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Research Article - (2013) Volume 0, Issue 0

Comparative Response and Structural Characterization of Sugarcane Bagasse, Straw and Bagasse-Straw 1:1 Mixtures Subjected to Hydrothermal Pretreatment and Enzymatic Conversion

Rondinele De Oliveira Moutta1,2, Maria Cristina Silva1, Roberta Cristina Novaes Reis Corrales1, Maria Alice Santos Cerullo2, Viridiana Santana Ferreira-Leitao1,3* and Elba Pinto Da Silva Bon2
1Biocatalysis Laboratory, Catalysis Division, National Institute of Technology, Ministry of Science and Technology (INT/MCT), 20081-312, Rio de Janeiro, RJ, Brazil
2Enzyme Technology Laboratory, Chemistry Institute, Federal University of Rio de Janeiro (UFRJ), 21941-909, Rio de Janeiro, Brazil
3Post-graduate Program in Biochemistry, Department of Biochemistry, Chemistry Institute, Federal University of Rio de Janeiro (UFRJ), 21941-909, Rio de Janeiro, Brazil
*Corresponding Author: Viridiana Santana Ferreira-Leitao, National Institute of Technology-Ministry of Science, Technology and Innovation Catalysis Division-Biocatalysis Laboratory, Av. Venezuela 82/302, CEP 20081-312, Rio de Janeiro, RJ, Brazil, Tel: + (55) 21-2123-1108, Fax: + (55) 21-2123-1166 Email:


The change of manually harvested sugarcane, after crop burning, to mechanically harvest green cane allows straw and bagasse to be available for further processing via either chemical or biochemical routes, which increases the sector’s energy efficiency. In this study, sugarcane bagasse, straw and a bagasse-straw 1:1 mixture were subjected, under comparative conditions, to hydrothermal pretreatment at 195°C for 10 minutes and to enzymatic conversion. We evaluated the individual responses of the three different materials regarding the effect of the pretreatment on hemicellulose and lignin extraction, the formation of furfural and hydroxymethylfurfural and cellulose enzymatic digestibility. The morphological, chemical and physical properties of the raw and pretreated materials were analyzed by Scanning Electron Microscopy (SEM), infrared spectroscopy (FTIR) and X-ray Diffraction (XRD). We observed a higher hemicellulose extraction from straw (93.3%) in comparison to bagasse (83.7%), and the hemicellulose extract that was obtained from straw contained a higher concentration of inhibitors. Intermediate values for hemicellulose extraction (88.5%) and inhibitor formation were observed for the bagasse-straw 1:1 mixture. The cellulose enzymatic hydrolysis yield was higher for straw (90.5%) in comparison to bagasse (68.2%), whereas an intermediate yield of 73.3% was observed for the mixture. According to the SEM images, the pretreatment altered the native biomass at the level of the structure of the cell wall, and consequently, the arrangement of the macromolecular components of the cell wall was closely related to the high degree of hemicellulose removal. FTIR data indicated chemical changes mostly in OH, OCH3 and C=O groups; these changes were most noticeable in the pretreated straw. Adjusted data for the crystallinity index suggested that the pretreated materials had decreased crystallinity. All of the results showed that straw had a lower recalcitrance.


In Brazil, the ethanol fuel industry, which is well established and economically competitive, is based on the fermentation of sugarcane juice and molasses. This industry generates considerable amounts of bagasse and straw, which are low-cost lignocellulosic residues. The exploitation of these materials, via their enzymatic hydrolysis, to produce C6 and C5 sugar syrups, ethanol and other value-added products in a cost-effective manner is challenging due to the need for the deployment of an efficient and cost-effective biomass pretreatment on an industrial scale.

The 2011/2012 sugarcane harvest in Brazil was estimated at 568.5 million tons [1], corresponding to 170.6 million tons of bagasse (50% moisture) and 193.3 million tons of straw (10% moisture) [2]. Currently, approximately 88% of sugarcane bagasse is burned in the sugar-ethanol industry for energy co-generation to supply the internal demand [1,2]. Sugarcane straw availability has been increasing because the traditional practice of burning the sugarcane crop prior to manual harvesting is becoming more restricted due to the Brazilian Federal Law 2661/98. In this scenario where the manual harvest has been gradually replaced by mechanical harvest [3,4], the available straw could be used as a feedstock for biorefinery platforms for the production of a wide spectrum of bio-based products (food, feed, chemicals and materials) and bioenergy (biofuels, power and/or heat) [4-7]. Nevertheless, the sugarcane stalks that are presently processed in the mills for sugarcane juice extraction contain residual straw and mechanical harvesting permits a substantial straw surplus. However, it is important to note that 30 to 50% of the straw should be left in the field because straw plays an important role in maintaining soil fertility [2,3].

The process of sugar release from lignocellulosic biomass in the biochemical platform includes biomass pretreatment, enzyme production and biomass enzymatic hydrolysis, which contribute as much as 40–45% of the total cost of cellulosic ethanol production [8-10]. The pretreatment step alone represents approximately 20% of this value [5].

Hydrothermal, steam explosion and dilute acid pretreatments have been widely studied and applied in the cellulosic ethanol plants in pilot and demonstration scales [11-13]. These three pretreatments result in a significant amount of hemicellulose removal, which improves the access of enzymes to the cellulose substrate in the subsequent enzymatic hydrolysis step [14].

During hydrothermal pretreatment, pressure maintains water in a liquid state at high temperatures (160–240°C) [15,16]. This process has been tested on a demonstration scale using a temperature range of 180–200°C for 5-15 minutes to process 460,000 tons of biomass per year [17]. Hydrothermal pretreatment is advantageous in comparison to other biomass pretreatment options because there is no requirement for special non-corrosive reactor materials or for preliminary feedstock size reduction [18]. This pretreatment has also some disadvantages, such as the down-stream processing of large volumes of water [19]. The biomass slurry that results from hydrothermal pretreatment can be separated into two fractions: a liquid fraction that is rich in hemicellulose oligomers and C5 sugars and an insoluble celluloselignin fraction. The first fraction can be used as a carbon source in fermentation processes for the production of chemicals such as organic acids, sorbitol and biopolymers in biorefinery platform. The enzymatic hydrolysis of the insoluble cellulose-lignin fraction generates glucose syrup that can be used as a carbon source in fermentation processes for the production of fuel ethanol; the glucose syrup can then be further used in a biorefinery platform for the production of a range of renewable bio-based products.

The residue that results from the enzymatic hydrolysis of the insoluble cellulose-lignin fraction, which is highly rich in the phenolic macromolecule lignin, has great potential as a chemical feedstock and for power generation due to its high caloric value of 27000 kJ kg-1, which matches the biorefinery concept that emphasizes the use of renewable chemicals and energy sources [2].

This work compared the responses of sugarcane bagasse, straw and a 1:1 mixture of both materials to the use of hydrothermal pretreatment under comparative pretreatment conditions, and changes in their structure and chemical composition were evaluated. All materials were subsequently subjected to enzymatic hydrolysis with commercial cellulases to evaluate their individual responses to the same pretreatment condition. All untreated and pretreated materials were submitted to structural, chemical and physical analysis by SEM, FTIR and XRD.

Materials and Methods

Materials and chemical characterization

The lignocellulosic feedstocks employed in this study were sugarcane bagasse, straw and a 1:1 mixture of both materials. Bagasse was kindly provided by Centro de Tecnologia Canavieira, São Paulo, Brazil, whereas straw was kindly provided by Dedini–Indústrias de Base (São Paulo, Brazil). The chemical characterization of the untreated materials and the solid and liquid fractions resulting from the pretreatment were performed as previously described [20] using milled and sieved samples with particles with sizes less than 2 mm. It is worth noting that the straw was previously washed for the determination of the content of its extractives. Carbohydrates were determined by HPLC using an Elite Lachrom chromatograph equipped with a Hitachi L-2490 refractive index detector using a Biorad HPX-87P column. Elution was carried out at 80°C with water using a flow rate of 0.6 mL min-1. Acid acetic, furfural, and hydroxymethylfurfural (HMF) were also analyzed by HPLC using a Biorad Aminex HPX-87H column equipped with a refractive index detector. Elution was carried out at 80°C with 0.005 M sulfuric acid using a flow rate of 0.6 mL min-1. The chemical composition of the pretreated materials was adjusted by mass yield according to equation 1:

Equation (1)

Where, Cadjusted is the composition value adjusted for cellulose, hemicellulose or lignin content in the pretreated materials; mf is the dry mass obtained after pretreatment; mi is the dry mass utilized in pretreatment (30 g); and Cpretreated is the composition value for cellulose, hemicellulose or lignin content in the pretreated materials.


Biomass was submitted to hydrothermal pretreatment in a 1 L reactor vessel PARR/4848 with temperature and pressure control. Bagasse, straw and the bagasse-straw 1:1 mixture were pretreated in three separate runs. Pretreatment was carried out at 195°C for 10 minutes using 30 g (dry weight) of biomass and 300 mL of distilled water, which corresponded to a solid:liquid ratio of 1:10. The pretreatment conditions were based on the experimental data obtained by Silva and co-workers (2011) for sugarcane bagasse [21].

The pretreated biomass was filtered and washed with water to remove residual hemicellulose-derived sugars and inhibitors. The water-insoluble materials and the liquid fractions were stored at 4°C for subsequent analyses and processing.

Biomass enzymatic hydrolysis

Commercial enzymes, Celluclast 1.5 L and β-glucosidase (Novozym 188) were used throughout this work. Filter Paper Activity (FPA) was determined as described by Ghose [22] and expressed in Filter Paper Units (FPU). FPA was determined using filter paper Whatman n°1 as a substrate for the incubation at 50°C for 60 min. Total reducing sugars were determined calorimetrically using the 3,5-dinitrosalicylic acid method [23]. The β-glucosidase activity was measured as reported [22] using 15 mM cellobiose as a substrate, and the glucose concentration was measured using a Biochemistry Analyser YSI 2700 Select.

The reaction mixture for biomass enzymatic hydrolysis, which was carried out in 50 mM citrate buffer, pH 4.8, contained the pretreated insoluble materials (bagasse, straw and the bagasse-straw 1:1 mixture) at 5% (dw/v) and an enzyme load of 10 FPU/g of biomass (1:3 FPU:β- glucosidase ratio). The glucan content of 5 g (dw) of bagasse was 2.45 g and the straw content was 1.80 g. Reaction mixtures, in 250 mL flasks, were incubated at 50°C and 200 rpm for 72 h. Periodic sampling of 0.5 mL was used for glucose quantification in a Biochemistry Analyser YSI 2700 Select. Untreated biomass was used in hydrolysis control experiments.

Infrared spectroscopy (FTIR)

Infrared spectra (wave numbers in cm-1) were obtained on a Magma-IR 560 E.S.P.–Nicolet spectrophotometer, using a KBr disk containing 3% finely ground samples. Thirty-two scans were taken of each sample recorded from 4000 to 400 cm-1 at a resolution of 4 cm-1.

X-ray diffraction (XDR)

The crystallinity of the cellulose fibers was evaluated by X-ray diffraction using a Rigaku MiniFlex diffractometer and filtered copper Kα radiation (λ=0.1542 nm) using a monochromator at 30 KV voltage and 15 mA electric current, with a speed of approximately 2º/min and scanning at an angle (2θ) in the range of 2-60°. The crystallinity index (CI) was obtained considering the intensity of the 002 peak (I002, 2θ=22.5°) and the minimum dip (Iam, 2θ=18.5°) between the 002 and the 101 peaks, according to equation 2 [24,25]:

Equation (2)

Where, I002 is the intensity of plane 002 and Iam is related to the amorphous structure.

The CI values were adjusted by mass yield according to equation 3:

Equation (3)

Where, mf is the dry mass obtained after pretreatment; mi is the dry mass utilized in pretreatment (30 g); and CIpretreated is the CI value that was obtained for pretreated materials.

Scanning electron microscopy (SEM)

Scanning electron microscopy (SEM-FEI/Inspect S50 model) was used to investigate the morphology of the untreated and treated materials. Samples were adhered to carbon tape, sputter coated with 28 nm gold, using an Emitech/K550 model and observed in the SEM through the use of an acceleration voltage of 20 KV and a working distance of approximately 19 mm. Several images were obtained on different areas of the samples (at least 20 images per sample) to guarantee the reproducibility of the results.

Results and Discussion

Chemical composition of untreated and pretreated materials

Table 1 presents the chemical composition of untreated sugarcane bagasse, straw and their pretreated counterparts. The chemical composition of the untreated and pretreated bagasse-straw 1:1 mixture was calculated as the arithmetic average composition of the untreated and treated corresponding materials. The untreated bagasse showed cellulose content of 40.0%, which was higher than that for straw, 29.4%. However, the straw hemicellulose content of 31.4% was higher than that for bagasse, which was 21.8%. Similar results were previously reported by Ferreira-Leitão and co-workers [5] who detected bagasse and straw cellulose contents of 41.1% and 33.3%, respectively. The lignin content for both materials was comparable, but the straw ash content was significantly higher in comparison to that of bagasse. After the hydrothermal pretreatment of 30 g (dry weight) bagasse, straw and the bagasse-straw mixture, 24.5 g, 22.0 g and 20.8 g (dry weight) were recovered as insoluble residues of the aforementioned materials, respectively indicating a different pattern of the extraction of the components into the liquid phase of the pretreatment. The lower than expected mass recovery of the bagasse-straw mixture, which nevertheless was within the acceptable 10% range, could be related to the sugarcane bagasse biomass heterogeneity because bagasse and straw are formed by different types of plant tissues. The analysis of the liquid and insoluble pretreatment fractions showed that 5.5 g (83.7%), 8.7 g (93.3%) and 7.1 g (88.5%) of hemicellulose were removed from bagasse, straw and the bagasse-straw mixture, respectively, indicating a higher susceptibility of straw to the hydrothermal pretreatment. These results are in agreement with those of Silva and co-workers [21], who reported 88.7% hemicellulose extraction from bagasse by hydrothermal pretreatment. Hemicellulose was mostly removed as xylan oligomers that corresponded to 52.1% (3.4 g), 78.4% (7.4 g) and 70.0% (5.6 g) of the total extracted hemicellulose from bagasse, straw and the bagassestraw mixture, respectively. Hemicellulose removal, predominantly in an oligomeric form, has also been observed by Ferreira-Leitão and coworkers [5] after sugarcane bagasse and straw pretreatment by steam explosion.

Components % (w/v) Untretated material Pretreated material
Bagasse Straw Bagasse Straw Bagasse-straw 1:1 mixture
Cellulose 39.99 ± 3.50 29.39 ± 8.20 48.97 ± 3.28 36.34 ± 3.44 42.66 ± 3.16
Hemicellulose 21.82 ± 4.43 31.38 ± 2.88 4.37 ± 0.42 2.62 ± 0.50  4.41 ± 0.42
Lignin 26.51 ± 1.45 26.00 ± 0.60 32.05 ± 0.53 30.76 ± 2.89 33.34 ± 2.95
Ashes 1.40 ± 0.04 4.83 ± 0.10 2.57 ± 0.50 6.28 ± 0.01 4.64 ± 0.60
Extractives*/others 7.41 ± 0.71 12.65 ± 0.09 9.94 ± 5.8 18,54 ± 4.81 8.37 ± 0.30
Total 97.14 ± 8.30 104.24 ± 6.97 97.90 ± 3.41 94.54 ± 6.81 100.20 ± 6.03

*The extractives content of the pretreated materials was estimated based on mass balance after pretreatment. The untreated straw extractives content, of 12.60%, was determined after washing the material. The straw used in the pretreatment was not washed and presented 17.55 ± 0.53 extractives content

Table 1: Chemical composition of sugarcane bagasse and straw and the bagasse-straw 1:1 mixture before and after hydrothermal pretreatment.

The hydrothermal pretreatment removed 1.3%, 5.6% and 11.9% lignin from bagasse, straw and the biomass mixture, respectively. The high lignin removal from the biomass mixture was in agreement with its lower than expected insoluble fraction recovery, as previously mentioned. Minimal removal of lignin from bagasse would generate, after the biomass enzymatic hydrolysis, a richer lignin solid byproduct, which would benefit the process energy balance upon its further use as solid fuel.

Table 2 presents the concentrations of acetic acid (resulting from xylan deacetylation), furfural and hydroxymethylfurfural (HMF) that are formed by the degradation of the xylose and glucose during the pretreatment. Similar concentrations of acetic acid of 2.03 g L-1, 1.93 g L-1 and 1.88 g L-1 were measured in the liquid stream from the bagasse, straw and bagasse-straw mixture hydrothermal pretreatment, respectively despite the noticeably higher straw hemicellulose content. The concentrations (g L-1) of furfural and HMF for bagasse (0.59 ± 0.10 and 0.03 ± 0.01) and straw (0.81 ± 0.15 and 0.33 ± 0.01) were 1.4 and 11 fold higher for straw in comparison to bagasse, which revealed a higher susceptibility of straw-derived sugars to the pretreatment. Moutta and coworkers [6] measured, in the liquid fraction from dilute sulfuric acid pretreatment of sugarcane straw, 3.19 g L-1 acetic acid, 0.56 g L-1 furfural and 0.17 g L-1 HMF. Nevertheless, the hydrothermal pretreatment conditions used in the present work resulted in a higher formation of furfural and HMF, while the acidic pretreatment resulted in a higher acetic acid concentration in the pretreatment liquid fraction. For both pretreatments, the concentration of HMF was more than 2 fold higher than that of furfural. The HMF, furfural and acetic acid contents in the liquid fraction from the bagasse-straw mixture roughly represented the average of the inhibitor contents that were determined for the corresponding materials. The hydrothermal pretreatment largely preserved the materials’ cellulose content, which is required to maximize the glucose yield upon the subsequent enzymatic saccharification step; the efficient use of the sugar from lignocellulosic biomass is a key factor for the economic feasibility of ethanol production [26].

Inhibitors Inhibitor content (g L-1)
Bagasse Straw Bagasse-straw 1:1 mixture
Furfural 0.59 ± 0.10 0.81 ± 0.15 0.67 ± 0.17
HMF 0.03 ± 0.01 0.33 ± 0.01 0.17 ± 0.02
Acetic Acid 2.03 ± 0.40 1.93 ± 0.10 1.88 ± 0.13

Table 2: Concentration of inhibitors (g L-1) in the liquid phase ofthe hydrothermal pretreatment of sugarcane bagasse, straw and the sugarcane bagasse-straw 1:1 mixture.

Enzymatic hydrolysis for untreated and pretreated sugarcane bagasse, straw and the bagasse-straw 1:1 mixture

Figure 1 shows the time course for glucose accumulation during the enzymatic hydrolysis of untreated and pretreated sugarcane bagasse, straw and the bagasse-straw 1:1 mixture. No significant cellulose conversion into glucose was observed for untreated bagasse, with a maximum glucose concentration of 0.73 g L-1 that corresponded to 4.0% cellulose hydrolysis. However, the maximum straw-derived glucose concentration, 3.24 g L-1, corresponding to 22.0% cellulose hydrolysis, was over 4-fold higher in comparison to that of the bagasse. As expected, biomass hydrolysis was significantly increased upon hydrothermal pretreatment, such that after 8 h the straw-derived glucose concentration, 13.4 g L-1, was more than 4-fold higher, and the bagasse-derived glucose concentration, 9.1 g L-1, was more than 13-fold higher in comparison to their untreated counterparts. After 24 h, the glucose concentrations from the pretreated straw and bagasse reached 17.2 g L-1 and 13.5 g L-1, which corresponded to cellulose hydrolysis yields of 84.4% and 49.1%, respectively. The straw cellulose conversion plateaued within 24 h, while that for bagasse showed a slow increasing trend such that within 72 h, the hydrolysis of bagasse-derived glucose reached 17.8 g L-1 (68.2% yield). The bagasse conversion was lower than the straw conversion, but this difference was balanced by the higher cellulose content of bagasse, such that comparable glucose concentrations were reached upon 72 h hydrolysis. This result is higher than the 56.9% of cellulose conversion into glucose after enzymatic hydrolysis of hydrothermal-pretreated bagasse that was reported by Silva and co-workers [21]. According to the aforementioned data for untreated and treated bagasse and straw cellulose conversion, which indicate higher hydrolysis yields for straw, the straw cellulose could present a distinctive structural arrangement that would favor enzymatic saccharification.


Figure 1: Time course for the enzymatic saccharification of hydrothermalpretreated bagasse (♦), straw (▲) and the bagasse-straw 1:1 mixture (). For comparison, data for untreated bagasse (-●-) and straw (-▲-) are also presented.

The enzymatic digestibility for the bagasse-straw 1:1 mixture showed a glucose concentration pattern and cellulose hydrolysis yield that were similar to the hydrolysis behaviors of the individual materials. Accordingly, the mixed materials did not either positively or negatively interfere with each other during the hydrolysis time course or in the final glucose syrup concentration. The kinetic profile of the enzymatic hydrolysis of the pretreated bagasse-straw 1:1 mixture was also similar to that of the kinetic behaviors of the individual materials, whereby the initial 8 h was greatly influenced by the straw hydrolysis rate, reaching a glucose concentration of 11.5 g L-1 and a yield of 41.9%. However, after 24 h, the hydrolysis trend was closer to that of bagasse, reaching a glucose concentration of 17.5 g L-1 (63.5% yield) within 72 h. A noticeable decrease in the hydrolysis reaction rate was observed for all materials after 8 h, which was even more pronounced after 24 h. After 48 h, low glucose productivities of 0.32 g L-1 h-1, 0.40 g L-1 h-1 and 0.35 g L-1 h-1 were detected for bagasse, straw and the mixed materials, respectively which would justify the interruption of the hydrolysis process to save costs during the process scale-up.

Structural analysis of sugarcane biomass by scanning electron microscopy

The SEM images did not permit the identification of morphological differences among untreated bagasse, straw or the bagasse-straw 1:1 mixture because in all cases, a high degree of heterogeneity was observed in terms of particle size, shape and plant tissue structure; however, as expected, noticeable differences were observed between the untreated and pretreated sugarcane bagasse and straw. Figure 2 shows the native structure and changes that bagasse and straw underwent upon hydrothermal pretreatment. Figure 2A represents a typical arrangement of bagasse or straw fibers, which are a category of sclerenchyma cells, organized as bundles. The cells are identified by their elongated shape with tapered ends and thick walls with simple pits (holes in the walls) [27]. The pits in this image are represented by highlighted spots where the plating was hampered by the edge effect. At a higher magnification, at the bottom of the figure, the primary wall of the cell can be observed, where the cellulose fibrils are relatively disordered, and in the upper portion of the figure, the inner region of another cell that has undergone a longitudinal cut showing the secondary wall with the aligned cellulose fibrils can be observed. The cell grouping and the composition of a cell wall that, aside from cellulose also contains lignin and hemicelluloses, increases the recalcitrance of the material. Therefore, cells must be disaggregated and the cell wall structure disrupted by pretreatment to increase the exposure of the cellulose fibrils [28]. Figure 2B presents the modifications in the fiber structure upon the hydrothermal pretreatment. A high degree of disorganization, rupture of the cell walls and decreased fiber adhesion can be observed. In figure 2C, a group of phloem parenchyma cells of untreated bagasse or straw is shown. The parenchyma cells are more fragile due to the absence of a secondary wall, which makes their cell walls thinner and less crystalline due to their lower cellulose content. Figure 2D shows parenchyma cells that are highly altered by the pretreatment, and at a higher magnification, the changes in the cell wall structure that result in exposed cellulose fibrils can be observed.


Figure 2: Scanning electron microscopy (SEM) images of sugarcane biomass tissues that are commonly found in bagasse and straw. Selected images showing (A) untreated and (B) hydrothermal-pretreated fibers and (C) untreated and (D) hydrothermal-pretreated parenchyma.

FTIR spectra

The infrared technique was used to identify the chemical changes that the sugarcane biomass undergoes after the pretreatment. The main features of the FTIR spectra were attributed to the chemical bonds that are present on the main biomass components: cellulose, hemicellulose and lignin. The infrared spectra (Figure 3) of untreated bagasse (A), pretreated bagasse (B), untreated straw (C), pretreated straw (D) and pretreated bagasse-straw mixture (E) were quite similar, showing that the pretreatment conditions did not promote significant and/or differentiated chemical changes in the samples studied. The relative absorbance values indicated a small increase in intensity for the pretreated samples (Table 3); the values were higher for pretreated straw in comparison to pretreated bagasse, indicating that straw is more susceptible to hydrothermal pretreatment. The bagasse-straw mixture had values that were very similar to those that were obtained for the pretreated straw [29,30].


Figure 3: FTIR spectra: (A) untreated sugarcane bagasse, (B) pretreated bagasse, (C) untreated straw, (D) pretreated straw and (E) pretreated bagasse-straw 1:1 mixture.

Assignment of FTIR absorption   Relative absorbance
wavenumber (cm-1) Untreated bagasse Pretreated bagasse Untreated straw Pretreated straw Pretreated bagasse-straw 1:1mixture
O-H stretching (H-bonded) 3396 0.64 0.65 0.62 0.73 0.68
O-H vibration of phenolic group 1371 0.93 0.97 0.89 0.94 0.95
O-H stretching of secondary alcohol 1163 0.81 0.84 0.78 0.82 0.83
O-H stretching of primary alcohol 1051 0.70 0.77 0.61 0.72 0.74
Mean value   0.77 0.81 0.72 0.80 0.80
C-H vibration of methoxyl group 2850 0.89 0.92 0.84 0.91 0.89
C-H angular deformation of methoxyl group 1462 0.97 0.99 0.91 0.96 0.98
Mean value   0.93 0.95 0.87 0.93 0.93
C-Ph vibration 1604 0.93 0.98 0.91 0.98 0.97
C=C aromatic skeletal vibration 1633 0.93 1.02 0.90 0.98 0.97
C-C and C-O stretching of aromatic skeletal 1334 0.95 0.97 0.92 0.94 0.96
C-O stretching of phenols 1253 0.88 0.96 0.88 0.94 0.94
Mean value   0.91 0.96 0.90 0.94 0.95
C-H aliphatic axial deformation 2912 0.83 0.86 0.76 0.84 0.84
C-H aliphatic angular deformation 1425 0.96 0.98 0.90 0.95 0.97
C-O-C stretching 1113 0.76 0.78 0.68 0.76 0.77
β-glycisidic linkages 897 1.11 1.18 1.01 1.08 1.08
C=O stretching 1735 0.98 1.15 1.01 1.07 1.08

Table 3: Relative intensity of bands in the infrared spectrum of different groups in the untreated and pretreated sugarcane bagasse, straw and bagasse-straw 1:1 mixture.

Within this context, some differences were identified in accordance with the assignments that were given to the absorption bands that are referred to in the literature [31-36]. The band at 1514 cm-1 was chosen as an internal standard because it was well defined despite being present in all spectra.

The absorption characteristics of the OH groups that are present in lignin and carbohydrates were related to the band at 3396 cm-1. Absorption changes at 3396 cm-1 were more noticeable in the pretreated straw. The phenolic OH group band can be observed at 1371 cm-1. Higher values were observed for both pretreated materials.

The bands of the primary and secondary OH groups can be observed at 1051 cm-1 and 1163 cm-1, respectively, whereas the absorption at 2912 cm-1 can be attributed to C-H aliphatic axial deformation in the CH2 and CH3 groups from cellulose, lignin and hemicellulose. According to Nada and coworkers [33], the band at 2850 cm-1 is assigned to the vibration of the OCH3 groups that are present in lignin. The C-H aliphatic angular deformation in CH3 and CH2 is observed at 1425 cm-1. The band at 1334 cm-1 is attributed to C-C and C-O skeletal vibrations.

The band at 1735 cm-1 that is associated with the acetyl groups of hemicellulose exhibited a higher relative absorbance in the pretreated samples in comparison to the untreated ones, which indicates that the pretreatment efficiency promoted a significant exposure to the biomass structure.

Noticeably, the pretreated bagasse showed a higher relative absorbance of the C=O group compared to the pretreated straw (Table 3) because the hemicellulose removal after the hydrothermal pretreatment of straw was higher (Table 1).

The band at 897 cm-1 is attributed to the ß-glycosidic linkages between monosaccharide units, and it was also higher for the pretreated samples (approximately 6.2%) (Table 3), suggesting a higher exposure of cellulose because hemicellulose was extensively removed by the pretreatment.

X-Ray diffraction (XRD)

The crystallinity of lignocellulosic biomass represents the relative amount of crystalline cellulose in the material, and it is strongly influenced by the composition of the biomass, which varies according to its nature. Diffractograms of untreated bagasse, pretreated bagasse, untreated straw, pretreated straw and pretreated bagasse-straw 1:1 mixture are shown in figure 4. As can be observed, all of the samples exhibited typical cellulose diffraction peaks (2θ=22.5° and 2θ=16.0°), the highest of which corresponded to the 002 crystallographic planes.


Figure 4: Diffractograms of the untreated and pretreated materials: (A) untreated sugarcane bagasse; (B) hydrothermal pretreated bagasse; (C) untreated straw; (D) hydrothermal pretreated straw and (E) hydrothermal pretreated bagasse-straw 1:1 mixture.

Table 4 shows the CI and the CI adjusted by mass yield for the pretreated materials. The untreated bagasse and straw showed lower CI values compared to the pretreated samples (unadjusted data). Many studies indicate that there is an increase in the value of the CI when biomass is subjected to pretreatment [37-41], which is related to the removal of the amorphous fractions (hemicellulose and lignin).

Samples CI (%)   CI (%) adjusted by mass yields of the pretreatment
untreated bagasse 53.5 -
pretreated bagasse 59.5 48.6
untreated straw 51.8 -
pretreated straw 61.5 45.1
pretreated bagasse-straw 1:1 mixture 60.6 42.0

Table 4: Crystallinity index (CI) of the untreated and pretreated sugarcane bagasse and straw and the adjusted CI (%) according to the mass yields after the pretreatment.

To evaluate the pretreatment effect on cellulose crystallinity, the CI was adjusted considering the mass yield after the pretreatment step [42]. Pretreated bagasse and straw had adjusted CI values of 48.6% and 45.1%, respectively (Table 4). These data show that pretreatment promoted reductions in the bagasse and straw cellulose CIs of 9.2% and 12.9%, respectively, which corroborates the higher susceptibility of straw cellulose.

As expected, an intermediate CI value was obtained for the pretreated bagasse-straw 1:1 mixture of 60.6% compared to the CIs of the pretreated bagasse and straw. Although a lower adjusted CI value was obtained for the pretreated bagasse-straw mixture (Table 4) that could be related to the lower mass recovery for the bagasse-straw mixture (Table 1), no significant improvement in enzymatic cellulose hydrolysis was observed (Figure 1).


This comparative study of the responses of sugarcane bagasse, straw and a bagasse-straw 1:1 mixture to hydrothermal pretreatment and enzymatic conversion, as well as to the chemical and structural characterization of treated and untreated materials, revealed distinctive features of this sugarcane biomass-derived feedstock. It was shown that raw bagasse had higher cellulose and lower hemicellulose content in comparison to straw, while the lignin content in both materials was comparable. Straw was markedly more responsive towards the hydrothermal pretreatment conditions as shown by its higher hemicellulose and lignin extraction to the pretreatment liquid phase, but it presented a higher concentration of inhibitors due to the degradation of the extracted sugars. Nonetheless, the pretreatment largely preserved the cellulose content of bagasse and straw in the solid phase. The straw cellulose from both untreated and pretreated material was more susceptible to enzymatic hydrolysis in comparison to untreated bagasse cellulose, which was due to its higher saccharification rates and yields. The results for the bagasse-straw 1:1 mixture regarding the pretreatment and enzymatic hydrolysis had an intermediate pattern for both materials, which indicates the feasibility of blending the two types of biomass for further processing via biotechnological routes for the production of chemical feedstock. This strategy could balance the lower bagasse cellulose digestibility with the higher straw cellulose digestibility and also the higher straw sugar degradation. Pretreatment did not significantly affect the materials’ CI, but a more accentuated CI reduction was observed for straw, which accounts for the lower recalcitrance of straw cellulose. FTIR data indicated chemical changes that occurred mostly in OH, OCH3 and C=O groups, which were more pronounced in straw. The SEM images did not allow the identification of morphological differences among untreated bagasse, straw or the bagasse-straw 1:1 mixture, but noticeable differences were observed between the untreated and pretreated sugarcane materials. Pretreatment decreased the fiber adhesion and altered the native biomass at the level of the cell wall structure, and consequently, the arrangement of its macromolecular components was closely linked to the high degree of hemicellulose removal.


The authors would like to thank the Brazilian Innovation Agency-(FINEP), the Ministry of Science, Technology and Innovation (MCTI) and the Brazilian Foundation for Graduate Students (CAPES) for financial support. The authors would like to express their gratitude to colleagues from CENANO/INT/MCTI (Center of Nanostructure Characterization), and in particular to Sheyla Santana de Carvalho and Fernanda C.S.C. dos Santos. Celso Sant’Anna from the National Institute of Metrology, Standardization and Industrial Quality is also acknowledged.


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Citation: de Oliveira Moutta R, Silva MC, Reis Corrales RCN, Santos Cerullo MA, Ferreira-Leitão VS, et al. (2013) Comparative Response and Structural Characterization of Sugarcane Bagasse, Straw and Bagasse-Straw 1:1 Mixtures Subjected To Hydrothermal Pretreatment and Enzymatic Conversion. J Microb Biochem Technol S12:005.

Copyright: © 2013 de Oliveira Moutta R, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.