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Dilute mixed-acid pretreatment of sugarcane bagasse for ethanol production
b i o m a s s a n d b i o e n e r g y 3 5 ( 2 0 1 1 ) 6 6 3 e6 7 0
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Dilute mixed-acid pretreatment of sugarcane bagasse for ethanol production 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
Laborato´rio Nacional de Cieˆncia e Tecnologia do Bioetanol e CTBE, P.O. Box 6170, 13083-970 Campinas, SP, Brazil Department of Chemistry and Chemical Engineering, University of Matanzas, Carretera Varadero Km 3 1/2, Matanzas, Cuba c Department of Chemical Engineering, Federal University of Pernambuco, P.O. Box 3447, 50100-100 Recife, PE, Brazil d Department of Antibiotics, Federal University of Pernambuco, P.O. Box 3447, 50100-100 Recife, PE, Brazil e Center of Sugarcane Technology, P.O. Box 162, 13400-970 Piracicaba, SP, Brazil b
article info
abstract
Article history:
Integral utilisation of bagasse is a high priority for the diversification of the sugarcane
Received 24 May 2010
industry. The application of a biorefinery philosophy to bagasse utilisation requires its
Received in revised form
fractionation into its main components: cellulose, hemicelluloses and lignin. The first stage
10 September 2010
in that process is the pretreatment, in which a considerable part of hemicelluloses is solu-
Accepted 18 October 2010
bilised, and cellulose is activated towards enzymatic hydrolysis. In this work, a pretreatment
Available online 20 November 2010
method using a mixture of sulfuric and acetic acid is investigated. Two different solid-toliquid ratios (1.5:10 and 1:10) were used in the pretreatment. Both conditions efficiently
Keywords:
hydrolysed the hemicelluloses giving removals above 90%. The extractive components were
Sugarcane bagasse
also effectively solubilised, and lignin was only slightly affected. Cellulose degradation was
Pretreatment
below 15%, which corresponded to the low crystallinity fraction. The analysis of the
Dilute-acid hydrolysis
morphology of pretreated bagasse confirmed the results obtained in the chemical
Ethanol
characterization.
Mixed acid
1.
Introduction
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 229 millions 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
ª 2010 Elsevier Ltd. All rights reserved.
technological improvements made to the boilers it is possible 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 bagasse as well 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]. Hydrolysis is crucial for the conversion of bagasse polysaccharides, mainly cellulose, into valuable products. However, the strong
* Corresponding author. Address: P.O. Box 116, 12600-970 Lorena, SP, Brazil. Tel.: þ55 12 31595030; fax: þ55 12 31533165. E-mail address: [email protected] (G. Jackson de Moraes Rocha). 0961-9534/$ e see front matter ª 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biombioe.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 pretreatment methods have been proposed. Pretreatment and delignification processes are aimed to disrupt the celluloseehemicelluloseselignin 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 and mild enough as to avoid a significant chemical degradation of biomass components 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 lignocellulosic 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 hydrochloric, 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 acidcatalyzed process with diluted sulfuric and acetic acids at two different liquid-to-solid ratios in a rotary reactor, especially designed for this purpose.
2.
Materials and methods
2.1.
Raw material
Sugarcane bagasse, kindly donated by a sugar mill (Usina ´ gua, Camutanga, Pernambuco, Brazil), was Central Olho D’A used. A portion of bagasse was milled 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 solidto-liquid ratios. The pretreatment was performed in a 20-L rotary reactor (Regmed EU/E 20, Regmed Indu´stria Te´cnica Ltda.). The reactor, with a built electrical heating device, was
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 atmospheric pressure and 100 C, and was subsequentially discharged. The pretreated slurry was vacuum-filtered, and the sugar-rich liquid fraction was separated and stored frozen. The solid fraction (pretreated bagasse) was exhaustively washed with four 15-L portions of warm water (70 C), and dried 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 enzymatic hydrolysis.
3.
Theory/calculation
3.1.
Analysis of chemical composition
Raw and pretreated bagasse were analysed using a methodology 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 chromatographic quantification of sugars and degradation products contained in the hydrolyzate and by the gravimetric determination 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 previously weighed filter paper for gelatinous solids. The hydrolyzate 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 C until 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 previously weighed crucible, incinerated first at 300 C during 40 min and then at 800 C during 2 h. The mass of the ashes was 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
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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 min1, and were detected with an RI detector (Shimadzu RID-6A). Furfural and hydroxymethilfurfural (HMF) were separated on an RP-18 (C-18) de 125 4 mm (Hawlett-Packard), and were detected with a UV detector (Shimadzu SPD-10A) at 25 C using a mobile phase composed of a 1-% acetic acid-containing 1:8 acetonitrile-water solution pumped at 0.8 mL min1. 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 UVspectroscopy 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)
(31 ¼ 146,85 cm1 g1 L e experimentally obtained); 32: HMF absortivity at 280 nm (32 ¼ 114 cm1 g1 Le experimentally obtained); 3lig: lignin absortivity at 280 nm (3lig ¼ 23.7 cm1 g1 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].
1
Parameters: Clig: concentration of soluble lignin (g L ); Alig280: absorbance of the solution at 280 nm; Apd280: absorbance of sugar-degradation products (furfural and HMF) Apd280 ¼ C1 31 þ C2 32
(2)
Parameters: C1: furfural concentration (g L1); C2: HMF concentration (g L1); 31: furfural absortivity at 280 nm
4.
Results
4.1.
Data from the different samples of the experiments
(Table 1)
Table 1 e Chemical composition of in natura bagasse, bagasse pretreated at 1.5:10 solid-to-liquid ratio and bagasse pretreated at 1:10 solid-to-liquid ratio. Components
Dry matter, % Cellulose Hemicelluloses Total lignin* Mineral compounds Ethanol extractives Total
in naturaa (%)
at 1.5:10 solid-to-liquid ratiob (%)
at 1:10 solid-to-liquid ratioc (%)
Content (w/w)
Content (w/w)
Mass yield (g)
Losses
Content (w/w)
Mass yield (g)
Losses
100.00 45.5 1.1 27.0 0.8 21.1 0.9 2.2 0.1 4.6 0.3 100.4 0.4
100.00 58.06 0.61 3.79 0.02 32.38 0.31 5.90 0.28 ND 99.3 0.70
65.00 37.74 0.61 2.46 0.02 21.04 0.31 3.83 0.02 ND 67.48 0.70
e 13.1 90.8 0.3 e e e
100.00 61.65 0.50 2.79 0.03 32.96 0.25 3.97 0.02 ND 101.37 0.80
63.00 38.84 0.50 1.87 0.03 20.76 0.25 2.50 0.02 ND 63.97 0.70
e 14.6 93.4 4.7 e e e
ND - Not Detected. a Chemical composition of sugarcane bagasse “in natura”. *Sum of Klason lignin and acid-soluble lignin. b Chemical composition of the liquid fraction obtained by pretreatment of sugarcane bagasse at 1.5:10 solid-to-liquid ratio. c Chemical composition of the solid fraction obtained by pretreatment of sugarcane bagasse at 1:10 solid-to liquid ratio. *Sum of Klason lignin and acid-soluble lignin.
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Fig. 1 e Heating-up and cooling-down curve for the Regmed AU/EL20 reactor.
4.2.
Fig. 2 e Panoramic micrograph showing a bundle of fibres in bagasse pretreated at 1.5:10 solid-to-liquid ratio. Magnification: 503.
Reactor heating data
(Fig. 1)
4.3. ratio
Data from pretreatment at 1.5:10 solid-to-liquid
(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 pretreated bagasse (Fig. 8)
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
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 hemicelluloses 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 potentiate 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 pretreatment method was tested. The reactor was designed after modifications of a prototype previously used for wood pulping.
Table 2 e Chemical composition of the liquid fraction obtained by pretreatment of sugarcane bagasse at 1.5:10 and 1:10 solid-to-liquid ratios. Concentration, g L1
Compound
SLR: 1.5:10 Xylose Arabinose Glucose Cellobiose Acetic acid Glucuronic acid Formic acid Furfural HMF
9.04 1.03 3.67 0.20 2.21 0.21 0.74 0.05 0.01
0.04 0.02 0.16 0.00 0.04 0.00 0.00 0.00 0.00
SLR: 1:10 9.33 1.00 3.09 0.33 2.89 0.30 0.80 0.10 0.02
0.03 0.00 0.01 0.00 0.02 0.00 0.00 0.00 0.00
Fig. 3 e Micrograph of a bundle of fibres in bagasse pretreated at 1.5:10 solid-to-liquid ratio. Magnification: 50003.
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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.
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].
5.1.
Pretreatment at 1.5:10 solid-to-liquid ratio
Parameters: ‘t’ and ‘T’ are the time (s) and the operating temperature (K), respectively. The pretreatment was performed at two different solid-toliquid 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.
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 pretreated 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 prehydrolyzate revealed that xylose was completely in monomeric
Fig. 5 e Panoramic micrograph bagasse pretreated at 1:10 solid-to-liquid ratio. Magnification: 503.
Fig. 7 e Amplification of another region of the same bundle of fibers. Magnification: 40003.
Z lnR0 ¼
T100
e 14:75 dt
(3)
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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).
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 significant 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 a magnification of 5000, shows a fiber with 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 hemicelluloses 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).
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, were of 90 and 93% respectively and induced a lignin loss of almost 5%. Experiments carried out with 1:10 solidto-liquid ratio, containing 0.1 g H2SO4/g dry bagasse, resulted in 70% of mass yield [29,30]. The same experiment carried without 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 oligosaccharides 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 solubilisation 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-crystalinity 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
The experiments on the enzymatic hydrolysis of the pretreated solids revealed that the pretreatment was effective in improving the enzymatic convertibility of cellulose. Pretreatment led to conversions above 76% after 72 h, whereas for pretreated bagasse only 6% of the initial cellulose was hydrolyzed (Fig. 8). The hydrolysis of the bagasse pretreated at 1:10 solid-to-liquid ratio had a higher initial rate, and a 75% cellulose was achieved in 48 h. However, no significant increase was 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.
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The chemical analyses revealed that both of the pretreatment 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 enzymatic 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-degradation inhibitory products. The morphological analyses revealed that the process was effective in disrupting the fibres and confirmed the results achieved in the chemical characterization.
Acknowledgments This work was possible thank to the financial support provided by the CNPq-MES cooperation program (grant No. 490830/ 2006-4). Sa˜o Carlos Chemistry Institute is acknowledged for the SEM analysis.
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[1] CONAB (Companhia Nacional de Abastecimento). Produc¸a˜o de cana no Brasil tera´ novo recorde. Available at: http//www. conab.gov.br. [Accessed Dec 10, 2009]. [2] Ga´lvez LO. Diversified productions in sugarcane agroindustry. Havana, Cuba. In: Ga´lvez LO, editor. Handbook of sugarcane derivatives. 3rd ed.; 2000. p. 3e17. [3] Fengel D, Wegener G. Wood chemistry, ultrastructure, reactions. Berlin: Walter de Gruyter; 1989. pp 14, 27e28. [4] Canetieri E, Rocha GJM, de Carvalho JR, Silva JBA. Optimization of acid hydrolysis from the hemicellulosic fraction of Eucalyptus grandis residue using response surface methodology. Bioresour Technol 2007;98:422e8. [5] Rocha GJM. 2000. Oxigen-assisted delignification of sugarcane bagasse. PhD Thesis, Institute of Chemistry, University of Sa˜o Paulo. [6] Chandra RP, Bura R, Mabee WE, Berlin A, Pan X, Saddler JN. Substrate pretreatment: the key to effective enzymatic hydrolysis of lignocellulosics? Adv Biochem Eng Biotechnol 2007;108:67e93. [7] Martı´n C, Rocha GJM, Pe´rez M, Lo´pez Y, Herna´ndez E, Plasencia Y. Acid prehydrolysis, alkaline delignification and enzymatic hydrolysis of rice hulls. Cell Chem Technol 2007; 41:129e35. [8] Mussatto S, Dragone G, Rocha GJM, Roberto I. Optimum operating conditions for brewer’s spent grain soda pulping. Carbohydr Polym 2006;64:22e8. [9] Saad M, Oliveira L, Ca´ndido L, Quintana G, Rocha GJM, Gonc¸alves A. Preliminary studies on fungal treatment of sugarcane straw for organosolv pulping. Enzyme Microb Technol 2008;43:220e5. [10] Yang B, Wyman CE. Pretreatment: the key to unlocking lowcost cellulosic ethanol. Biofuels Bioprod Biorefin 2008;2:26e40.
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de Biomassa para Hidro´lise Enzima´tica e Fermentac¸a˜o Alcoo´lica. Trabalho de Conclusa˜o de Curso de Cieˆncias Biolo´gicas Universidade Federal de Pernambuco; 2008. [30] Lins TO, Gouveia CK, Oliveira FMV, Souto-Maior AM, Rocha GJM, Pinheiro IO. Pre´-Tratamento Hidrote´rmico de Bagac¸o de Canade-Ac¸u´car para Produc¸a˜o de Bioetanol. Site. In: XVII Sinaferm Simpo´sio Nacional de Bioprocessos, Natal-RN, Brasil, http:// www.sinaferm2009.com.br/acessado; 2009. em 10/08/2010.
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Available at www.sciencedirect.com
http://www.elsevier.com/locate/biombioe
Dilute mixed-acid pretreatment of sugarcane bagasse for ethanol production 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
Laborato´rio Nacional de Cieˆncia e Tecnologia do Bioetanol e CTBE, P.O. Box 6170, 13083-970 Campinas, SP, Brazil Department of Chemistry and Chemical Engineering, University of Matanzas, Carretera Varadero Km 3 1/2, Matanzas, Cuba c Department of Chemical Engineering, Federal University of Pernambuco, P.O. Box 3447, 50100-100 Recife, PE, Brazil d Department of Antibiotics, Federal University of Pernambuco, P.O. Box 3447, 50100-100 Recife, PE, Brazil e Center of Sugarcane Technology, P.O. Box 162, 13400-970 Piracicaba, SP, Brazil b
article info
abstract
Article history:
Integral utilisation of bagasse is a high priority for the diversification of the sugarcane
Received 24 May 2010
industry. The application of a biorefinery philosophy to bagasse utilisation requires its
Received in revised form
fractionation into its main components: cellulose, hemicelluloses and lignin. The first stage
10 September 2010
in that process is the pretreatment, in which a considerable part of hemicelluloses is solu-
Accepted 18 October 2010
bilised, and cellulose is activated towards enzymatic hydrolysis. In this work, a pretreatment
Available online 20 November 2010
method using a mixture of sulfuric and acetic acid is investigated. Two different solid-toliquid ratios (1.5:10 and 1:10) were used in the pretreatment. Both conditions efficiently
Keywords:
hydrolysed the hemicelluloses giving removals above 90%. The extractive components were
Sugarcane bagasse
also effectively solubilised, and lignin was only slightly affected. Cellulose degradation was
Pretreatment
below 15%, which corresponded to the low crystallinity fraction. The analysis of the
Dilute-acid hydrolysis
morphology of pretreated bagasse confirmed the results obtained in the chemical
Ethanol
characterization.
Mixed acid
1.
Introduction
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 229 millions 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
ª 2010 Elsevier Ltd. All rights reserved.
technological improvements made to the boilers it is possible 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 bagasse as well 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]. Hydrolysis is crucial for the conversion of bagasse polysaccharides, mainly cellulose, into valuable products. However, the strong
* Corresponding author. Address: P.O. Box 116, 12600-970 Lorena, SP, Brazil. Tel.: þ55 12 31595030; fax: þ55 12 31533165. E-mail address: [email protected] (G. Jackson de Moraes Rocha). 0961-9534/$ e see front matter ª 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.biombioe.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 pretreatment methods have been proposed. Pretreatment and delignification processes are aimed to disrupt the celluloseehemicelluloseselignin 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 and mild enough as to avoid a significant chemical degradation of biomass components 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 lignocellulosic 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 hydrochloric, 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 acidcatalyzed process with diluted sulfuric and acetic acids at two different liquid-to-solid ratios in a rotary reactor, especially designed for this purpose.
2.
Materials and methods
2.1.
Raw material
Sugarcane bagasse, kindly donated by a sugar mill (Usina ´ gua, Camutanga, Pernambuco, Brazil), was Central Olho D’A used. A portion of bagasse was milled 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 solidto-liquid ratios. The pretreatment was performed in a 20-L rotary reactor (Regmed EU/E 20, Regmed Indu´stria Te´cnica Ltda.). The reactor, with a built electrical heating device, was
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 atmospheric pressure and 100 C, and was subsequentially discharged. The pretreated slurry was vacuum-filtered, and the sugar-rich liquid fraction was separated and stored frozen. The solid fraction (pretreated bagasse) was exhaustively washed with four 15-L portions of warm water (70 C), and dried 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 enzymatic hydrolysis.
3.
Theory/calculation
3.1.
Analysis of chemical composition
Raw and pretreated bagasse were analysed using a methodology 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 chromatographic quantification of sugars and degradation products contained in the hydrolyzate and by the gravimetric determination 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 previously weighed filter paper for gelatinous solids. The hydrolyzate 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 C until 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 previously weighed crucible, incinerated first at 300 C during 40 min and then at 800 C during 2 h. The mass of the ashes was 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
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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 min1, and were detected with an RI detector (Shimadzu RID-6A). Furfural and hydroxymethilfurfural (HMF) were separated on an RP-18 (C-18) de 125 4 mm (Hawlett-Packard), and were detected with a UV detector (Shimadzu SPD-10A) at 25 C using a mobile phase composed of a 1-% acetic acid-containing 1:8 acetonitrile-water solution pumped at 0.8 mL min1. 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 UVspectroscopy 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)
(31 ¼ 146,85 cm1 g1 L e experimentally obtained); 32: HMF absortivity at 280 nm (32 ¼ 114 cm1 g1 Le experimentally obtained); 3lig: lignin absortivity at 280 nm (3lig ¼ 23.7 cm1 g1 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].
1
Parameters: Clig: concentration of soluble lignin (g L ); Alig280: absorbance of the solution at 280 nm; Apd280: absorbance of sugar-degradation products (furfural and HMF) Apd280 ¼ C1 31 þ C2 32
(2)
Parameters: C1: furfural concentration (g L1); C2: HMF concentration (g L1); 31: furfural absortivity at 280 nm
4.
Results
4.1.
Data from the different samples of the experiments
(Table 1)
Table 1 e Chemical composition of in natura bagasse, bagasse pretreated at 1.5:10 solid-to-liquid ratio and bagasse pretreated at 1:10 solid-to-liquid ratio. Components
Dry matter, % Cellulose Hemicelluloses Total lignin* Mineral compounds Ethanol extractives Total
in naturaa (%)
at 1.5:10 solid-to-liquid ratiob (%)
at 1:10 solid-to-liquid ratioc (%)
Content (w/w)
Content (w/w)
Mass yield (g)
Losses
Content (w/w)
Mass yield (g)
Losses
100.00 45.5 1.1 27.0 0.8 21.1 0.9 2.2 0.1 4.6 0.3 100.4 0.4
100.00 58.06 0.61 3.79 0.02 32.38 0.31 5.90 0.28 ND 99.3 0.70
65.00 37.74 0.61 2.46 0.02 21.04 0.31 3.83 0.02 ND 67.48 0.70
e 13.1 90.8 0.3 e e e
100.00 61.65 0.50 2.79 0.03 32.96 0.25 3.97 0.02 ND 101.37 0.80
63.00 38.84 0.50 1.87 0.03 20.76 0.25 2.50 0.02 ND 63.97 0.70
e 14.6 93.4 4.7 e e e
ND - Not Detected. a Chemical composition of sugarcane bagasse “in natura”. *Sum of Klason lignin and acid-soluble lignin. b Chemical composition of the liquid fraction obtained by pretreatment of sugarcane bagasse at 1.5:10 solid-to-liquid ratio. c Chemical composition of the solid fraction obtained by pretreatment of sugarcane bagasse at 1:10 solid-to liquid ratio. *Sum of Klason lignin and acid-soluble lignin.
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Fig. 1 e Heating-up and cooling-down curve for the Regmed AU/EL20 reactor.
4.2.
Fig. 2 e Panoramic micrograph showing a bundle of fibres in bagasse pretreated at 1.5:10 solid-to-liquid ratio. Magnification: 503.
Reactor heating data
(Fig. 1)
4.3. ratio
Data from pretreatment at 1.5:10 solid-to-liquid
(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 pretreated bagasse (Fig. 8)
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
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 hemicelluloses 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 potentiate 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 pretreatment method was tested. The reactor was designed after modifications of a prototype previously used for wood pulping.
Table 2 e Chemical composition of the liquid fraction obtained by pretreatment of sugarcane bagasse at 1.5:10 and 1:10 solid-to-liquid ratios. Concentration, g L1
Compound
SLR: 1.5:10 Xylose Arabinose Glucose Cellobiose Acetic acid Glucuronic acid Formic acid Furfural HMF
9.04 1.03 3.67 0.20 2.21 0.21 0.74 0.05 0.01
0.04 0.02 0.16 0.00 0.04 0.00 0.00 0.00 0.00
SLR: 1:10 9.33 1.00 3.09 0.33 2.89 0.30 0.80 0.10 0.02
0.03 0.00 0.01 0.00 0.02 0.00 0.00 0.00 0.00
Fig. 3 e Micrograph of a bundle of fibres in bagasse pretreated at 1.5:10 solid-to-liquid ratio. Magnification: 50003.
b i o m a s s a n d b i o e n e r g y 3 5 ( 2 0 1 1 ) 6 6 3 e6 7 0
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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.
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].
5.1.
Pretreatment at 1.5:10 solid-to-liquid ratio
Parameters: ‘t’ and ‘T’ are the time (s) and the operating temperature (K), respectively. The pretreatment was performed at two different solid-toliquid 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.
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 pretreated 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 prehydrolyzate revealed that xylose was completely in monomeric
Fig. 5 e Panoramic micrograph bagasse pretreated at 1:10 solid-to-liquid ratio. Magnification: 503.
Fig. 7 e Amplification of another region of the same bundle of fibers. Magnification: 40003.
Z lnR0 ¼
T100
e 14:75 dt
(3)
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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).
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 significant 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 a magnification of 5000, shows a fiber with 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 hemicelluloses 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).
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, were of 90 and 93% respectively and induced a lignin loss of almost 5%. Experiments carried out with 1:10 solidto-liquid ratio, containing 0.1 g H2SO4/g dry bagasse, resulted in 70% of mass yield [29,30]. The same experiment carried without 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 oligosaccharides 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 solubilisation 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-crystalinity 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
The experiments on the enzymatic hydrolysis of the pretreated solids revealed that the pretreatment was effective in improving the enzymatic convertibility of cellulose. Pretreatment led to conversions above 76% after 72 h, whereas for pretreated bagasse only 6% of the initial cellulose was hydrolyzed (Fig. 8). The hydrolysis of the bagasse pretreated at 1:10 solid-to-liquid ratio had a higher initial rate, and a 75% cellulose was achieved in 48 h. However, no significant increase was 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.
b i o m a s s a n d b i o e n e r g y 3 5 ( 2 0 1 1 ) 6 6 3 e6 7 0
The chemical analyses revealed that both of the pretreatment 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 enzymatic 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-degradation inhibitory products. The morphological analyses revealed that the process was effective in disrupting the fibres and confirmed the results achieved in the chemical characterization.
Acknowledgments This work was possible thank to the financial support provided by the CNPq-MES cooperation program (grant No. 490830/ 2006-4). Sa˜o Carlos Chemistry Institute is acknowledged for the SEM analysis.
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