Bioresource Technology 102 (2011) 7887–7895

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Bioresource Technology
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Production of bioethanol, methane and heat from sugarcane bagasse

in a biorefinery concept
S.C. Rabelo a,b,⇑, H. Carrere b, R. Maciel Filho a, A.C. Costa a
a
Faculdade de Engenharia Química, Universidade Estadual de Campinas (UNICAMP), Caixa Postal 6066, 13083-970 Campinas, São Paulo, Brazil
b
INRA, UR50, Laboratoire de Biotechnologie de l’Environnement, Avenue des Etangs, Narbonne F-11100, France

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

Article history: The potential of biogas production from the residues of second generation bioethanol production was
Received 23 November 2010 investigated taking into consideration two types of pretreatment: lime or alkaline hydrogen peroxide.
Received in revised form 26 May 2011 Bagasse was pretreated, enzymatically hydrolyzed and the wastes from pretreatment and hydrolysis
Accepted 30 May 2011
were used to produce biogas. Results have shown that if pretreatment is carried out at a bagasse concen-
tration of 4% DM, the highest global methane production is obtained with the peroxide pretreatment:
72.1 L methane/kg bagasse. The recovery of lignin from the peroxide pretreatment liquor was also the
Keywords:
highest, 112.7 ± 0.01 g/kg of bagasse. Evaluation of four different biofuel production scenarios has shown
Sugarcane bagasse
Bioethanol
that 63–65% of the energy that would be produced by bagasse incineration can be recovered by combin-
Anaerobic digestion ing ethanol production with the combustion of lignin and hydrolysis residues, along with the anaerobic
Lignin digestion of pretreatment liquors, while only 32–33% of the energy is recovered by bioethanol production
Biorefinery alone.
Ó 2011 Elsevier Ltd. Open access under the Elsevier OA license.

1. Introduction remainder is stockpiled (UNICA, 2011). Due to the amount of this

In recent years, efforts have increased toward the commercial
production of ethanol, considered the most promising biofuel from
renewable resources. The development of second generation bio-
ethanol made from lignocellulosic biomass can increase the sus-
tainability of feedstock production without competing with food
production or the cultivation of farmland. Unfortunately, this pro-
cess is very costly in terms of the energy input required. However,
the possibility of using the lignocellulosic material in its entirety,
thus linking bioethanol production with the coproduction of ther-
mochemical fuels and/or power, can reduce production costs by
minimizing the utilization of fossil energy sources and reusing
the excess material and by-products of the technology employed
(Laser et al., 2009).
Among the various agricultural crop residues, sugarcane ba-
gasse is the most abundant lignocellulosic material in tropical
countries. In Brazil, as a general rule, 1 ton of sugarcane generates
280 kg of bagasse and the estimate for the 2011/2012 sugarcane
harvest is of 568.50 million tons (UNICA, 2011). About 50% of this
residue is used in distillery plants as a source of energy; the
⇑ Corresponding author. Present address: Laboratório Nacional de Ciência e
Tecnologia do Bioetanol (CTBE), Caixa Postal 6170, 13083-970 Campinas, São Paulo,
Brazil. Tel.: +55 19 3518 3191; fax: +55 19 3518 3164.
E-mail address: sarita.rabelo@bioetanol.org.br (S.C. Rabelo).
biomass as an industrial waste, there is great interest in developing
in a biorefinery concept, methods for the production of fuels and
chemicals that offer economic, environmental, and strategic
advantages.
A biorefinery integrates biomass conversion processes to pro-
duce fuels, electrical power and chemicals from biomass and, as
such, is analogous to a petroleum refinery (Cherubini, 2010). By
producing multiple products, a biorefinery can take advantage of
the differences in biomass constituents and intermediates and
maximize the value derived from the biomass feedstock according
to the market situation and biomass availability (Luo et al., 2011).
Bagasse, like all lignocellulosic materials, has three major constitu-
ents: cellulose, hemicellulose and lignin which, due to its recalci-
trant nature, cannot be easily separated into readily utilizable
components. Thus, second generation bioethanol production in-
volves four steps: pretreatment, to render the cellulose accessible;
hydrolysis with the addition of enzymes or an acid catalyst to re-
lease the monomeric sugars; fermentation to convert sugars into
ethanol; and finally, distillation for product recovery (Margeot
et al., 2009).
Pretreatment is one of the most expensive and least technolog-
ically-mature steps in the process of converting biomass to
fermentable sugars. Hence, it offers a great potential for improve-
ment in efficiency and the reduction of costs through research
and development (Mosier et al., 2005). In this work, two promising
pretreatment technologies were used: pretreatment with lime

0960-8524 Ó 2011 Elsevier Ltd. Open access under the Elsevier OA license.
doi:10.1016/j.biortech.2011.05.081
7888 S.C. Rabelo et al. / Bioresource Technology 102 (2011) 7887–7895
(Fuentes et al., 2011; Rabelo et al., 2008) and pretreatment with
alkaline hydrogen peroxide (Rabelo et al., 2008; Rivera et al.,
2010). They were chosen because they can be carried out in condi-
tions of moderate temperature and pressure and without acids.
Lime pretreatment has low formation of fermentation inhibi-
tors, increases pH and provides a low-cost alternative for lignin
solubilization, removing approximately 33% of lignin and 100% of
acetyl groups. The action of lime is slower than that of other pre-
treatment chemicals but its low cost and safe handling makes it
attractive (Wyman et al., 2005).
Furthermore, lime can be easily recovered as calcium carbonate
by neutralization with carbon dioxide, although this is an energy-
intensive process and as yet, is not economically feasible. The cal-
cium hydroxide can be subsequently regenerated using established
kiln technology (Kaar and Holtzapple, 2000).
Hydrogen peroxide is a well-known reagent in the paper and
cellulose industry, where it is used as a bleaching agent. It also
has the great advantage of not leaving residues in the biomass as
it degrades into oxygen and water. Furthermore, the formation of
secondary products is practically inexistent. This agent has been
shown to be an excellent choice for the pretreatment of sugarcane
bagasse, leading to almost 100% recovery of the cellulose as glu-
cose after enzymatic hydrolysis (Rivera et al., 2010).
Biomass ethanol production generates great amounts of residu-
als, both liquid and solid. Among the liquid residuals are the pre-
treatment liquor, rich in pentoses, soluble and insoluble lignin;
and the vinasse, the effluent after ethanol recovery. There are also
solid wastes from the hydrolysis process, composed mainly of lig-
nin and hemicelluloses that are not solubilized during pretreat-
ment and hydrolysis.
One of the great challenges of ethanol production from bio-
mass is the use of all wastes left over from the process. It is prob-
ably only in this way that the process can be made economically
viable as well as environmentally sustainable. The best use for the
pentoses in the pretreatment liquor is ethanol production; how-
ever, current processes using existing microorganisms lead to ex-
tremely low yields (Kaparaju et al., 2009). The development of
genetically-modified microorganisms that can convert pentoses
into ethanol is being assessed by different research groups but
no viable industrial process has been developed up to now. There
exist some methods for vinasse re-use, such as thermal concen-
tration, land application, for cattle feed, fungi production and
recycling of vinasse into partially dilute molasses in fermentation
(Andrade et al., 2009).
Another alternative for the use of these wastes is biogas produc-
tion, which can be a sustainable solution for the organic matter re-
moval from effluents. Another further advantage here is the
possibility of using the new effluent resulting from biogas produc-
tion as fertilizer on agricultural soils (Liu et al., 2006). Before using
the residual liquid, lignin can be precipitated and used for heat and
energy production (Sassner et al., 2008), though this requires addi-
tional energy input to reduce the water content. In the current
study, several scenarios for bioethanol production and co-produc-
tion of electrical power and biogas using dry bagasse as substrate
were investigated and compared in terms of gross energy output.
Ethanol was produced from bagasse pretreated with either lime
or hydrogen peroxide-pretreated bagasse, using sequential hydro-
lysis and fermentation (SHF). Methane and power were produced
from the process residues of ethanol production. The primary
aim was to compare the energy output per unit of bagasse mass
for the different scenarios (1) Combustion of untreated bagasse,
(2) bioethanol from pretreated bagasse, (3) bioethanol from pre-
treated bagasse, lignin combustion, and biogas from pretreatment
liquor and hydrolysis residue, (4) bioethanol from pretreated ba-
gasse, lignin and hydrolysis residue combustion, biogas from liquor
pretreatment.
2. Methods
2.1. Process description
Based on the above considerations, the bagasse biorefinery con-
cept was developed to produce biofuel (bioethanol) and the pro-
cess effluents were characterized and utilized for the production
of additional biofuel (methane) and power (lignin and/or enzy-
matic hydrolysis residue) to improve the overall recovery of bioen-
ergy from sugarcane. The scheme of the integrated process
adopted in this paper can be seen in Fig. 1.
2.2. Raw material
Sugarcane bagasse (Saccharum officinarum) from a single har-
vest was obtained from the Usina São Luiz-Dedini S/A sugar plant
(Pirassununga/SP, Brazil). It was dried at 45 °C for 48 h, left for 48 h
at room temperature, put into plastic bags and kept in a storage
room. The dry matter content (DM) was approximately 95%.
2.3. Pretreatment methods
The pretreatments were carried out in optimal conditions as
determined in previous studies (Fuentes et al., 2011; Rabelo
et al., 2008; Rivera et al., 2010). Biomass concentrations in each
pretreatment were varied to determine the maximum solids con-
centration compatible with good performance.
2.3.1. Calcium hydroxide (lime) pretreatment
The studies were done with solids concentrations of 4%, 5%, 6%,
7% and 8% DM. The material was treated with a lime solution pre-
pared by dissolving 0.47 g/g DM in 100.0 mL distilled water. In all
the assays, a certain amount of lime remained insoluble although it
continued to dissolve during pretreatment. The flasks were incu-
bated in an orbital shaker MA-832 (Marconi, Piracicaba, SP, Brazil),
agitated at 150 rpm, 90 °C for 90 h (Fuentes et al., 2011; Rabelo
et al., 2008).
2.3.2. Alkaline hydrogen peroxide pretreatment (AHP)
The pretreatment was performed with solids concentrations of
4%, 5%, 6%, 7%, 8%, 9%, 10% and 15% DM. A hydrogen peroxide solu-
tion was prepared by dissolving 7.36% (v/v) of H2O2 in 100.0 mL
distilled water and adjusting the pH to 11.5 with sodium hydrox-
ide. The flasks were incubated in an orbital shaker MA-832 (Mar-
Sugarcane
bagasse
Pretreatment
Solid fraction Liquid fraction
Enzimac Hydrolysis
Anaerobic
Solid fraction
digeson
Lignin
Liquid fraction
Fermentaon Methane
Boilers/energy
Disllaon
Vinasse
Second generaon Ferlizer
bioehtanol
Fig. 1. Integrated process for second generation bioethanol and biogas production.
 S.C. Rabelo et al. / Bioresource Technology 102 (2011) 7887–7895
coni, Piracicaba, SP, Brazil), agitated at 150 rpm, at 25 °C for 1 h
(Rabelo et al., 2008; Rivera et al., 2010).
2.4. Pretreatment liquor and precipitated lignin
After the pretreatment step, the liquors were separated from
the solid fraction by filtration and then reserved for the precipita-
tion of lignin.
The solids fraction was washed several times for removal of the
water-soluble solids (WS) and used to determine the solids recov-
ery yield and chemical composition (Sluiter et al., 2008a,b).
The insoluble lignin was precipitated using a 1% (v/v) solution
of hydrochloric acid down to pH 2 (Ibrahim and Chuah, 2004)
and the liquor was centrifuged at 3000 rpm to obtain the liquid
fraction (soluble sugars) that was used for biogas production.
2.5. Enzymatic hydrolysis
The enzymatic hydrolysis of the washed material was per-
formed using a substrate concentration of 3.0% (w/w) WIS
(water-insoluble solids) in flasks incubated in an orbital shaker
MA-832 (Marconi, Piracicaba, SP, Brazil) agitated at 100 rpm at
50 °C. The pH was adjusted to 4.8 with 0.05 mol/L sodium citrate
buffer.
Cellulase from T. reesei (Sigma–Aldrich Corporation, St. Louis,
MO, USA) was added at a concentration corresponding to 50 FPU/
g WIS for the material pretreated with lime and 3.5 FPU/g WIS
for material pretreated with hydrogen peroxide. b-glucosidase
from Aspergillus niger (Sigma–Aldrich Corporation, St. Louis, MO,
USA) was added at a concentration corresponding to 25 UI/g WIS
for both pretreated materials.
Cellulase activity was determined as filter paper units per mil-
liliter, as recommended by the International Union of Pure and Ap-
plied Chemistry (Ghose, 1987). b-glucosidase activity was
determined through a solution of cellobiose 15 mmol/L and ex-
pressed in units per milliliter (IU/mL) (Wood and Bhat, 1988). En-
zyme activity was 47.44 FPU/mL for cellulases and 343.63 IU/mL
for b-glucosidase.
After enzymatic hydrolysis, the liquid fraction rich in glucose
was used for ethanol production by fermentation of the liquor by
Saccharomyces cerevisiae and the solid residue was used for biogas
production. In this study, only the residues remaining after enzy-
matic hydrolysis of bagasse pretreated at a solids concentration
of 4% DM were used to produce biogas, as this was the pretreat-
ment condition that led to the best glucose yields after hydrolysis.
2.6. Analytical methods
2.6.1. Solid fraction after pretreatment (bioethanol production)
Extractives, structural carbohydrates and lignin were analyzed
in accordance with Sluiter et al. (2008a,b). Prior to compositional
analysis the samples were finely ground in a knife mill MA-630/
1/E (Marconi, Piracicaba, SP, Brazil).
Sugars and organic acids, HMF and furfural were analyzed with
an HPLC system (Waters Corporation, Milford, MA, USA) equipped
with a refractive index detector. Cellobiose, glucose, xylose, arabi-
nose and mannose were separated on a Sugar-Pak I column
(Waters Corporation, Milford, MA, USA) run at a flow rate of
0.5 mL/min at 70 °C, with water as the eluent. Acetic acid, HMF,
furfural, lactic acid, formic acid and levulinic acid were separated
using an Aminex HPX-87H column (Bio-Rad Laboratories Inc., Her-
cules, CA, USA) at 65 °C with 0.5 mL/min of H2SO4 at 5 mM as the
eluent.
7889
2.6.2. Pretreatment liquors and solids residues (methane production)
2.6.2.1. Chemical Oxygen Demand (COD). COD was determined
using SpectroquantÒ test kits (Merck, Darmstadt, Germany) based
on the oxidation of the sample through a heated sulfuric acid solu-
tion of potassium dichromate, using silver sulfate as the catalyst.
Samples were analyzed in triplicate by adding 2.0 mL of diluted
pretreatment liquor to tubes containing the oxidant solution and
heated for 2 h at 150 °C. Thereafter, the tubes were cooled to ambi-
ent temperature and the solution was analyzed in a spectropho-
tometer HACH DR/2000 (Hach Company, Loveland, CO, USA) at
620 nm. The results obtained have been expressed in mg O2/L.
For the solid fraction, the COD was determined, as previously
described, by adding 0.2 g of hydrolysis residue in the Spectro-
quantÒ test kits (Merck, Darmstadt, Germany).
2.6.2.2. Sugar concentration. The pretreatment liquor was analyzed
by an HPLC equipped with a refractive index detector, in accor-
dance with the National Renewable Energy Laboratory (NREL)
standard procedure (Sluiter et al., 2008b). Approximately 5.0 mL
of liquor was subjected to acid hydrolysis after the addition of sul-
furic acid to a pH level of 2. The solution was hydrolyzed at 121 °C
for 1 h. This step was necessary to ensure that all the oligosaccha-
rides present were hydrolyzed to monosaccharides and could thus
be quantified.
The separation was performed in a Sugar-Pak I column (Waters
Corporation, Milford, MA, USA) at 70 °C with a flow rate of 0.5 mL/
min, using filtered deionized water as the mobile phase.
2.6.2.3. Chemical composition. The chemical composition of the res-
idues resulting from the hydrolysis was determined by the Van So-
est method (Van Soest, 1963). The bagasse residue after enzymatic
hydrolysis was analyzed by the FiberBag system (Gerhardt Analyt-
ical Systems, Königswinter, Germany). This involves a sequential
extraction under neutral and acid detergent, followed by strong
acid extraction. The different fractions were: (i) soluble in neutral
detergent fraction (SND); (ii) hemicellulose (HEMI), which was ex-
tracted by acid detergent; (iii) cellulose (CELL), which was ex-
tracted by 76% sulfuric acid; (iv) lignin (LIGN).
2.6.2.4. Total solids (TS), dry matter (DM) and volatile solids
(VS). Total solids (TS) or dry matter (DM) and volatile solids (VS)
were determined in accordance with the Standard Methods (APHA,
1995). To determine TS or DM, approximately 20.0 mL of liquor or
2.00 g of hydrolysis residue were dried at 105 °C to constant
weight. After this, to quantitate VS, the material was burnt in an
oven at 550 °C for two hours.
2.6.3. Lignin characterization
Lignin samples obtained by the two pretreatments were ana-
lyzed for moisture content in accordance with the NREL standard
procedure (Sluiter et al., 2008a). Samples of lignin were also
analyzed by Differential Scanning Calorimetry (DSC) (Mettler-
Toledo, Columbus, OH, USA) and had their molar weight distribu-
tion determined by Gel Permeation Chromatography (GPC) using
a CLASS-LC10 data analyzer and a series of 500, 103 and 104 Å
PLGel columns (Shimadzu, Kyoto, Japan).
2.7. Biomethane potential (BMP) tests
Methane production was assessed by batch biochemical meth-
ane potential (BMP) in mesophilic conditions (35 °C) in 100 mL
reactors, samples of the pretreatment liquor and solid residues
from hydrolysis were added to a solution of macroelements
(sources of N, P, Mg, Ca, K), a solution of oligoelements, a solution
of bicarbonate (buffer solution) and the innoculum (sludge from an
anaerobic digester at a sugar factory, Marseille, France; its compo-
7890 S.C. Rabelo et al. / Bioresource Technology 102 (2011) 7887–7895

sition was 91.3 ± 0.002 g TS/L and 76.6 ± 0.001 g VS/L). The samples that the residue from the hydrolysis of lime-pretreated bagasse
were added to obtain a concentration of 0.5 g COD/g TS of sludge. had more hemicellulose, since this pretreatment solubilizes only
Once the reactors had been prepared, a degasification with nitro- a small part of this polymer. Although peroxide pretreatment is
gen was carried out to obtain anaerobic conditions. more efficient in solubilizing hemicelluloses, 40% of the residue
Biogas production without the addition of liquor or hydrolysis after hydrolysis was hemicellulose, highlighting that it would be
residue was also carried out in order to quantitate endogenous advantageous to use the residue for biogas production. There was
production and served as a negative control. In addition, biogas almost no cellulose left in the residues; its amount was a little
production with the addition of ethanol, a completely degradable higher in the residue from lime-pretreated bagasse while the lignin
material replacing pretreatment liquor or hydrolysis residue, was composition was similar for the two residues.
used as a positive control. BMP tests were duplicated and lasted
up to 40 days. Liquor biodegradability was calculated on the basis 3.2. COD analysis and sugar concentration
of a theoretic production yield of 350 NmL CH4/g COD (Angelidaki
and Sanders, 2004). Results of COD in the samples of pretreatment liquor and
hydrolysis residues are presented in Table 1 In addition, the table
2.8. Volume and composition of the produced biogas shows sugar concentration in the pretreatment liquor.
It can be seen that COD values for the pretreatment liquor from
Biogas volume was measured by vertical displacement of water alkaline hydrogen peroxide were higher that those for the pretreat-
in a gasometer and its composition was measured by a gas chro- ment with lime. Besides the higher concentration of sugars in the
matograph Varian GC-CP4900 (Agilent Technologies, Santa Clara, liquor, which led to higher values of COD, the excess of hydrogen
CO, USA) equipped with two columns. The first one, a Molsieve peroxide in the reaction medium could have further increased this
5A PLOT (Sigma–Aldrich Corporation, St. Louis, MO, USA), was used value. Indeed, the residual concentration of hydrogen peroxide
to separate O2, N2 and CH4 and the second, a HayeSep A column modifies the COD value by consuming the oxidizing agent potas-
(Vici Valco Instruments Co. Inc., Houston, TX, USA), to separate sium dichromate (K2Cr2O7), as shown in Eq. (1) (Talinli and Ander-
CO2 from other gases. The detection of gaseous compounds was son, 1992).
done using a thermal conductivity detector with an injected vol- Cr2 O2- þ 3þ
7 þ 3H2 O2 þ 8H ! 2Cr þ 3O2 þ 7H2 O ð1Þ
ume of 1.0 mL. Calibration was performed with a standard gas
composed of 25.00% of CO2, 1.98% of O2, 10.00% of N2 and 63.02% This interference can be corrected by knowing the residual con-
of CH4. centration of hydrogen peroxide in each pretreatment liquor.
According to Lin and Lo (1997), a concentration of 1.0 g/L of hydro-
gen peroxide is equivalent to 270 mg/L of COD. Similar results were
3. Results and discussion
obtained by Dantas (2005), who recorded 263 mg/L of COD in
1.0 g/L of hydrogen peroxide. Thus, even though there was residual
3.1. Chemical composition of solids fraction
peroxide in the pretreatment liquor, interference in the final COD
was very low in comparison with the values given in Table 1.
Fig. 2 depicts the chemical composition of untreated bagasse,
In Brazil, vinasse, obtained after distillation, presents a COD
the solid fraction after pretreatment (4% DM) and solid residues
range from 15 to 33 g O2/L vinasse when the fermentation is car-
after hydrolysis with the two catalysts.
ried out with sugarcane juice, from 40 to 50 g O2/L vinasse when
One ton of bagasse (95% DM) gave a yield of 560.5 kg DM after
the juice and molasses are blended in fermentation and from 60
lime pretreatment. Much of the cellulose was preserved in the so-
to 75 g O2/L vinasse when fermentation is performed with molas-
lid fraction, representing 66.2% of pretreated material. Part of the
ses (Lamo, 1991).
hemicelluloses were solubilized, and this constituent represented
It can also be noted from Table 1 that higher sugar concentra-
20.4% of the chemical composition of pretreated material. For ba-
tions were observed for liquors from alkaline peroxide pretreat-
gasse pretreated with alkaline hydrogen peroxide, a yield of
ment as this pretreatment solubilizes more hemicelluloses than
433.2 kg DM was obtained, while the pretreated material was
the pretreatment with lime (Rabelo et al., 2008).
composed primarily of cellulose which represented 80.8% of the
material. Much of the hemicelluloses was solubilized during pre-
3.3. Volume and composition of the biogas produced
treatment such that after pretreatment this constituent repre-
sented only 11.7% of the solid fraction. It can be seen from Fig. 2
In this study, biomethane potential tests were carried out esti-
mate the potential production of biogas from the liquors from pre-
treatment and from the solid residues of hydrolysis. Methane
1.5 7.5
100 13.3 Table 1
25.0 25.1 25.1 COD values and sugar concentration.
80 7.2 3.2 Concentration of solids in COD (g O2/L or g O2/g Sugar
pretreatment (% DM) biomassa) concentration
60 38.4 66.2 80.8 39.5 (g/L)
50.8
40 Lime AHP Lime AHP
23.2 4 10.0 ± 0.1 21.4 ± 0.4 1.504 7.565
20 32.1 5 12.6 ± 0.6 23.2 ± 0.0 2.196 8.625
20.4 17.0
11.9 11.7 6 11.6 ± 0.3 27.7 ± 0.4 1.787 12.773
0 7 16.8 ± 0.9 37.0 ± 0.4 2.933 16.598
Untreated Lime* AHP* Lime** AHP** 8 21.2 ± 0.1 43.5 ± 0.4 2.904 19.731
9 – 45.2 ± 0.7 – 16.920
10 – 58.2 ± 0.3 – 20.330
15 – 67.4 ± 0.9 – 21.759
Fig. 2. Chemical composition of untreated and pretreated bagasse () and 4⁄ 1.1 ± 0.0 1.1 ± 0.0 – –
enzymatic hydrolysis residues (). SND represents the soluble matter in the
a
residue. Solid residues from hydrolysis.
 S.C. Rabelo et al. / Bioresource Technology 102 (2011) 7887–7895 7891

potential can be expressed specifically per amount of waste (L CH4/ 4

kg-waste), per volume of waste (L CH4/L-waste), per mass of vola-
(A)
tile solids added (L CH4/kg-VS) or per COD added (L CH4/kg-COD).
The volume is usually expressed in standard pressure (1 atm) 3
and temperature (0 °C) conditions (STP conditions) (Angelidaki
and Sanders, 2004). In this work, the theoretic methane yields
were based on the COD. To calculate the biodegradability, the the- 2
oretic methane production (350.0 NL/kg of COD) was considered.
Table 2 shows the composition of biogas and the methane yield
(NL/kg of COD). The highest methane yield was obtained by work- 1
ing with 4% DM solids for both types of pretreatment. Considering
the solid residue (residue after enzymatic hydrolysis), 162 ± 2 L/kg
COD were produced for the hydrolysis residue of bagasse pre- 0
0 10 20 30 40
treated with lime in comparison to 152 ± 1 L/g COD for the hydro-
Time (days)
lysis residue of bagasse pretreated with hydrogen peroxide.
Fig. 3 shows the amount of methane produced per liter of pre-
7 (B)

CH4/pretreatment liquor (NL/L) or

treatment liquor, considering pretreatments performed with dif-

hydrolysis residue (NL/kgx103)

ferent solids concentrations (4–15% DM) and the amount of 6
methane produced per ton of hydrolysis residue for the two types
of pretreatment considered. 5
It can be noted from Fig. 3 that, in general, higher solids concen- 4
trations in the pretreatment step lead to larger volumes of meth-
ane produced, as higher solids concentrations produce higher 3
concentrations of sugars and volatile acids in the reaction medium
2
(not shown).
The highest production of methane was observed for the pre- 1
treatments with the highest solids concentration: 15% DM (perox-
ide pretreatment) and 8% DM (lime pretreatment). 6.5 ± 0.3 NL of 0
0 10 20 30 40
methane/L pretreatment liquor were produced using the liquor Time (days)
from the pretreatment with peroxide and 3.13 ± 0.06 NL of meth-
ane/L pretreatment liquor using the liquor from the pretreatment Fig. 3. Volume of methane produced relative to the volume of pretreatment liquor
with lime. introduced (NL/L) or to the mass of hydrolysis residue from bagasse (NL/kg  103)
pretreated with lime (A) or alkaline hydrogen peroxide (B), with different solids
Fig. 3 also gives the volume of methane produced relative to the
concentrations (DM): 4% ( ), 5% ( ), 6% ( ), 7% ( ), 8% ( ), 9%
mass of hydrolysis residue put into the reactor for each pretreat- ( ), 10% ( ), 15% ( ) and solid residue ( ). N represents the Normal
ment. It can be seen that the results are similar for both pretreat- Temperature and Pressure (0 °C and 1 atm).
ments: 167 ± 1 NL of methane/kg of residue was obtained with the
residue of hydrolysis of bagasse pretreated with lime and
166 ± 2 NL of methane/kg of when the pretreatment was done with treatment liquor and 0.0275 kg of hydrolysis residue. As 165.6 L of
alkaline hydrogen peroxide. methane were produced per kg of hydrolysis residues and 2.7 L
In this work, experiments to determine the potential of biogas biogas were produced per liter of pretreatment liquor, it is possible
production from vinasse were not carried out, although the pro- to calculate a volume of 4.5 L of methane/kg bagasse from
posed integrated process, shown in Fig. 1, considers this alterna- the hydrolysis residue and 67.5 L of methane/kg bagasse from
tive. Even using only the wastes from pretreatment and the pretreatment liquor. Thus, the total biogas production was
hydrolysis, however, the results are promising. In fact, nowadays 72.1 L methane/kg bagasse.
the vinasse is used in soil irrigation as a source of potassium but When the pretreatment was with lime (4% DM) 1 kg of bagasse
part of the process stream could be diverted to biogas production. resulted in the same volume of pretreatment liquor but 0.08225 kg
When pretreatment with alkaline hydrogen peroxide carried of hydrolysis residue. 167.2 L of methane/kg of hydrolysis residue
out with 4% DM is considered, 1 kg bagasse resulted in 25 L of pre- and 1.8 L methane/L of pretreatment liquor were obtained, which

Table 2
Biogas composition and methane yield from anaerobic digestion.

Concentration of solids in pretreatment (% DM) Biogas composition (%) CH4 yield (NL CH4/kg COD) Biodegradabilityb
Lime AHP Lime AHP Lime AHP
CO2 CH4 CO2 CH4
4 33.7 ± 1.4 66.3 ± 0.4 30.3 ± 1.1 69.7 ± 1.1 180 ± 2 127 ± 2 0.51 0.36
5 31.1 ± 0.0 68.9 ± 0.2 33.2 ± 0.2 66.8 ± 2.1 173 ± 1 98 ± 0 0.49 0.28
6 31.4 ± 0.3 68.6 ± 0.2 35.6 ± 0.2 64.4 ± 0.0 183 ± 1 97 ± 3 0.52 0.28
7 29.8 ± 0.5 70.2 ± 0.6 34.9 ± 0.6 65.1 ± 0.4 170 ± 2 85 ± 2 0.49 0.24
8 31.0 ± 0.3 69.0 ± 0.5 39.7 ± 0.4 60.3 ± 1.0 148 ± 2 89 ± 1 0.42 0.25
9 – – 32.9 ± 0.4 67.1 ± 1.4 – 85 ± 2 – 0.24
10 – – 33.2 ± 0.2 66.8 ± 0.1 – 84 ± 1 – 0.24
15 – – 33.9 ± 0.1 66.1 ± 0.2 – 96 ± 2 – 0.27
4a 38.2 ± 0.0 61.8 ± 0.2 37.4 ± 0.9 62.6 ± 0.3 162 ± 2 152 ± 1 0.46 0.43
a
Solid residues from hydrolysis.
b
The ratio of measured CH4 yield/theoretic CH4 yield.
7892 S.C. Rabelo et al. / Bioresource Technology 102 (2011) 7887–7895
resulted in a production of 13.7 L of methane/kg bagasse from the
hydrolysis residue and 45.0 L of methane/kg bagasse from the pre-
treatment liquor, making a total methane production of 58.7 L of
methane/kg bagasse.
Lu et al. (2009), assessing the co-production of hydrogen and
methane from cornstalks pretreated by steam explosion, obtained
63.7 and 114.6 L of hydrogen and biogas per kg of biomass, respec-
tively. Bauer et al. (2009) evaluated a process for ethanol and
methane production from steam-pretreated wheat straw. They
proposed optimizing the second generation ethanol production
process by using vinasse after ethanol production to produce bio-
gas. They obtained 183 L of methane/kg of wheat straw from the
vinasse. Kaparaju et al. (2009) evaluated the production of bioeth-
anol, biohydrogen and biogas from hydrothermally-pretreated
wheat straw. Pretreated biomass was enzymatically hydrolyzed
and fermented to ethanol and biohydrogen. The effluents from
the two processes were further used to produce methane with
yields of 324 and 381 L/kg of volatile solids, respectively.
3.4. Lignin characterization
The lignin mass precipitated and its moisture content are given
in Table 3 for both types of pretreatment studied. Moisture content
directly influences the calorific value of a compound. Table 3 also
shows the values of molecular weight average Mw and number
average Mn molecular weights for the lignin obtained by precipita-
tion of the pretreatment liquors from both reagents used. Polydis-
persity was calculated according to Eq. (2):
Polydispersity ¼ Mw=Mn
From Table 3 it can be seen that lignin isolated from peroxide
pretreatment liquor presented fragments with higher weight aver-
age molecular weights Mw and had more fragments with different
molecular weights, which is evidenced by the higher polydispersity.
Fig. 4 shows the curves of differential scanning calorimetry for
samples of lignin precipitated from both pretreatment liquors.
The exotherm of degradation of the lignin from lime pretreatment
occured around 413.5 °C with enthalpy of 67.9 J/g and the exo-
therm for the lignin from the peroxide pretreatment occured at
400.6 °C and enthalpy of 52.5 J/g.
3.5. Mass balance and energy output
The mass flows for the two biorefinery alternatives are pre-
sented in Fig. 5 for both the lime (A) and alkaline hydrogen perox-
ide (B) pretreatments. Mass balance was expressed in terms of dry
matter (DM) and the mass loss during chemical pretreatment of
the biomass was considered in the calculation.
After pretreatment of one ton of bagasse with hydrogen perox-
ide, 45.6% DM remained in the solid fraction and the remaining
54.4% were extracted as hydrolysate. For lime pretreatment,
59.0% of DM remained in the solid fraction and 41.0% were ex-
tracted as hydrolysate.
For biomass pretreated with peroxide and lime, Fig. 6 intro-
duces four possible scenarios proposed for optimizing bagasse
use as well as the different contributions of individual biofuels in
each scenario. It should be noted that the present study was car-
ried out with the aim of comparing two pretreatment methods. It
Table 3
Lignin characterization.
Lignin Lignin recovered (g/kg of bagasse) Moisture content (%)
Lime 139.63 ± 0.01 22.72 ± 0.5
AHP 147.28 ± 0.01 19.43 ± 0.1
Lignin - lime
1
Lignin - hydrogen peroxide
-1
Heat flux (mW)
-3
EXO
-5
-7
0 100 200 300 400 500
Temperature (°C)
Fig. 4. DSC profile representative of lignin degradation.
is a simplified analysis in which the energy requirements involved
in biofuel production processes such as farming, harvesting, trans-
porting, feedstock processing, fermentation, alcohol recovery, alco-
hol purification, biogas process, etc. were not considered. However,
the energy requirements of these unit operations can be assumed
to be the same whatever the pretreatment used.
The combustion of the 1 ton of bagasse (DM) produced
15,130.6 MJ of useful energy and was considered as the reference
scenario (Scenario 1) (Dias et al., in press).
Among the scenarios studied (2, 3 and 4), the highest energy
output for both types of pretreatments was obtained with scenario
4. The total energy output for this scenario was 10,731.2 MJ/ton
ð2Þ bagasse and 10,281.7 MJ/ton bagasse when the pretreatment was
with respectively alkaline hydrogen peroxide and lime.
In scenario 4, the hydrolyzed cellulose was used for bioethanol
production leading to an energy generation of 5389.3 MJ/ton ba-
gasse when bagasse was pretreated with peroxide and
5278.4 MJ/ton bagasse for pretreatment with lime. Ethanol pro-
duction represented around 32% of the energy that can be pro-
duced from bagasse combustion and 50–51% of the total energy
recovered in scenario 4. Part of the lignin was precipitated from
the pretreatment liquor and used together with the hydrolysis res-
idue of each pretreatment process for energy generation through
direct combustion in boilers. The energy provided by burning the
lignin was obtained through the mass of lignin recovered in each
pretreatment, taking into account the calorific value of the lignin
of 25.689 kJ/kg (Baker, 1983). By burning lignin, it was possible
to obtain an energy output of 2895.9 MJ/ton bagasse for the lignin
obtained from the pretreatment liquor from peroxide-pretreated
bagasse and 2633.4 MJ/ton bagasse when pretreatment was with
lime.
This energy from lignin represented 16–18% of the energy that
can be produced from bagasse combustion and 26–27% of the en-
ergy recovered in scenario 4. Burning of the hydrolysis residue led
to a generation of energy of 572.5 MJ/ton bagasse for peroxide pre-
treatment and 1458.5 MJ/ton bagasse for pretreatment with lime.
Energy from burning hydrolysis residues represented 4–9% of en-
ergy that can be produced from bagasse combustion and 5–14%
of energy recovered in scenario 4. Finally, the soluble fraction of
the pretreatment liquor was used for biogas production, generating
1873.5 MJ/ton bagasse and 911.4 MJ/ton bagasse of energy for the
liquor of bagasse pretreated with peroxide and lime, respectively.
 
Weight average Mw Number average Mn Polydispersity Mw=Mn
907 460 3.66
952 502 3.88
 S.C. Rabelo et al. / Bioresource Technology 102 (2011) 7887–7895 7893

Fermentation
Hydrolyzate
Hydrolysis liquor (DM kg) fermentation (DM kg)
469.0 Saccharomyces
377.5
Enzymatic hydrolysis Glucose 372.5 cerevisiae
Xylose 75.5
Xylose 75.5 CO2 182.1
Fibers (DM kg)
50.0 FPU/g Glycerol 9.3
560.5
Sugarcane bagasse (DM kg) 25.0 CBU/g Ethanol 190.3
Cellulose 369.2
1000 950 Lime pretreatment Hydrolysis residue (DM kg)
Hemicellulose 112.9 Anaerobic digestion (DM kg)
Cellulose 384.8 Lignin 75.1 91.5
Innoculum 22.3
Hemicellulose 232.8 90 h Others 3.4 Cellulose 6.6
Methane 9.3
Lignin 250.8 90◦C Hemicellulose 46.5
Carbonic gas 13.0
Ash 15.2 0.4 (g/g) Lignin 22.9
Pretreatment liquor (DM kg)
Other 66.5 Others 15.5
389.5
Cellulose 14.8
Anaerobic digestion (DM kg)
Hemicellulose 119.9 Innoculum Fertilizer
73.5
Lignin 162.5
Methane 30.7
Others 92.3
Carbonic gas 42.8

102.5 kg precipitable lignin (recovered)

(A) Fermentation
Hydrolyzate
Hydrolysis liquor (DM kg) fermentation (DM kg)
390.7 Saccharomyces
348.2
Enzymatic hydrolysis Glucose 394.4 cerevisiae
Xylose 39.1
Xylose 39.1 CO2 192.9
Fibers (DM kg)
3.5 FPU/g Glycerol 9.9
433.2
Sugarcane bagasse (DM kg) Hydrogen peroxide 25.0 CBU/g Ethanol 201.5
Cellulose 356.3
1000 950 pretreatment Hydrolysis residue (DM kg)
Hemicellulose 51.3 Anaerobic digestion (DM kg)
Cellulose 384.8 Lignin 32.3 42.5
Innoculum 6.8
Hemicellulose 232.8 1h Cellulose 1.4
Methane 3.1
Lignin 250.8 25◦C Hemicellulose 16.8
Carbonic gas 3.7
Ash 15.2 7.35 % (v/v) Lignin 10.7
Pretreatment liquor (DM kg)
Other 66.5 Others 13.7
516.8
Cellulose 10.5
Anaerobic digestion (DM kg)
Hemicellulose 166.3 Innoculum Fertilizer
100.9
Lignin 184.3
Methane 46.0
Others 155.8
Carbonic gas 54.9

112.7 kg precipitable lignin (recovered)

(B)
Fig. 5. Mass flow in the biorefinery process for pretreatment with lime (A) and hydrogen peroxide (B).

16000
Energy (MJ/ton bagasse)

14000
12000
10000
8000
6000
4000
2000
0
1 2* 2** 3* 3** 4* 4**
Scenario

Fig. 6. Energy output from each scenario studied in the biorefinery concept using bagasse pretreated with hydrogen peroxide () or lime (). (1) Untreated bagasse –
combustion, (2) pretreated bagasse – bioethanol, (3) pretreated bagasse – bioethanol, lignin combustion, biogas from pretreatment liquor and hydrolysis residue, (4)
pretreated bagasse – bioethanol, lignin and hydrolysis residue combustion, biogas from liquor pretreatment.

This energy represented 6–11% of the overall potential from ba- which was more than twice the energy recovered by ethanol pro-
gasse combustion and 9–18% of energy recovered in scenario 4. duced alone from hexoses: 4400–5100 MJ/ton processed.
Kaparaju et al. (2009), studying the hydrothermal pretreatment Comparing scenarios 3 and 4, there is a decrease in the energy
of wheat straw, obtained their best result for energy generation, on production of 3.93% and 11.22%, respectively when using bagasse
the basis of multiple biofuels, of 9364 MJ/ton wheat straw of en- pretreated with peroxide and lime, in scenario 3, the residue of
ergy during the production of biogas, bioethanol and hydrogen. enzymatic hydrolysis was directed to the production of biogas,
The authors also showed that when using biomass to produce only which generated less energy than the straightforward burning of
bioethanol, the energy output was 3572 MJ/ton wheat straw. this residue. It is thus more advantageous to burn hydrolysis resi-
Using steam-pretreated hemp as the substrate, Kreuger et al. dues rather than to use them to produce biogas. Production of bio-
(2011) studied the co-production of ethanol and methane, obtain- ethanol alone (scenario 2) was of course the least energy-efficient
ing a high yield: 11,100–11,700 MJ/ton processed dry matter process.
7894 S.C. Rabelo et al. / Bioresource Technology 102 (2011) 7887–7895
This study has not considered using vinasse for biogas produc-
tion. According to Salomon and Lora (2009), for each liter of first
generation bioethanol obtained after distillation, approximately
13 L of vinasse are produced and this is, in fact, the amount usually
found in large-scale production plants in Brazil. This effluent is
highly polluting but as high fertilizing value due to being rich in or-
ganic matter and having high BOD (Biochemical Oxygen Demand).
The chemical composition of vinasse depends on the characteris-
tics of the soil, the variety of sugarcane, the period of the harvest
and the industrial process used for the production of ethanol.
According to Lettinga and Haandel (1993), 1 L of vinasse yields
about 14.23 L of methane, which could represent a significant in-
crease in useful energy production. Along with the methane pro-
duced by the anaerobic digestion of vinasse, several other
products are released. The production of 6–8 kg granular sludge
per m3 of bioethanol can be sold as inoculum of UASB digesters
(Upflow Anaerobic Sludge Blanket). Also, there is a release of a sig-
nificant amount of solids which settle spontaneously (10 g/L of
vinasse).
In Brazil, the vinasse is directly applied to the soil as a fertilizer
and source of potassium. But for this practice to be efficient, a thor-
ough soil analysis is necessary, so that the appropriate amount of
vinasse can be used. Environmental agencies are limiting the quan-
tity of vinasse per hectare (Salomon and Lora, 2009). In the case of
vinasse obtained from second generation bioethanol production, it
will not contain potassium and phosphorus. Thus, ultimately it will
lose its advantage as a fertilizer, both in the case of direct applica-
tion of the vinasse to the soil or when the solid residue after biogas
production is used.
Despite the low energy output from the production of biofuels
such as ethanol, methane and burning lignin when compared to
the incineration of biomass, there are advantages in the conversion
of biomass to biofuels, mainly related to environmental gains
resulting from greenhouse gas reduction.
Brazil has a great interest in cellulosic ethanol, the aims being to
making ethanol from sugarcane more competitive and expanding
production without having to increase the area under sugarcane.
By combining first and second generations, more ethanol is pro-
duced per planted area. Studies conducted indicate that a distillery
that today produces 1 million liters of ethanol per day using sugar-
cane juice could, with lignocellulose biorefinery technology, gener-
ate an additional 150,000 L of ethanol from bagasse. In 2025, with
the technique improved, it could show an increase in production of
400,000 L, recovered from bagasse (Revista Pesquisa Fapesp, 2007).
Sugarcane straw is another potential source for bioethanol produc-
tion and, with the prohibition of burning, it has the potential for
use as a source of cellulose.
In the future, with many options for the construction of an ener-
getic matrix, bagasse could either be used for burning to generate
bioelectricity or for the production of cellulosic ethanol, and the
decision will be the market, emphasizing the most profitable
option.
4. Conclusions
The residues from second generation bioethanol production
were used for biogas production and for heat generation using
two different types of pretreatment: alkaline hydrogen peroxide
and lime, and different solids concentrations.
Simulation of different scenarios has shown that the use of ba-
gasse for energy generation by incineration was more efficient, the
energy recovery being about 1.6 times higher than for the best
multiple fuels production scenario. However, this greater energy
potential is realised only by the production of heat. Other energy
vectors, in particular liquid fuels, are of great interest and justify
the development of these processes.
Acknowledgements
The authors acknowledge the financial support provided by Fa-
pesp, CNPq and INRA.
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