ACCELERATED PUBLICATION Fractionating Recalcitrant Lignocellulose at Modest Reaction Conditions

Yi-Heng Percival Zhang,1 Shi-You Ding,2 Jonathan R. Mielenz,3 Jing-Biao Cui,4 Richard T. Elander,5 Mark Laser,5 Michael E. Himmel,2 James R. McMillan,2 Lee R. Lynd5 Biological Systems Engineering Department, Virginia Tech, Blacksburg, Virginia 24061; telephone: (540) 231-7414; fax: (540) 231-3199; e-mail: ypzhang@vt.edu 2 National Bioenergy Center, National Renewable Energy Laboratory, Golden, Colorado 80401 3 Life Science Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37831 4 Center for Nanomaterials Research, Dartmouth College, Hanover, New Hampshire 03755 5 Thayer School of Engineering, Dartmouth College, Hanover, New Hampshire 03755
Received 23 December 2006; accepted 5 February 2007 Published online 22 February 2007 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bit.21386
1

Introduction
ABSTRACT: Effectively releasing the locked polysaccharides from recalcitrant lignocellulose to fermentable sugars is among the greatest technical and economic barriers to the realization of lignocellulose bioreneries because leading lignocellulose pre-treatment technologies suffer from low sugar yields, and/or severe reaction conditions, and/or high cellulase use, narrow substrate applicability, and high capital investment, etc. A new lignocellulose pre-treatment featuring modest reaction conditions (508C and atmospheric pressure) was demonstrated to fractionate lignocellulose to amorphous cellulose, hemicellulose, lignin, and acetic acid by using a non-volatile cellulose solvent (concentrated phosphoric acid), a highly volatile organic solvent (acetone), and water. The highest sugar yields after enzymatic hydrolysis were attributed to no sugar degradation during the fractionation and the highest enzymatic cellulose digestibility ($97% in 24 h) during the hydrolysis step at the enzyme loading of 15 lter paper units of cellulase and 60 IU of betaglucosidase per gram of glucan. Isolation of high-value lignocellulose components (lignin, acetic acid, and hemicellulose) would greatly increase potential revenues of a lignocellulose biorenery. Biotechnol. Bioeng. 2007;97: 214223. 2007 Wiley Periodicals, Inc. KEYWORDS: biorenery; cellulose hydrolysis; cellulosic ethanol; lignocellulose fractionation

Correspondence to: Y.-H.P. Zhang Contract grant sponsor: ACS Petroleum Research Fund Contract grant sponsor: Oak Ridge Associated Universities Contract grant sponsor: DOE Ofce of the Biomass Program Contract grant sponsor: UT-Battelle Contract grant sponsor: DOE Contract grant sponsor: NIST

Lignocellulose, the most abundant renewable biomass produced from photosynthesis, has a yearly supply of approximately 200 billion metric tons worldwide (Ragauskas et al., 2006; Zhang et al., 2006b). Transition from a fossilfuel-based economy to a renewable carbohydrate economy will inevitably take place in the foreseeable future because of the depletion of fossil fuel reserves and the resulting accumulation of atmospheric greenhouse gases (Caldeira et al., 2003; Demain et al., 2005; Farrell et al., 2006; Ragauskas et al., 2006). Lignocellulose consists primarily of plant cell wall materials; it is a complicated natural composite with three main biopolymerscellulose, hemicellulose, and lignin (Ding and Himmel, 2006; Zhang and Lynd, 2004). Lignocellulose structures and compositions vary greatly, depending on plant species, plant parts, growth conditions, etc. (Ding and Himmel, 2006; Zhang and Lynd, 2004). Lignocellulose bioreneries via biological conversion generally have three main steps: (1) lignocellulose pretreatment, which converts the recalcitrant lignocellulose structure to reactive cellulosic intermediates; (2) enzymatic cellulose hydrolysis, by which cellulases hydrolyze reactive intermediates to fermentable sugars (e.g., glucose and xylose); and (3) fermentation, which produces cellulosic ethanol or other bio-based chemicals (e.g., lactic acid, succinic acid) (Ragauskas et al., 2006; Wyman et al., 2005b; Zhang et al., 2006b).

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Effectively overcoming the recalcitrance structure of lignocellulose and releasing the locked polysaccharides is one of the most important and urgent R&D priorities for the emerging cellulosic ethanol and bio-based chemical industries (Biomass Research and Development Technical Advisory Committee, 2002; Ofce of Energy Efciency and Renewable Energy and Ofce of Science, 2006), because lignocellulose pre-treatment is among the most costly steps and has a major inuence on the costs of both prior operation (e.g., lignocellulose particle size reduction) and subsequent operations (e.g., enzymatic hydrolysis and fermentation) (Wooley et al., 1999; Wyman et al., 2005b). A number of lignocellulose pre-treatment technologies are under intensive investigation on both laboratory scale and as pilot plants, for example, dilute acid, ow-through, ammonia ber explosion, ammonia recycle percolation, lime, steam explosion, and organosolv (OS) pre-treatment (Mosier et al., 2005; Wyman et al., 2005a,b). All these processes suffer from relatively low sugar yields, severe reaction conditions (high temperature and/or high pressure), accompanied by large capital investment, high processing costs, and great investment risks (Eggeman and Elander, 2005). Development of a lignocellulose pretreatment featuring modest reaction conditions is highly desired because it would not only decrease utility consumption and initial capital investment but also reduce sugar degradation and inhibitor formation (McMillan, 1994). We hypothesized that the root causes of the recalcitrance of lignocellulose to enzymatic hydrolysis could be mainly attributed to: (1) low accessibility of (micro-) crystalline cellulose bers, which restricts cellulase activity, and (2) the presence of lignin (mainly) and hemicellulose on the surface of cellulose, which prevents cellulase from accessing the substrate. As for pure cellulose, our functionally based mathematical model suggests that increasing cellulose accessibility to cellulase is the most inuential for increasing the rate of enzymatic cellulose hydrolysis (Zhang and Lynd, 2006). After we studied a number of chemical and physical methods that can break up orderly hydrogen bonds among glucan chains in crystalline cellulose, we found that concentrated phosphoric acid (>81%) was an ideal cellulose solvent (Zhang et al., 2006a), because (1) cellulose dissolution by phosphoric acid occurs at low temperatures, (2) phosphoric acid can dissolve cellulose in the presence of water, (3) the regenerated cellulose remains in an amorphous form suitable for hydrolysis, and (4) the residual phosphorous acid has no inhibitory effects on the sequential hydrolysis and fermentation. The regenerated amorphous cellulose has a high reactivity because its enzymatic hydrolysis pattern is completely different from that of crystalline cellulose and the synergy among exoglucanase and endoglucanase is unimportant to amorphous cellulose hydrolysis (Zhang and Lynd, 2005, 2006). Recently, similar results were also reported by using cellulose solventsionic liquids (Dadi et al., 2006).

As for natural lignocelluloselignin-hemicellulosecellulose composite, the current leading lignocellulose pre-treatments cannot efciently disrupt orderly hydrogen bonds among glucan chains in crystalline cellulose, partially resulting in slow hydrolysis rates and low cellulose digestibility (i.e., modest sugar yields) (Wyman et al., 2005a,b). Furthermore, after most lignocellulose pretreatments, a majority of lignin and hemicellulose remains on the surface of cellulose. Here we present a novel method which combines a nonvolatile cellulose solvent and a second highly volatile organic solvent to separate lignocellulose components and recycle both solvents easily.

Materials and Methods

Chemicals and Materials All chemicals were reagent-grade and purchased from Sigma (St. Louis, MO), unless otherwise noted. Microcrystalline cellulose, Avicel PH105 (20 mm) was obtained from FMC Corp. (Philadephia, PA). Corn stover, switchgrass, and hybrid poplar were graciously provided by the National Renewable Laboratory (NREL, Golden, CO); Douglas-r was graciously gifted from Prof. Jack Saddler at UBC. All lignocellulosic materials were knife-milled and screened. The lignocellulose particles, smaller than a 40-mesh screen and bigger than a 60-mesh screen, were used for pretreatment experiments.

Lignocellulose Fractionation One gram of dry lignocellulose was placed in a 50-mL disposable plastic centrifuge tube, and then mixed with 8 mL of pre-calibrated concentrated phosphoric acid (e.g., 83$85.9%) using a glass rod. The solid/liquid slurry was incubated in a rotary water bath at 90 rpm and 50 0.28C. After the reaction, $20 mL of pre-cold acetone was added into the tube for quenching the reaction, and then mixed well by force. After centrifugation by a Thermo Electron CL3R benchtop centrifuge with swing buckets (3,400 rpm, 20 min, room temperature), the supernatant was collected. The solid pellet was suspended by $40 mL of the acetone and centrifuged three times. The supernatants from acetone washes were collected for separation of lignin, acetic acid, and acetone. After acetone removal from the supernatant by simple evaporation, the lignin precipitated from aqueous phosphoric acid was centrifuged, washed by water, dried, and weighed. The solid pellets after acetone washes were suspended in 40 mL of distilled water and centrifuged three times, the supernatants were combined. The acetone in the combined supernatant was evaporated in a chemical fume hood and the residual hemicellulose sugar components after post-hydrolysis were measured by HPLC (Liu and Wyman, 2003). The cellulosic pellets after acetone and water washes were diluted to 10 g glucan/L in 50 mM citric

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acid buffer (pH 4.8) and 0.5 g/L sodium azide for enzymatic hydrolysis.

Assays The composition of lignocellulosic feedstock was determined through application of NREL LAP 001, 002, and 012 procedures as described by NREL elsewhere (Liu and Wyman, 2005; Sluiter et al., 2006). The soluble sugars after enzymatic cellulose hydrolysis, acidication by addition of sulfuric acid, and centrifugation, were measured by HPLC using a Bio-Rad HPX-87H column operated at 558C with 0.01% (v/v) H2SO4 as the mobile phase (Zhang and Lynd, 2003).

Enzymatic Cellulose Hydrolysis All enzymatic hydrolysis experiments were carried out in a rotary shaker at 50 0.28C. Enzyme loadings for hydrolysis were 15 Genencor Spezyme CP cellulase FPU and 60 IU novozyme 188 b-glucosidase per gram of glucan. The enzymatic cellulose digestibility (Xreal) for pre-treated lignocellulosic samples was calculated as described elsewhere (Zhang et al., 2007) because this new method can effectively reduce the measurement randomness effect on the accuracy of enzymatic cellulose hydrolysis at high enzymatic cellulose digestibility and the results obtained are more accurate and conservative. The enzymatic hydrolysate was centrifuged once and washed by 20 mL of distilled water twice. The freeze dried pellet was measured for sugar, lignin, and ash composition, according to the standard protocol (Sluiter et al., 2006). Cellulose digestibility was calculated based on the adjustment of the reaction volume decrease due to the samples taken. An atomic force microscope (AFM) was used to image the intact and pre-treated materials, respectively (Ding and Himmel, 2006).

Results
In order to effectively deal with two root causes of the recalcitrance of lignocellulosebreaking up orderly hydrogen bonds in crystalline cellulose and removing lignin and hemicellulose from the surface of cellulose, we designed a novel chemical process that not only fractionated lignocellulose components based on the signicantly different solubility of cellulose, hemicellulose, and lignin in cellulose solvent, organic solvent, and water, but also easily recycled solvents due to the large difference in volatility between phosphoric acid and acetone. Figure 1 shows the conceptual processes of the lignocellulose fractionation technology, by using cellulose solvent (concentrated phosphoric acid) and organic solvent (acetone) with reagent recycling. The mechanisms for each unit operation are:

(1) in the digestion tank, concentrated H3PO4 (>82%) is used to treat (wet) lignocellulose at 508C for $30 60 min, where phosphoric acid is used to: (i) disrupt the lignin-carbohydrate complex bonds; (ii) dissolve cellulose brils and hemicellulose by breaking up orderly hydrogen bonds among sugar chains; (iii) weakly hydrolyze cellulose and hemicellulose to low degree of polymerization (DP) fragments; and (iv) remove acetyl groups from hemicellulose to form acetic acid. (2) in the precipitation tank, acetone is added to precipitate the dissolved cellulose and hemicellulose and to dissolve partial lignin in the liquid phase; (3) in the washer-1 (solid/liquid separator), acetone is used to wash out $99.5% of phosphoric acid from the precipitated solids and to remove the dissolved lignin; (4) in the washer-2 (solid/liquid separator), water is used to wash out acetone in the solids and to remove watersoluble hemicellulose from the solid cellulose; (5) in the hydrolysis reactors, enzymatic cellulose hydrolysis is carried out at 508C; (6) in the distiller, the black liquor containing phosphoric acid, acetone, acetone-soluble lignin, and acetic acid from hemicellulose can be separated. Highly volatile acetone and modestly volatile acetic acid can be easily separated based on different volatility, and the precipitated lignin, after removal of acetone, can be separated from non-dilute concentrated phosphoric acid for dry biomass or slightly dilute concentrated phosphoric acid for wet biomass at the bottom of the column by a solid/liquid separator; and (7) in the ash tank, the light liquor containing acetone, a small amount of phosphoric acid, and water-soluble hemicellulose can be separated. Acetone can be recovered by ashing; the precipitated Ca3(PO4)2, after the reaction with CaCO3, can be used to regenerate concentrated phosphoric acid by adding concentrated sulfuric acid (the wet phosphoric acid production method); water soluble hemicellulose remains in the liquid phase. In all, this technology can fractionate lignocellulose into amorphous cellulose (mainly glucose after hydrolysis), lignin, hemicellulose, and acetic acid at modest reaction conditions (508C, atmospheric pressure) with easy recycling of acetone and phosphoric acid. Figure 2 shows the enzymatic hydrolysis proles for the cellulosic samples with and without the pre-treatment. In addition to Avicel (microcrystalline cellulose) and acellulose (a mixture of cellulose [$96%] and hemicellulose [$4%]), four representative lignocellulosic materials were tested: corn stover, the most abundant agricultural residue in the USA; switchgrass, a potential biofuel herbaceous plant; hybrid poplar, a potential biofuel hardwood plant; and Douglas r, a popular softwood plant in Canada. Pretreated Avicel and a-cellulose were completely converted to soluble sugars (a transparent solution) within 3 h

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Figure 1. Conceptual owchart of cellulose-solvent and organic-solvent lignocellulose fractionation with recycling of concentrated phosphoric acid and acetone. [Color gure can be seen in the online version of this article, available at www.interscience.wiley.com.] (Fig. 2A and B), suggesting that regenerated amorphous cellulose without lignin can be hydrolyzed very quickly and efciently. For herbaceous cellulose (corn stover and switchgrass) and hardwood lignocellulose, the pre-treated cellulosic samples were hydrolyzed by $94% at the 12th hour and by $9697% at the 24th hour. The data of hydrolysis rates and digestibility were the highest as compared to the literature (McMillan, 1994; Mosier et al., 2005; Wyman et al., 2005a,b). Although herbaceous and hardwood lignocellulose have different inherent physical structures and chemical compositions, this technology can convert their crystalline cellulose to amorphous cellulose and remove major hemicellulose and lignin, resulting in increases in digestibility by factors of 4.7, 7.4, and 13.4 for corn stover, switchgrass, and hybrid poplar, respectively. Residual lignin on the surface of amorphous cellulose slows hydrolysis rates and decreases digestibility as compared to amorphous cellulose without lignin. This technology seems not to be efcient for treating softwood Douglas r ($75% digestibility) because of inefcient lignin removal. Mass ow for key lignocellulose components of corn stover after lignocellulose fractionation and enzymatic hydrolysis is shown in Figure 3. The total yields of glucose, including glucose in the hemicellulose solution and enzymatic hydrolysate, and xylose, representing all hemicellulose sugars, are as high as 0.40 wt glucose ($95% of the original glucose) and 0.189 wt xylose equivalent ($79% of the original xylose). With improvement in technologies (e.g., a hemicellulase supplement for enzymatic cellulose hydrolysis, optimization of reaction conditions, washing methods and conditions (e.g., solvent temperatures, ow rates), higher xylose recovery yields are anticipated without the sacrice of glucose yields. The dramatic changes in the supramolecular structures of corn stover before and after pre-treatment are shown by atomic force microscopy (AFM) images (Fig. 4). Before pretreatment, the plant cell wall structures of corn stover and elementary cellulose bers were clearly identied (Fig. 4A). After pre-treatment, no bril structures were observed (Fig. 4B), indicating that concentrated phosphoric acid not only disrupted all linkages among cellulose, hemicellulose, and lignin, but also broke up the orderly hydrogen bonds among glucan chains. These images are completely different from those after such treatments as dilute acid, ammonia

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Figure 2.

Prole of enzymatic cellulose hydrolysis for the cellulosic samples (A, Avicel; B, a-cellulose; C, corn stover; D, switchgrass; E, hybrid poplar; F, Douglas r) at a 10 g/L glucan and a cellulase loading 15 lter paper unit/g glucan and cellobiase 60 IU/g glucan at 508C. The open and closed symbols denote the samples with and without the pretreatment, respectively. Avicel and a-cellulose were pre-treated by 81.7% phosphoric acid at room temperature for a half-hour; corn stover and switch grass were pre-treated by 84% phosphoric acid at 508C for 45 min; hybrid poplar and Douglas r were pre-treated by 85% phosphoric acid at 508C for 60 min. All solid/liquid separations were conducted by centrifugation. [Color gure can be seen in the online version of this article, available at www.interscience.wiley.com.]

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Figure 3. Mass ow through lignocellulose fractionation technology and enzymatic cellulose hydrolysis based on corn stover. [Color gure can be seen in the online version of this article, available at www.interscience.wiley.com.] recycle percolation, etc. (Kim and Lee, 2005; Ofce of Energy Efciency and Renewable Energy and Ofce of Science, 2006). cellulose solvents (Pereira et al., 1988). For example, regenerated cellulose dissolved by concentrated sulfuric acid or phosphoric acid loses its original crystalline structure completely (Xiang et al., 2003; Zhang et al., 2006a). Using concentrated acid saccharication (CAS) for lignocellulose has a long history but has three big technical obstacles acid/soluble sugar separation, acid recovery, and acid reconcentration (U.S. Department of Energy; Fengel and Wegener, 1984).

Discussion
Concentrated inorganic acids (e.g., sulfuric acid, hydrochloric acid, nitric acid) have been well known to work as

Figure 4. Images of corn stover before treatment (A) and after treatment (B) by AFM. [Color gure can be seen in the online version of this article, available at www.interscience.wiley.com.]

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Ladisch et al. (1978) invented a method using a nonhydrolysis cellulose solvent to disrupt linkages among cellulose, hemicellulose, and lignin as well as break up the orderly hydrogen bonds in crystalline cellulose. But this method cannot efciently remove lignin and hemicellulose from amorphous cellulose, resulting in lower cellulose digestibility and slower hydrolysis rates as compared to this technology. Recently, use of ionic liquids to treat cellulosic materials is becoming a hot research topic (Dadi et al., 2006; Swatloski et al., 2002), but it is still challenging to deal with real lignocellulose. Concentrated phosphoric acid used here must be primarily regarded as a cellulose solvent and secondarily as a cellulose hydrolysis catalyst. In Unit 3, we can easily separate solid (precipitated) amorphous cellulose with phosphoric acid so that we solve a technical obstacle of CASsugar/acid separation. Since concentrated phosphoric acid is a modest acid, the hydrolysis degree of hemicellulose and cellulose can be easily controlled at modest temperatures. Here modest hydrolysis was preferred because we can separate oligo-hemicellulose sugars (DP $10) from amorphous cellulose (DP $ several hundreds) based on their different solubility in organic solvent and water. We rst introduced a second solventacetone, between concentrated phosphoric acid and water. Use of acetone has four goals: (1) to precipitate dissolved cellulose and hemicellulose to amorphous forms, resulting in an easy separation of solid saccharides from liquid acid; (2) to dissolve partial lignin in acetone and recover solid lignin after removal of acetone because acetone-dissolved lignin is insoluble in acidic aqueous solutions; and (3) to recycle concentrated phosphoric acid by avoiding acid dilution using water and easy acid re-concentration; and (4) to fractionate oligo-hemicellulose sugars from cellulose because the former has some water solubility but poor solubility in the acetone/water mixture (Gray et al., 2003). As compared to CAS, this technology not only solves the three main technical challenges: acid/sugar separation, acid recovery, and acid re-concentration, but also isolates lignin and acetic acid, and decreases hemicellulose degradation. This lignocellulose fractionation has something in common with OS, which is well known as an environmentally friendly technology and has been fully tested in pilot plants (Aziz and Sarkanen, 1989; Holtzapple and Humphrey, 1984; McDonough, 1993; Pan et al., 2005). As compared to OS, this technology offers relative advantages: higher yields of glucose and xylose, lower utility consumption for lignocellulose cooking (508C vs. $1608C), less capital investment for high temperature and pressure corrosion-resistance reactors, easier organic solvent recycling (acetone vs. ethanol), as well as amorphous cellulose formation. This technology has broad substrate applicability, including corn stover, switchgrass, and hardwood hybrid poplar, but it seems not to be efcient for treating softwood Douglas r ($75% digestibility). Digestibility of Douglas r ($75%) was much higher than when pre-treated by SO2

steam explosion (44%) (Yang et al., 2002). Further softwood lignin removal could be achieved by using hot acetone or other solvents to solubilize more lignin, hot water to wash out lignin via ltration (Nagle et al., 2002), countercurrent washing or ow-through to remove lignin (Liu and Wyman, 2003), or even the relatively costly alkaline peroxide method (Yang et al., 2002). Board substrate applicability of this technology suggests that the most important thing for bioenergy plants could be to increase biomass productivity rather than to decrease lignin contents (Sticklen, 2006). Amorphous cellulose with little presence of lignin and hemicellulose offers benets in: the highest sugar yields, the fastest hydrolysis rates, the least use of enzyme, possible enzyme recycling, lower electricity consumption for hydrolysis mixing, and lower capital investment for smaller size hydrolysis reactors. For example, a relatively costly cellulase, which is released in the liquid phase after hydrolysis, can be re-used by adding new solid cellulase substrate (Gregg and Saddler, 1996) because of a much shorter hydrolysis time (e.g., 1224 h vs. usually 72 h). In addition, engineering cellulases based on amorphous cellulose will be much simpler than before, since hydrolysis of amorphous cellulose can be conducted by only endoglucanase and b-glucosidase without exoglucanase (Zhang et al., 2006b; Zhang and Lynd, 2006). Production of biodegradable plastics and chemicals from hemicellulose, acetic acid, and lignin will increase potential revenues (Table I). Hemicellulose and its derivatives with the 5$20-fold selling price of glucose will have large markets. Hemicellulose has been used as plant gum for thickeners, adhesives, protective colloids, emulsiers, and stabilizers (Kamm and Kamm, 2004). Recently, a very promising application is biodegradable oxygen barrier lms (Grondahl et al., 2004; Jonas Hartman, 2006). Partially hydrolyzed hemicellulose oligosaccharides provide a source of higher value co-products, such as animal feed additives (Davis et al., 2002; Fernandez et al., 2002). Monomeric xylose from hemicellulose has a selling price of $$1.2/kg. Xylose can also be further fermented to another value-added productxylitol, a sweetener and nutritional additive (Demetrakopoulos and Amos, 1978; Levine, 1986). Furfural, a chemically degraded product of xylose with a selling price of $$1/kg, is used to produce lubricants, coatings, adhesives, plastics, and polytetramethylene ether glycerol for production of Lycra and Spandex (Arato et al., 2005; Kadam and McMillan, 2003; Kamm and Kamm, 2004). Another emerging huge market of furfural will be Nylon 6,6 and Nylon 6; early Nylon production started with hemicellulose furfural but it was abandoned in the United States in 1961 for economic reasons (Kamm and Kamm, 2004). As prices of oils are soaring, it is time to revive this old production method, especially when an abundance of hemicellulose can be isolated from lignocellulose. Lignin is not being fully utilized because of limited supplies of high-quality lignin (Lora and Glasser, 2002). Historically high-quality lignin from OS pulping plants was always sold at decent prices (Pye, 2006). High purity, low

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Table I. Economic analysis based on the values of lignocellulose intermediates and the potential revenues from the nal productsethanol production and other co-products. Values of Lignocellulose intermediates Simple Comp. Glucan Xylan Arabinan Lignin Acetyl Sum
a b

Final revenuesd Complete Simple Rev. $/ton 67.40 39.95 6.72 7.28 121.4 Partial Rev. $/ton 67.4 39.95 6.72 108.2 32.0 254.27 Compl. Rev. $/ton 67.4 256.8 43.2 228.8 32.0 628.2

Partialc Value $/ton 43.32a 25.68 4.32 7.28 80.60 Price $/kg 0.12 0.12 0.12 0.52 Value $/ton 43.32 25.68 4.32 108.2 32.0 213.5 Price $/kg 0.12 1.20 1.20 1.10 1.00

Wt. kg/ton 361 214 36 208 32

Price $/kg 0.12 0.12 0.12 0.04b

Value $/ton 43.32 256.8 43.20 228.8 32.0 604.1

Sugar yields are estimated to be 90% of theoretical yields. Lignin price is estimated based on coal price ($35/short ton). c Half of lignin is burned as a fuel, and the other half is used for a high value co-product. d Ethanol selling prices $1.5/gallon.

molecular weight lignin from organic solvent extraction can be used as bio-dispersants, polyurethane foams, expoxy and phenolic powder resins, controlled-released carriers, and raw chemical sources for phenol and ethylene (Lora and Glasser, 2002; Reddy and Yang, 2005). In addition, lignin can be converted to carbon ber (Kadla et al., 2002; Shimizu et al., 1998). Figure 5 shows the scheme of a multi-product lignocellulose biorenery based on this lignocellulose fractionation technology. Modern oil reneries require full utilization of each component of crude oil by increasing the revenues from main product and co-product. Similarly, lignocellulose bioreneries must make the most of major lignocellulose components because hemicellulose, lignin, and acetic acid have $510-fold selling prices of glucose (Pan et al., 2005; Pye, 2006). Take corn stover as an example, Table II presents

that the inherent values of corn stover components are $80.6, $213.5, and $604.1 for simple utilization (all sugars glucose and lignin as burning fuels), partial utilization (all sugars glucose, a half of lignin as burning fuels, the other half of lignin as materials, and acetic acid as a commodity), and complete utilization (each sugar as its high price, all lignin as polymeric materials, and acetic acid as a commodity), respectively. Clearly, co-utilization of lignocellulose components clearly improves the economy of a biorenery dramatically. If all fermentable sugars are fermented to ethanol, the potential revenues of simple lignocellulose-ethanol bioreneries could be $121 per ton of corn stover, far lower than the case of partial co-utilization ($252/ton) and complete co-utilization ($628/ton). Lignocellulose-ethanol bioreneries with partial co-utilization would be very promising, based on economic and societal

Figure 5. The scheme of multi-product lignocellulose bioreneries based on lignocellulose fractionation. [Color gure can be seen in the online version of this article, available at www.interscience.wiley.com.]

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Table II. Features

The features of the lignocellulose fractionation technology and the relevant benets. Technical benets No sugar degradation No inhibitor formation No special reactor Co-utilization Applications for lignin and hemicellulose Economic benets High revenue from high sugar yield No detoxication needed Less capital investment Low utility consumption High revenue from co-products Small-size biorenery Lower crude oil consumption and CO2 emissions Biodegradable materials Highest revenue from glucose Less use of cellulase Less use of cellulase Smaller hydrolysis reactors Easy recovery of phosphoric acid Easy recovery of phosphoric acid Easy recovery of acetone Lower transportation costs

Modest reaction conditions (508C, atmospheric pressure)

Composition fractionation

Amorphous cellulose

Highest cellulose digestibility Fast hydrolysis rate Enzyme recycle Separation of solid sugars/liquid acid No acid re-concentration Use of highly volatile acetone Good for herbaceous and hardwood Full utilization of various biomass

Efcient solvent recycling

Broad substrate applicability

concerns and would reduce investors risk against uctuating markets of a sole product. The experimental data clearly justify the hypothesis about the root causes of lignocellulose recalcitrance(1) low accessibility of crystalline cellulose and (2) the presence of lignin and hemicellulose on the cellulose surface. Because the root causes of recalcitrant lignocellulose were addressed, the locked sugars from herbaceous and hardwood lignocellulose were effectively released after hydrolysis by this means with the nearly theoretical sugar yields and the fastest hydrolysis rates. The use of combinatorial cellulose solvent and organic solvent for lignocellulose fractionation is very promising because of their unique features and much better potential economy (Table II). This lignocellulose fractionation technology will have great impact on utilization of lignin and hemicellulose, cellulase engineering, and bioenergy plant development. The lignocellulose fractionation technology is in its infant stage. Prior to industrialization, intensive efforts of research and development are urgently needed, for example, conducting an economic analysis as compared to other lignocellulose pre-treatments, validating solvent recycling technologies on relatively large scales, developing more economical solvent recycling systems, signicantly decreasing solvent use volume, minimizing lignocellulose size reduction, nding out the possible better combination of cellulose solvent and organic solvent, identifying the most economical solid/liquid separators, characterizing the properties of isolated lignin, developing new applications and markets for lignin and hemicellulose, testing enzyme recoveries among each hydrolysis cycle, etc. We believe that substantial progress can be made in this new direction and the principles of lignocellulose fractionation will make contributions to realization of lignocellulose-based bioreneries.
We appreciated the useful discussion and suggestions from Charles Wyman, Wolfgang Glasser, Jerzy Nowak, and Colin South. We thanked the nancial supports partially from the ACS Petroleum

Research Fund and Oak Ridge Associated Universities (to YHPZ); DOE Ofce of the Biomass Program (to coauthors at NREL); and UTBattelle (to coauthors at ORNL); DOE and NIST (to LRL).

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