Chemical composition and enzymatic digestibility of sugarcane clones selected for varied lignin content
© Masarin et al; licensee BioMed Central Ltd. 2011
Received: 16 September 2011
Accepted: 6 December 2011
Published: 6 December 2011
The recalcitrance of lignocellulosic materials is a major limitation for their conversion into fermentable sugars. Lignin depletion in new cultivars or transgenic plants has been identified as a way to diminish this recalcitrance. In this study, we assessed the success of a sugarcane breeding program in selecting sugarcane plants with low lignin content, and report the chemical composition and agronomic characteristics of eleven experimental hybrids and two reference samples. The enzymatic digestion of untreated and chemically delignified samples was evaluated to advance the performance of the sugarcane residue (bagasse) in cellulosic-ethanol production processes.
The ranges for the percentages of glucan, hemicellulose, lignin, and extractive (based on oven-dry biomass) of the experimental hybrids and reference samples were 38% to 43%, 25% to 32%, 17% to 24%, and 1.6% to 7.5%, respectively. The samples with the smallest amounts of lignin did not produce the largest amounts of total polysaccharides. Instead, a variable increase in the mass of a number of components, including extractives, seemed to compensate for the reduction in lignin content. Hydroxycinnamic acids accounted for a significant part of the aromatic compounds in the samples, with p-coumaric acid predominating, whereas ferulic acid was present only in low amounts. Hydroxycinnamic acids with ester linkage to the hemicelluloses varied from 2.3% to 3.6%. The percentage of total hydroxycinnamic acids (including the fraction linked to lignin through ether linkages) varied from 5.0% to 9.2%, and correlated to some extent with the lignin content. These clones released up to 31% of glucose after 72 hours of digestion with commercial cellulases, whereas chemically delignified samples led to cellulose conversion values of more than 80%. However, plants with lower lignin content required less delignification to reach higher efficiencies of cellulose conversion during the enzymatic treatment.
Some of the experimental sugarcane hybrids did have the combined characteristics of high biomass and high sucrose production with low lignin content. Conversion of glucan to glucose by commercial cellulases was increased in the samples with low lignin content. Chemical delignification further increased the cellulose conversion to values of more than 80%. Thus, plants with lower lignin content required less delignification to reach higher efficiencies of cellulose conversion during the enzymatic treatment.
List of abbreviations
filter paper unit
gas chromatography/mass spectrophotometry
high-performance liquid chromatography
Sugarcane residue (bagasse) is an abundant agricultural residue and a promising substrate for ethanol production . Although it contains enough cellulose to be an excellent source of sugars for ethanol production, the conversion of cellulose to glucose by enzymes is very limited without the use of an efficient lignocellulose pretreatment. The recalcitrance of lignocellulosic materials is related to several factors, including the close association of cellulose with hemicellulose and lignin in the cell wall, which hinders the cellulase action . One of the ways to increase the cellulose conversion by enzymes in such materials involves lignin degradation or removal by pretreatment [3–6], which increases the cell-wall porosity, facilitating enzyme infiltration and cellulose hydrolysis [7, 8].
Some recent work has focused on depleting lignin by breeding new cultivars or transgenic plants as a way to diminish lignocellulose recalcitrance [9–13]. Grabber et al.  reported that artificially lignified cell walls from maize had their digestibility to ruminal biota decreased as a function of increased lignification, and the authors concluded, on the basis of the use of different lignin precursors, that the engineering of plants for reduced lignification or ferulate-lignin crosslinking improves fiber digestibility to a greater degree than does shifting lignin composition (for example, by selecting high syringyl content in lignified plants). This finding is relevant because in grasses, part of the recalcitrance is associated not only with the occurrence of lignin in cell walls but also with the presence of hydroxycinnamic acids linked primarily to the hemicelluloses [8, 14, 15]. It is also known that cell-wall digestibility varies significantly among plant varieties . Part of this variation is often associated with the total lignin concentration and the presence of hydroxycinnamic acid crosslinks in the cell walls [12, 15].
Recently, a sugarcane breeding program in Brazil, RIDESA (Academic Network for the Development of Sugar-Alcohol Sector; http://www.ridesa.com.br) has been making efforts to select sugarcane plants with low lignin content and/or altered lignin composition , using a recurrent selection method to increase the frequency of favorable alleles through repeated cycles of crossing and selection. In this paper, we report the chemical composition and some agronomic characteristics of 11 experimental hybrids. Enzymatic digestion of the untreated sugarcane bagasse from those hybrids and of some chemically delignified samples was evaluated in an attempt to enhance the performance of the sugarcane bagasse in cellulosic-ethanol production programs.
Results and discussion
Chemical composition and field productivity of the sugarcane clones
Chemical composition* of sugarcane bagasse samples obtained from experimental sugarcane hybrids ranked† by their lignin content
16.8 ± 0.1a
27.3 ± 0.3a,c,d,f,i,j,k,l,m
40.3 ± 0.1a,b,c,d,f,g,h,i,j,k,l,m
7.5 ± 0.1a
18.6 ± 0.1b,c,d
31.6 ± 0.8b,e,g,h
40.9 ± 0.3b,c,d,e,f,g,h,i,j,l
2.4 ± 0.1b,c,d,e,g,h,i,l,m
18.6 ± 0.1c,d
26.3 ± 0.1c,d,f,i,j,k,l,m
40.9 ± 0.3c,d,e,f,g,h,i,j,l
2.6 ± 0.2c,d,e,h,i,l,m
19.4 ± 0.5d,e,f,g
27.1 ± 0.4d,f,i,j,k,l,m
42.2 ± 0.5d,e,f,g,h,i,l
2.7 ± 0.1d,e,h,i,l,m
19.6 ± 0.5e,f,g,h,i
31.5 ± 0.1e,g,h
43.2 ± 0.4e,f,h,l
1.9 ± 0.1e,g,h,l,m
19.7 ± 0.1f,g,h,i,j
27.3 ± 0.8f,i,j,k,l,m
42.2 ± 0.3f,g,h,i,l
3.9 ± 0.4f,i
20.2 ± 0.4g,h,i,j
31.0 ± 1.0g,h
40.4 ± 0.5g,h,i,j,k,l,m
1.6 ± 0.3g,l
20.5 ± 0.1h,i,j
30.0 ± 2.0h,j
42.0 ± 1.0h,i,l
2.5 ± 0.1h,i,l,m
20.5 ± 0.4i,j
26.6 ± 0.7i,j,k,l,m
40.0 ± 1.0i,j,k,l,m
3.2 ± 0.4i,m
20.6 ± 0.1j,k
28.2 ± 0.5j,k,l
39.0 ± 1.0j,k,m
4.9 ± 0.2j,k
21.5 ± 0.2k
27.0 ± 0.3k,l,m
38.2 ± 0.5k,m
5.1 ± 0.5k
24.0 ± 0.1l,m
26.0 ± 1.0l,m
42.0 ± 2.0l
2.2 ± 0.4l,m
24.5 ± 0.5m
25.2 ± 0.4m
38.2 ± 0.2m
2.6 ± 0.1m
The hemicellulose content was calculated on the basis of the monomeric sugars and acetic acid released after acid hydrolysis. Under the analytical conditions used, xylose, mannose, and galactose eluted at the same retention time, and appeared as a single peak. To assess the levels of these individual sugars in the sugarcane samples, some of the experimental clones and the mill bagasse were analyzed using a pulsed amperometric detector . The chromatograms showed no detectable mannose and galactose in the mill bagasse sample, whereas small peaks of galactose were detected in clones 87, 89, and 140, corresponding, respectively, to 0.90%, 0.83%, and 0.71% of the oven-dry mass of the plant material. These data suggest that the hemicellulose content detected in the evaluated sugarcane samples consisted mainly of xylan backbones ramified with arabinose and acetic acid. The hemicellulose probably contains 4-O-methyl-glucuronic acid also, as this is well documented in the literature for hemicelluloses from sugarcane [17, 20]. The molar ratios of xylose and arabinose were similar for all samples, whereas acetic acid varied slightly, giving a substitution pattern of 10 xylose to 1 arabinose to 3 or 4 acetic acid for the xylan structures. This pattern of branching in the structure of xylan in sugarcane is in the same range as previously reported data [17, 20].
The content of extractives also varied significantly between the samples (Table 1). Bagasse from commercial sugarcane varieties generally contains ethanol-soluble extractives in the range of 1.5% to 3.0% [21, 22]. We found results within this range for the mill bagasse and the reference cultivar (2.2% and 2.6%, respectively), but some of the clones contained very high levels of extractives (for example, 5.1% and 7.5% in clones 140 and 89, respectively). The ethanol-soluble fraction is characterized by the presence of waxy materials and low molar mass aromatics, but extraction with 95% ethanol can also dissolve small amounts of oligosaccharides present in the sugarcane samples .
Chemical characteristics of the fraction extracted with 95% ethanol from sugarcane bagasse samples
Total extractives, %
Comparison of the entire dataset (Table 1) using one-way analysis of variance showed that the lignin content differed between samples, as follows: 1) the reference cultivar and mill bagasse had the highest lignin content (these samples did not overlap with the clone containing the highest lignin content, which was clone 140); 2) clone 89 had the lowest lignin content (differing significantly from all other samples); and 3) clones 58 and 146 had the second lowest lignin content but they overlapped with clone 53 (which overlapped with the next lowest. There was more overlap for the polysaccharide content, thus the samples could not be classified into groups on this basis. However, the extractives content could also be classified, as follows: 1) clone 89 had the highest extractives content; 2) clones 121 and 140 had the second highest extractives content but differed from clone 89; and 3) most of the clones had extractives content ranging from 1.6% to 3.9%.
The overall assessment of the chemical composition data showed that the samples with the smallest amounts of lignin did not contain the largest amounts of total polysaccharides (Table 1). This lack of correlation suggests that hybrids depleted in lignin do not have a corresponding increase in the amount of a single major component (such as polysaccharides); instead, there seems to be a variable increase in the mass of a number of components, including extractives, to compensate for the reduction in lignin content. These results differ from previous results obtained with transgenic alfalfa, which showed that plants with the lowest lignin content had the highest total carbohydrate levels .
Hydroxycinnamic acid content
Hydroxycinnamic acid composition of sugarcane bagasse samples obtained from experimental sugarcane hybrids ranked by their lignin content
Hydroxycinnamic acids, g/100g of untreated bagasse
Released by mild alkali treatment
Released by severe alkali treatment
0.50 ± 0.01a,b,c,d,f,h,i,j,k,l
1.8 ± 0.1a,d,f,h,l
2.3 ± 0.1a,d,f,l
1.1 ± 0.1a,b,c,d,e,f,g,h,i,j,k,l
3.9 ± 0.5a,c,d,f,h,j,k
5.0 ± 0.5a,c,d,f,h,i,j,k
0.59 ± 0.02b,c,e,g,h,i,j,k,m
2.5 ± 0.2b,c,d,e,f,g,h,i,j,k,l,m
3.1 ± 0.2b,c,d,e,f,g,h,i,j,k,m
1.4 ± 0.1b,e,f,g,h,l,m
6.2 ± 0.5b,c,d,e,f,g,h,i,j,k,l,m
7.6 ± 0.5b,e,f,g,h,i,j,k,l,m
0.59 ± 0.01c,e,g,h,i,j,k,m
2.6 ± 0.1c,d,e,f,g,h,i,j,k,m
3.2 ± 0.1c,d,e,g,h,i,j,k,m
0.8 ± 0.2c,d,f,i,j,k
4.4 ± 1.2c,d,e,f,h,i,j,k,l
5.2 ± 1.2c,d,f,h,i,j,k
0.49 ± 0.02d,f,h,i,j,k,l
2.2 ± 0.1d,e,f,h,i,j,k,l,m
2.7 ± 0.1d,f,h,i,j,k,l
0.9 ± 0.1d,f,h,i,j,k
4.6 ± 0.9d,e,f,h,i,j,k,l
5.5 ± 0.9d,f,h,i,j,k,l
0.63 ± 0.05e,g,h,i,m
2.8 ± 0.3e,g,h,i,j,k,m
3.4 ± 0.3e,g,h,i,j,k,m
1.4 ± 0.1e,f,g,h,l,m
6.2 ± 0.2e,f,g,h,i,j,k,l,m
7.6 ± 0.2e,f,g,h,i,j,k,l,m
0.42 ± 0.04f,j,l
2.1 ± 0.3f,h,i,k,l
2.5 ± 0.3f,h,k,l
1.1 ± 0.1f,g,h,i,j,k,l
5.7 ± 0.9f,g,h,i,j,k,l,m
6.8 ± 0.9f,g,h,i,j,k,l
0.65 ± 0.01g,m
2.9 ± 0.1g,h,i,j,k,m
3.6 ± 0.1g,h,i,j,k,m
1.4 ± 0.1g,h,l,m
6.8 ± 0.5g,h,i,j,k,l,m
8.2 ± 0.5g,h,i,j,k,l,m
0.55 ± 0.03h,i,j,k,l,m
2.4 ± 0.2h,i,j,k,l,m
3.0 ± 0.2h,i,j,k,l,m
1.2 ± 0.1h,i,j,k,l
5.2 ± 0.4h,i,j,k,l
6.4 ± 0.4h,i,j,k,l
0.55 ± 0.07i,j,k,l,m
2.7 ± 0.4i,j,k,m
3.3 ± 0.4i,j,k,m
1.0 ± 0.1i,j,k
6.0 ± 0.2i,j,k,l,m
7.0 ± 0.2i,j,k,l
0.50 ± 0.04j,k,l
2.8 ± 0.3j,k,m
3.3 ± 0.3j,k,m
0.9 ± 0.1j,k
5.8 ± 0.5j,k,l,m
6.7 ± 0.5j,k,l
0.53 ± 0.02k,l,m
2.6 ± 0.1k,m
3.0 ± 0.1k,l,m
1.0 ± 0.1k
5.2 ± 0.3k,l
6.2 ± 0.3k,l
0.47 ± 0.01l
1.9 ± 0.1l
2.4 ± 0.1l
1.4 ± 0.2l,m
6.1 ± 0.8l,m
7.5 ± 0.8l,m
0.62 ± 0.01m
2.8 ± 0.1m
3.4 ± 0.1m
1.7 ± 0.2m
7.5 ± 1.0m
9.2 ± 1.0m
The clones were ranked as a function of the total lignin content (Table 1) because this work focused on the evaluation of sugarcane hybrids as candidate feedstock for cellulosic-ethanol production. It is well documented that lignin acts as a barrier for enzyme infiltration in lignified cell walls, and that it unproductively binds the cellulases during enzymatic conversion of lignocellulosic substrates [2, 5, 8]. In principle, plants with low lignin content are easier to pre-treat and to hydrolyze by cellulases compared with plants with high lignin content [2, 6, 9, 28].
Relationship between plant, biomass and sucrose production with lignin content
Plant productivity parameters, biomass content and sucrose yield of experimental sugarcane hybrids ranked by their lignin content
Plant productivity, wet ton/hectare
Diameter at internode, mm
Plant bending score*
Dry biomass content (bagasse), kg/ton of wet plant
Sucrose yield, kg/ton of wet plant
Plant bending is another relevant characteristic in sugarcane, because a high degree of bending makes either manual or mechanized cutting difficult. In general, the data suggest that a low lignin content in the plants favors plant bending during growth, with a clear exception being clone 89, which displayed moderate (level 2) plant bending even though it contained a very low lignin concentration.
Enzymatic hydrolysis of the sugarcane samples
The digestibility data of the sugarcane samples showed that the enzymatic hydrolysis of cellulose increased in clones with diminished lignin content. However, the overall efficiency of the process was still low (31% after 72 hours of hydrolysis) for the clone with the lowest lignin content (16.8%). To challenge the effect of lignin depletion through breeding with lignin depletion by chemical removal from the cell walls, two of the studied samples were further delignified by a selective chemical process using sodium chlorite under acidic pH. Several levels of lignin removal were obtained, depending on the reaction time (Figure 3). This selective chemical delignification produced samples in which the cellulose conversion was significantly enhanced. There was an almost linear correlation between the cellulose conversion levels after enzymatic hydrolysis and the lignin content of these samples. Considering that the slope for the curve obtained with data from clone 146 was higher than that for the reference cultivar (slopes of 8.1 and 6.7, respectively), it is clear that plants bred to contain less lignin will require less delignification to reach a defined level of cellulose hydrolysis. For example, to reach an 80% level of cellulose hydrolysis, clone 146 and the reference cultivar required delignification levels of 37% and 44%, respectively.
The data for enzymatic hydrolysis of the untreated and delignified samples showed that in delignified samples, lignin removal enhances enzymatic digestibility not only because the pretreated material has a lower lignin content, but also because the chemical pretreatment should increase the porosity of the cell walls and the reactivity of the substrates, thus facilitating the hydrolysis of the constituent polysaccharides, as previously reported [8, 31]. This finding is in close agreement with previous studies on the evaluation of the recalcitrance in untreated and chemically pretreated lignocellulosic materials [2, 5, 8, 31].
The current evaluation of 11 sugarcane experimental hybrids selected for varied lignin content showed that some plants had the combined characteristics of high biomass and sucrose productivity with low lignin content. However, the samples with the smallest amounts of lignin did not produce the largest amounts of total polysaccharides. Instead, there was a variable increase in the mass of a number of components, including the group of extractives, which seems to reflect compensation for the reduction in the lignin content.
After enzymatic digestion of the bagasse samples, the cellulose conversion levels increased in the samples with lower lignin content. In non-pretreated samples, the cellulose conversion reached a maximum of 31% after 72 hours of hydrolysis for the clone with the lowest lignin content, 16.8%. By contrast, chemically delignified samples led to cellulose conversion values of more than 80%. Interestingly, plants with lower lignin levels required lower levels of delignification to reach higher efficiencies of cellulose conversion during the enzymatic treatment.
Raw material and biomass preparation
Eleven experimental sugarcane hybrids were selected from the breeding program developed by RIDESA associated with the Federal University of Viçosa, Viçosa, MG, Brazil . Seeds obtained after hybridization were planted in trays and maintained in a greenhouse for 30 days. The seedlings were first transferred to tubs and then to the field to generate initiating plants for subsequent vegetative propagation. The clone plantation was set in May 2007 using rows 5 m in length in an experimental field in Oratórios, MG, Brazil (20°25'50'' latitude south, 42°48'20" longitude west).
The first clonal crop was obtained in July 2008. The plant material that regrew after the first cut (second clonal crop) was harvested in July 2009 (12-month-old plants) and used for the evaluation reported in this paper. Ten stalks taken randomly from each row were used for estimating field-productivity parameters. Plant productivity was estimated from the total number of stalks per row and the wet weight of 10 stalks. Stalk diameter was measured at the fifth internode from the plant base. Plant bending was estimated from a five-point approximate scale varying from 1 (straight; less than 5° of the angle between stalks) to 5 (bent; more than 150° of the angle between stalks). Dry biomass values were obtained after juice extraction of 500 g samples crushed in a hydraulic press. Sucrose yield was determined from polarimetric determination of sugars in the extracted juice .
In addition to the 11 experimental hybrids, two reference crops were included in the study. The first was a widely grown sugarcane cultivar often planted in family farms in the state of São Paulo to produce sugar syrup with a high sucrose concentration. This sample included 18-month-old stalks harvested in a farm located in Lorena, SP, Brazil (22°43'51" latitude south, 45°07'29" longitude west), and was termed the 'reference cultivar'. The second reference material was a sugarcane bagasse sample from a sugar and ethanol mill that crushes a mix of commercial sugarcane cultivars. This material, termed the 'mill bagasse' sample, was collected at the mill as freshly crushed material and air-dried to a final humidity of 12%, then stored in plastic bags until used.
For chemical characterization and enzymatic hydrolysis studies, approximately 15 stalks of the harvested hybrids or the reference cultivar were cut by a reaper machine into 5 to 10 mm long fragments. The cut material was blended in water and washed to remove sucrose. To avoid loss of fine particles (< 0.2 mm in length), the material was washed inside a PVC column 1 m long and 150 mm in diameter, with a 200-mesh screen at the bottom. Any particles passing through the screen were pumped back to the top of the column. Filtrate recirculation permitted the formation of a fiber mat at the column base that retained these particles. Water recirculation was stopped when the washing water was clear. After this point, additional blended biomass was applied to the column, and fresh water was passed through the column until the wash produced a negative color result in a phenol/sulfuric acid assay. The obtained biomass material was stored at -18°C until use. For the mill bagasse, the sample biomass was washed with water, air-dried, and stored at room temperature until use.
The reference cultivar and hybrid sample 146 were delignified with a sodium chlorite/acetic acid aqueous solution to evaluate the effect of selective lignin removal on the sample performance under enzymatic hydrolysis. Samples were milled to pass through a 0.84-mm screen, and delignified for reaction periods from 0.25 to 4 hours as described previously  to produce samples with progressively decreased lignin content.
Chemical composition of the samples
Ethanol-soluble extractives were determined by extraction with 95% (v/v) ethanol in a Soxhlet apparatus. The samples were air-dried and milled to pass through a 0.84-mm screen. Approximately 3 g of the milled sample was extracted with 95% ethanol for 6 hours in a Soxhlet apparatus. The percentage of extractives was determined on the basis of the dry weights of the extracted and non-extracted milled samples (data are provided as mean ± standard deviation (SD)). This procedure was conducted in triplicate. Some of the ethanolic extracts were dried under vacuum in a rotary evaporator at 60°C. The obtained solids were then further dried to a constant weight at 60°C, and maintained under vacuum over phosphorus pentoxide in a desiccator. The level of aromatic compounds in these solids was estimated by UV spectroscopy. The solids were suspended in 0.5 mol/l sodium hydroxide at a concentration of approximately 1 mg/ml, and filtered through 0.45 μm membranes, then the absorbance of the resulting solutions was measured at 280 nm. The concentration of aromatic compounds in this solution was estimated to have an average absorptivity of 20 l/cm.g . The total sugar content in the same solution was determined by the sulfuric acid/phenol method using sucrose as the calibration standard .
Ethanol-extracted or chlorite-delignified milled samples were hydrolyzed with 72% (w/w) sulfuric acid at 30°C for 1 hour (3 ml of acid to 300 mg of sample) as described previously . The acid hydrolysate was diluted with 79 ml of water, and the mixture was autoclaved at 121°C for 1 hour. The residual material was cooled, and filtered through a porous glass filter (number 3). Solids were dried to a constant weight at 105°C, and assessed as the insoluble lignin component. The concentration of soluble lignin in the aqueous fraction was determined by measuring the absorbance of the solute at 205 nm, using the value of 105 L/cm.g as the absorptivity of soluble lignin . The concentrations of monomeric sugars in the soluble fraction were determined by HPLC (HPX87H column; Bio-Rad, Hercules, CA, USA) at 45°C and an elution rate of 0.6 mL/min with 5 mmol/l sulfuric acid. Sugars were detected in a temperature-controlled refractive index detector at 35°C. Under these conditions, xylose, mannose and galactose eluted at the same time, and appeared as a single peak. Glucose, xylose, arabinose and acetic acid were used as external calibration standards. No corrections were performed because of the sugar-degradation reactions that take place during acid hydrolysis . The factors used to convert sugar monomers to anhydromonomers were 0.90 for glucose and 0.88 for xylose and arabinose. Acetyl content was calculated as the acetic acid content multiplied by 0.72. This procedure was conducted in duplicate (data shown as mean ± SD). Glucose was reported as glucan after correction by the hydrolysis factor, and the other sugars and acetic acid were used to calculate the hemicellulose content.
Some of the acid hydrolysates were also analyzed with a pulsed amperometric detector (871 Advanced BioScan, Metrohm AG, Herisau, Switzerland) to detect hemicellulose sugars as separate peaks, using an analytical column (Carbopac PA10; Dionex Co., Chelmsford, MA, USA) with an elution rate at 1.0 mL/min using a mixture of two eluents: NaOH 50 mmol/l and deionized water. The solvents were mixed automatically to give a ratio of 10% NaOH to 90% H2O during the first 20 minutes, then 45% NaOH to 55% H2O for 1 minute, followed by 90% NaOH to 10% H2O for 9 minutes. Finally, the solvent mixture was set to the initial composition (10% NaOH to 90% H2O) for 10 minutes to wash out the system .
Hydroxycinnamic acid extraction and determination
Hydroxycinnamic acids were determined after extraction by alkaline solutions under mild or severe reaction conditions. Mild reaction conditions release hydroxycinnamic acids that are ester-linked to hemicelluloses, whereas severe conditions release the total amount of hydroxycinnamic acids, either ester-linked or ether-linked to the lignocellulose components .
Mild reactions were performed with 50 mg of the biomass sample and 5 ml of 1 mol/l NaOH. The sample was incubated at 30°C for 24 hours, using rotary agitation at 120 rpm, then the reaction mixture was acidified to pH 2 with 6 mol/l HCl, and water was added to give a final volume of 10 ml. The mixture was then stored at 4°C for 16 hours, filtered through a 0.45 μm membrane, and analyzed for p-coumaric (3-(4-hydroxyphenyl)prop-2-enoic acid) and ferulic (3-(4-hydroxy-3-methoxy-phenyl)prop-2-enoic acid) acids by HPLC. Severe reaction conditions were conducted with 200 mg of the biomass sample and 2 mg of anthraquinone with 16 ml of 4 mol/l NaOH inside 100 mL stainless-steel reactors heated to 170°C for 2 hours . After cooling the reactors, the reaction mixture was acidified to pH 2 with 6 mol/l HCl, and water was added to give a final volume of 100 ml. The mixture was stored at 4°C for 16 hours, and treated as described for the mild-condition samples.
Filtrates of both reaction conditions were analyzed by chromatography (AKTA10 chromatograph; Amersham Biosciences, Piscataway, NJ, USA) equipped with a column 250 mm in length and 4 mm outside diameter (Hypersil; Thermo-Scientific) eluted at 0.8 mL/min with acetonitrile:water (1:4) containing 1% (v/v) of acetic acid. Hydroxycinnamic acids were detected in line at 315 nm.
External calibration was performed using authenticated p-coumaric and ferulic acid standards. To correct for partial degradation of hydroxycinnamic acids during the reactions, two calibration curves were built using the same reaction procedures as previously described. Hydroxycinnamic acids were not decomposed under mild reaction conditions. However, severe reaction conditions resulted in 55% and 47% decomposition of ferulic and p-coumaric acids, respectively. To determine these degradation levels, authenticated standards were initially dissolved in diethyl ether and quantitatively transferred to stainless-steel reactors filled with 200 mg of filter paper. After the diethyl ether was evaporated, the filter paper-adsorbed compounds were treated under identical reaction conditions as previously described, and the soluble fractions were analyzed by HPLC.
Samples of the reaction mixtures were also evaluated by GC/MS analysis to confirm the presence of the hydroxycinnamic acids. For this evaluation, 4 ml of the acidified mixture was filtered and extracted with an equal volume of ethyl ether (three successive extractions). The ether extracts were combined and dried over anhydrous Na2SO4, then concentrated under vacuum, and dissolved in 100 μl of pyridine. This solution was left to react with 100 μl of BSTFA (N,O-bis-(trimethylsilyl)-trifluoroacetamide) at 60°C for 1 hour . After silylation, the solution was analyzed in an ion trap mass gas chromatograph (Finnigan MAT-GCQ; Thermo Fisher Scientific Inc., Rockford, IL, USA) equipped with a column 30 m long, with inside diameter 0.25 mm and a 0.25 μm film (BPX-5MS; SGE International, Melbourne, VIC, Australia). Column temperature was initially maintained at 80°C for 2 minutes and then heated to 280°C at 10°C/min. This final temperature was maintained for 15 minutes. Helium at 33.0 cm/s was used as the carrier gas. Injector and transfer line temperatures were 240°C and 275°C, respectively. Samples (1 μl) were injected using a split of 1:30. Retention time in the GC and the mass spectrum of each identified peak were identical to that of silylated authenticated standards. The compounds, their retention time (RT, shown in brackets) and main mass spectrum information (m/z, relative intensity) were as follows: p-coumaric acid (RT 17.56 minutes), 308 (61; M+), 293 (100), 249 (46), 219 (18), 73 (8); ferulic acid (19.05), 338 (100; M+), 323 (85), 308 (66), 293 (47), 249 (18), 219 (9), 73 (3); sinapic acid (3-(4-hydroxy-3,5-dimethoxyphenyl) prop-2-enoic acid) (20.43), 368 (91; M+), 353 (54), 338 (100), 323 (42), 279 (14), 209 (6), 73 (1).
Enzymatic hydrolysis of the sugarcane samples
Enzymatic hydrolysis experiments were performed using a mixture of commercial enzyme preparations (Celluclast and Novozym 188; Novozymes, Denmark) at a dosage of 20 FPU plus 40 IU of β-glucosidase per gram of bagasse (oven-dry weight). Each hydrolysis experiment was conducted in 125-ml Erlenmeyer flasks containing 1 g of milled sample (oven-dry weight) and 10 ml of 50 mmol/l sodium-acetate buffer pH 4.8 in addition to the enzyme solution. The flasks were incubated at 45°C with rotary agitation at 120 rpm. The reaction was stopped at defined periods from 24 to 72 hours by heating the flask to 100°C for 5 minutes, followed by centrifugation of the material at 7800 g for 15 minutes. The soluble fractions were assayed for glucose and xylose by HPLC using a column (HPX87H; Bio-Rad) at 45°C, with an elution rate of 0.6 mL/min with 5 mmol/l sulfuric acid. Sugars were detected using a temperature-controlled infrared detector set at 35°C. The cellulose and xylan conversion levels reported in the text refer to the conversion of the polysaccharides to their monomers. Values (mean ± SD) for the hydrolysis of the untreated sugarcane samples were estimated from triplicate runs performed with the reference cultivar and experimental hybrid number 58, 140, and 146. The SD values for the hydrolysis experiments of the chlorite-delignified samples were estimated from triplicate runs for each evaluated sample.
We thank J. M. Silva and J. C. Tavares for their technical assistance. This work was supported by FAPESP (contract number 08/56256-5) and CNPq (contract number 55274/2007-8). FM and DG thank, respectively, FAPESP and CNPq for their post-doctoral fellowships.
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