Dilute mixed-acid pretreatment of sugarcane bagasse for ethanol production

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Dilute mixed-acid pretreatment of sugarcane bagasse forethanol production

798081828384858687888990

George Jackson de Moraes Rocha a,*, Carlos Martin b, Isaias Barbosa Soares c,Ana Maria Souto Maior d, Henrique Macedo Baudel e, Cesar Augusto Moraes de Abreu c

a Laboratorio Nacional de Ciencia e Tecnologia do Bioetanol e CTBE, P.O. Box 6170, 13083-970 Campinas, SP, BrazilbDepartment of Chemistry and Chemical Engineering, University of Matanzas, Carretera Varadero Km 3 1/2, Matanzas, CubacDepartment of Chemical Engineering, Federal University of Pernambuco, P.O. Box 3447, 50100-100 Recife, PE, BrazildDepartment of Antibiotics, Federal University of Pernambuco, P.O. Box 3447, 50100-100 Recife, PE, BrazileCenter of Sugarcane Technology, P.O. Box 162, 13400-970 Piracicaba, SP, Brazil

919293949596979899

100101102103104105

a r t i c l e i n f o

Article history:

Received 24 May 2010

Received in revised form

10 September 2010

Accepted 18 October 2010

Available online xxx

Keywords:

Sugarcane bagasse

Pretreatment

Dilute-acid hydrolysis

* Corresponding author. Address: P.O. Box 11E-mail address: [email protected]

106107

Please cite this article in press as: Jacksonethanol production, Biomass and Bioene

0961-9534/$ e see front matter ª 2010 Publidoi:10.1016/j.biombioe.2010.10.018

a b s t r a c t

Integral utilisation of bagasse is a high priority for the diversification of the sugarcane

industry. The application of a biorefinery philosophy to bagasse utilisation requires its

fractionation into its main components: cellulose, hemicelluloses and lignin. The first stage

in that process is the pretreatment, in which a considerable part of hemicelluloses is solu-

bilised, and cellulose is activated towards enzymatic hydrolysis. In thiswork, a pretreatment

method using a mixture of sulfuric and acetic acid is investigated. Two different solid-to-

liquid ratios (1.5:10 and 1:10) were used in the pretreatment. Both conditions efficiently

hydrolysed the hemicelluloses giving removals above 90%. The extractive components were

also effectively solubilised, and lignin was only slightly affected. Cellulose degradation was

below 15%, which corresponded to the low crystallinity fraction. The analysis of the

morphology of pretreated bagasse confirmed the results obtained in the chemical

characterization.

ª 2010 Published by Elsevier Ltd.

108

Bagasse is the solid residue remaining after crushing the

sugarcane for stripping the juice to be used for sugar or

ethanol production. The enormous sugar and ethanol

production in Brazil generates huge amounts of bagasse.

During the 2008/2009 harvest more than 629 millions tons of

sugar cane was crushed, which generated around 229millions

tons of solid residues [1]. Sugarcane bagasse is currently used

as the main source of the energy required in sugar mills and

ethanol distilleries and also for generating electricity to be

sold to the grids. However, an important part of the produced

bagasse is underutilised. It is well documented that with the

6, 12600-970 Lorena, SP,(G. Jackson de Moraes Ro

de Moraes Rocha G, etrgy (2010), doi:10.1016/j.

shed by Elsevier Ltd.

to satisfy the energy requirements of the plants with only half

of the produced bagasse. The surplus of bagasse can be used in

more than forty different applications, such as production of

ethanol, pulp and paper, boards, animal feed and furfural [2].

The integral utilisation of bagasse components is desirable

both from economical and environmental reasons.

Sugarcane bagasseaswell as other types of plant biomass is

composed of cellulose, hemicelluloses, lignin, and small

amounts of extractives and mineral salts. The structural

components are distributed in a lamellar structure [3]. Hydro-

lysis is crucial for the conversion of bagasse polysaccharides,

mainly cellulose, into valuable products. However, the strong

Brazil. Tel.: þ55 12 31595030; fax: þ55 12 31533165.cha).

126127128129130

al., Dilute mixed-acid pretreatment of sugarcane bagasse forbiombioe.2010.10.018

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crystalline arrangement of cellulose, and the protective effects

by lignin and hemicelluloses difficult the access of enzymes

and acid catalysts to the b1/4 glycosidic bonds, which

constitute a serious obstacle to hydrolysis [4].

In order to facilitate cellulose hydrolysis different

kinds of chemical, physical and physico-chemical pretreat-

ment methods have been proposed. Pretreatment and

delignification processes are aimed to disrupt the cellulo-

seehemicelluloseselignin complex, and they are important

technological steps for the fractionation of lignocellulosic

materials in their main components for their utilisation

according to a biorefinery philosophy [5e9].

The development of pretreatment processes strong enough

as to separate the cell wall arrangement andmild enough as to

avoid a significant chemical degradation of biomass compo-

nents is a challenge for today’s chemical industry [4]. For the

novel pretreatment methods it is advisable to use cheap

and easily recoverable chemicals and low-cost equipment.

The use of environmentally friendly and low energy-intensive

approaches is highly desired.

Traditional pretreatment processes are severe, destructive

and not efficient enough. Recently, innovative methods able

to separate the three main polymeric constituents of ligno-

cellulosic biomass and to decrystalinize cellulose with

minimal chemical alteration of hemicellulose and lignin have

been investigated [10].

Different approaches of dilute acid prehydrolysis have

been shown to be effective pretreatment processes [11,12].

Sulfuric acid is the most commonly used acid in pretreatment

of sugarcane bagasse [13], but other reagents, such as hydro-

chloric, nitric and phosphoric acids can also be used [14,15].

Recently, the use of acetic acid combined with hydrogen

peroxide was reported as a way of removing lignin prior to

enzymatic hydrolysis of bagasse [16].

In this context, the current work is aimed to study

a pretreatment method for sugarcane bagasse using an acid-

catalyzed process with diluted sulfuric and acetic acids at two

different liquid-to-solid ratios in a rotary reactor, especially

designed for this purpose.

239240241242243244245246247248249

2. Materials and methods

2.1. Raw material

Sugarcane bagasse, kindly donated by a sugar mill (Usina

Central Olho D’Agua, Camutanga, Pernambuco, Brazil), was

used. A portion of bagassewasmilled to a particle size of 16/60

mesh and used for raw material analysis.

2.2. Pretreatment

Bagasse, with a moisture content of 12%, was mixed in the

pretreatment reactor with 10 L of a solution containing 1% (w/

v)sulfuric acid and 1% (w/v) acetic acid. Bagasse samples

of 1 and 1.5 kg (DW) were used to reach 1.5:10 and 1:10 solid-

to-liquid ratios. The pretreatment was performed in a 20-L

rotary reactor (Regmed EU/E 20, Regmed Industria Tecnica

Ltda.). The reactor, with a built electrical heating device, was

Please cite this article in press as: Jackson de Moraes Rocha G, etethanol production, Biomass and Bioenergy (2010), doi:10.1016/j.

especially projected for the pretreatment method assayed in

this work.

When bagasse and the acid mixture were loaded, the

reactor was closed and heated up to 190 �C for 10 min. After

that, the reactor was gradually depressurized until atmo-

spheric pressure and 100 �C, and was subsequentially

discharged. The pretreated slurry was vacuum-filtered, and

the sugar-rich liquid fractionwas separated and stored frozen.

The solid fraction (pretreated bagasse) was exhaustively

washed with four 15-L portions of warm water (70 �C), anddried at room temperature. A portion of the pretreated bagasse

was stored for subsequent chemical analyses, and the rest

of the material was used for further delignification and enzy-

matic hydrolysis.

3. Theory/calculation

3.1. Analysis of chemical composition

Raw and pretreated bagasse were analysed using a method-

ology adapted by Rocha et al. [17] and validated by Gouveia

et al. [18]. The methodology is based on a two-step acid

hydrolysis of extractive-free material followed by chromato-

graphic quantification of sugars and degradation products

contained in the hydrolyzate and by the gravimetric deter-

mination of acid-insoluble lignin using a modification of the

Klason method [17].

Milled bagasse was extracted with 95% ethanol for 8 h in

a Soxhlet apparatus. A 2-g aliquot of the extractive-free

material was treated with 10 mL of 72-% H2SO4 in a 100-mL

beaker maintained in a thermostated bath at 45.0 � 0.5 �C for

7 min with vigorous shaking. The reaction was stopped by

addition of 50 mL of distilled water, and the resulting mixture

was quantitatively transferred to a 500-mL erlenmeyer flask,

where the acid was diluted by water addition up to a final

volume of 275 mL. For completing the hydrolysis of the

unhydrolyzed olygosaccharides, the flask was closed with an

aluminium foil and autoclaved for 30 min at 1.05 atm.

After elapsing the reaction time and depressurizing the

autoclave the hydrolysis mixture was filtered using a previ-

ously weighed filter paper for gelatinous solids. The hydroly-

zate was collected in a 500-mL volumetric flask, and the

filtration residue was washed with 50-mL portions of distilled

water until completing the flask volume. The lignin retained

in the filter was thoroughly washed until washing out the

sulphate anions (approximately 1500 mL), and dried at 105 �Cuntil constant weight. For the pretreated bagasse the same

treatment was applied, except that no ethanol extraction was

performed since the pretreatment practically remove most of

the extractives.

For determination of the ash content in acid-insoluble

lignin, the dry residue was quantitatively transferred to

a previouslyweighed crucible, incineratedfirst at 300 �Cduring

40minand thenat 800 �Cduring 2h. Themass of the asheswas

determined in an analytical balance after cooling down the

crucible with the incinerated sample in a desiccator [19].

Sugars and degradation products in the hydrolyzate were

analysed by HPLC (Shimadzu C-R7A). Cellobiose, glucose,

xylose, arabinose and acetic acid were separated with an

al., Dilute mixed-acid pretreatment of sugarcane bagasse forbiombioe.2010.10.018

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Aminex HPX 87H (300 � 7.8 mm, BIO-RAD, Hercules, CA) at

45 �C using 5 mM H2SO4 as mobile phase at a flow rate of

0.6 mL min�1, and were detected with an RI detector (Shi-

madzu RID-6A).

Furfural and hydroxymethilfurfural (HMF) were separated

on an RP-18 (C-18) de 125� 4mm (Hawlett-Packard), and were

detected with a UV detector (Shimadzu SPD-10A) at 25 �Cusing a mobile phase composed of a 1-% acetic acid-contain-

ing 1:8 acetonitrile-water solution pumped at 0.8 mL min�1.

The concentrations of glucose, cellobiose and HMF were used

for calculating the cellulose content, whereas the content of

hemicelluloses was calculated based on the concentrations of

xylose, arabinose, glucuronic acid, acetic acid and furfural.

Conversion factors of 0.9, 0.95 and 1.29, respectively, were

used for glucose, cellobiose and HMF, whereas for xylose,

arabinose, acetic acid and furfural the conversion factors

were, respectively, 0.88, 0.88, 0.72 and 1.37 [17].

For determination of the concentration of sugar oligomers,

50-mL samples of the hydrolyzates were adjusted to pH 1.0

with H2SO4, and the mixture was autoclaved at 121 �C for

30 min. The resulting material was filtered, diluted to 100 mL

in a volumetric flask and analysed by HPLC as described

previously.

Soluble lignin in the hydrolyzate was determined by UV-

spectroscopy in a 5-mL aliquot of the hydrolyzate, which was

adjusted to pH 12 with 6 M NaOH and 10-fold diluted. The

absorbance of the solution was read at 280 nm (Perkin Elmer

LAMBDA 25). The concentration was calculated using the

following expression and previously determined absortivity

values [5,17,18,20]:

Clig ¼�Alig280 � Apd280

��3lig (1)

Parameters: Clig: concentration of soluble lignin (g L�1); Alig280:

absorbance of the solution at 280 nm; Apd280: absorbance of

sugar-degradation products (furfural and HMF)

Apd280 ¼ C131 þ C232 (2)

Parameters: C1: furfural concentration (g L�1); C2: HMF

concentration (g L�1); 31: furfural absortivity at 280 nm

Table 1 e Chemical composition of in natura bagasse, bagassepretreated at 1:10 solid-to-liquid ratio.

Components in naturaa (%) at 1.5:10 solid-to-liq

Content(w/w)

Content(w/w)

Mass y(g)

Dry matter, % 100.00 100.00 65.00

Cellulose 45.5 � 1.1 58.06 � 0.61 37.74 �Hemicelluloses 27.0 � 0.8 3.79 � 0.02 2.46 � 0

Total lignin* 21.1 � 0.9 32.38 � 0.31 21.04 �Mineral compounds 2.2 � 0.1 5.90 � 0.28 3.83 � 0

Ethanol extractives 4.6 � 0.3 ND ND

Total 100.4 � 0.4 99.3 � 0.70 67.48 �

ND - Not Detected.

a Chemical composition of sugarcane bagasse “in natura”. *Sum of Klaso

b Chemical composition of the liquid fraction obtained by pretreatment

c Chemical composition of the solid fraction obtained by pretreatment of

and acid-soluble lignin.

Please cite this article in press as: Jackson de Moraes Rocha G, etethanol production, Biomass and Bioenergy (2010), doi:10.1016/j.

(31 ¼ 146,85 cm�1 g�1 L e experimentally obtained); 32:

HMF absortivity at 280nm (32¼ 114 cm�1 g�1 Le experimentally

obtained); 3lig: ligninabsortivityat280nm(3lig¼23.7cm�1 g�1 Le

experimentally obtained).

3.2. Analysis of the morphology

The morphology of raw and pretreated bagasse was analysed

by scanning electron microscopy (SEM). The SEM pictures

of raw and pretreated bagasse were taken at different

magnifications such as 50, 500, 700, 1000, 1500, 2000, 4000 and

5000 times using LEO 440 equipment with a Oxford detector

operating at 20 kV, 2.82 A and 950 pA. The samples were

coated with 20 nm of gold in a metalizator (Coating System

BAL-TEC MED 020) and kept in a desiccator until analysis.

3.3. Enzymatic convertibility

The enzymatic convertibility of cellulose in the pretreated

bagasse was determined according to the protocol described

by Carrasco et al. [21]. Commercial enzyme preparations

(Celluclast 1.5 L (65 FPU/mL and 17 IU/mL of b-glucosidase) and

Novozym 188 (376 IU/g of b-glucosidase)) kindly donated by

Novozymes A/S (Bagsværd, Denmark) were used. The filter

paper and b-glucosidase activities were determined according

to Mandels et al. [22] and Berghem and Pettersson [23],

respectively. The enzymatic hydrolysis was conducted with

10 g of washed solids (WIS), 2.32 g of Celluclast 1.5 L and 0.52 g

of Novozym 188. Acetate buffer (pH 4.8) was added to give

500 g of the reaction mixture with a 2-% consistency. The WIS

determination was performed according to the NREL standard

assay [24].

4. Results

4.1. Data from the different samples of the experiments

(Table 1)

pretreated at 1.5:10 solid-to-liquid ratio and bagasse

uid ratiob (%) at 1:10 solid-to-liquid ratioc (%)

ield Losses Content(w/w)

Mass yield(g)

Losses

e 100.00 63.00 e

0.61 13.1 61.65 � 0.50 38.84 � 0.50 14.6

.02 90.8 2.79 � 0.03 1.87 � 0.03 93.4

0.31 0.3 32.96 � 0.25 20.76 � 0.25 4.7

.02 e 3.97 � 0.02 2.50 � 0.02 e

e ND ND e

0.70 e 101.37 � 0.80 63.97 � 0.70 e

n lignin and acid-soluble lignin.

of sugarcane bagasse at 1.5:10 and 1:10 solid-to-liquid ratios.

sugarcane bagasse at 1:10 solid-to liquid ratio. *Sum of Klason lignin

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al., Dilute mixed-acid pretreatment of sugarcane bagasse forbiombioe.2010.10.018

Fig. 1 e Heating-up and cooling-down curve for the

Regmed AU/EL20 reactor. Fig. 2 e Panoramic micrograph showing a bundle of fibres

in bagasse pretreated at 1.5:10 solid-to-liquid ratio.

Magnification: 503.

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4.2. Reactor heating data

(Fig. 1)

4.3. Data from pretreatment at 1.5:10 solid-to-liquidratio

(Table 2; Figs. 2e4)

4.4. Data from pretreatment at 1:10 solid-to-liquid ratio

(Figs. 5e7)

4.5. Data from enzymatic hydrolysis of the pretreatedbagasse

(Fig. 8)

495496497498499500501502503

5. Discussion

The chemical composition of bagasse in natura is shown in

Table 1. The high cellulose content (above 45%) indicates the

high potential of the material for its conversion through the

saccharification route. In general, the results of the chemical

Table 2 e Chemical composition of the liquid fractionobtained by pretreatment of sugarcane bagasse at 1.5:10and 1:10 solid-to-liquid ratios.

Compound Concentration, g L�1

SLR: 1.5:10 SLR: 1:10

Xylose 9.04 � 0.04 9.33 � 0.03

Arabinose 1.03 � 0.02 1.00 � 0.00

Glucose 3.67 � 0.16 3.09 � 0.01

Cellobiose 0.20 � 0.00 0.33 � 0.00

Acetic acid 2.21 � 0.04 2.89 � 0.02

Glucuronic acid 0.21 � 0.00 0.30 � 0.00

Formic acid 0.74 � 0.00 0.80 � 0.00

Furfural 0.05 � 0.00 0.10 � 0.00

HMF 0.01 � 0.00 0.02 � 0.00

Please cite this article in press as: Jackson de Moraes Rocha G, etethanol production, Biomass and Bioenergy (2010), doi:10.1016/j.

analyses indicate that the content of the main components in

the raw bagasse used in this study is in good agreement with

the content of other sorts of bagasse previously reported

[25,26].

If bagasse is going to be hydrolyzed with enzymes, it is

necessary to pretreat the material for removing the hemi-

celluloses and enhancing the enzymatic convertibility of

cellulose. Acid pretreatment, mainly using sulfuric acid, and

hydrothermal methods, based on the autocatalytic action of

acetic acid released by hydrolytic cleavage of acetyl groups,

have shown to be effective in improving the enzymatic

hydrolysis of cellulose. In the pretreatment method proposed

in this work, externally-added acetic acid is aimed to poten-

tiate the catalytic effect of in situ generated acetic acid, and

thus facilitating the hydrolysis of hemicelluloses and

enhancing the enzymatic hydrolysis of cellulose. A reactor

especially projected for the assayed pretreatmentmethodwas

tested. The reactor was designed after modifications of

a prototype previously used for wood pulping.

Fig. 3 e Micrograph of a bundle of fibres in bagasse

pretreated at 1.5:10 solid-to-liquid ratio. Magnification:

50003.

504505506507508509510511512513514515516517518519520

al., Dilute mixed-acid pretreatment of sugarcane bagasse forbiombioe.2010.10.018

Fig. 4 e Micrography of a region of vascular fibers in

pretreated bagasse. Magnification: 15003.

Fig. 6 e Micrograph of a longitudinal section of bagasse

pretreated at 1:10 solid-to-liquid ratio. Magnification:

10003.

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The reactor displayed an excellent behaviour, and the

heating was rather fast and regular (Fig. 1). After reaching the

190 �C, the reactor held that temperature steadily during

the isothermal treatment period. The overall severity factor of

the whole process was 5.37. Previous results by this group

have shown that severities below 5.1 lead to hydrolytic

conversion of hemicellulose under 60% and do not especially

increase the enzymatic convertibility of cellulose [27]. The

severity factor was calculated according to the expression

proposed by Overend and Chornet [28].

lnR0 ¼Z

eT�10014:75 dt (3)

Parameters: ‘t’ and ‘T’ are the time (s) and the operating

temperature (K), respectively.

The pretreatment was performed at two different solid-to-

liquid ratios, 1.5:10 and 1:10. Both experiments are discussed

separately in order to give a detailed discussion of the results

of the analysis of the chemical composition and the

morphology of the pretreated material.

Fig. 5 e Panoramic micrograph bagasse pretreated at 1:10

solid-to-liquid ratio. Magnification: 503.

Please cite this article in press as: Jackson de Moraes Rocha G, etethanol production, Biomass and Bioenergy (2010), doi:10.1016/j.

5.1. Pretreatment at 1.5:10 solid-to-liquid ratio

The experiment at 1.5:10 solid-to-liquid ratio resulted in a 65%

mass yield of pretreated solids indicating that 35% of bagasse

mass was solubilised (Table 1). The hemicelluloses were the

main solubilised component. Their mass yield in the pre-

treated solids was only 2.46 g down from 27.0% in the raw

bagasse, revealing that 90.9% of the hemicelluloses initially

contained in the raw material were solubilised. This is an

indication of the effectiveness of the pretreatment for

removing the hemicelluloses. Since hemicelluloses are

a physical barrier that surrounds cellulose fibers and protect

them from enzymatic attack [11], their removal indicates the

potential of the used method for activation of bagasse for

enzymatic hydrolysis.

As result of hydrolysis of hemicelluloses a rather high

concentration of xylose was detected in the liquid fraction

(Table 2). The acid posthydrolysis experiments performed in

order to detect the presence of sugar oligomers in the prehy-

drolyzate revealed that xylose was completely in monomeric

Fig. 7 e Amplification of another region of the same bundle

of fibers. Magnification: 40003.

630631632633634635636637638639640641642643644645646647648649650

al., Dilute mixed-acid pretreatment of sugarcane bagasse forbiombioe.2010.10.018

Fig. 8 e Cellulose conversion during enzymatic hydrolysis

of bagasse pretreated at 1:10 solid-to-liquid-ratio (rhombs),

1.5:10 solid-to-liquid-ratio (squares) and untreated

bagasse (triangles).

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form. Other products of hemicellulose hydrolysis, such as

arabinose, acetic acid and glucuronic acid, were also found in

the liquid fraction. The concentrations of sugar-degradation

products, such as furfural, HMF and formic acid, were rather

low, suggesting the potential good fermentability of the

prehydrolyzate.

The analysis of the pretreated solids did not reveal signif-

icant removal of lignin (Table 1). This is a very interesting

feature of the investigated acid pretreatment. Typically, acid

pretreatments, remove, in addition to hemicelluloses, a small,

but not insignificant, part of the lignin fraction [12].

Around 13.1% of cellulose was solubilised (Table 1). It is

assumed that most of the solubilised cellulose corresponded

to its amorphous or low-crystalinity fraction. The relatively

low concentration of glucose in the prehydrolyzate is in

accordance with the low cellulose solubilisation (Table 2). By

visual inspection of the pretreated solids their porosity was

evident, especially when the material became fragile like

a rotted wood after being manually compressed.

For elucidating the physical changes occurred during

pretreatment, the morphology of the pretreated bagasse was

investigated using SEM. In the Fig. 2, a panoramic micrograph

of the pretreated bagasse is shown. Although apparently the

material seems to be similar to raw bagasse, its porosity is

clearly observed. In the bottom of the picture, the smooth

surface reveals a fragment of the external region of the bark.

The Fig. 3, with amagnification of 5000�, shows a fiberwith

thick walls and gives a good resolution of the pits, which are

distributed in high amount along the whole surface of the

fibers. The sizes of the pits were approximately 1 mm.

A region of the pretreated sample composed of vascular

fibers is exposed in Fig. 4. Those fibers present thin walls with

rounded, flattened or branched endings. With a magnification

degree of 1500�, it is revealed that although the fibers are still

grouped in bundles, individual fibers are separated from each

other. That can be attributed to the removal of the hemi-

celluloses and extractives occurred during pretreatment.

5.2. Pretreatment at 1:10 solid-to-liquid ratio

The pretreatment at 1:10 solid-to-liquid ratio, containing 0.2 g

acid mix/g dry bagasse, produced 63% of mass yield (Table 1).

Please cite this article in press as: Jackson de Moraes Rocha G, etethanol production, Biomass and Bioenergy (2010), doi:10.1016/j.

The pretreatment at 1.5:10 solid-to-liquid ratio, containing

0.13 g acid mix/g dry bagasse, produced 65% of mass yield

(Table 1). The hydrolysis of the hemicelluloses for these to

processes,wereof 90and93%respectivelyand induceda lignin

loss of almost 5%. Experiments carried out with 1:10 solid-

to-liquid ratio, containing 0.1 gH2SO4/gdrybagasse, resulted in

70%ofmassyield [29,30].Thesameexperimentcarriedwithout

acids, resulted in a mass yield of 67.5% [31].

Both pretreatments presented xylose as the main sugar

detected in the liquid fraction (Table 2), and no oligosac-

charides were found. However, the concentration of xylose

at the 1:10 solid-to-liquid ratio experiment was slightly

higher than the one found for 1.5:10 solid-to-liquid ratio

experiment. This is in total agreement with the higher sol-

ubilisation of hemicelluloses detected in the chemical

analysis of the solid fraction, which is also backed by the

higher concentration of acetic acid. The slightly higher

concentrations of furfural, HMF and formic acid reveal that

the degradation of sugars, albeit still low, was higher than in

the previous pretreatment.

The panoramic micrograph shown in Fig. 5 shows bundles

of fibers in bagasse pretreated at 1:10 solid-to-liquid ratio. In

the central part of the image pith flocks are visible. The results

of the pretreatment are evident in the micrograph of the

longitudinal section of the vascular bundle shown in Fig. 6. It

is obvious that bagasse was considerably exposed to the

action of the hydrolyzing agents as can be deduced from

the fragments of pith flocks distributed on the whole surface

of this bundle. It is also possible to find empty spaces between

the fibers, as consequence of the removal of hemicelluloses

and low-crystalinity cellulose flocks. Pith flocks are easily

hydrolyzed since they are constituted of parenchyma cells,

which have a high content of amorphous and/or low-crysta-

linity cellulose [32]. This is also evident in another region

of the same bundle of fibers, where similar morphological

elements are observed (Fig. 7). The high interfibrilar porosity is

a consequence of the high hemicellulose solubilisation and

the total removal of extractives occurred during pretreatment.

5.3. Enzymatic hydrolysis of the pretreated bagasse

Theexperimentson theenzymatichydrolysis of thepretreated

solids revealed that the pretreatment was effective in

improving the enzymatic convertibility of cellulose. Pretreat-

ment led to conversions above 76% after 72 h, whereas for

pretreated bagasse only 6% of the initial cellulose was hydro-

lyzed (Fig. 8). The hydrolysis of the bagasse pretreated at 1:10

solid-to-liquid ratio had a higher initial rate, and a 75% cellu-

losewasachieved in 48h.However, no significant increasewas

observed in the subsequent 24 h, and the final conversions

were comparable for both pretreatment conditions.

6. Conclusions

The fast and practically linear heating-up time, the stability

upon constant temperature and the possibility of safe manual

depressurization ensured a moderate-severity process and

demonstrated the feasibility of the reactor for the assayed

pretreatment method.

al., Dilute mixed-acid pretreatment of sugarcane bagasse forbiombioe.2010.10.018

Q2

b i om a s s a n d b i o e n e r g y x x x ( 2 0 1 0 ) 1e8 7

781782783784785786787788789790791792793794795796797798799800801802803804805806807808809810811812813814815816817818819820821822823824825826827828829830831832833834835836837838839840841842843844845

846847848849850851852853854855856857858859860861862863864

JBB2101_proof ■ 8 November 2010 ■ 7/8

The chemical analyses revealed that both of the pretreat-

ment conditions investigated were effective for preparing

the bagasse for cellulases-based hydrolysis processes since

more than 90% of the hemicelluloses were removed, cellulose

was only marginally affected during pretreatment and enzy-

matic conversions above 76% were achieved. The degraded

cellulose corresponded to the low-crystallinity fraction.

A high solubilisation of the extractive compounds was

also achieved.

The obtained hemicellulose hydrolyzates are potentially

good substrates for fermentation processes since the

pretreatment led to low formation of sugar- and lignin-degra-

dation inhibitory products.

The morphological analyses revealed that the process

was effective in disrupting the fibres and confirmed the

results achieved in the chemical characterization.

865866867868869870871872873

Acknowledgments

Thisworkwas possible thank to the financial support provided

by the CNPq-MES cooperation program (gs1) (grant No. 490830/

2006-4). SaoCarlosChemistry Institute is acknowledged for the

SEM analysis.

874875876877878879880881882883884885886887888889890891892893894895896897898899900901902903904905906907908909910

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