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Optimization of sugarcane bagasse conversion by hydrothermal treatment for the recovery of xylose
Bioresource Technology 100 (2009) 6537–6542
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Optimization of sugarcane bagasse conversion by hydrothermal treatment for the recovery of xylose Houda Boussarsar, Barbara Rogé, Mohamed Mathlouthi * Laboratoire de Chimie Physique Industrielle, UMR 614 Fractionnement des Agroressources et Emballage, Université de Reims Champagne Ardenne, Centre Europol’Agro, B.P. 1039, F-51687 Reims Cedex 2, France
a r t i c l e
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Article history: Received 6 April 2009 Received in revised form 8 July 2009 Accepted 8 July 2009 Available online 6 August 2009 Keywords: Sugarcane bagasse Biorefinery Hydrothermal treatment Hemicellulose Xylose
a b s t r a c t This work aims at the valorization of sugarcane bagasse by extracting xylose which is destined to the production of xylitol after purification and hydrogenation. Our approach consists in applying the principle of biorefinery to sugarcane bagasse because of its hemicellulose composition (particularly rich in xylan: (92%)). Optimizing of the thermal treatment was investigated. A treatment at 170 °C for 2 h was found optimal, with higher solubilzation of hemicellulose than that at 150 °C and lower degradation of sugar monomers than 190 °C. Recovery of xylose was high and the purity of xylose solution (78%) allows expecting an easy purification and separation of xylose before hydrogenation. Analysis of thermal hydrolyzates shows the presence of xylan oligomers and polymers with large distribution of DPs. This fraction should be submitted to enzymatic treatment to recover more xylose monomer. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Lignocellulosic biomass is one of the most available and renewable resources which represent a promising low cost raw material for the production of biofuel, bioenergy and added value biomolecules. Hemicellulosic materials such as agricultural residues (sugarcane bagasse, cereals bran and straw, corncob, brewer’s spent grain, etc.) thanks to their cellulose (40–60%), lignin (10–25%) and hemicellulose (20–40%) rich composition offer possibilities of valorization (Shleser, 1994). Current technologies for biomass exploitation are based on ‘‘destructive strategy” to obtain highly pure components like cellulose. Other biomass fractions generated by these technologies are either abandoned or burnt to produce electricity. This type of technology besides being environmentally harmful does not produce added value chemicals. Oppositely, the biorefinery concept constitutes an alternative approach for lignocellulosic materials utilization with interesting economic and environmental perspectives. Biorefinery consists in the fractionation of biomass to obtain different added value chemicals with an optimal exploitation of renewable resources on the basis of a sustainable agro-industry. Among biomass components, hemicelluloses which are mainly composed of xylans, provide an important source of interesting molecules such as xylose and xylo-oligosaccharides which have
* Corresponding author. Fax: +33 326 91 33 04. E-mail address: [email protected] (M. Mathlouthi). 0960-8524/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2009.07.019
potential applications in different areas, notably in chemical, food and pharmaceutical industries (Fooks et al., 1999). Several technologies were proposed for fractionation or extraction of hemicelluloses from feedstock. Some of these technologies were previously applied in industry especially in paper manufacturing. Conventional techniques for biomass fractionation were generally based on chemical treatments (Shevchenko et al., 2000; Garrote et al. 2001a,b; Montané et al., 2002). These processes became non competitive, because of the high cost of chemical reagents and the release of a large number of co-products and undesirable compounds which need an important purification step and may act as inhibitors in fermentation during the production of ethanol (Shah et al., 1984). As a consequence, chemical processes are replaced by enzymatic ones, which are specific and environment friendly. As the complex structure of lignocellulosic biomass limits enzyme accessibility, there is a need of a pretreatment to improve cellulose and/or hemicellulose susceptibility to enzymatic hydrolysis. Such pretreatment may be chemical, mechanical, hydrothermal or the combination of two or more of these processes (Saha & Bothast, 1999; Vazquez et al., 2001; Ballesteros et al., 2002; Saha, 2003). Recently, fractionation and solubilization studies of lignocellulosic materials by hydrothermal treatments have shown the efficiency of this technology to improve the yields of extraction of hemicelluloses (Allen et al., 2001; Laser et al., 2002; Kabel et al., 2002; Garrote et al., 2002; Carvalheiro et al., 2004; Parajó et al., 2004; Nabarlatz et al., 2007). Moreover, these treatments are compatible with sustainability and biorefinery concepts.
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Biomass hydrothermal treatment is based on the use of water (liquid water in high pressure reactor or water vapor) and heat (150–230 °C) for the treatment of lignocellulosic biomass (Laser et al., 2002). These treatments allow the selective obtaining of hydrolysates composed essentially of hemicellulose derivatives and solid pulp composed of cellulose and lignin residue (Van Walsum et al., 1996; Garrote et al., 1999). The advantage of this treatment is the prevention of equipment corrosion observed for acid hydrolysis and the absence of neutralization and acid recycling steps, which simplifies process conduction. Economic estimations show that autohydrolysis can be a useful and advantageous technique as compared to other alternative methods (Kubikova et al., 1996). In the present study, we report results of application of a hydrothermal treatment to lignocellulosic material aimed at the extraction of pentoses. The raw material used is sugarcane bagasse. The hydrothermal treatment of sugarcane bagasse permitted solubilization of the hemicellulosic fraction. An acidic hydrolysis was also performed to obtain the maximum extractible sugars and used as a reference standard in the evaluation of fractionation yield. Optimizing of the treatment was based on the minimization of impurity content in the obtained xylose juice.
2. Methods 2.1. Raw material Sugarcane bagasse used in this work was provided by CERF (Centre d’Essai, de Recherche et de Formation sur la canne à sucre, 97494, Sainte Clotilde- La Réunion). It was ground to obtain grain size below 1 cm. Before treatment, bagasse samples were swollen under mixing overnight in doubly distilled water. 2.2. Physico-chemical characterization of sugarcane bagasse 2.2.1. Chemical composition Carbohydrate composition of sugarcane bagasse was determined by high performance anion exchange chromatography (HPAEC). Solid sample of bagasse (10 mg) was pre-hydrolyzed using H2SO4 (12 M, 2 h, 25 °C under agitation) diluted to 2 M and heated (2 h, 100 °C) (Seaman et al., 1954). The sample was diluted 50-fold, filtered and injected onto a Carbo Pac PA1 anion-exchange column (4 250 mm, Dionex). Neutral monosaccharides were separated in 0.1 M NaOH. The flow rate was 1 ml/min and detection was performed by pulsed amperometry (PAD2, Dionex). Lignin content was determined by acetyl bromide procedure used by Iiyama and Wallis (1990) and water was obtained by oven drying of a sample of bagasse (2 g) at 105 °C until constant weight. Ash content was also determined gravimetrically as the residual mass of a bagasse sample after incineration at 500 °C during 4 h. 2.2.2. Determination of Water absorbency and Swollen Volume of bagasse The capacity of bagasse to absorb water was determined using the method of Gould et al. (1989) with some modifications. A dry sample (3 g) of bagasse was mixed with an excess of doubly distilled water and allowed to the hydrate for 2 h. The excess water was then removed by draining on a fine-meshed wire screen. A portion of the wet sample on the screen was removed, weighed, and dried to constant weight at 105 °C. The water absorbency (g of absorbed water/g dry material) was defined as (wet weight– dry weight)/dry weight (Sangnark and Noomhorm, 2003). The volume occupied by the fully hydrated bagasse sample (swollen volume) was measured by mixing 1 g of bagasse with a large excess of doubly distilled water in a graduated cylinder.
The suspension was mixed intermittently for several hours to ensure complete hydration of the sample and allow its settling overnight. The volume in the cylinder occupied by the swollen particles was taken as the swollen volume in ml/g of dry bagasse (Gould et al., 1989). 2.3. Hydrothermal treatment of bagasse Hydrothermal treatment consists in submitting a sample of bagasse to high temperature and pressure in presence of water. A special batch reactor was set to achieve bagasse thermal treatment, where a suspension of bagasse (5% dry masse/volume) was introduced. The desired temperature in reactor was obtained with a thermostated oil bath (Bioblock Scientific 06 Huber CC41). Several hydrothermal treatments were conducted by varying treatment temperature (150 °C, 170 °C and 190 °C) and time. Treatment stopping was obtained by a progressive cooling at ambient temperature. Then, reactor is open and the solubilized fraction separated from solid residue by vacuum filtration. Hydrolysates were stored at 4 °C before analysis. 2.4. Acid hydrolysis of bagasse The acid hydrolysis of bagasse (5% (w/v)) was conducted under the same operational conditions as hydrothermal treatment, with adding of sulfuric acid H2SO4 (1% v/v). The acid hydrolysis was achieved at 150 °C and different treatment durations. 2.5. Sugar analysis by HPAEC Monosaccharides composition of the hydrolyzates obtained by hydrothermal and acid treatments was determined by high performance anion exchange chromatography (HPAEC) as described in Section 2.2.1. Before analysis, hydrothermal soluble fractions were hydrolyzed in H2SO4 (2 M, 1 h at 100 °C) (Seaman et al., 1954). Monosaccharides (xylose, arabinose, glucose and galactose) were used as references and fucose was added as internal standard. 2.6. Reducing sugar determination The amount of reducing sugar extremities released from bagasse by acid hydrothermal treatment was measured by the Kidby and Davidson method (Kidby and Davidson, 1973). One hundred microlitres aliquots of hydrolysates were added to 1.5 ml of Kidby reagent in Eppendorf testing tube. The mixture was heated to boiling point during 5 min, and then cooled 5 min in tap water at room temperature. The absorbance at 420 nm was measured using a UV–vis spectrophotometer (Shimadzu UV-2101 PC). Concentrations of free reducing sugar released by hydrolysis in the reaction medium were determined after establishing of a calibration curve with xylose at different concentrations (ranging from 0 to 400 lg/ml). 2.7. Hydrolysate dry mass determination The dry mass of fractions solubilized by hydrothermal treatment was determined by drying 10 ml of hydrolysate overnight at 105 °C. The obtained residue was then weighed (Bobleter et al., 1989). The difference of weight between hydrolyzate dry mass and the xylose content in the same sample gives the impurities content. 2.8. Structural characterization of hemicelluloses 2.8.1. Thin layer chromatography (CCM) Hydrolysates obtained at selected operational conditions are diluted and analyzed by thin layer chromatography according to the
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protocol used by Boussarsar et al. (2007). Xylose, xylobiose, xylotriose and xylotetraose are used as references. 2.8.2. High performance size exclusion chromatography (HPSEC) Molecular characterization of xylan solubilized was performed using a multi-detector high performance size exclusion chromatographic (HPSEC) system, connected on line to a UV (Waters 2996), a refractive index (RI) (Waters 410) and a Multi-Angle Laser Light Scattering (MALLS) detector (Dawn MALLS; 632.8 nm; Wyatt corporation). Solution of NaNO3 50 mM containing 0.02% NaN3 was used as eluent. Samples (200 ll) are filtered on 0.45 lm PTFE filter. Chromatographic separation of injected solutions was achieved on a thermostatically controlled Shodex KD (802, 803 and 805 M) (each 4.6 300 mm) column used at a flow rate of 1 ml/min. The MALLS photometer was calibrated with toluene and the responses of the detector signal using cow molecular weight monodisperse pullulan (Shodex standard). On-line data collection and post-collection treatment (calculation of molecular weight, distribution and averages) were performed using Astra for Windows 4.73 (Wyatt technology, Santa Barbara, CA). 3. Results and discussion 3.1. Physico-chemical properties of bagasse 3.1.1. Chemical composition Sugarcane bagasse polysaccharide composition is presented in Table 1. Chemical composition confirms that lignocellulosic biomass is mainly formed by cellulose, hemicellulose and lignin. The hemicellulosic fraction is 26%, with preponderance of xylose (92%) as monosaccharide which suggests the availability of only slightly branched xylan in the hemicellulosic fractions. Bagasse also contains a relatively high amount of lignin (20%) comparable to that in wheat straw (Rémond-Zilliox, 1996). Lignin strengthens the bagasse structure and influences its suspension texture in water. Moreover, lignin hydrophobic character considerably reduces the bagasse moisturizing facility. 3.1.2. Water absorbency and swollen volume Bagasse water absorbency was found equal to 6.16 ± 0.6 g water/g dry mass and the swollen volume is 1.5 ml/g dry bagasse. The low bagasse density (492 ± 15 kg/m3 (Rasul et al., 1999) and the low volume of water associated to bagasse make it necessary to use a low solid/liquid ratio to ensure the efficiency of the hydrothermal treatment.
Fig. 1. Concentrations of xylose, glucose and reducing sugars released by acid treatment (H2SO41% at 150 °C) from sugarcane bagasse versus time.
tion dry mass. This maximal yield was obtained after 20 min of treatment. After this duration the amount of xylose and reducing sugars start decreasing very likely because of the progressive degradation of the solubilized compounds under the action of heat and acid. Fig. 1 also shows a rapid decrease of reducing sugars. A delayed neutralization of the reaction medium can considerably affect the extraction yield. Therefore, the necessity of use of a moderate treatment like the hydrothermal method becomes evident. 3.3. Sugarcane bagasse hydrothermal treatment optimization Bagasse hydrothermal treatment permitted obtaining of two fractions: a soluble fraction containing hemicellulose derivates and a residual solid fraction of the treated bagasse. Hydrothermal treatment optimization step was achieved by varying treatment time and temperature to select operational conditions which allow obtaining of a high xylan solubilization with a minimum of impurities. Bagasse biomass was treated at three temperatures 150 °C, 170 °C and 190 °C and different durations. The obtained solubilized fractions were then analyzed by HPAEC in order to determine sol-
3.2. Maximum extractible xylose: acid hydrolysis of bagasse The highest extractible xylose concentration obtained by acid treatment is reported in Fig. 1. Glucose and the other liberated reducing sugars concentrations are summarized in the same figure. Results confirm that acid treatment of bagasse allows release of a maximal concentration of reducing sugars (15.65 g/l including 11 g/l xylose), which corresponds to a conversion yield of 0.22 g xylose/g dry bagasse corresponding to 88% of hemicellulose frac-
Table 1 Chemical composition of sugarcane bagasse. Polysaccharides
Content (%)
Cellulose Hemicellulose (xylose) Lignin Ash Other compounds (of which salt)
45 26 (92) 20 2.1 9
Fig. 2. HPAEC analysis of xylose composition of hydrolysates obtained by hydrothermal treatment of sugarcane bagasse at different temperatures (150 °C, 170 °C and 190 °C).
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Fig. 3. Variation of concentration of non-xylose sugars. arabinose (a) glucose (b) et le galactose (c) obtained after thermal treatments at 150 °C, 170 °C and 190 °C.
Fig. 4. Variation of hydrolyzate dry masses and xylose concentrations versus hydrothermal treatment time at three temperatures. 150 °C (a) 170 °C (b) and 190 °C (c).
ubilized sugar percentage. Dry masses of the different fractions were analyzed. 3.3.1. HPAEC analysis Fig. 2 presents HPAEC analysis results of the hydrolysates at the three studied temperatures. We observe at 150 °C a minimal extraction and a slow solubilization in function of treatment time. We obtain a comparable extraction rate at 170 °C and 190 °C, but xylose concentration seems to decrease after two hours at 190 °C. This concentration drop can be explained by a progressive degradation of the monomeric sugars liberated at high tempera-
ture. For this reason, we select 170 °C as optimal temperature for sugarcane bagasse hydrothermal treatment. Maximal yields obtained were 22.6 ± 0.22% (w/w) at 150 °C, 53.56 ± 0.22% (w/w) at 190 °C. At 170 °C, a maximal yield of 55 ± 0.22% (w/w) is obtained after 4 h, but 2 h permitted to reach 48.8 ± 0.22% of extraction yield. Therefore, 2 h were selected as optimal treatment duration. In terms of efficiency, it does not seem worthy adding 2 h of treatment to obtain only 7% as supplementary solubilization.
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Fig. 5. Purity degrees of sugarcane bagasse hydrothermal hydrolysates obtained at different time and temperature conditions.
Concentration of non xylose sugars (arabinose, glucose and galactose) was determined after thermal treatment at each of the 3 temperatures applied. Results are reported in Fig. 3. These monosaccharides are considered as impurities in presence of the target molecule (xylose). However, the percentage of monosaccharides obtained after thermal treatment remains low (<1 g/l) as compared to acid hydrolysis. Apart from generating more impurities in the xylose solution, acid treatment poses the problem of feasibility because of the technical constraints of recycling the acid reagent and using acid resistant material for the reactor. 3.3.2. Dry mass analysis Analyses of hydrolyzates dry masses for the three studied temperatures are represented in Fig. 4. The three graphs present the dry masses and xylose concentrations of the different hydrolyzates. The difference between dry masses and xylose concentrations represents impurities contents. All solubilized compounds other than xylose and xylo-oligosaccharides, such as solubilized lignin, phenolic acids in addition to the eventual degradation products are considered as impurities. Treatment at 150 °C is not exploited because it does not permit a good solubilization of xylan. For 170 °C and 190 °C extraction rates are above 50%, but hydrolyzates dry masses analyses show a higher concentration of impurities at 190 °C as compared to 170 °C. 3.3.3. Hydrothermal hydrolysates purity analysis The purity of hydrolyzates is calculated as the ratio of xylose concentration to the total sugar concentrations released during processing. The corresponding purity degrees obtained for the different hydrothermal treatment carried out in different conditions are given in Fig. 5 showing a maximal purity degree of 78%. The increase in purity in extreme operating conditions could be explained by the high degradation of sugars. 3.4. Hydrothermal treatment product characterization 3.4.1. Thin layer chromatography The thin layer chromatogram presented in Fig. 6 shows the different bands corresponding to a hydrolyzate resulting from a hydrothermal treatment at the selected operational conditions (170 °C/2 h) diluted 5 times. Results reveal a large distribution of DPs of released products. The low resolution of bands separation can be attributed to the large variety of saccharides obtained. We note the relative importance of the xylose band which demonstrates the ability of the hydrothermal treatment to liberate xylose.
Fig. 6. Thin layer chromatography analysis of hydrothermal hydrolysate obtained at 170 °C/2 h.
3.4.2. High performance size exclusion chromatography (HPSEC) Several hydrothermal hydrolyzates (4 samples) obtained in the same operating conditions of the selected treatment were analyzed by high performance size exclusion chromatography (HPSEC) to characterize the macromolecular hydrolyzates composition. Data calculated from chromatograms obtained are given in Table 2, which summarizes the results of the chromatographic size exclusion high-performance analysis for the four repeated hydrothermal hydrolyzates. The analysis of the polydispersity index shows values close to 1.5 for the four samples, indicating that their composition tends to be uniform. The distribution of lengths of chain is low with a relatively homogeneous population. This result is consistent, as it is known that the adopted method allows mainly to release long chains of xylan and a low quantity short chains and monomeric xylose. The hydrolyzate (1) has the highest polydispersity with a value of 1.78 which remains relatively low (below 2). The analysis of the polydispersity index permits to confirm the repeatability of hydro-
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Table 2 Macromolecular characteristics of hydrothermal hydrolysates (4 repetitions) determined by high performance size exclusion chromatography (HPSEC).
(1) (2) (3) (4) a b c
Retention time (min)
Mwa (g/mol)
Mnb (g/mol)
Pc = Mw/Mn
26.202 26.215 26.204 26.176
4825 3418 3416 4971
2697 2156 2173 3297
1.789344 1.585411 1.572338 1.507685
Mw: Average molecular weight. Mn: Average weight of constituents with low molecular weight. P: Polydispersity index = Mw/Mn.
thermal treatment. The analysis of the average molecular weight (Mw) and the average mass of the constituents of low molecular weight (Mn) confirms results obtained by the analysis of the polydispersity. Indeed Mn gives an idea about the composition on short chains whereas Mw on the longest one. The sample (1) with the highest difference between its Mw and Mn present the highest recorded polydispersity index. Hydrolyzates (2) and (3) present almost equal Mw and Mn confirming the repeatability of the hydrothermal treatment. The retention time, for all analyzed samples was about 26 min confirming the similarity of the composition of the four hydrolyzates and therefore the repeatability of the hydrothermal treatment. 4. Conclusion A hydrothermal treatment of sugarcane bagasse during 2 h at 170 °C permitted obtaining of an acceptable xylose extraction yield. Xylose and dry mass analysis allow supporting the choice of 170 °C for 2 h as an optimal treatment for xylose recovery with a minimum of impurities. Another advantage of the chosen moderate operational conditions is the utilization of water as the sole reagent. Structural characterization of hydrothermal hydrolysates obtained at 170 °C show a large distribution of DPs of released xylo-oligosaccharides. This work will be followed by the enzymatic hydrolysis of oligomers to increase the recovery of xylose monomer. Acknowledgements The authors acknowledge the analytical facilities provided by the team ‘‘Transformation of lignocelluloses” at INRA of Reims (UMR 614) and the support by Roquette Frères of this work. References Allen, S.G., Schulman, D., Laser, M., Lichwa, J., Antal, J.R., Spencer, M.J., Lynd, L.R., 2001. A comparison between hot liquid water and steam fractionation of corn fiber. Ind. Eng. Chem. Res. 40, 2934–2941. Ballesteros, I., Oliva, J.M., Negro, M.J., Manzanares, P., Ballesteros, M., 2002. Enzymic hydrolysis of steam exploded herbaceous agricultural waste (Brassica carinata) at different particule sizes. Process Biochem. 38 (2), 187–192. Bobleter, O., Vidotti, R., Zemann, A., Prutsch, W., 1989. Hydrothermal pretreatment of bagasse and wheat straw. Biomass for energy and industry. In: Grassi, G., Gross, G., dos Santos, (Eds.), Proceedings of the Fifth EC Conference, vol. 2, Elsevier, New York, pp. 31–37.
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Optimization of sugarcane bagasse conversion by hydrothermal treatment for the recovery of xylose Houda Boussarsar, Barbara Rogé, Mohamed Mathlouthi * Laboratoire de Chimie Physique Industrielle, UMR 614 Fractionnement des Agroressources et Emballage, Université de Reims Champagne Ardenne, Centre Europol’Agro, B.P. 1039, F-51687 Reims Cedex 2, France
a r t i c l e
i n f o
Article history: Received 6 April 2009 Received in revised form 8 July 2009 Accepted 8 July 2009 Available online 6 August 2009 Keywords: Sugarcane bagasse Biorefinery Hydrothermal treatment Hemicellulose Xylose
a b s t r a c t This work aims at the valorization of sugarcane bagasse by extracting xylose which is destined to the production of xylitol after purification and hydrogenation. Our approach consists in applying the principle of biorefinery to sugarcane bagasse because of its hemicellulose composition (particularly rich in xylan: (92%)). Optimizing of the thermal treatment was investigated. A treatment at 170 °C for 2 h was found optimal, with higher solubilzation of hemicellulose than that at 150 °C and lower degradation of sugar monomers than 190 °C. Recovery of xylose was high and the purity of xylose solution (78%) allows expecting an easy purification and separation of xylose before hydrogenation. Analysis of thermal hydrolyzates shows the presence of xylan oligomers and polymers with large distribution of DPs. This fraction should be submitted to enzymatic treatment to recover more xylose monomer. Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction Lignocellulosic biomass is one of the most available and renewable resources which represent a promising low cost raw material for the production of biofuel, bioenergy and added value biomolecules. Hemicellulosic materials such as agricultural residues (sugarcane bagasse, cereals bran and straw, corncob, brewer’s spent grain, etc.) thanks to their cellulose (40–60%), lignin (10–25%) and hemicellulose (20–40%) rich composition offer possibilities of valorization (Shleser, 1994). Current technologies for biomass exploitation are based on ‘‘destructive strategy” to obtain highly pure components like cellulose. Other biomass fractions generated by these technologies are either abandoned or burnt to produce electricity. This type of technology besides being environmentally harmful does not produce added value chemicals. Oppositely, the biorefinery concept constitutes an alternative approach for lignocellulosic materials utilization with interesting economic and environmental perspectives. Biorefinery consists in the fractionation of biomass to obtain different added value chemicals with an optimal exploitation of renewable resources on the basis of a sustainable agro-industry. Among biomass components, hemicelluloses which are mainly composed of xylans, provide an important source of interesting molecules such as xylose and xylo-oligosaccharides which have
* Corresponding author. Fax: +33 326 91 33 04. E-mail address: [email protected] (M. Mathlouthi). 0960-8524/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2009.07.019
potential applications in different areas, notably in chemical, food and pharmaceutical industries (Fooks et al., 1999). Several technologies were proposed for fractionation or extraction of hemicelluloses from feedstock. Some of these technologies were previously applied in industry especially in paper manufacturing. Conventional techniques for biomass fractionation were generally based on chemical treatments (Shevchenko et al., 2000; Garrote et al. 2001a,b; Montané et al., 2002). These processes became non competitive, because of the high cost of chemical reagents and the release of a large number of co-products and undesirable compounds which need an important purification step and may act as inhibitors in fermentation during the production of ethanol (Shah et al., 1984). As a consequence, chemical processes are replaced by enzymatic ones, which are specific and environment friendly. As the complex structure of lignocellulosic biomass limits enzyme accessibility, there is a need of a pretreatment to improve cellulose and/or hemicellulose susceptibility to enzymatic hydrolysis. Such pretreatment may be chemical, mechanical, hydrothermal or the combination of two or more of these processes (Saha & Bothast, 1999; Vazquez et al., 2001; Ballesteros et al., 2002; Saha, 2003). Recently, fractionation and solubilization studies of lignocellulosic materials by hydrothermal treatments have shown the efficiency of this technology to improve the yields of extraction of hemicelluloses (Allen et al., 2001; Laser et al., 2002; Kabel et al., 2002; Garrote et al., 2002; Carvalheiro et al., 2004; Parajó et al., 2004; Nabarlatz et al., 2007). Moreover, these treatments are compatible with sustainability and biorefinery concepts.
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Biomass hydrothermal treatment is based on the use of water (liquid water in high pressure reactor or water vapor) and heat (150–230 °C) for the treatment of lignocellulosic biomass (Laser et al., 2002). These treatments allow the selective obtaining of hydrolysates composed essentially of hemicellulose derivatives and solid pulp composed of cellulose and lignin residue (Van Walsum et al., 1996; Garrote et al., 1999). The advantage of this treatment is the prevention of equipment corrosion observed for acid hydrolysis and the absence of neutralization and acid recycling steps, which simplifies process conduction. Economic estimations show that autohydrolysis can be a useful and advantageous technique as compared to other alternative methods (Kubikova et al., 1996). In the present study, we report results of application of a hydrothermal treatment to lignocellulosic material aimed at the extraction of pentoses. The raw material used is sugarcane bagasse. The hydrothermal treatment of sugarcane bagasse permitted solubilization of the hemicellulosic fraction. An acidic hydrolysis was also performed to obtain the maximum extractible sugars and used as a reference standard in the evaluation of fractionation yield. Optimizing of the treatment was based on the minimization of impurity content in the obtained xylose juice.
2. Methods 2.1. Raw material Sugarcane bagasse used in this work was provided by CERF (Centre d’Essai, de Recherche et de Formation sur la canne à sucre, 97494, Sainte Clotilde- La Réunion). It was ground to obtain grain size below 1 cm. Before treatment, bagasse samples were swollen under mixing overnight in doubly distilled water. 2.2. Physico-chemical characterization of sugarcane bagasse 2.2.1. Chemical composition Carbohydrate composition of sugarcane bagasse was determined by high performance anion exchange chromatography (HPAEC). Solid sample of bagasse (10 mg) was pre-hydrolyzed using H2SO4 (12 M, 2 h, 25 °C under agitation) diluted to 2 M and heated (2 h, 100 °C) (Seaman et al., 1954). The sample was diluted 50-fold, filtered and injected onto a Carbo Pac PA1 anion-exchange column (4 250 mm, Dionex). Neutral monosaccharides were separated in 0.1 M NaOH. The flow rate was 1 ml/min and detection was performed by pulsed amperometry (PAD2, Dionex). Lignin content was determined by acetyl bromide procedure used by Iiyama and Wallis (1990) and water was obtained by oven drying of a sample of bagasse (2 g) at 105 °C until constant weight. Ash content was also determined gravimetrically as the residual mass of a bagasse sample after incineration at 500 °C during 4 h. 2.2.2. Determination of Water absorbency and Swollen Volume of bagasse The capacity of bagasse to absorb water was determined using the method of Gould et al. (1989) with some modifications. A dry sample (3 g) of bagasse was mixed with an excess of doubly distilled water and allowed to the hydrate for 2 h. The excess water was then removed by draining on a fine-meshed wire screen. A portion of the wet sample on the screen was removed, weighed, and dried to constant weight at 105 °C. The water absorbency (g of absorbed water/g dry material) was defined as (wet weight– dry weight)/dry weight (Sangnark and Noomhorm, 2003). The volume occupied by the fully hydrated bagasse sample (swollen volume) was measured by mixing 1 g of bagasse with a large excess of doubly distilled water in a graduated cylinder.
The suspension was mixed intermittently for several hours to ensure complete hydration of the sample and allow its settling overnight. The volume in the cylinder occupied by the swollen particles was taken as the swollen volume in ml/g of dry bagasse (Gould et al., 1989). 2.3. Hydrothermal treatment of bagasse Hydrothermal treatment consists in submitting a sample of bagasse to high temperature and pressure in presence of water. A special batch reactor was set to achieve bagasse thermal treatment, where a suspension of bagasse (5% dry masse/volume) was introduced. The desired temperature in reactor was obtained with a thermostated oil bath (Bioblock Scientific 06 Huber CC41). Several hydrothermal treatments were conducted by varying treatment temperature (150 °C, 170 °C and 190 °C) and time. Treatment stopping was obtained by a progressive cooling at ambient temperature. Then, reactor is open and the solubilized fraction separated from solid residue by vacuum filtration. Hydrolysates were stored at 4 °C before analysis. 2.4. Acid hydrolysis of bagasse The acid hydrolysis of bagasse (5% (w/v)) was conducted under the same operational conditions as hydrothermal treatment, with adding of sulfuric acid H2SO4 (1% v/v). The acid hydrolysis was achieved at 150 °C and different treatment durations. 2.5. Sugar analysis by HPAEC Monosaccharides composition of the hydrolyzates obtained by hydrothermal and acid treatments was determined by high performance anion exchange chromatography (HPAEC) as described in Section 2.2.1. Before analysis, hydrothermal soluble fractions were hydrolyzed in H2SO4 (2 M, 1 h at 100 °C) (Seaman et al., 1954). Monosaccharides (xylose, arabinose, glucose and galactose) were used as references and fucose was added as internal standard. 2.6. Reducing sugar determination The amount of reducing sugar extremities released from bagasse by acid hydrothermal treatment was measured by the Kidby and Davidson method (Kidby and Davidson, 1973). One hundred microlitres aliquots of hydrolysates were added to 1.5 ml of Kidby reagent in Eppendorf testing tube. The mixture was heated to boiling point during 5 min, and then cooled 5 min in tap water at room temperature. The absorbance at 420 nm was measured using a UV–vis spectrophotometer (Shimadzu UV-2101 PC). Concentrations of free reducing sugar released by hydrolysis in the reaction medium were determined after establishing of a calibration curve with xylose at different concentrations (ranging from 0 to 400 lg/ml). 2.7. Hydrolysate dry mass determination The dry mass of fractions solubilized by hydrothermal treatment was determined by drying 10 ml of hydrolysate overnight at 105 °C. The obtained residue was then weighed (Bobleter et al., 1989). The difference of weight between hydrolyzate dry mass and the xylose content in the same sample gives the impurities content. 2.8. Structural characterization of hemicelluloses 2.8.1. Thin layer chromatography (CCM) Hydrolysates obtained at selected operational conditions are diluted and analyzed by thin layer chromatography according to the
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protocol used by Boussarsar et al. (2007). Xylose, xylobiose, xylotriose and xylotetraose are used as references. 2.8.2. High performance size exclusion chromatography (HPSEC) Molecular characterization of xylan solubilized was performed using a multi-detector high performance size exclusion chromatographic (HPSEC) system, connected on line to a UV (Waters 2996), a refractive index (RI) (Waters 410) and a Multi-Angle Laser Light Scattering (MALLS) detector (Dawn MALLS; 632.8 nm; Wyatt corporation). Solution of NaNO3 50 mM containing 0.02% NaN3 was used as eluent. Samples (200 ll) are filtered on 0.45 lm PTFE filter. Chromatographic separation of injected solutions was achieved on a thermostatically controlled Shodex KD (802, 803 and 805 M) (each 4.6 300 mm) column used at a flow rate of 1 ml/min. The MALLS photometer was calibrated with toluene and the responses of the detector signal using cow molecular weight monodisperse pullulan (Shodex standard). On-line data collection and post-collection treatment (calculation of molecular weight, distribution and averages) were performed using Astra for Windows 4.73 (Wyatt technology, Santa Barbara, CA). 3. Results and discussion 3.1. Physico-chemical properties of bagasse 3.1.1. Chemical composition Sugarcane bagasse polysaccharide composition is presented in Table 1. Chemical composition confirms that lignocellulosic biomass is mainly formed by cellulose, hemicellulose and lignin. The hemicellulosic fraction is 26%, with preponderance of xylose (92%) as monosaccharide which suggests the availability of only slightly branched xylan in the hemicellulosic fractions. Bagasse also contains a relatively high amount of lignin (20%) comparable to that in wheat straw (Rémond-Zilliox, 1996). Lignin strengthens the bagasse structure and influences its suspension texture in water. Moreover, lignin hydrophobic character considerably reduces the bagasse moisturizing facility. 3.1.2. Water absorbency and swollen volume Bagasse water absorbency was found equal to 6.16 ± 0.6 g water/g dry mass and the swollen volume is 1.5 ml/g dry bagasse. The low bagasse density (492 ± 15 kg/m3 (Rasul et al., 1999) and the low volume of water associated to bagasse make it necessary to use a low solid/liquid ratio to ensure the efficiency of the hydrothermal treatment.
Fig. 1. Concentrations of xylose, glucose and reducing sugars released by acid treatment (H2SO41% at 150 °C) from sugarcane bagasse versus time.
tion dry mass. This maximal yield was obtained after 20 min of treatment. After this duration the amount of xylose and reducing sugars start decreasing very likely because of the progressive degradation of the solubilized compounds under the action of heat and acid. Fig. 1 also shows a rapid decrease of reducing sugars. A delayed neutralization of the reaction medium can considerably affect the extraction yield. Therefore, the necessity of use of a moderate treatment like the hydrothermal method becomes evident. 3.3. Sugarcane bagasse hydrothermal treatment optimization Bagasse hydrothermal treatment permitted obtaining of two fractions: a soluble fraction containing hemicellulose derivates and a residual solid fraction of the treated bagasse. Hydrothermal treatment optimization step was achieved by varying treatment time and temperature to select operational conditions which allow obtaining of a high xylan solubilization with a minimum of impurities. Bagasse biomass was treated at three temperatures 150 °C, 170 °C and 190 °C and different durations. The obtained solubilized fractions were then analyzed by HPAEC in order to determine sol-
3.2. Maximum extractible xylose: acid hydrolysis of bagasse The highest extractible xylose concentration obtained by acid treatment is reported in Fig. 1. Glucose and the other liberated reducing sugars concentrations are summarized in the same figure. Results confirm that acid treatment of bagasse allows release of a maximal concentration of reducing sugars (15.65 g/l including 11 g/l xylose), which corresponds to a conversion yield of 0.22 g xylose/g dry bagasse corresponding to 88% of hemicellulose frac-
Table 1 Chemical composition of sugarcane bagasse. Polysaccharides
Content (%)
Cellulose Hemicellulose (xylose) Lignin Ash Other compounds (of which salt)
45 26 (92) 20 2.1 9
Fig. 2. HPAEC analysis of xylose composition of hydrolysates obtained by hydrothermal treatment of sugarcane bagasse at different temperatures (150 °C, 170 °C and 190 °C).
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Fig. 3. Variation of concentration of non-xylose sugars. arabinose (a) glucose (b) et le galactose (c) obtained after thermal treatments at 150 °C, 170 °C and 190 °C.
Fig. 4. Variation of hydrolyzate dry masses and xylose concentrations versus hydrothermal treatment time at three temperatures. 150 °C (a) 170 °C (b) and 190 °C (c).
ubilized sugar percentage. Dry masses of the different fractions were analyzed. 3.3.1. HPAEC analysis Fig. 2 presents HPAEC analysis results of the hydrolysates at the three studied temperatures. We observe at 150 °C a minimal extraction and a slow solubilization in function of treatment time. We obtain a comparable extraction rate at 170 °C and 190 °C, but xylose concentration seems to decrease after two hours at 190 °C. This concentration drop can be explained by a progressive degradation of the monomeric sugars liberated at high tempera-
ture. For this reason, we select 170 °C as optimal temperature for sugarcane bagasse hydrothermal treatment. Maximal yields obtained were 22.6 ± 0.22% (w/w) at 150 °C, 53.56 ± 0.22% (w/w) at 190 °C. At 170 °C, a maximal yield of 55 ± 0.22% (w/w) is obtained after 4 h, but 2 h permitted to reach 48.8 ± 0.22% of extraction yield. Therefore, 2 h were selected as optimal treatment duration. In terms of efficiency, it does not seem worthy adding 2 h of treatment to obtain only 7% as supplementary solubilization.
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Fig. 5. Purity degrees of sugarcane bagasse hydrothermal hydrolysates obtained at different time and temperature conditions.
Concentration of non xylose sugars (arabinose, glucose and galactose) was determined after thermal treatment at each of the 3 temperatures applied. Results are reported in Fig. 3. These monosaccharides are considered as impurities in presence of the target molecule (xylose). However, the percentage of monosaccharides obtained after thermal treatment remains low (<1 g/l) as compared to acid hydrolysis. Apart from generating more impurities in the xylose solution, acid treatment poses the problem of feasibility because of the technical constraints of recycling the acid reagent and using acid resistant material for the reactor. 3.3.2. Dry mass analysis Analyses of hydrolyzates dry masses for the three studied temperatures are represented in Fig. 4. The three graphs present the dry masses and xylose concentrations of the different hydrolyzates. The difference between dry masses and xylose concentrations represents impurities contents. All solubilized compounds other than xylose and xylo-oligosaccharides, such as solubilized lignin, phenolic acids in addition to the eventual degradation products are considered as impurities. Treatment at 150 °C is not exploited because it does not permit a good solubilization of xylan. For 170 °C and 190 °C extraction rates are above 50%, but hydrolyzates dry masses analyses show a higher concentration of impurities at 190 °C as compared to 170 °C. 3.3.3. Hydrothermal hydrolysates purity analysis The purity of hydrolyzates is calculated as the ratio of xylose concentration to the total sugar concentrations released during processing. The corresponding purity degrees obtained for the different hydrothermal treatment carried out in different conditions are given in Fig. 5 showing a maximal purity degree of 78%. The increase in purity in extreme operating conditions could be explained by the high degradation of sugars. 3.4. Hydrothermal treatment product characterization 3.4.1. Thin layer chromatography The thin layer chromatogram presented in Fig. 6 shows the different bands corresponding to a hydrolyzate resulting from a hydrothermal treatment at the selected operational conditions (170 °C/2 h) diluted 5 times. Results reveal a large distribution of DPs of released products. The low resolution of bands separation can be attributed to the large variety of saccharides obtained. We note the relative importance of the xylose band which demonstrates the ability of the hydrothermal treatment to liberate xylose.
Fig. 6. Thin layer chromatography analysis of hydrothermal hydrolysate obtained at 170 °C/2 h.
3.4.2. High performance size exclusion chromatography (HPSEC) Several hydrothermal hydrolyzates (4 samples) obtained in the same operating conditions of the selected treatment were analyzed by high performance size exclusion chromatography (HPSEC) to characterize the macromolecular hydrolyzates composition. Data calculated from chromatograms obtained are given in Table 2, which summarizes the results of the chromatographic size exclusion high-performance analysis for the four repeated hydrothermal hydrolyzates. The analysis of the polydispersity index shows values close to 1.5 for the four samples, indicating that their composition tends to be uniform. The distribution of lengths of chain is low with a relatively homogeneous population. This result is consistent, as it is known that the adopted method allows mainly to release long chains of xylan and a low quantity short chains and monomeric xylose. The hydrolyzate (1) has the highest polydispersity with a value of 1.78 which remains relatively low (below 2). The analysis of the polydispersity index permits to confirm the repeatability of hydro-
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Table 2 Macromolecular characteristics of hydrothermal hydrolysates (4 repetitions) determined by high performance size exclusion chromatography (HPSEC).
(1) (2) (3) (4) a b c
Retention time (min)
Mwa (g/mol)
Mnb (g/mol)
Pc = Mw/Mn
26.202 26.215 26.204 26.176
4825 3418 3416 4971
2697 2156 2173 3297
1.789344 1.585411 1.572338 1.507685
Mw: Average molecular weight. Mn: Average weight of constituents with low molecular weight. P: Polydispersity index = Mw/Mn.
thermal treatment. The analysis of the average molecular weight (Mw) and the average mass of the constituents of low molecular weight (Mn) confirms results obtained by the analysis of the polydispersity. Indeed Mn gives an idea about the composition on short chains whereas Mw on the longest one. The sample (1) with the highest difference between its Mw and Mn present the highest recorded polydispersity index. Hydrolyzates (2) and (3) present almost equal Mw and Mn confirming the repeatability of the hydrothermal treatment. The retention time, for all analyzed samples was about 26 min confirming the similarity of the composition of the four hydrolyzates and therefore the repeatability of the hydrothermal treatment. 4. Conclusion A hydrothermal treatment of sugarcane bagasse during 2 h at 170 °C permitted obtaining of an acceptable xylose extraction yield. Xylose and dry mass analysis allow supporting the choice of 170 °C for 2 h as an optimal treatment for xylose recovery with a minimum of impurities. Another advantage of the chosen moderate operational conditions is the utilization of water as the sole reagent. Structural characterization of hydrothermal hydrolysates obtained at 170 °C show a large distribution of DPs of released xylo-oligosaccharides. This work will be followed by the enzymatic hydrolysis of oligomers to increase the recovery of xylose monomer. Acknowledgements The authors acknowledge the analytical facilities provided by the team ‘‘Transformation of lignocelluloses” at INRA of Reims (UMR 614) and the support by Roquette Frères of this work. References Allen, S.G., Schulman, D., Laser, M., Lichwa, J., Antal, J.R., Spencer, M.J., Lynd, L.R., 2001. A comparison between hot liquid water and steam fractionation of corn fiber. Ind. Eng. Chem. Res. 40, 2934–2941. Ballesteros, I., Oliva, J.M., Negro, M.J., Manzanares, P., Ballesteros, M., 2002. Enzymic hydrolysis of steam exploded herbaceous agricultural waste (Brassica carinata) at different particule sizes. Process Biochem. 38 (2), 187–192. Bobleter, O., Vidotti, R., Zemann, A., Prutsch, W., 1989. Hydrothermal pretreatment of bagasse and wheat straw. Biomass for energy and industry. In: Grassi, G., Gross, G., dos Santos, (Eds.), Proceedings of the Fifth EC Conference, vol. 2, Elsevier, New York, pp. 31–37.
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