Pretreatment of Miscanthus x giganteus Using the Ethanol Organosolv

Jul 31, 2009 - A two-step procedure involving a dilute-acid presoaking step and an aqueous-ethanol organosolv treatment has been evaluated and optimiz...
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Ind. Eng. Chem. Res. 2009, 48, 8328–8334

Pretreatment of Miscanthus x giganteus Using the Ethanol Organosolv Process for Ethanol Production Nicolas Brosse,*,† Poulomi Sannigrahi,‡ and Arthur Ragauskas‡ Laboratoire d’Etude et de Recherche sur le Materiau Bois, Faculte´ des Sciences et Techniques, Nancy-UniVersite´, BouleVard des Aiguillettes, F-54500 VandoeuVre-le`s-Nancy, France, and School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332

A two-step procedure involving a dilute-acid presoaking step and an aqueous-ethanol organosolv treatment has been evaluated and optimized for the conversion of Miscanthus x giganteus (MxG). This process allowed for the efficient fractionation of the raw material into a cellulose-rich residue, an ethanol organosolv lignin fraction, and a water-soluble fraction mainly containing hemicellulose sugars. It was found that the presoaking step not only allowed a better recovery of xylans, but also enhanced the dissolution of lignin in the aqueous ethanol and the digestibility of the remaining cellulose by enzymes. The optimized conditions yielded a solid residue containing about 95% of the initial glucans, from which 98% was recovered after 48 h of enzymatic hydrolysis. In addition, 71% of the lignin was recovered as ethanol organosolv lignin (EOL), and the recovery of the xylans was equivalent to 73% of the xylose present in the raw Miscanthus. 1. Introduction Considering the growing demand for and decreasing reserves of fossil fuels and the necessity to protect our environment, lignocellulosic biomass has obvious potential as a renewable starting material for the production of fuel-grade ethanol and materials according to the biorefinery philosophy.1,2 Lignocellulosic feedstock is mainly composed of three biopolymers (i.e., cellulose, hemicellulose, and lignin), and its effective utilization necessitates the development of cost-effective pretreatment technologies that are necessary to separate the polymers and, from the perspective of ethanol production, to enhance the enzyme digestibility of cellulose.3,4 Several types of physical and/or chemical pretreatments for lignocellulosic biomass conversion have been proposed, including steam explosion,5,6 sulfuric acid,7,8sulfur dioxide,9,10 organosolv,11,12 ammonia,13 aqueous lime and sodium hydroxide,14 and wet oxidation.15-17 These technologies can provide good results in terms of sugar conversion, but a full recovery of the feedstock through optimum utilization of all lignocellulosic components, including nonsugar compounds, as marketable products should be one of the major goals of optimizing a biomass-to-ethanol process. Thus, the key issues concerning the process of pretreatment are manifold: (1) to optimize hydrolysis of the hemicelluloses by limiting their degradation to furans, which act as fermentation inhibitors; (2) to recover the cellulose in a less recalcitrant form, thereby facilitating its reactivity to cellulases; (3) to recover the lignin and to avoid its degradation and recondensation from the perspective of valorization; and (4) to develop a process that can be applied at the pilot, demonstration, and commercial scales. In this context, among the various pretreatment methods currently studied, the ethanol organosolv process seems to be very promising. Indeed, recently, Pan et al.12,18,19 successfully developed this pretreatment technology for hybrid poplar and lodgepole pine killed by mountain pine beetle, producing * To whom correspondence should be addressed. E-mail: [email protected]. Tel.: +333 83 68 48 62. Fax: +333 83 68 44 98. † Nancy-Universite´. ‡ Georgia Institute of Technology.

substrates with very good enzymatic digestibility. This procedure also produces a large amount of a high-quality lignin that is relatively pure, primarily unaltered, and less condensed than other pretreatment lignins. It is soluble in many organic solvents and could find future applications in the fields of adhesives,20 films,21 and biodegradable polymers.22,23 Among biomass feedstocks, Miscanthus x giganteus (MxG) has attracted considerable attention as a possible dedicated energy crop. Trials for bioenergy production commenced in Denmark in 1983, spreading to Germany in 1987 before more widespread evaluation throughout Europe. Field trials are also currently underway in the United States. Indeed, MxG presents some valuable advantages: it is a perennial grass that requires little nitrogen or herbicide, it can grow over 3 m tall per year to produce from 20 to 25 tons of dry matter per hectare, and it is noninvasive.24 Moreover, it is a rhizomatous C4 grass species that has a high carbon dioxide fixation rate. Miscanthus could be an interesting raw material for industrial bioconversion processes given that it is rich in carbohydrates, which constitute approximately 75% of the dry matter content. Recently, a small number of studies have been published on the pretreatment and enzymatic hydrolysis of Miscanthus. The treatments applied include ammonia fiber expansion,25 one-step extrusion/NaOH pretreatment,26 and wet explosion.24 In view of the high xylose content of Miscanthus, soaking with dilute sulfuric acid prior to other pretreatments has also been explored.24 In this article, we describe the optimization of the ethanol organosolv pretreatment of Miscanthus x giganteus for ethanol production. The optimized conditions, which included a presoaking step before treatment, permitted a good separation of hemicelluloses in a water-soluble fraction, lignin as (i.e., ethanol organosolv lignin), and cellulose in the solid residue. This resulting cellulose-rich material was evaluated by enzymatic hydrolysis and fermentation. The results from this two-step pretreatment have been compared to those obtained from dilute sulfuric acid pretreatment, which is an established method for the pretreatment of lignocellulosic biomass.

10.1021/ie9006672 CCC: $40.75  2009 American Chemical Society Published on Web 07/31/2009

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from the pretreatment were sampled through the reactor’s sampling valve and kept for furans analysis. The pretreated Miscanthus was washed thoroughly with water and air-dried. 2.4. Combined Severity (CS) Determination. The organosolv treatments were evaluated with the severity correlation,28 which describes the severity of the pretreatment (in terms of the severity factor Ro) as a function of treatment time (t, min) and temperature (T, °C), where Tref ) 100 °C log(Ro) ) log{t exp[(T - Tref)/14.7]} The effect of pH was taken into consideration through the combined severity, defined as combined severity (CS) ) log(Ro) - pH

Figure 1. Schematic of the two-step presoaking and ethanol organosolv pretreatment.

2. Materials and Methods Raw Miscanthus x giganteus was harvested in spring 2008 from Trier, Germany; the air-dried Miscanthus straw was sent by air mail to the United States, where it was milled to a particle size of 1-3 mm using a Wiley mill and stored at -5 °C during the course of this study. All chemical reagents used in this study were purchased from VWR International and used as received. The enzymes were obtained from Sigma Aldrich (St. Louis, MO) and the K-EtOH ethanol measurement kit was obtained from from Megazyme International (Bray, County Wicklow, Ireland). 2.1. Organosolv Treatment. Twenty-five grams (oven-dry matter) of Miscanthus was treated with aqueous ethanol with sulfuric acid as a catalyst (see Table 2, below, for pretreatment conditions), following the method outlined in Pan et al.18 and depicted in Figure 1. The solid-to-liquid ratio used was 1:8. The pretreatments were carried out in a 1.0-L glass-lined pressure Parr reactor with a Parr 4842 temperature controller (Parr Instrument Company, Moline, IL). The reaction mixture was heated at a rate of ∼3 °C/min with continuous stirring. The pressure increased to 15-20 bar, depending on the temperature and ethanol concentration. At the end of the treatment, the effluents were sampled directly through the reactor’s sampling valve, after being passed through an ice-cooled condensation chamber, and an aliquot was stored for furans analysis. The pretreated Miscanthus was washed with 60 °C ethanol-water (8: 2, 3 × 50.00 mL) and then air-dried overnight. The washes were combined, and three volumes of water were added to precipitate the ethanol organosolv lignin (EOL), which was collected by centrifugation and then air-dried. A portion of each the solid residue and the liquid was separated and stored in a freezer at -5 °C before analysis. 2.2. Presoaking. In the presoaking step, 55 g (dry weight, dry matter content of about 90%) of Miscanthus, 500.0 mL of water, and 40.0 mL of 2 M sulfuric acid (concentration of H2SO4 ) 0.15 mol · L-1) were mixed in a flask and heated to reflux for 17 h. At the end of the reaction, the residue was filtered and air-dried. A portion of each the solid residue and the liquid was separated and stored in a freezer at -5 °C before analysis. 2.3. Dilute Sulfuric Acid Treatment. Miscanthus was soaked overnight with dilute H2SO4 (0.9%, w/w, based on the dry matter content, solid-to-liquid ratio of 1:8) and treated at 180 °C for 2 min.27 The pretreatments were carried out in the 1.0-L Parr pressure reactor previously described. The effluents

As a first approximation, the pH of the liquor can be employed as a measure of the hydrogen ion concentration for sulfuric acid ethanol-water solutions.29 2.5. Analytical Procedures. Oven-dried weights were determined using a moisture analyzer (Mettler HR73). Carbohydrate and lignin contents were measured on extractive-free (Soxhlet extracted with dichloromethane overnight) material, ground to pass a 40-mesh screen, according to the laboratory analytical procedure (LAP) provided by the National Renewable Energy Laboratory (NREL). Samples were hydrolyzed with 72% sulfuric acid for 1 h and then autoclaved after being diluted to 3% sulfuric acid through the addition of water. The autoclaved samples were filtered, and the dried residue was weighed to give the Klason lignin content. Monosaccharide contents in the filtrate were quantified using high-performance anion-exchange chromatography with pulsed amperometric detection (HPAECPAD).30 The acid-soluble lignin content was determined from absorbance at 205 nm according to Lin and Dence.31 To determine the composition of the water-soluble fractions, an aliquot (10.00 mL) was freeze-dried and redissolved in deionized water (10.00 mL), and the monosaccharide contents were quantified using HPAEC-PAD before and after hydrolysis in order to determine the amount of oligomers. The latter was accomplished by addition of 72% H2SO4 to obtain a 3% sulfuric acid solution (348 µL). Furan contents were estimated in the first effluent (obtained through the sampling valve) and in the water-soluble fraction from the absorbance at 284 and 305 nm according to Martinez et al.32 2.6. Solid-State CP/MAS 13C NMR Analysis. Miscanthus samples (untreated and treated), ground to pass a 40-mesh screen, were packed in 4-mm-diameter zirconium oxide rotors fitted with Kel-F caps for solid-state nuclear magnetic resonance (NMR) analysis. Cross-polarization/magic angle spinning (CP/ MAS) 13C NMR experiments were performed on a Bruker Avance-400 spectrometer operating at a 13C frequency of 100.59 MHz. Glycine was used for the Hartman-Hahn matching procedure and as an external standard for the calibration of the chemical shift scale relative to tetramethylsilane. All spectra were acquired at ambient temperature using a Bruker 4-mm MAS probe. 2.7. Enzymatic Hydrolysis. Enzymatic hydrolysis of the pretreated Miscanthus was performed using a commercial cellulase mixture (Celluclast 1.5 L) supplemented with β-glucosidase (Novozym 188) at loadings of 20 FPU (filter paper activity units) and 40 IU (international units) per gram of cellulose, respectively. Enzymatic hydrolyses were performed at 2% (cellulose w/v) in 100 mL of 50 mM acetate buffer, pH 4.8.18 For the experiment supplemented with nonionic surfactant, 250 mg of Tween 80 (polyoxyethylene sorbitan monooleate,

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Table 1. Compositions of Untreated Miscanthus and the Solid Residue and Soluble Fraction after the First Presoaking Stepa presoaked material b

component Klason lignin acid-soluble lignin glucose xylose mannose galactose arabinose

raw material (%)

solid residue (%)

25.1 ( 0.1 23.2 ( 0.5 1.2 ( 0.03 0.4 ( 0.02 carbohydrate 37.7 ( 0.8 37.1 ( 1.5 33.8 ( 0.6 8.8 ( 0.9 0.1 ( 0.05 0.1 ( 0.05 0.6 ( 0.1 0.01 ( 0.01 2.8 ( 0.09 0.03 ( 0.01

soluble fraction (%) 0.8 ( 0.08 1.2 ( 0.4 17.8 ( 2.6 0.2 ( 0.05 0.7 ( 0.2 2.6 ( 0.6

a All data are yields of components (g) per 100 g of oven-dried Miscanthus. b CH2Cl2 extracted.

2.5 g · L-1) was added to the reaction medium. The reaction mixtures were incubated at 45 °C in a shaker at 150 rpm, sampled periodically, and centrifuged to remove the insoluble materials for analysis. Glucose contents of the aqueous phase were quantified using a Shimadzu high-performance liquid chromatograph (HPLC), equipped with an evaporative light scattering (ELS) detector and a Prevail Carbohydrates ES column. Acetonitrile/water (75%, v/v) was used as the eluent. Hydrolysis data are reported as averages from duplicate experiments. 2.8. Preliminary Fermentation Experiments. The aqueous phase from the enzymatic hydrolysis experiments were fermented using baker’s yeast (i.e., Saccharomyces cereVisiae). The method outlined in So¨derstrom et al.35 was followed for these experiments. The solutions were filtered through a 0.45µm membrane, and their pH was adjusted to ∼5.5 by the addition of 5% NaOH solution. The fermentation experiments were carried out in 250-mL Erlenmeyer flasks with a working volume of 100 mL consisting of 92.5 mL of the liquid, 2.5 mL of nutrient solution, and 5.0 mL of yeast inoculum. The flasks were closed with rubber stoppers vented with syringe needles to release the CO2 generated during fermentation. A nutrient solution containing (NH4)2PO4, NaH2PO4, MgSO4 · 7H2O, and yeast extract was prepared and sterilized in an autoclave prior to use. The flasks were incubated for 48 h in a shaking incubator set at 30 °C and 140 rpm, and samples were withdrawn periodically. Ethanol concentrations at different time points were measured using the Megazyme K-EtOH assay kit. Glucose concentrations in these samples were measured using the same HPLC technique as for the enzymatic hydrolysis samples. Percent theoretical ethanol yields were calculated from the equations given in Keating et al.33 2.9. Error Analysis. Analytical errors for Klason lignin and carbohydrate measurements were calculated from analysis of duplicate samples. Error values of 0.8-1.2% (standard deviation) and 8-14% (standard deviation) were determined for the Klason lignin and carbohydrate analyses, respectively. The compositions of the raw and presoaked materials (solid and liquid fractions; Table 1) were established from two independent experiments performed using the same conditions. The standard deviations of the compositions of the fractions after organosolv treatment were obtained from four independent experiments performed under the same conditions (assays 14-17, Table 2). 3. Results and Discussion 3.1. Raw Material Composition and Effects of Presoaking. The composition of raw material used in this study is given in the Table 1. Compared to literature reports on the composition of Miscanthus,24,26 the sample used in this study has a

comparable amount of Klason lignin, a substantially higher proportion of xylose, more arabinose, and less glucose. It has been reported that Miscanthus hemicelluloses are mainly composed of arabinoxylan with a high xylose content,24,34 as is usual in monocotyledonous plants. Because of this high xylose content, the effect of an acid presoaking was investigated. Sørensen et al.24 found that presoaking Miscanthus using a 0.75% sulfuric acid solution at 100 °C for 14 h resulted in the extraction of 63.2% of the xylans. This corresponds to the removal of the easily hydrolyzable fraction of the hemicelluloses. The composition of the presoaked material obtained using this procedure is given in Table 1. We observed the removal of 74% of the xylans and almost all of the arabinans and galactans. It is important to note that 80% of the extracted xylans and a large part of the other sugars were recovered in the water effluents in the form of monosaccharides. Patel and Varshney35 studied the effects of dilute hydrogen peroxide and dilute sulfuric acid presoaking on sugar cane bagasse prior to organosolv pretreatment. In their study, bagasse was soaked in 0.1% sulfuric acid at 125 °C and elevated pressures (45 psi) for 4 h in a pressure reactor. This resulted in the removal of 41.0% of the total hemicelluloses from sugar cane bagasse and aided in organosolv delignification of this material. However, the presoaking procedure employed by Patel and Varshney34 differs from that followed by Sørensen et al.24 and in this study, where the presoaking is carried out under milder pressure and temperature conditions for a longer duration. 3.2. Ethanol Organosolv Pretreatment. The influence of four reaction parameters (i.e., temperature, reaction time, sulfuric acid concentration, and ethanol/water ratio) on the compositions of the solid residue and filtrate and the amount of EOL was examined. According to previously described ethanol organosolv pretreatment studies, among these four parameters, temperature and sulfuric acid concentration showed a more significant effect on the pretreatment.18 It was also demonstrated on softwood and hardwood that experimental conditions corresponding to the optimum from the carbohydrates and lignin recovery point of view were in a temperature range between 170 and 190 °C, at a sulfuric acid concentration between 0.5% and 1.2% and an ethanol concentration of 65%, for a reaction time of around 60 min.18,19 The condition sets used in the present study are given in Table 2 and were selected considering these previously described results. Table 2 presents all of the results obtained, including the combined severity (CS) of each assay (see section 2.4), the composition of the solid residues and liquid phases, and the amount of organosolv lignin recovered. The composition of the liquid phases includes the filtrate and water washes from the presoaking step and he ethanol-water phase after precipitation of EOL in the organosolv step. The amounts of monomeric and oligomeric sugars of glucose, xylose, and arabinose were determined before and after a posthydrolysis step using 3% H2SO4 (see Materials and Methods). The experimental conditions for assays 1-9 are given in the Table 2. As a comparison, we present in the Table 2 the data obtained with an aqueous dilute sulfuric acid pretreatment (SAP) according to a described protocol designed for a biomass-to-ethanol process with optimum xylose yields.27 The experimental conditions used were as follows: 180 °C, 0.9% sulfuric acid/water solution, 2 min. With these conditions (which were not optimized), almost all of the glucans and the lignin were recovered in the solid. whereas 57% of the xylans and 73% of the arabinans were hydrolyzed and present, to a large extent, in the water effluent as monomeric and oligomeric sugars. The very low concentra-

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Table 2. Experimental Conditions for the Different Pretreatments Evaluated for Miscanthus and Compositions of the Solid and Soluble Fractions Isolated after the Pretreatmentsa,b conditions assay

T

t

SA

solid C

MxG SAP 180 2 0.9 0 1 170 60 0.5 0.65 2 170 60 0.9 0.65 3 170 60 0.9 0.75 4 170 60 0.9 0.5 5 170 60 1.2 0.65 6 170 80 1.2 0.65 7 180 60 0.9 0.65 8 180 60 1.2 0.65 9 190 60 1.2 0.65 d 10 170 60 0.9 0.65 d 180 60 0.9 0.75 11 12d 180 60 1.2 0.65 180 60 0.9 0.8 13d 14d 170 60 0.5 0.8 14-17d 170 60 0.5 0.8 standard deviation, assays 14-17:

water-soluble fraction c

CS

KL

ASL

Glu

Xyl

Ara

EOL

SL

furans

Glu

Xylc

Man

Gal

Arac

1.03 1.75 2.07 1.99 2.26 2.27 2.39 2.36 2.56 2.86 2.07 2.28 2.56 2.23 1.69 1.69

25.1 24.1 15.5 11 14.5 15 9.4 7.8 8.5 7.7 8.4 9.2 6.8 10.4 5.7 7.6 7.75 0.32

1.17 0.53 0.85 0.65 0.68 0.38 0.6 0.73 0.43 0.34 0.5 0.2 0.31 0.31 0.3 0.15 0.12 0.02

37.7 37.1 30.8 30.07 38.4 38.1 37.4 36 33 36 35.2 35.3 29.8 22.4 21.21 35.8 35.48 1.07

33.8 14.6 31.1 25.04 22.2 12.7 17.4 6.2 12.7 4.7 3.4 3.1 2.6 0.3 0.98 3.8 3.2 0.43

2.8 0.78 1.35 0.69 1.12 0.5 0.53 0.3 0.5 0.2 0.2 0 0 0 0.1 0.1 0.09 0.05

3.7 10.7 8.8 4.4 10.8 11 10.8 11 12.8 14.8 20.8 12 24 19.2 18.1 1.61

0.58 0.63 0.58 0.5 0.68 0.68 0.55 0.83 0.86 0.51 0.57 0.87 0.29 0.16 0.18 0.02

0.12 0.35 0.36 0.39 0.47 0.59 1.03 0.67 0.97 1.89 0.87 1.48 4.46 2.1 0.44 0.41 0.07

0.44 (0.22) 0.19 (0.12) 0.14 (0.1) 0.21 (0.19) 0.8 (0.72) 0.53 (0.25) 0.93 (0.26) 0.53 (0.51) 0.85 (0.53) 0.92 (0.25) 3.33 (1.57) 6.24 (2.1) 9.08 (6.13) 5.54 (3.1) 1.74 (1.14)

15.3 (8.67) 3.96 (3.69) 7.53 (4.69) 3.9 (3.1) 10.26 (5.83) 9.33 (5.01) 5.89 (1.85) 10.19 (6.8) 9.34 (5.0) 5.89 (1.85) 22.33 (1.97) 21.8 (1.04) 20.37 (0.49) 20.62 (0.51) 21.7 (1.32)

0.4 0.06 0.05 0.07 0.1 0.08 0.07 0.08 0.09 0.06 0.15 0.14 0.28 0.14 0.13

0.4 0.25 0.28 0.26 0.4 0.35 0.23 0.27 0.34 0.23 0.64 0.65 0.65 0.68 0.72

1.7 (0.22) 0.98 (0.49) 0.93 (0.52) 0.81 (0.51) 1.23 (0.75) 1.03 (0.65) 0.59 (0.26) 0.81 (0.48) 1.01 (0.77) 0.60 (0.27) 2.38 (0.14) 2.51 (0.12) 2.4 (0.07) 2.43 (0.08) 2.51 (0.12)

a All data are yields of components (g) per 100-g dry weight of Miscanthus. b Abbreviations used: ASL ) acid-soluble lignin, C ) ethanol/water ratio, CS ) combined severity, EOL ) ethanol organosolv lignin, KL ) Klason lignin, MxG ) Miscanthus x giganteus (raw material), SA ) sulfuric acid concentration (%, based on weight of dry Miscanthus), SAP ) sulfuric acid pretreatment, SL ) soluble lignin, T ) temperature (°C), t ) time (min). c Values in parnetheses correspond to the fraction of oligosaccharides. d Assays 10-17 include the presoaking step.

tion of furans detected attested to the low degradation of sugars under these experimental conditions. In the case of organosolv pretreatments, for the assays without presoaking (assays 1-9), almost all of the glucans were recovered in the solid residue, but depending on the conditions used, 30-60% of the lignin and 10-93% of the hemicelluloses were not removed. The relatively high xylan content in our raw material can explain the amount of xylan retained in the pretreated material. It appears from assays 2-4 in Table 2 that an ethanol concentration of 65% gave the best result for delignification, which is in accordance with the conditions previously defined by Pan et al.19 for lodgepole pine. This concentration of ethanol appears to be a good compromise between the optimum conditions for the depolymerization of lignin and sugars and the solubilization of lignin. Increasing the severity of the reaction conditions (assays 5-9) resulted in a weak reduction of the Klason lignin content. It was noted that the use of more severe conditions (combined severity CS > 2.5, assays 8 and 9) permitted the removal of a large part of the hemicellulose content from the solid phase. On the other hand, under these conditions, higher contents of soluble lignin and furans were observed, suggesting greater degradation of the lignin and sugars. As shown in Table 2 and in Figure 2, a large part of the dissolved lignin was recovered as EOL, but it appears that a two-step protocol including a presoaking was better for hemicellulose recovery. In assays 10-14, a recovery of sugars in the liquor of about 30% of the dry mass was obtained, compared to 5-10% without presoaking (assays 1-9; see Table 2 and Figure 2). These higher overall yields can be explained by the less harsh conditions of the presoaking steps compared to the organosolv treatment, preventing the degradation of the xylose. More surprisingly, starting from presoaked material, better results were obtained in terms of delignification, and a decrease of the cellulose content in the solid residue was observed, with a significant amount of glucose detected in the liquid phase. An extensive degradation of the cellulose was obtained with more severe conditions (assay 12, CS ) 2.56) supported by the high glucose and furans concentrations in the liquid phase. These observations suggest that the removal of a part of the hemicelluloses during the presoaking step disrupted enough of the remaining polymers to enhance the hydrolysis of cellulose

Figure 2. Composition of the ethanol-water-soluble fractions resulting from organosolv pretreatment of MxG. All data are yields of components (g) per 100 g of oven-dried Mxg. The amounts of the monomeric sugars of glucose, xylose, and arabinose were determined after a posthydrolysis step using 3% H2SO4 (see section 2.5).

and lignin during the organosolv process. In fact, it was reported35 that acid hydrolysis of more than 50% of the hemicelluloses in bagasse leads to significant modification of the lignin and cellulose, which could facilitate a subsequent delignification reaction. As a consequence, a low sulfuric acid concentration and/or high ethanol content (low combined severity) was required to obtain a good solubilization of the partially disrupted lignin and to avoid an extensive degradation of the cellulose. Under the optimized conditions obtained with a low combined severity (Table 2, assay 14, CS ) 1.69), the yield of solid fraction was 62% w/w; the composition of this solid residue was 11.2% Klason lignin and 81.5% cellulose (corresponding to 7.6% and 35.8%, respectively, of the initial dry mass). About 70% of the lignin was dissolved and recovered as EOL after precipitation of the ethanol-rich fractions. It appears that our procedure allowed for a clean fractionation of the three main constituents of Miscanthus with high recovery yields. The reproducibility of the results was evaluated by statistical analysis of four independent experiments (assays 14-17) performed under the same conditions.

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Figure 4. Cellulose-to-glucose conversion (%) at different time points during the enzymatic hydrolysis experiments. SAP ) sulfuric acid pretreated Miscanthus. Figure 3. 13C CP/MAS NMR spectra of Miscanthus: (A) untreated, (B) pretreated with ethanol organosolv process. Assignment of the signals: (a) CdO of acetate groups (lignin and hemicellulose); (b) aromatic carbons (lignin); (c) hemicelluloses and celluose C-1; ( d) crystalline cellulose C-4; (e) amorphous cellulose C-4; (f) hemicelluloses and cellulose C-2, C-3, and C-5; (g) cellulose C-6; (h) OMe (lignin and hemicellulose); (i) Me of acetate groups (lignin and hemicellulose).

3.3. Solid-State CP/MAS 13C NMR Analysis. Solid-state CP/ MAS 13C experiments were carried out on organosolv-treated (assay 14) and untreated Miscanthus to gain information on their chemical compositions. The spectra are shown in Figure 3. The NMR resonances were assigned according to data from the literature.36-38 In the spectrum of untreated Miscanthus in Figure 3A, the region between 65 and 120 ppm includes peaks from O-alkyl carbons. In particular, the resonance at 83 ppm is due to the C-4 carbons of amorphous cellulose and hemicelluloses. The shoulders at 65 and 89 ppm correspond to the C-6 and C-4 carbons of crystalline cellulose, respectively. The intense peaks at 73 and 75 ppm are overlapping signals due to the C-2, C-3, and C-5 carbons of all polysaccharides. The resonance at 105 ppm is assigned to the anomeric carbon (C-1) of cellulose. The signals at 56 ppm, 130-155 ppm and 168-178 ppm are associated respectively with the methoxyl, aromatic, and carbonyl groups of hemicelluloses and lignin. Finally, the peak at 20 ppm is due to the acetyl groups of hemicelluloses. In the NMR spectrum of treated Miscanthus (Figure 3B), the presence of very little lignin and hemicellulose in the sample is revealed, with signals of very low intensity at 20, 55, and 130-190 ppm. Moreover, the decrease in the peak at 83 ppm (C-4 in amorphous carbohydrate moieties) relative to the peak at 89 ppm (C-4 in crystalline cellulose) suggests a degradation of amorphous carbohydrates (mainly hemicelluloses). All of these observations confirm the high cellulose content of the solid residue. 3.4. Enzymatic Hydrolysis. Enzymatic hydrolyses of the solid residues of two selected experiments, assays 6 and 14 in Table 2, were performed using standard literature conditions,39 and these results are shown in Figure 4. It appears that the enzymatic hydrolysis of the solid fraction obtained by dilute sulfuric acid pretreatment showed a low cellulose-to-glucose conversion yield of 40% after 72 h. On the other hand, by means of the organosolv pretreatment, the cellulose was made more amenable to enzymatic hydrolysis, yielding ∼78-98% cellulose-to-glucose conversions after 48 h of incubation. The lower lignin content of the organosolv-treated material is presumably one of the reasons that the pretreated substrate exhibited a better hydrolysability. The dilute sulfuric acid pretreatment did not

significantly delignify the raw material, and most of the lignin remained in the resulting substrate, which continued to be very resistant to enzymatic hydrolysis. High lignin content hinders enzymatic hydrolysis through the nonproductive binding of cellulase enzymes to lignin.40 Moreover, presoaked Miscanthus exhibited better hydrolysibility than nonpresoaked material: a complete conversion was obtained for assay 14 compared to 80% yield for assay 6 in Figure 4. This observation is in accordance with the results described by Sørensen et al.24 Thus, the presoaking step makes the biomass more susceptible to the pretreatment by a partial disintegration of the cellulose. This hypothesis is supported by the slight degradation of the cellulose observed starting from the presoaked Miscanthus during the organosolv pretreatment. It has been reported that addition of nonionic surfactants, particularly Tween species, effectively improves the cellulase activity, especially in the case of steam-exploded and acid-pretreated biomass, which have poor digestibility due to high lignin contents.41 It is interesting to note that, in assay 6, which was performed without presoaking, addition of Tween 80 to the medium enhanced the enzymatic hydrolysis. 3.5. Fermentation Test. In a preliminary study, the filtrate resulting from the enzymatic hydrolysis of assay 14 in Table 2 was fermented using ordinary baker’s yeast (S. cereVisiae) to investigate the fermentability of our liquor and the potential inhibitory effects of degradation products, such as furfural and 5-hydroxymethyl-2-furfural (HMF), on fermentation. The experimental conditions used were described by Soederstroem et al.39 Figure 5 shows a plot of the ethanol yield over the course of fermentation. In this experiment, a theoretical ethanol yield of 70% was obtained in 48 h, demonstrating the fermentability of the liquor. The hemicellulose-rich liquid fraction after the first presoaking step should also be fermentable using yeasts capable of utilizing both C5 and C6 sugars. Successful fermentation of this fraction will greatly contribute to making the overall process of converting Miscanthus to bioethanol more economically attractive. 4. Conclusions In the present study, we have examined the pretreatment of Miscanthus using an ethanol organosolv process. A protocol in two steps, including a presoaking step, allowed for a clean fractionation of the three main constituents of Miscanthus with high recovery yields. Our results show that the presoaking step not only allowed for a better recovery

Ind. Eng. Chem. Res., Vol. 48, No. 18, 2009

Figure 5. Ethanol yield during fermentation at different time points.

of the xylose (recovery of ∼10% of the dry mass without presoaking, ∼20% with presoaking), but also enhanced the dissolution of lignin in aqueous ethanol. The presoaking improved enzymatic digestibility of the residue after organosolv treatment (80% cellulose-to-glucose conversion without presoaking and 98% conversion with presoaking). Moreover, the organosolv treatment performed after a presoaking step can be carried out at a lower severity, thus producing a low concentration of furans (sugar degradation products