1.0% H2SO4 did not affect the glu-
cose concentration in SCB hemicellulose hydrolysate, but when
H2SO4 concentration was between 1.0 and 5% H2SO4 glucose con-
centration decreased. Xylose was found as the main sugar in SCB
hemicellulose hydrolysate. In order to increase the reducing sugar
production from in the SCB and acid recovering, Cheng et al. (2008)
has proposed an acid recycle process and detoxication of hydroly-
sate performed by electrodialysis. The main problem encountered
when treating the lignocellulose with acids is the formation of fur-
an derivatives and other non identied toxic products. This is par-
ticularly true in the case of xylans, very easily leading to furfural
production.
2.1.2. Acid pretreatment using hydrochloric acid (HCl)
Hydrochloric acid has been used for pretreatment of different
lignocellulosics (e.g. sorghum straw, SCB, ryegrass and palm oil
wastes), however, environmental impact and corrosive properties
strongly limits its application. SCB hydrolysis with HCl shows high-
er yields (see Table 1) compared to other lignocellulosics (Hernn-
dez-Salas et al., 2009) and converting more than 30% by weight to
reducing sugars.
2.1.3. Acid pretreatment using phosphoric acid (H3PO4)
The interest in the use of H3PO4 is that after neutralization of
hydrolysates with NaOH, the salt formed is sodium phosphate
(Gmez et al., 2006). This salt can remain in the hydrolysates be-
cause it is used as nutrient by microorganisms. Therefore, a ltra-
tion operation of is not needed with the consequent advantages:
improvement of process protability (avoiding salts removal and
decreasing the amount of nutrients needed for fermentation) and
positive impact to the environment (the salt formed is not a
waste). Gmez et al. (2006) have evaluated the hydrolysis of SCB
with phosphoric acid under mild conditions (see Table 1). Using
these conditions, 17.6 g of xylose/L; 2.6 g of arabinose/L; 3.0 g of
glucose/L, 1.2 g furfural/L and 4.0 g acetic acid/L were obtained.
The efciency in these conditions was 4.46 g sugars/g inhibitors
and the mass fraction of sugars for dissolved solids in liquid phase
was up to 55%. The rate of xylose release increased with the phos-
phoric acid concentration. Xylose concentrations in hydrolysates at
Table 1
Implemented pretreatments for sugarcane bagasse exploitation.

Pretreatment Agent Conditions Yield Remarks References

%w/w g/L
of SCBa
Dilute acid HCl Acid concentration (1.2% v/v) mL of acid solution/g of 37.21 ND For depithed bagasse more Hernndez-Salas et al.
bagasse by weight: 15:1. Operation at 121 C and 1.1 kg/cm2 for 4 h than 30% by weight was (2009)
converted to reducing sugars
Acid concentration (1.2% v/v) mL of acid solution/g of 35.37 ND For pith bagasse Hernndez-Salas et al.
bagasse by weight: 15:1. Operation at 121 C and 1.1 kg/cm2 for 4 h (2009)
Acid concentration (2.5% v/v) bers size between 2.2 ND 30.29 Chandel et al. (2007)

C.A. Cardona et al. / Bioresource Technology 101 (2010) 47544766

and 10 mm. Operation at 140 C for 30 min. Solid to liquid ratio of 1:10
H2SO4 Acid concentration (1.25%, w/w). Operation at 121 C during 2 h. ND 59.1 Cheng et al. (2008)
The biomass at a solid loading of 10% (w/w)
Acid concentration (0.5%). Operation at 121 C, 1.5 kg/cm2 ND 24.5 Pattra et al. (2008)
during 60 min
H3PO4 Acid concentration (4%). Operation at 122 C during 300 min. ND 23.2 Gmez et al. (2006)
Water/solid ratio of 8 (g water/g sugarcane bagasse on dry basis)
HNO3 Acid concentration (6%). Operation at 122 C for 9.3 min ND 23.51 Rodriguez-Chong et al.
(2004)
Alkalineenzyme NaOH Base concentration (2% w/v) mL of solution/g of bagasse: 5:1 1318 ND Hernndez-Salas et al.
pretreatment NaOH: 50 mg/g of bagasse. Operation at 121 C, 1.1 kg/cm2 (2009)
during 4 h. 0.19 mL of enzyme per gram of bagasse
Alkaline pretreatment NaOH Base concentration 3%, solid to liquid ratio of 1:25 (g/mL) Operation at 27.65 ND For dewaxed sugarcane bagasse. Peng et al. (2009)
50 C for 3 h 74.9% of the original hemicelluloses were
hydrolyzed. Xylose was the predominant
sugar (79.296.7% of total sugars)
Steam explosion Water Operation at 121 C and 1.1 kg/cm2 for 4 h ND ND Hernndez-Salas et al.
(2009)
Water, SO2 and H2SO4 SO2 concentration 2% by weight of water in the bagasse. Acid ND ND Glucose and xylose yields in average Sendelius (2005)
concentration 0.25 g H2SO4 per 100 g dry matter. 180 C during 5 min 86.3% and 72.0%, respectively
Wet oxidation Water and oxygen Operation at 195 C during 15 min, alkaline pH. Oxygen pressure: 12 bar 11.6 ND Yielding a solid material with nearly 70% Martn et al., 2007a
cellulose content, hemicelluloses
solubilization: 93%
of and 50% of lignin. Enzymatic
convertibility of
cellulose of around 75%
Water and oxygen Operation at 185 C, 5 min, acidic pH. Oxygen pressure: 12 bar 16.1 ND Xylose was the main sugar obtained Martn et al. (2007a)
Organosolv pretreatment Supercritical CO2 and 1- Operation at 7 MPa and 190 C. 60% of butanol in the solvent mixture. ND ND Delignication extent: 94.5% Pasquini et al. (2005a)
butanol-water mixture Reaction time 105 min
Supercritical CO2 and Operation at 16.0 MPa and 190 C. Ethanolwater (1:1/v:v). Reaction ND ND Pulping yield: 32.7%; Residual Klason Pasquini et al. (2005b)
ethanolwater mixture times in the range: 30150 min lignin: 8.7%. Delignication extent: 88.4%
Dimethyl formamide Operation at 200210 C for 150 min and 4060% DMF ND ND Delignication extent: 82.7%. Solid Rezayati-Charani et al.
(DMF) material with nearly 83.53% of a-cellulose (2006)
a
SCB: Sugarcane bagasse; ND: non-data available.

4757
4758 C.A. Cardona et al. / Bioresource Technology 101 (2010) 47544766
60 min of reaction were 6.1, 7.3 and 8.6 g/L using H3PO4 concentra-
tions of 2%, 4% and 6%, respectively.
2.1.4. Acid pretreatment using nitric acid (HNO3)
Comparison of results obtained using sulphuric and hydrochlo-
ric acids (see Table 1) demonstrated that the nitric acid presents
similar results for hydrolysis under the evaluated conditions by
Rodriguez-Chong et al. (2004) (acid concentration, 26%; reaction
time, 0300 min; and temperature, 100128 C). Optimal condi-
tions obtained from kinetic models by Rodriguez-Chong et al.
(2004) were: 122 C, 6% HNO3 and 9.3 min. Using these conditions,
18.6 g xylose/L; 2.04 g arabinose/L; 2.87 g glucose/L; 0.9 g acetic
acid/L and 1.32 g furfural/L were obtained. The highest xylose con-
centration (21.0 g/l) was reached after 180 min.
2.2. Alkaline pretreatment
Alkaline pretreatment of SCB digests the lignin matrix and
makes cellulose and hemicellulose available to enzyme degrada-
tion (Pandey et al., 2000). Alkali treatment of lignocellulosic sub-
stances such as cereal straw and bagasse disrupts the cell wall by
dissolving hemicelluloses, lignin, and silica, by hydrolyzing uronic
and acetic esters, and by swelling cellulose. Last decreases the crys-
tallinity of cellulose. By this process, straw and bagasse can be sim-
ply fractionated into alkali-soluble lignin, hemicelluloses and
residue, which makes easy to utilize them for more valuable prod-
ucts. The end residue (mainly cellulose) can be used to produce
either paper or cellulose derivatives. Recently, some important
applications for hemicelluloses, such as the production of xylans,
have been proposed (Peng et al., 2009). They have evaluated the
sequential treatments of dewaxed bagasse with water and 1%
and 3% NaOH aqueous solutions yielded 25.1% hemicelluloses from
bagasse and accounted for 74.9% of the original hemicelluloses.
These results indicated that 1% and 3% NaOH aqueous solutions un-
der these conditions promoted a substantial dissolution of the
hemicellulosic polysaccharides and lignin macromolecules.
2.3. Thermal pretreatment
Fractionation and solubilization studies of lignocellulosic mate-
rials by thermal treatments have shown the efciency of this tech-
nology to improve the yields of extraction of hemicelluloses.
Boussarsar et al. (2009) have evaluated the SCB conversion by
hydrothermal treatment. Optimal conditions were 170 C for 2 h,
reaching higher solubilization of hemicellulose than that at
150 C and lower degradation of sugar monomers than 190 C.
However, analysis of thermal hydrolysates shows the presence of
xylan oligomers and polymers with large chains. On the other
hand, Sendelius (2005) has evaluated the steam pretreatment con-
ditions with respect to nal ethanol yield, using SCB as feedstock.
The variables considered were temperature (180, 190 and
205 C), time (5 and 10 min) and impregnating agents (water, 2%
SO2 by weight of water in the bagasse and 0.25 g H2SO4 per 100 g
dry matter). The most prominent tested pretreatment condition
was: SO2-impregnation with a temperature of 180 C during
5 min, which gave a glucose yields in average 86.3% and xylose
yields in average 72.0%. The fermentation of these hydrolyzed
materials gave an overall ethanol yield of 80%, based on theoretical
value.
2.4. Biological pretreatment
It is generally known that microorganisms degrade untreated
bagasse slowly; therefore, isolation of efcient strains is regarded
as an important research area for lignin degradation in SCB. The
most promising microorganisms for biological pretreatment are
the white rot fungi, microorganism that belong to the class Basid-
iomycetes and that are capable of degrading a lignocellulose sub-
strate (Pan et al., 2005). Camassola and Dillon (2009) pretreated
SCB with the white rot fungus Pleurotus sajor-caju PS2001. Subse-
quently, they evaluated the use of this biologically pretreated ba-
gasse for the production of cellulases and xylanases by the
fungus Penicillium echinulatum. Despite the environmental advan-
tages offered by this type of pretreatment, biological pretreatment
using the fungus P. sajor-caju PS2001 was not effective since the
enzymatic activities with biologically pretreated SCB were lower
than the control treatments carried out with untreated SCB and
cellulose. Also, although the enzymatic activities of the culture
with biologically pretreated bagasse were lower than the cultures
carried out with untreated SCB, it should be noted that the produc-
tion of enzymes of the cellulose and hemicellulase complex after
the production of the mushrooms is another way to add value to
this agricultural residue.
A marine fungus, Phlebia sp. MG-60, which has been screened
from mangrove stands, proved to have excellent lignin degradation
ability and selectivity. Li et al. (2002) have incubated this marine
fungus, with SCB. With this pretreatment more than 50% of lignin
in the SCB was degraded by Phlebia sp. MG-60, and less than 10% of
the holocellulose was lost. Without Kirk medium addition, Phlebia
sp. MG-60 did not show higher delignication ability or better del-
ignication selectivity than the other white rot fungi. However,
when Kirk medium was added to the culture instead of sterilized
water, outstanding delignication capability and excellent selec-
tive property to delignify SCB were observed. Thus, with proper
addition of a nutrient such as Kirk medium, Phlebia sp. MG-60
could efciently degrade lignin in SCB while holocellulose was
scarcely damaged. Kirk medium composition was: 1% (w/v) glu-
cose, 1 g/L KH2PO4, 1 g/L Ca(H2PO4), 221 mg/L ammonium tartrate,
500 mg/L MgSO47H2O, 1 mg/L thiamineHCl and 10 ml Kirk min-
eral solution (Kirk et al., 1978). Other microorganisms evaluated
in the degradation of bagasse are several white rot fungi: Phanero-
chaete chrysosporium ME-466, Phanerochaete sordida YK-624, and
Ceriporia sp. MZ-340.
2.5. Wet oxidation
Wet oxidation (WO) is the process of treating material with
water and either air or oxygen at temperatures above 120 C.
Two types of reactions occur during WO: a low-temperature
hydrolytic reaction and a high-temperature oxidative reaction. It
has been demonstrated that combination of alkali and WO reduces
the formation of toxic furaldehydes and phenol aldehydes (Klinke
et al., 2002). In a recent work, the enzymatic convertibility and
the fermentability of bagasse pretreated by WO at different pH val-
ues were investigated (Martn et al., 2006). Martn et al. (2007a)
have investigated different conditions of wet oxidation (WO) pre-
treatment on fractionation and enzymatic convertibility of SCB.
Variable factors studied were pH, temperature and reaction time,
while pressure (12 bar) was kept constant. The pH was adjusted
by adding Na2CO3 or H2SO4. The highest cellulose content, nearly
70%, was obtained in the pretreatment at 195 C, 15 min and alka-
line pH. The highest sugar yield in the liquid fraction, 16.1 g/100 g,
was obtained at 185 C; 5 min and acidic pH (see Table 1). Cellu-
lose enrichment was reached due to removal of hemicelluloses
and lignin, as can be deduced from the high degrees of solubiliza-
tion of hemicelluloses and lignin achieved in the pretreatments
leading to bers with higher cellulose content. Although the anal-
ysis of the solid fraction in most of the pretreatments showed high
degrees of hemicelluloses solubilization, the content of free sugars
in the liquid fraction was very low. It is known that wet oxidation
mainly catalyses the transfer of hemicelluloses from the solid
phase to the liquid phase, but it does not catalyse the hydrolysis
 C.A. Cardona et al. / Bioresource Technology 101 (2010) 47544766 4759

of the liberated hemicelluloses molecules. The products of drawbacks include the formation of furan derivatives and other
hemicelluloses hydrolysis during WO are not monosaccharides, toxic products and the need of an additional concentration step.
but sugar oligomers. Reactive oxygen species such as N-methyl- Alkaline pretreatment decreases the polymerization degree and
morpholine-N-oxide (Kuo and Lee, 2009), sodium hypochlorite crystallinity of cellulose by the destruction of links between lignin
and hydrogen peroxide (Lee et al., 2009) in solution have been and other polymers, and breakdown of lignin. Its costs are so high
investigated for its ability to oxidize sugarcane bagasse. that these methods are not competitive for large-scale plants. Bio-
logical pretreatment has low energy requirements and mild envi-
ronmental conditions. However, these processes are too slow
2.6. Organosolv pretreatment
limiting its application at industrial level.
Wet oxidation and organosolv pretreatment are the most per-
Organic solvent or organosolv pulping processes are alterna-
spective technologies for SCB hydrolysis at the near future, because
tives to soda or kraft pulping to delignify lignocellulosic materials
both lead to high degree of solubilization of hemicelluloses and lig-
for the production of paper pulp. For the industrial processes (Kraft
nin, and formation of degradation products is avoided. Last implies
and Soda), the burning step is of fundamental importance to re-
the elimination of the detoxication stage. However in the case of
cover the inorganic chemicals employed in the pulping. In the
organosolv pretreatment large reaction time and high pressure are
organosolv process, the exclusive utilization of organic solvent/
needed. Moreover, for wet oxidation, products of hydrolysis are
water mixtures eliminates the need to burn the liquor and allows
oligomers.
the isolation of the lignins (by distillation of the organic solvent)
(Pereira et al., 2007). Formic acid, a typical organosolv system,
has been examined under atmospheric pressure to pulp bagasse -
3. Cellulose hydrolysis
bers. Tu et al. (2008) showed that efcient bagasse pulping was
achieved when the formic acid concentration was limited to 90%
Cellulose obtained from pretreatment should be degraded into
(v/v). The delignication of bagasse by 90% formic acid was almost
glucose (saccharication) using acids or enzymes. In the former
completed after approximately 80 min. Dimethyl formamide has
case, concentrated or dilute acids can be used. If dilute acids
been also used for organosolv pulping of bagasse (Rezayati-charani
(H2SO4 and HCl) are employed, temperatures of 200240 C at
and Mohammadi-Rovshandeh, 2005; Rezayati-Charani et al.,
1.5% acid concentrations are required to hydrolyze the crystalline
2006). Other organosolv alternative is its combination with super-
cellulose, but the degradation of glucose into HMF and other
critical carbon dioxide. Organosolv-CO2 pulping consists in the uti-
non-desired products is unavoidable under these conditions. One
lization of pressurized carbon dioxide as an important part of the
variant of the acid hydrolysis is the use of extremely low acid
pulping liquor (50% alcohol/water mixture and 50% carbon diox-
and high-temperature conditions during batch processes (Ojumu
ide). This process combines the utilization of a lower amount of or-
and Ogunkunle, 2005). However, cellulose hydrolysis is currently
ganic solvent and facilitates the lignin recovery, by the release of
carried out using microbial cellulolytic enzymes. Enzymatic hydro-
pressure after pulping. This process produces pulp with lower
lysis has demonstrated better results for the subsequent fermenta-
strength properties but in similar yields and in shorter times when
tion because no degradation components of glucose are formed
compared with the industrial processes (Pereira et al., 2007).
although the process is slower.
SCB delignication was studied combining the utilization of car-
Commercial enzymes have been used to convert SCB to fer-
bon dioxide at high pressures and solvent mixtures, methanol/
mentable sugars. Enzymatic hydrolysis of cellulosic materials by
water, ethanol/water and n-propanol/water (Pasquini et al.,
cellulase enzymes is the most promising approach to get high
2005b). The utilization of these different alcohols produced pulps
product yields critical to economic success (Lynd et al., 1996). To
with similar delignication extent but with a continuous decrease
help the enzymes to perform well and degrade the lignocellulose
in pulp yield with the increase of the alcohols chain length (Pasqu-
efciently, the bers in the raw material need to be accessible to
ini et al., 2005a). To extent the study of the effect of the co-solvent
the enzymes. A pretreatment in some way is needed to expose
(alcohol/water) in the delignication process Pasquini et al.
the bers. If the pretreatment is too harsh, liberated sugars can
(2005a, b) have described the utilization of CO2 at sub- and super-
be degraded to enzyme- and yeast-inhibiting compounds lowering
critical conditions with 1-butanol/water and ethanol/water as co-
the overall yields. On the other hand, if too weak pretreatment
solvents in the delignication of SCB. For 1-butanol/water case
conditions are used this will result in low enzyme accessibility
the higher delignication extent (94.5%) was obtained at 7 MPa,
and the same drawbacks. Several pretreatment methods have been
190 C, 105 min and 60% 1-butanol in the co-solvent mixture.
evaluated jointly with enzymatic hydrolysis (saccharication).
The results also indicate a low selectivity of the process once the
Among them are alkaline pretreatment (Hernndez-Salas et al.,
lignin removal was accomplished by an extensive hydrolysis of
2009), steam explosion (Sendelius, 2005; Hernndez-Salas et al.,
the polysaccharide fraction. The best compromise between lignin
2009) and wet oxidation (Martn et al., 2007a).
removal and polysaccharide preservation was obtained at high
Hernndez-Salas et al. (2009) had optimized an enzyme formu-
pressures and low content of 1-butanol in the co-solvent mixture.
lation to process SCB and agave bagasse, which contained Cellu-
For ethanol/water mixture the best results were obtained at
clast, Novozyme and Viscozyme L. From alkalineenzymatic
16.0 MPa and 190 C. Under these conditions the delignication
hydrolysis of SCB samples, a reduced level of reducing sugar yield
extent was in the order 88.4% for SCB.
was obtained (1120%) compared to agave bagasse (1258%). Glu-
cose concentration was higher in hydrolysates derived from the
2.7. Final remarks alkalineenzymatic treatment. Martn et al. (2002a) used a mixture
of endo-glucanases and cellobiases to saccharify steam pretreated
Dilute sulphuric acid pretreatment has been successfully devel- SCB. The obtained hydrolysate had a sugar composition similar to
oped given that high reaction rates can be achieved improving sig- that reported from chemically treated bagasse.
nicantly the subsequent process of cellulose hydrolysis. However, Martn et al. (2007a) has evaluated the effect of wet oxidation
the costs of this type of pretreatment are still higher. The main pretreatment on fractionation and enzymatic convertibility of
advantage of dilute acid pretreatment is the higher recovery of SCB. Pretreatment conditions improved the enzymatic convertibil-
sugars derived from hemicelluloses, but concentration of reducing ity of cellulose. The highest convertibility, 74.9% was achieved in
sugars is relatively low due to high liquid to solid ratio. Other the hydrolysis of the material obtained by pretreatment at
4760 C.A. Cardona et al. / Bioresource Technology 101 (2010) 47544766

195 C, 15 min and alkaline pH. Some xylan convertibility was also
observed. Doubling the hydrolysis time from 24 to 48 h led only to
some additional conversion, since most of cellulose was already
hydrolyzed during the rst 24 h. The low increase of the convert-
ibility at 48 h might be an indication of some degree of denatur-
ation or inactivation of the cellulases. This pretreatment gave
also the highest overall glucose yield, 68.9%, which takes into ac-
count not only the glucose formed during the enzymatic hydroly-
sis, but also the losses occurred during the pretreatment. The
increase of the enzymatic convertibility is probably related to the
low content of lignin and hemicelluloses and the high cellulose
content of the remaining solid material. The solubilization of hemi-
celluloses and lignin and the destruction of their association with
cellulose have certainly led to an increase of the accessibility of cel-
lulose to enzymes. Some destruction of the crystalline structure of
cellulose and the decrease of its degree of polymerization are other
events leading to improvements of the enzymatic convertibility
that occurred during pretreatment. Lignin acts as a competitive
adsorbent for cellulases and reduces the activity of the adsorbed
enzymes (Martn et al., 2007a).
4. Detoxication
During pretreatment of lignocellulosics, in addition to the sug-
ars, aliphatic acids (acetic, formic and levulinic acid), furan deriva-
tives furfural and HMF, and phenolic compounds are formed. The
existence of these substances is more probably when acid and/or
high-temperatures are used. These compounds are known to affect
ethanol fermentation performance. Furfural could be generated as
a degradation product from pentoses. It was found that furfural
contents increase with the concentration of the acid catalysts such
as H2SO4 (Pattra et al., 2008).
Another inhibitory substances founded in SCB hemicellulose
hydrolysate is acetic acid. Acetic acid can be generated when the
hydrolysis reaction takes place at the acetyl group of hemicellulose
(Rodriguez-Chong et al., 2004). Generally, acetic acid is inhibitory
to yeast when its concentration is between 4 and 10 g/L. Maximum
concentration of acetic acid obtained for SCB hydrolysates from
acid hydrolysis pretreatment with 6% H2SO4 during 60 min was
2.72 g/L, value lower than that for a toxic effect (Pattra et al.,
2008). While using 4% H3PO4 during 300 min, the highest value
was 4.0 g acetic acid/L (Gmez et al., 2006). On the other hand, a
relative low furfural concentration (1.5 g/L) was obtained using
6% H3PO4 at 300 C, although it is over the limit (1.0 g/L) for yeast
inhibition. This shows that the decomposition of pentoses to furfu-
ral is low and conrms the selectivity of this treatment using phos-
phoric acid.
Several detoxication methods like neutralization, overliming
with calcium hydroxide, activated charcoal, ion exchange resins
(Carvalheiro et al., 2005) and enzymatic detoxication using
laccase (Chandel et al., 2007) are known for removing various
inhibitory compounds from lignocellulosic hydrolysates. Table 2
presents main detoxication methods implemented in SCB hydrol-
ysates with their corresponding operation conditions. Percentages
of toxic compounds removal are shown. Few methods can remove
enough quantities of all toxic substances.
4.1. Neutralization
In the operation of neutralization, it is usual to add chemicals
that neutralize the acids of the hydrolysates, forming salts. These
salts have low solubility and are normally removed by ltration.
The concentration of hydrolysates by evaporation is usual to in-
crease the sugar concentration. In this operation, besides water,

Table 2
Methods for sugarcane bagasse hydrolysate detoxication.

Method Agents Previous Conditions Removal (%) Remarks References

Pretreatment
Alkaline detoxication Overliming Steam- pH 910.5 then pH Furfural (51%), Snchez and Cardona
with Ca(OH)2 explosion adjustment to 5.56.5 HMF (51%), (2008)
dilute acid with H2SO4 or HCl phenolic
compounds (41%),
Acetic acid (0%)
Overliming Acid ND Furans (45.8%), Chandel et al. (2007)
hydrolysis phenolics
(35.87%)
Combined alkaline KOH and Acid pH 10, then pH ND Reduction of ketones and Palmqvist and Hahn-
detoxication sodium sulte hydrolysis adjustment to 6.5 with aldehydes, removal of volatile Hgerdal (2000)
HCl and addition of 1% compounds
sodium sulte at 90 C
Microbial Trichoderma Steam- ND Phenolic Palmqvist and Hahn-
detoxication reesei explosion compounds (80%) Hgerdal (2000)
Electrodialysis Charged Acid Pre-evaporation at Furfural (45%), Losses of sugar are less of 5% Cheng et al. (2008)
membranes hydrolysis 100 C during 15 min. acetic acid (90%)
and an Electrodialysis
electrical operation at 20 V. Flow
potential rate 50 L/h
difference
Ion exchange resin Commercial Acid Resin to hydrolisate Furans (63.4%), Chandel et al. (2007)
anion hydrolysis ratio (w/w): 1:10. phenolics (75.8%),
exchange resin Regular stirring for 1 h acetic acid (85.2%)
at room temperature
Activated charcoal Activated Acid ND Furans (38.7%), Chandel et al. (2007)
charcoal hydrolysis phenolic (57%),
acetic acid (46.8%)
Enzymes treatment Laccase from C. Acid Incubated in orbital Phenolics (77.5%) Does not affect furans and Chandel et al. (2007)
stercoreus hydrolysis shaker at 100 rpm for acetic acid content. Negligible
4 h at 30 C loss in total sugars and
maximum removal phenolic
compounds

ND: non-data available.

 C.A. Cardona et al. / Bioresource Technology 101 (2010) 47544766
small amounts of growth inhibitors such as acetic acid, furfural and
HMF are removed (Carvalho et al., 2002). A detoxication opera-
tion by adsorption on charcoal can remove the inhibitors. In this
operation, phenolic compounds proceeding from lignin can also
be removed.
4.2. Overliming
Overliming the hydrolysate has been effective as a detoxica-
tion process due to partial removal of toxic inhibitors, such as fur-
fural and 5-hydroxymethylfurfural, although the whole
mechanism is not well understood. During overliming, sulphuric
acid is removed from the initial hydrolysate by adding lime to ad-
just the pH and precipitation as gypsum. However, it has been ob-
served that the concentrations of acetic acid before and after the
detoxifying treatment were not altered signicantly (Keikhhosro
et al., 2006). Another potential drawback of overliming is sugar loss
due to hydroxide-catalysed degradation reactions and conversion
of sugars into unfermentable compounds (Carvalho et al., 2005).
Moreover, the acid cannot be reused any more because it has be-
come salt (Ali et al., 2006). Chandel et al. (2007) have also demon-
strated that acetic acid concentration is not altered using
overliming but this method led to the removal of furans (45.8%)
and phenolics (35.87%).
4.3. Adsorption with activated charcoal
Charcoal adsorption decreases the concentrations of both acetic
acid and phenolics derived from the SCB hydrolysate. Treatment
with activated charcoal caused 38.7%, 57% and 46.8% reduction in
furans, phenolics and acetic acid, respectively (Chandel et al.,
2007).
4.4. Ion exchange resins
Ion exchange treatment has demonstrated to be an efcient
method for removing furans (63.4%), total phenolics (75.8%) and
acetic acid (85.2%) from a SCB hydrolysate.
4.5. Enzymatic detoxication
Treatment with the enzymes like laccase, obtained from the lig-
ninolytic fungus Trametes versicolor, has been shown to increase
the ethanol productivity in a hemicellulose hydrolysate of SCB
(Chandel et al., 2007). The laccase treatment led to selective re-
moval of total phenolics by 77.5% without affecting furans and ace-
tic acid content of the hydrolysate.
4.6. Electrodialysis
Another detoxication method more currently used is electro-
dialysis (ED), which is an electrochemical separation process in
which electrically charged membranes and an electrical potential
difference are applied to separate ionic species from an aqueous
solution and other uncharged components. Cheng et al. (2008)
have evaluated the detoxication of SCB acid hydrolysate by boil-
ing and electrodialysis resulted in a better fermentability of the
hydrolysate (see Table 3). Volatile compounds, such as furfural,
were stripped by boiling, while acetic acid and sulphuric acid were
removed by electrodialysis. After treatment by electrodialysis, 90%
of acetic acid in hydrolysate was removed. The losses of glucose,
xylose, arabinose, galactose, mannose and cellobiose were lower
than 5%.
The sulphuric acid and acetic acid in concentrated compartment
of ED device are collected and separated by distillation and the sul-
phuric acid can be reused, which will save the operation cost and
4761
have no environmental impact. ED process reduces the loss of su-
gar and makes the production of ethanol easier, however, due to
the instrument cost, the economical evaluation of ED detoxica-
tion is required to be studied further in the actual production of
ethanol (Cheng et al., 2008).
Chandel et al. (2007) have evaluated the efciency of various
detoxication methods (ion exchange treatment, activated char-
coal, laccase, overliming and neutralization) (see Table 2) for the
removal of inhibitors from SCB hydrolysate and eventually for
improving the fermentation of hydrolysate to ethanol using Can-
dida shehatae. Overliming and laccase did not cause any affect on
acetic acid levels. Laccase treatment brought about negligible loss
in total sugars and maximum removal of phenolic compounds
present in acid hydrolysate. Ion exchange treated hydrolysate gave
maximum ethanol concentration (8.67 g/L), followed by activated
charcoal (7.43 g/L), laccase treatment (6.50 g/L), overliming
(5.19 g/L), and neutralized hydrolysate (3.46 g/L). The neutraliza-
tion of acid hydrolysate alone did not remove toxic compounds
to the desired levels, resulting in poor ethanol yield of 0.22 g/g
(see Table 3).
5. Ethanol production by fermentation
5.1. Production technologies
The conguration employed for fermenting biomass hydroly-
sates involves a sequential process where the hydrolysis of cellu-
lose and the fermentation are carried out in different units
(Snchez and Cardona, 2008). This conguration is known as sep-
arate hydrolysis and fermentation (SHF). When this sequential pro-
cess is employed, solid fraction of pretreated lignocellulosic
material undergoes hydrolysis (saccharication). This fraction con-
tains the cellulose in a form accessible to acids or enzymes. Once
hydrolysis is completed, the resulting cellulose hydrolysate is fer-
mented and converted into ethanol. Saccharomyces cerevisiae is
the most employed microorganism for fermenting the hydroly-
sates of lignocellulosic biomass. This yeast ferments the hexoses
contained in the hydrolysate but not the pentoses. One of the main
features of SHF process is that each step can be performed at its
optimal operating conditions (especially temperature and pH).
When a technological owsheet involving a SHF process is em-
ployed, the detoxied hemicellulose hydrolysate can be unied
with the cellulose hydrolysate coming from the enzymatic reactor.
To increase the amount of sugars converted into ethanol, yeast
assimilating the xylose besides glucose can be employed, but in
this case the biomass utilization rates are lower than that of micro-
organisms that only assimilate hexoses. This is explained by the
diauxic growth of this type of yeast. To offset this effect, sequential
fermentations are employed and both fermentations are per-
formed independently (co-fermentation) (see Fig. 1). One of the
main challenges in pentose fermentation lies in the fact that the
productivities of pentose utilizing microorganisms are less than
those of hexose-fermenting ones.
The co-fermentation of lignocellulosic hydrolysates represents
other technological option for utilizing all the sugars released dur-
ing biomass pretreatment and cellulose hydrolysis. This kind of
cultivation process aims at the complete assimilation of all the sug-
ars resulting from lignocellulosic degradation by the microbial
cells and consists in the use of a mixture of two or more compatible
microorganisms that assimilate both the hexoses and pentoses
present in the medium. This means that the fermentation is carried
out by a mixed culture.
Conversion of cellulose into ethanol can be carried out through
SSF (see Fig. 1) as in the case of starch. For this conversion, several
enzymes with cellulolytic activity (basically endo-glucanases, cel-
4762 C.A. Cardona et al. / Bioresource Technology 101 (2010) 47544766

Table 3
Sugarcane bagasse hydrolisate fermentation.

Previous Detoxication Microorganism Conditions Ethanol yield Remarks References

Pretreatment method
% (w/w of RSa) g/L
Acid Hydrolysis Electrodialysis Pachysolen Batch fermentation at 34 19 Table 4. Sugarcane bagasse hydrolisate Cheng et al. (2008)
With H2SO4 tannophilus 30 C for 14 h with a fermentation. Productivity of 0.53 g/
DW06 pH of 5. Air ow L h. Xylose consumption was total and
0.1 vvm. Agitation: only 60% of arabinose was assimilated
150 rpm.
Without Pachysolen Batch fermentation at 0.03 1.9 Sugar fermented was only 9% Cheng et al. (2008)
detoxication tannophilus 30 C for 14 h with a
DW06 pH of 5. Air ow
0.1 vvm. Agitation:
150 rpm.
Acid hydrolysis Without Non- Batch fermentation at 14.11 5 For depithed bagasse Hernndez-Salas
with HCl detoxication recombinant 30 C during 48 h. et al. (2009)
Saccharomyces
cerevisiae
Without Non- Batch fermentation at 15.72 4.7 For pith bagasse Hernndez-Salas
detoxication recombinant 30 C during 48 h. et al. (2009)
Saccharomyces
cerevisiae
Ion exchange Candida Batch fermentation at 48 8.67 Chandel et al. (2007)
resin shehatae NCIM 30 C during 24 h and
3501 150 rpm.
Activated Candida Batch fermentation at 42 7.43 Chandel et al. (2007)
charcoal shehatae NCIM 30 C during 24 h and
3501 150 rpm.
Enzymes Candida Batch fermentation at 37 6.50 Chandel et al. (2007)
(laccase from shehatae NCIM 30 C during 24 h and
C. stercoreus) 3501 150 rpm.
Acid hydrolysis Overliming Candida Batch fermentation at 30 5.19 Chandel et al. (2007)
with HCl shehatae NCIM 30 C during 24 h and
3501 150 rpm
Neutralization Candida Batch fermentation at 22 3.46 Chandel et al. (2007)
shehatae NCIM 30 C during 24 h and
3501 150 rpm
Alkaline treatment Without Non- Operation at 30 C 32.57 12.5 For depithed bagasse Hernndez-Salas
and enzymatic detoxication recombinant during 48 h et al. (2009)
saccharication Saccharomyces
cerevisiae
Without Non- Operation at 30 C 25.76 12.9 For pith bagasse Hernndez-Salas
detoxication recombinant during 48 h et al. (2009)
Saccharomyces
cerevisiae
H2SO4-catalysed Without Adapted xylose- Operation at 30 C 38 ND Pretreatment was followed by Martn et al. (2007b)
steam detoxication utilizing during 24 h enzymatic hydrolysis. Strain was
pretreatment recombinant isolated from an adaptation culture
Saccharomyces with increasing concentrations of
cerevisiae inhibitors. Ethanol productivity:
2.55 g/(g h)
Without Non-adapted Operation at 30 C 18 ND Pretreatment was followed by Martn et al. (2007b)
detoxication xylose-utilizing during 24 h enzymatic hydrolysis. Ethanol
recombinant productivity: 1.15 g/(g h)
Saccharomyces
cerevisiae
a
RS: Reducing sugars; ND: non-data available.

lobiohydrolases and b-glucosidase) are added to the suspension
obtained by mixing water with the solid fraction resulting from
the pretreatment step and that contains cellulose and lignin. In
the same way, process microorganisms (yeasts) are added to this
mixture in the bioreactor where SSF is accomplished for immedi-
ately converting the formed glucose into ethanol. The increased
ethanol concentration in the culture broth allows the reduction
of energy costs during distillation. In addition, SSF offers an easier
operation and a lower equipment requirement than the sequential
process since no hydrolysis reactors are needed. Nevertheless, SSF
has the inconvenient that the optimal conditions for hydrolysis and
fermentation are different, which implies a difcult control and
optimization of process parameters (Claassen et al., 1999). In addi-
tion, larger amounts of exogenous enzymes are required (Cardona
and Snchez, 2007).
In the case of lignocellulosic biomass, a very promising inte-
grated conguration for bioethanol production is the inclusion of
pentose fermentation in the SSF. This process is known as simulta-
neous saccharication and co-fermentation (SSCF) (see Fig. 1). This
conguration implies a higher degree of intensication through its
reactionreaction integration. In this case, the hydrolysis of cellu-
lose, the fermentation of glucose released, and the fermentation
of pentoses present in the feed stream is simultaneously accom-
plished in a same single unit. Besides the effectiveness of employed
cellulases, the key factor in SSCF is the utilization of an efcient
ethanol-producing microorganism with the ability of assimilating
not only hexoses (mainly glucose), but also pentoses (mainly
xylose) released during the pretreatment step as a result of the
hemicellulose hydrolysis. Therefore, genetically modied microor-
ganisms has been developed and successfully proven in SSCF
 C.A. Cardona et al. / Bioresource Technology 101 (2010) 47544766
processes for ethanol production from lignocellulosic materials
(Cardona and Snchez, 2007). Recombinant strains of Escherichia
coli, Zymomonas mobilis and S. cerevisiae capable of hexose and
pentose catabolism and high ethanol production have also been
constructed. Their use has made the conversion of lignocellulose
to ethanol economically feasible (Mohagheghi et al., 2002; Martn
et al., 2002b).
5.2. Production results
SCB has proven to be a feasible raw material for fuel ethanol
production due its relative low lignin content and high production
of sugars by appropriate pretreatments. Some of the more cur-
rently advances in fuel ethanol production using bagasse reported
alcohol yields up to 48% (% w/w of reducing sugars) (see Table 3).
Hernndez-Salas et al. (2009) have pretreated the whole SCB and
different fractions of it by dilute acid (HCl) and alkaline pretreat-
ment (NaOH). Selected hydrolysates were fermented with a non-
recombinant strain of S. cerevisiae and maximum alcohol yield by
fermentation (32.6%) was obtained from the hydrolysate of sugar-
cane depithed bagasse. Yields for other fractions are shown in Ta-
ble 3.
By the other side, using Pachysolen tannophilus DW06 for fer-
menting SCB hydrolysate obtained from acid pretreatment
(H2SO4) and detoxied with electrodialysis, it was possible to ob-
tain an ethanol yield of 34% (Cheng et al., 2008). Higher yields were
obtained by Chandel et al. (2007) with C. shehatae NCIM 3501 fer-
menting hydrolysates of SCB obtained with dilute acid (HCl) pre-
treatment and different detoxication methods: 48% with an
industrial ion exchange resin (DIAION HPA 25, Mitsubishi Chemical
Corporation, Japan), 42% with activated charcoal and 37% with lac-
case (from C. stercoreus). Lower ethanol yields were obtained with
overliming (30%) and neutralization (22%).
6. Energy cogeneration
Energy cogeneration is well established process in sugar indus-
try, due to the high quantity of SCB available, which is composed of
50% bre, 48% moisture and 2% sugars. It is normally burnt to gen-
erate steam and electricity to meet the energy requirements of the
cane sugar factory. The bagasse has a gross caloric value of
19.25 MJ/kg at zero moisture and 9.95 MJ/kg at 48% moisture.
The net caloric value of bagasse at 48% moisture is around 8 MJ/
kg. The fact that the sugar cane plant provides its own source of en-
ergy from sugar production in the form of bagasse has long been a
special feature of the sugar industry. In the traditional approach,
sugar factories co-generate just enough steam and electricity to
meet their needs. With the availability of advanced co-generation
technologies, sugar factories today can produce surplus electricity
for sale to the national grid or directly to other electricity users
(Quintero et al., 2008; Ramjeawon, 2008). However, in some coun-
tries bagasse is usually burned in low-efciency boilers to avoid
the need to handle surplus bagasse, and the cogeneration systems
work on back pressure steam turbines (BPST) with low pressure at
low-temperature (typically 1.921 MPa, 573 K), the process does
not produce energy in an efcient, cost-effective manner (Ramjea-
won, 2008).
Other potential lignocellulosic by-product from sugar factory
are the sugar cane agricultural residues (SCAR), which as a rule,
are burned just before the harvest in order to facilitate the easier
harvesting of cane stalks. Production of each million ton of raw su-
gar could mean 50,000 ton of SCAR (that is, SCAR with a moisture
content of 30% of sugarcane weight) with a lower heat value (LHV)
of 10.5 MJ/kg and a bulk density of 180 kg/m3. This amount of SCAR
could be substituted for the same quantity of bagasse, leaving the
4763
surplus bagasse to be converted into another kind of energy carrier,
such as ethanol, bio-oil, etc. (Ramjeawon, 2008).
Main reason for considering fuel ethanol production from SCB is
the social and industrial pressure about nding alternative raw
materials and agro-industrial residues like SCB that are offered in
high quantities in tropical countries. Table 4 shows an example
of revenues (based on range values) that a sugar industry placed
in a tropical country like Colombia, could obtain by using SCB for
producing either ethanol of electricity. As it can be see revenues
obtained from ethanol production are higher than that obtained
from electricity cogeneration even at low ethanol prices in the
market. However, this alternative is not protable at industrial le-
vel for the existing mills, because of the high capital investment
needed and the low maturity of this technology. In addition, elec-
tricity generated at cogeneration systems is very cheap for the mill
and heat energy is used in the whole process. Revenue obtained
from electricity cogeneration can be higher in other countries like
Brazil due to the lowest production cost (0.001090.00885 US$/
kWh) (Moreira, 2000).
7. Xylanases and cellulases production
High cost commercial xylanases and cellulases used in the sac-
charication step for SCB transformation to ethanol can be
produced from the same bagasse. Many microorganisms, including
lamentous fungi, yeasts and bacteria, have been cultivated in
media containing SCB or its hydrolysate. The use of SCB as low cost
raw material for xylanase production by Bacillus circulans D1 in
submerged fermentation has been investigated (Bocchini et al.,
2005). The microorganism was cultivated in a mineral medium
containing hydrolysate of bagasse or grass as carbon source. High
production of enzymes was obtained during growth in media with
bagasse hydrolysates (8.4 U/mL) and in media with grass hydroly-
sates (7.5 U/mL). Xylanase production in media with hydrolysates
was very close to that obtained in xylan containing media
(7.0 U/mL); and this fact conrms the feasibility of fermenting this
agro-industrial byproducts by B. circulans D1 as an alternative to
save costs on the enzyme production process.
The media containing hydrolysates of SCB, with initial sugar
concentration from 2.5 to 10.0 g/L, can be employed in place of
the control medium, since they afforded xylanase productions
equal or higher than that obtained in the medium with xylan. This
replacement implies economical advantages for xylanase produc-
tion process, mainly regarding to the commercial xylan high cost
and the availability and low cost of sugarcane (Bocchini et al.,
2005). SCB hydrolysates is an efcient alternative to reduce the
costs of xylanase production in submerged fermentation, since
these materials are often available in tropical countries, as an inex-
pensive source of components that propitiate the bacterial growth
and the enzyme production.
Table 4
Revenue obtained from using 1 ton of sugarcane bagasse for fuel ethanol production
or electricity cogeneration in Colombia.
Ethanol Units Electricity Units
a a
Yield per ton of SCB 150236 L 200600 kWh
Production Cost 0.260.33b US$/L 0.0280.034c US$/kWh
Selling price* 0.9300.980d US$/L 0.0330.07c,e US$/kWh
Revenue 0.60.720 US$/L 0.0010.042 US$/kWh
Revenue per ton of SCB 90170 US$ 0.225.2 US$
a
Botha and Blottnitz (2006).
b
Luo et al. (2009).
c
Ministry of the Environment (2005).
d
Proexport Colombia (2008).
e
Federacin de Biocombustibles (2009).
4764 C.A. Cardona et al. / Bioresource Technology 101 (2010) 47544766
Other strains like Penicillium janthinellum NCIM 1171 and Trich-
oderma viride NCIM 1051 have been evaluated in production of cel-
lulase and xylanase enzymes from chemically treated SCB (Adsul
et al., 2004). Higher xylanase and b-glucosidase activities were de-
tected in the medium with bagasse as compared to the values ob-
tained with pure cellulose powder. Bagasse treated with NaClO2
during 4 h at 70 C gave high yields of xylanase (130 IU/ml) and
b-glucosidase activities (2.3 IU/ml) for both P. janthinellum and T.
viride (Adsul et al., 2004).
8. Mathematical modeling
The modeling of the hydrolysis of a polysaccharide is very com-
plicate. Multiple factors related to the lignocellulosic material
(size, particle shape, structure, accessibility of proton to heterocy-
clic ether bond, etc.) and to the reaction medium (type of acid, con-
centration, temperature, time, etc.) affect the hydrolysis. The
solution of compromise between the complexity of a rigorous
model and the search of equations modeling the empirical data
in a simple and satisfactory way have conducted to the general
use of pseudohomogeneous, irreversible and rst order kinetics
that make easy the calculations without to sacrice the theory ba-
sis. The simplied models for the study of the kinetics in hydrolysis
process using acids began with the work of Saeman for the hydro-
lysis of douglas r wood using sulphuric acid (Saeman, 1945). The
hydrolysis of cellulose was studied establishing the following
model:
k1 k2
Cellulose ! Glucose ! Decomposition products HMF
where, k1 (min1) is the rate for release of glucose from cellulose
and k2 (min1) is rate for glucose decomposition. This model con-
siders the hydrolysis of cellulose to release glucose that in severe
conditions is decomposed into HMF. Both reactions (release and
decomposition) were considered irreversible and rst order. The
model of Saeman was also applied to the hydrolysis of the hemicel-
lulosic fraction (Tllez-Luis et al., 2002). The model of Saeman can
be applied to other polysaccharides; therefore the model can be
generalized for the decomposition of any polymer. The generalized
polymer could be cellulose, xylan, araban, etc. Kinetic parameters of
the above mentioned models for hydrolysis pretreatment with
phosphoric acid (Gmez et al., 2006) and nitric acid (Rodriguez-
Chong et al., 2004) has been reported. Other efforts have been made
in modeling delignication process of bagasse with formic acid (Tu
et al., 2008).
9. Stability of fermentation systems based on SCB
In general, more efcient pretreatment technologies, detoxica-
tion methods and the construction of microorganism strains capa-
ble to ferment lignocellulosic materials have different advances
during last years. However, other restrictions of the fermentation
processes related to original microorganism have not been passed.
One of them is the existence of nonlinear phenomena such as mul-
tiplicity and oscillation. The complexity of stability can be in-
creased as a result of the inhibition problems during
fermentation when furfural and formic acid from SCB hydrolysis
are present in the bioreactor. It is generally considered that the
nonlinear phenomena are unfavorable for stable operations in
industrial fermentation. The nonlinear analysis of oscillatory fer-
mentations with Z. mobilis indicates that with a change in the
parameters, these simple oscillations bifurcate in more complex
phenomena such as totally developed chaos (Garhyan and Elnas-
haie, 2004). Recombinant strains of Z. mobilis are common microor-
ganisms used in SCB transformation to ethanol (Mohagheghi et al.,
2002) and oscillations can be expected as an important issue in the
bioreactor design and control.
From different studies about behavior of continuous cultures of
S. cerevisiae, it has been established that various operation vari-
ables inuence the stability of processes with this microorganism.
In anaerobic cultures, it was seen that the inhibitory action of eth-
anol leads to unstable states. Efforts for using yeast strains with the
ability to ferment all lignocellulose-derived sugars include the use
and modication of S. cerevisiae with high inhibitor tolerance to
acetic acid, furfural and formic acid (Hahn-Hgerdal et al., 2007).
In the case of SCB hydrolysis, acetic acid is the key inhibitor formed
at higher proportion. There is no study in literature about the inu-
ence of this type of inhibitors on stability, but massive production
of bioethanol from SCB needs this type of information for process
design and performance. If this inhibition is increased by acetic
acid during fermentation of sugars from SCB hydrolysis not only
ethanol production can be reduced, but also stability characteris-
tics of the process get more complex. For example, addition of ace-
tate (10 g/L) or furfural (2 g/L), in concentrations similar to those
found in SCB hydrolysates, decreased cell mass formation and
growth rate in almost all strains of industrial S. cerevisiae (Garay-
Arroyo et al., 2004). The wide variability of responses to the
different environmental stress conditions tested show that no
general rules can be assumed for different S. cerevisiae strains,
and that these responses are highly dependent on their genetic
and environmental backgrounds. From here stability of the process
can be predicted to be complex.
Trends in bioethanol production from SCB and other lignocellu-
losic materials show the high potential of process integration. The
development of processes to produce ethanol by coupled sacchar-
ication and fermentation of SCB can be analyzed. Here, the acetic
acid and furfural removal should be coupled to the entire process
(simultaneous sacharication and cofermentation process SSCF)
and stability problems derived from the presence of these inhibi-
tors must be accounted. The study of steady states would allow
determining the optimal operation conditions of both processes.
Until now, there are not reports about stability studies for
SCB hydrolysis and fermentation to ethanol. It is an important
challenge in the development of technologies for ethanol pro-
duction from SCB. Moreover, nowadays, due to wide variety of
feedstocks, industrial yeast strains are exposed to constant envi-
ronmental changes including ethanol accumulated along the pro-
cess, solute concentration, medium ionic strength, and toxins or
inhibitory substances that can affect the stability of fermentation
(Garay-Arroyo et al., 2004; Zhao and Bai, 2009). Therefore, the
design of new processes that involve fermentation processes
must be accompanied of a stability study to formulate suitable
operation strategies.
10. Perspectives, challenges and conclusions
An increased use of biofuels would contribute to sustainable
development by reducing greenhouse-gas emissions and the use
of non-renewable resources. In recent years it has been suggested
that, instead of traditional feedstocks, cellulosic biomass (cellulose
and hemicellulose), including SCB could be used as an ideally inex-
pensive and abundantly available source of sugar for fermentation
into transportation fuel ethanol. The efciency of biomass conver-
sion to ethanol depends upon the ability of the microorganism
used in the process to utilize these diverse carbon sources and
amount of fraction present in biomass. The cost of ethanol produc-
tion from SCB is relatively high based on current technologies.
Pretreatment continues to be the most important step in etha-
nol production from SCB. It is expected, in the next future, that
technologies can impact more easily this step rather than genetic
 C.A. Cardona et al. / Bioresource Technology 101 (2010) 47544766
modication to sugarcane strains (with the purpose of reducing
lignin in the stalks). This is explained by the fact that the develop-
ment and transfer of this type of sugarcane strains to the agricul-
ture should take long time. Additionally, sugar factories linked to
the sugarcane growing sector (usually owning most of the crops)
can choice the status quo instead of investing in new projects.
Promising pretreatment methods as organosolv and wet oxidation
will be the most studied in the coming years. Cellulose hydrolysis
developments are high dependent on the pretreatment method
from the point of view of the obtained enzyme accessibility and
inhibitors production. New developments are more related to the
efcient in situ cellulases production from the SCB to be used in
the hydrolysis step.
Detoxication developments in the next years will be weak
(appointing to integration in one step of different detoxication
methods) in contrast to the genetic modications of fermenting
microorganisms that can tolerate desired concentrations of inhibi-
tors. Integrated congurations as SSF and SSCF are the top efcient
technologies to be analyzed and conrmed at pilot and industrial
levels before its wide use in industry. Energy cogeneration as well
as xylanases and cellulases production from SCB will stay as a real
alternative for adding high value to this residue. At the same way,
energy cogeneration for the sugar factory needings and electricity
supply to the grid will be always the main barrier to the use of SCB
for ethanol production. Other perspective of research and develop-
ment in ethanol production from SCB is the analysis of the stability
in bioreactors in the form of single units as well as integrated con-
gurations. Most of the sugarcane juice and molasses based etha-
nol production process suffer a lot of problems regarding
stability. The existence of new inhibitors in the ethanol process
from SCB supposes new stability problems for this technology. So
the design and introduction of high scale SCB use for ethanol pro-
duction in the industry requires serious analysis of this problem.
According to the above mentioned perspectives rst main chal-
lenge for successful use of SCB as raw material in fuel ethanol pro-
duction is to reduce hydrolysis costs to make SCB a cheaper
substrate like molasses and other directly fermentable materials.
Second challenge is process optimization, including detoxication
technologies and in situ cellulase enzyme production. Third chal-
lenge includes maintaining a stable performance of the genetically
engineered microorganisms in commercial scale fermentation
operations. The future trends for improving the pretreatment of
lignocellulosic feedstocks also include the production of geneti-
cally modied plant materials with higher carbohydrate content
or modied plant structure to facilitate pretreatment in milder
conditions or using hemicellulases. Other challenges are not de-
scribed in this paper, but the most important concern is how to
propose bioethanol production from SCB as a real, economical
and environmental alternative to burning or cogeneration in sugar
mills. Although bioethanol production has been greatly improved
by new technologies, there are still challenges that need further
investigations and developing more efcient pretreatment tech-
nologies for the lignocellulosic biomass and integrating the opti-
mal components into economic ethanol production systems.
Acknowledgements
The authors express their acknowledgments to the National Uni-
versity of Colombia at Manizales for funding different research pro-
jects in fuel ethanol production and lignocellulosics exploitation.
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