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.