bioalcohol production || thermochemical pretreatment of lignocellulosic biomass

2.1 Introduction

The need for alternative liquid transportation fuels is imperative due to increas-

ing demand from rapidly industrializing nations, dwindling petroleum and

natural gas supplies, exclusive monopoly by politically volatile countries and

detrimental effects of carbon dioxide emissions from fossil fuels on climate

change. The sustainable development of economically viable bio-fuels from

lignocellulosic biomass is one suitable alternative to the impending global

energy crisis.

However, lignocellulosic biomass is naturally recalcitrant to biological

degradation due to several inherent characteristics of plant cell walls (Himmel et

al., 2007). The enzymatic digestibility of native and pretreated lignocellulosics

depends primarily on two types of factors: (a) substrate-related and (b) enzyme-

related (Mansfield et al., 1999; Zhang and Lynd, 2004).

Substrate-related factors are typically cellulose crystallinity and degree of

polymerization (Fan et al., 1980; Stalbrand et al., 1998; Klein and Snodgrass,

1993; Ramos et al., 1993), hemicellulose side-chain branching (Chang and

Holtzapple, 2000), lignin monomer type and content (Besle et al., 1994),

2Thermochemical pretreatment of lignocellulosic

biomass

S . P . S . C H UNDAWAT , V . B A LAN , L . D A CO S T A

S OU SA a n d B . E . D A L E , Michigan State University, USA

Abstract: The development of an economically viable and environmentallysustainable bio-based chemical industry has been largely impeded by thenative recalcitrance of lignocellulosic feedstock to thermochemical (e.g.,chemical pretreatment) and biological processing (e.g., enzymatic hydrolysisand fermentation). This chapter explores various thermochemicalpretreatments that enhance enzymatic digestibility of lignocellulosics bysolubilizing, hydrolyzing and chemically modifying individual cell-wallcomponents like lignin, hemicellulose and cellulose. Substrate andpretreatment related factors that influence the effectiveness of the entirebiorefinery process, based on the ultimate enzymatic digestibility andfermentability of the treated biomass, are closely examined from aneconomic and environmental point of view.

Key words: thermochemical pretreatment, plant cell wall recalcitrance,enzymatic hydrolysis, cellulosic ethanol.

ßWoodhead Publishing Limited, 2010

coumarate and ferulate cross-linking (Besle et al., 1994; Laureano-Perez et al.,

2005), cell wall porosity (Ishizawa et al., 2007; Stone and Scallan, 1969; White

and Brown, 1981; Thompson et al., 1992) and biomass particle size (Zadrazil

and Puniya, 1995).

Enzyme-related characteristics that influence hydrolysis are enzyme specific

activity (Zhang et al., 2006b), enzyme synergy (Reese et al., 1950; Rosgaard et

al., 2007; Bhat and Hazlewood, 2001; Gow and Wood, 1988; Wood, 1968;

Wood and McCrae, 1986; Wood et al., 1989; Bhat and Bhat, 1997), enzyme

inactivation due to non-productive protein binding to lignin and cellulose during

hydrolysis (Eriksson et al., 2002a, 2002b; Yang et al., 2006; VaÈljamaÈe et al.,

1999; Holtzapple et al., 1994; De La Rosa et al., 1994), and inhibition due to

end-products or pretreatment degradation compounds (Gusakov and Sinitsyn,

1992; Selig et al., 2007).

Depending on the pretreatment catalyst and solvent used, it is possible to

solubilize, hydrolyze and physically separate cellulose, hemicellulose and lignin

(Weil et al., 1994). Several types of pretreatment chemicals/solvents have been

used, such as, concentrated acid (Goldstein and Easter, 1992), dilute acid (Saha

and Bothast, 1999), caustic soda (Koullas et al., 1993), sulfur dioxide (Clark and

Mackie, 1987), hydrogen peroxide (Gould, 1984), steam (FernaÂndez-BolanÄos et

al., 2001), liquid ammonia (Dale et al., 1996), alkali-peroxide (Schmidt and

Thomsen, 1998), lime (Kaar and Holtzapple, 2000), liquid hot water (Laser et

al., 2002), carbon dioxide (Dale and Moreira, 1982), and several other organic

solvents (Chum et al., 1988).

Thermochemical pretreatment of lignocellulosic biomass is known to

enhance the yield of fermentable sugars during enzymatic hydrolysis 3±10-

fold, depending on the nature of the substrate and type of pretreatment. This

review closely examines the leading thermochemical pretreatments that have

been tested on lignocellulosic feedstocks, such as, agricultural residues (e.g.

corn stover) and energy crops (e.g. poplar). Substrate and pretreatment related

factors affecting the viability of the process, based on the extent of enzymatic

digestibility and ethanologenic fermentability of treated biomass hydrolyzate,

are also examined.

2.2 Why is pretreatment necessary forlignocellulosics?

Thermochemical pretreatments (like ammonia fiber expansion or AFEX) help

reduce lignocellulose recalcitrance by unwinding and leaching the tightly woven

cell wall ultra structure (Chundawat et al., 2006; 2007; 2008b; Chundawat,

2009; Donohoe et al., 2008). The actual mechanism of `unwinding' the cell wall

is unique to each pretreatment, closely dependent on the pretreament chemistry

and nature of the substrate. Most flow-through-based pretreatments (employing

high liquid solvent to solid biomass loadings) physically extract lignin and

Thermochemical pretreatment of lignocellulosic biomass 25


hemicellulose into separate liquid process streams, hence improving cellulase

accessibility to residual cellulose (Liu and Wyman et al., 2005; Chandra et al.,

2007; FernaÂndez-BolanÄos et al., 2001; OÈ hgren et al., 2007; Wu and Lee, 1997;

Donohoe et al., 2008; Kristensen et al., 2008). However, certain pretreatments

like AFEX that do not physically extract lignin and hemicellulose are thought to

modify the cell wall ultra-structure through a mechanism that is currently not

well understood (Chundawat, 2009). Before exploring what parameters are

responsible for an effective pretreatment, it would be necessary to closely

examine the cell wall architecture in order to overcome their native

recalcitrance.

2.2.1 Native plant cell wall recalcitrance

The primary constituents of lignocellulosic biomass are 30±50% cellulose

(glucose polymer), 15±35% hemicellulose (hetero-sugar polymer), 10±30%

lignin (phenyl propanoid polymer) and other minor constituents that include

proteins (3±10%), lipids (1±5%), soluble sugars (1±5%) and minerals (5±10%)

(Pauly and Keegstra, 2008).

Cellulose is a linear homo-polysaccharide that consists of glucose (D-gluco-

pyranose) units linked together by �-(1-4) glycosidic linkages (�-D-glucan). The

degree of polymerization (DP) of cellulose varies depending on its source.

Cellulose from Avicel (PH-101) has 20-fold lower DP compared to untreated

corn stover (300 vs. 7000, respectively) (Wyman et al., 2006; Kumar et al.,

2009; Marx-Figini, 1969). The adjacent glucan chains form an elementary

microfibril (3±5 nm diameter) of water-insoluble aggregates of varying length

and width in the primary cell wall of corn stover (Ding and Himmel, 2006).

These aggregates contain ordered (crystalline) and less-ordered (amorphous)

regions of cellulose (Fengel and Wegener, 1989). The lattice forces responsible

for maintaining the crystalline regions are a result of extensive inter- and intra-

molecular hydrogen bonding. It is this solid, crystalline morphology of cellulose

that results in the slow saccharification kinetics largely due to steric hindrance of

accessible glucan chains that are hidden below each other (Zhou et al., 2009).

The cellulose polymorph typically seen in higher plants is I�, which is a more

tightly packed crystal structure, compared to other cellulose polymorphs due to

differences in hydrogen bonding patterns (O'Sullivan, 1997). Chemical treat-

ment of cellulose with sodium hydroxide or anhydrous liquid ammonia can

modify the native crystal structure of cellulose I� to cellulose II and III,

respectively (Wada et al., 2008). The rate of enzymatic hydrolysis of cellulose is

known to be closely dependent on its crystal structure and varies in the following

order for various cellulose allomorphs, based on ease of saccharification;

Amorphous > Cellulose III > Cellulose II ~ Cellulose I (Weimer et al., 1991;

Igarashi et al., 2007). However, more work is needed to better understand the

transformations between the various polymorphs during chemical pretreatments

26 Bioalcohol production


and the effect of different enzyme systems (free vs. complexed) on cellulose

allomorph digestion kinetics.

Hemicelluloses are complex hetero-polysaccharides whose chemical com-

position, unlike cellulose, varies between cell tissues and plant species

(Ebringerova, 2006; Ebringerova and Heinze, 2000). These polysaccharides

are formed by a wide variety of sugar building blocks including pentoses

(xylose, arabinose), hexoses (glucose, mannose, galactose) and uronic acids (4-

O-methyl-glucuronic, galacturonic acids) (Fengel and Wegener, 1989).

Generally, hemicelluloses fall into two classes: (a) un-branched chains such as

�-(1-4)-linked xylans or mannans; and (b) branched chains such as (1-4)-linked

galactoglucomannans and arabinoxylans (O2/O3 xylosyl linkages). The most

abundant hemicelluloses found in agricultural residues like corn stover are

arabinoxylans and arabino-(glucurono)-xylans (Buranov and Mazza, 2008;

Ebringerova and Heinze, 2000). The ratio of pentosans (xylose, arabinose) and

acidic sugars (uronic acids) varies considerably between tissues, but typically

corn stover cell walls contain at least 20±25% xylan, 4±5% arabinan and 3±5%

uronic acids.

Lignin is a phenyl-propanoid-based macromolecular network that is

primarily formed through the free-radical polymerization of p-hydroxy

cinnamyl alcohol units of varying methoxyl content (Palonen, 2004). The

chemical structure of lignin is complex and is largely based on its three

phenolic precursors: coniferyl alcohol, sinapyl alcohol, and p-coumaryl alcohol.

The ratios and absolute amounts of these precursors vary significantly between

species, phenotypes, organs (leaf, stem, sheath), tissues (xylem, sclerenchyma,

parenchyma, epidermis) and hence tremendously affect the physicochemical

nature of lignocellulosic cell walls (Besle et al., 1994). Lignification of cell

walls helps provide strength to plant tissues preventing collapse of water-

conducting elements and provides defense against various pathogens (fungi and

bacteria).

These three cell wall components are organized together in a complex matrix

(model schematic shown in Fig. 2.1) depending on the plant clade (monocots vs.

dicots), type of cell (sclerenchyma vs. parenchyma cells) and type of cell wall

(primary vs. secondary). Several simplified models of the primary cell wall have

been presented over the years (McCann and Carpita, 2008; Carpita and Gibeaut,

1993), with little work on understanding the ultra-structural architecture of

secondary cell walls (Bidlack et al., 1992; Ruel et al., 2006). Secondary cell

walls (e.g., xylem vessels, sclerenchyma cells) are more recalcitrant to

enzymatic hydrolysis than primary cell walls. Therefore, a better understanding

of the chemical composition and ultra-structure architecture of the secondary

wall before/after thermochemical pretreatment would help identify the

fundamental rate-limiting steps to cell wall deconstruction.



2.2.2 Effect of thermochemical pretreatment on cell wallcomposition and ultra-structure

Most chemical pretreatments modify cell wall ultra-structures through certain

physicochemical modifications, depending on the pretreatment chemistry and

type of biomass, which ultimately helps enhance the enzymatic digestibility.

The primary goal of any pretreatment is to enhance the rate of enzymatic

hydrolysis, which is typically achieved through improving enzyme accessibility

to the cell wall polysaccharides by removal of lignin and/or hemicellulose.

Hemicellulose and lignin form physical barriers within the cell wall that limit

enzymatic accessibility to cellulose. Most pretreatments target lignin-

carbohydrate complex (LCC) linkages between lignin and hemicellulose to help

physically extract the amorphous matrix components while revealing the under-

lying cellulosic fibrils (Koshijima and Watanabe, 2003; Koshijima et al., 1989).

One of the common LCC linkages is the ester bonds between arabinose side-

chains and ferulic acid (Lapierre et al., 2001; Saulnier and Thibault, 1999).

2.1 Untreated grass cell wall structural components depicted by theintertwined matrix of cellulose, hemicellulose and lignin. Crystalline (straight,parallel lines) and amorphous cellulose (curved, wavy lines) microfibrils arecomposedof�-1,4 linkedglucanchains.Hemicellulose (thick, short lines)withno side-chains is hydrogen-bonded to cellulose, while branches with side-chains (of arabinosyl, acetyl, glucuronyl groups shown as circles and squares)help in cross-linking cellulose microfibrils. Lignin carbohydrate complexes(between arabinose and ferulic/coumaric acids) further restrict access ofenzymes to cell wall polysaccharides. Cross-linked lignin monomers(consisting of hydroxyphenyl, syringyl, guaicyl units) constitute the core-ligninfraction.



While, deacetylation helps improve accessibility of debranched hemicellulose to

endoxylanases (Kong et al., 1992; Chang and Holtzapple, 2000), acidic pre-

treatments also hydrolyze polysaccharide-based glycosidic linkages to form

gluco- and xylo-oligomers depending on the severity of pretreatment (Yang and

Wyman, 2008). Certain pretreatments modify the crystallinity and accessibility

of cellulose to enzymes/microbes (Laureano-Perez et al., 2005; Weimer et al.,

1991). Lignin-based ether linkages are prone to cleavage during acidic pre-

treatments (Shevchenko et al., 2001). While, alkaline pretreatments like wet-

alkali oxidation and ammoniation are also thought to chemically alter lignin

structure, but the chemistries are not well understood (Sewalt et al., 1997; Gould,

1984; Klinke et al., 2002). Certain pretreatments physically extract lignin along

with the liquid stream, primarily depending on the solvent to biomass loading

employed (Yang and Wyman, 2004; Papatheofanous et al., 1996; Kim and Lee,

2005a). Chemical characterization of pretreated lignocellulosics is relatively

better understood compared to the effect of thermochemical pretreatment on the

three-dimensional ultra-structural network of cellulose-hemicellulose-lignin.

There have been efforts in recent years to study the effect of dilute acid and

steam explosion-based pretreatments on ultra-structural modifications within

lignocellulosic grass cell walls using high resolution transmission/scanning

electron (TEM, SEM) and atomic force microscopy (AFM)-based techniques

(Donohoe et al., 2008; Selig et al., 2007; Kristensen et al., 2008). Dilute-acid

pretreatment has been shown to cause lignin coalescence into droplets that

migrate out of corn stover cell walls. The migrating droplets redeposit within

and outside the cell walls at the end of pretreatment, ranging in size from 5 nm to

10�m (Donohoe et al., 2008). These spherical droplets are composed essentially

of lignin and were found to severely inhibit enzymatic activity (Selig et al.,

2007). Lignin re-localization away from cellulose microfibrils helps improve

enzyme accessibility, however, complete lignin removal might cause extensive

collapse of microfibrils. Extensive pretreatment may hence impede accessibility

of cellulose microfibrils to enzymes (Weimer et al., 1986; Chou, 1986). Alkaline

extraction and delignification of spruce-based softwood result in considerable

nanostructural changes of the hemicellulose-lignin matrix surrounding cellulose

microfibrils depending on the severity of pretreatment (Jungnikl et al., 2008).

Alkali pulping typically results in swelling of cellulose crystallites as seen via

AFM imaging (Fahlen and Salmen, 2003) and causes significant modification of

cell wall morphology. Treating hard woods with liquid ammonia has shown to

result in extensive fiber defibrillation with no significant extraction of lignin/

hemicellulose (O'Connor, 1972). Recent work on ammonia pretreatment

(AFEX) has shown a unique mechanism by which the plant cell walls are

modified resulting in enhanced enzymatic digestibility and fermentability

(Chundawat, 2009; Lau and Dale; 2009).

Localization of lignin and hemicellulose residues around cellulosic micro-

fibrils seems to be an important rate-limiting step to the hydrolysis of plant cell



walls. This would suggest that engineering plant cell walls with artificial LCC

linkages (and/or `zipper' lignin containing ester linkages) that can be easily

cleaved during pretreatment would help reduce thermochemical severity to

maximize digestibility (Mansfield, 2009). However, the final hurdle for rapid

cell wall hydrolysis would be the breakdown of crystalline cellulose. This is

essentially due to the spatially coupled organization of densely, packed glucan

chains in cellulose microfibrils that results in steric obstruction of cellulases

(Zhou et al. 2009). Despite the tremendous advances in high-resolution imaging

of acid-treated plant cell walls (Singh et al., 2009; Donohoe et al., 2008), there is

still much to be learned on understanding the rate-limiting steps that affect cell

wall hydrolysis, especially for alkaline-based pretreatments (like AFEX).

2.2.3 Glycosyl hydrolases necessary for saccharification aredependent on type of pretreatment, thermochemicalseverity and cell wall composition

There are essentially three classes of cellulase enzymes (typically extracellular

fungal enzymes) that have been extensively studied over the past five decades

(Bhat and Hazlewood, 2001; Bayer et al., 1998); namely:

(a) Cellobiohydrolases (CBH) or exoglucanases that act at the ends (reducing

or non-reducing) of cellulose, processively cleaving cellobiose from the

glucan chain ends. These enzymes typically belong to glycosyl hydrolase

(GH) family 6, 7 and 9.

(b) Endoglucanases (EG) act randomly to hydrolyze easily accessible, interior

�-1,4-glucan linkages of the cellulose chain, breaking it into smaller units

and providing more `ends' for the exo-enzymes to act on. These enzymes

typically belong to GH families 5, 7, 9, 12, 45 and 61.

(c) �-Glucosidases which hydrolyze cellobiose and short chain oligosaccharides

into monomeric glucose units. These enzymes typically belong to GH family

1 and 3.

Though, there have been several studies to optimize cellulase mixtures for

hydrolyzing model cellulosic substrates like avicel and bacterial micro-

crystalline cellulose (Baker et al., 1998; Boisset et al., 2001), very little work

has been conducted on pretreated lignocellulosic biomass. Baker and colleagues

(1998) found that a ternary combination of CBH I: CBH II: EG I at 60 : 20 : 20

(0.4 mg/g glucan total enzyme loading) gave the highest glucan conversion for

crystalline cellulose (i.e. sigmacell). Boisset and colleagues (2001) found a

ternary combination of Cel7A: Cel6A: Cel 45A (native Humicola enzymes,

analogous to CBH I, CBH II and EG I, respectively) at 68.75:30:1.25 (100 mg/g

glucan total enzyme loading) gave the highest glucan conversion on crystalline

cellulose. These two findings, among many others, indicate that the complex

nature of synergy between similar endo- and exo-enzymes is dependent on the



total protein loading, substrate characteristics and individual protein binding

properties to substrate active sites. Rosgaard et al. (2007) have studied the

synergistic hydrolysis of acid, steam and hot-water pretreated barley straw using

quaternary mixtures of purified CBH I, CBH II, EG I and EG II. The optimal

ratio of the four enzymes required for higher severity (acid impregnation-steam

explosion) pretreated straw was different from the lower severity pretreated

straws (water impregnation-steam explosion and hot water extraction). The

higher severity pretreated straw, required lesser amount of EG I (and greater

amount of CBH II, with nearly constant CBH I) compared to lower severity

material. The optimal ratio of CBH I: CBH II: EG I (Trichoderma enzymes) for

high and low severity steam explosion pretreated barley straw was found to be

50:25:25 and 45:20:35, respectively. This is not surprising considering that acid

pretreatment results in removal of amorphous cellulose giving a more crystalline

polymer that requires lesser synergy between cellobiohydrolase (CBH I) and

endoglucanases (EG I) (VaÈljamaÈe et al., 1999). Rosgaard et al. also found

significant interaction effects between EG (EG I or EG II) with CBH I in the

hydrolysis of lower severity pretreatments, possibly due to cross-activity of EG I

on xylan (Gao et al., 2010).

The complete degradation of side-chain decorated hemicelluloses requires

the concerted action of several enzymes (Saha, 2003; Bhat and Hazlewood,

2001). The important classes of hemicellulose degrading enzymes are as

follows:

(a) Endoxylanases (EX) hydrolyze interior �-1,4-xylosidic linkages of the

xylan backbone. These enzymes typically belong to GH family 10 (acidic

pI, high molecular weight) and 11 (basic pI, low molecular weight).

(b) �-Xylosidase (�X) hydrolyzes xylobiose dimers and short chain xylooligo-

saccharides to xylose. These enzymes typically belong to GH family 3.

(c) �-Arabinofuranosidase hydrolyzes terminal non-reducing �-arabino-

furanose from arabinoxylan side-chains. These enzymes typically belong

to GH family 51, 54 and 62.

(d) �-Glucuronidase releases glucuronic acid from glucuronoxylan side-

chains. These enzymes typically belong to GH family 67.

(e) Acetyl xylan esterases hydrolyzes acetylated ester linkages from the xylan

backbone. These enzymes typically belong to carbohydrate esterase family 1.

(f) Phenolic acid esterases hydrolyze feruloyl and p-coumaroyl ester bonds in

lignin-hemicellulose complexes. These enzymes typically belong to carbo-

hydrate esterase family 1.

Purified accessory hemicellulase synergy studies have been conducted for low

severity, hot-water pretreated corn stover (Selig et al., 2008b; Knoshaug et al.,

2008). It was found that glucan conversions to cellobiose (enzyme loading = 15

and 50mg CBH I/g glucan) by CBH I increased by 13±84% in the presence of a

suitable acetyl xylan and/or feruloyl esterase (enzyme loading = 2.5mg/g



glucan). The greatest synergistic enhancements to glucan and xylan conversion

were observed at lower cellobiohydrolase loading (15mg/g glucan). Despite,

significant removal of hemicellulose during dilute acid pretreatment, hemi-

cellulases have been shown to significantly enhance the rate of hydrolysis (Selig

et al., 2008a). This would suggest a significant enhancement in the hydrolysis

rates for lower acid severity (OÈ hgren et al., 2007) and alkaline (e.g. AFEX)-

based treatments (Chundawat et al., 2006), containing a significant fraction of

unhydrolyzed hemicellulose via supplementation of suitable hemicellulases and

other accessory enzymes (Selig et al., 2008b). Recent findings for AFEX treated

corn stover have shown synergistic interactions between cellulases (CBH I,

CBH II and EG I) and hemicellulases (EX, �X), as function of total enzyme

loading, that maximize both glucan and xylan digestibility by several folds (Gao

et al., 2010).

Most commercially produced enzymes (typically from Trichoderma) do not

have the correct ratio and total amounts of critical enzymes necessary to hydrolyze

pretreated lignocellulosic biomass, since there has been no evolutionary pressure

on microbes to grow on pretreated lignocellulosics (pretreatment also modifies the

cell wall ultra-structure) to produce the optimum ratios of cellulases and

hemicellulases (Rosgaard et al., 2007; Eriksson et al., 2002b). This is one of the

reasons that crude protein mixtures should be optimized for maximizing sugar

yield from pretreated biomass (Chundawat et al., 2008a). This would eventually

lead to the construction of multigene expression systems that produce a balanced

set of necessary enzymes for a particular feedstock-pretreatment combination.

Several studies have looked at optimizing commercially available crude enzyme

mixtures on acid pretreated (Berlin et al., 2007), steam exploded and organosolv

treated lignocellulosic biomass (Berlin et al., 2005; 2006; Gusakov et al, 2007).

Optimizing crude enzyme mixtures for acid pretreated corn stover helped reduce

total protein loading by two-fold for equivalent hydrolysis yields (Berlin et al.,

2007). Several covalent lignin-polysaccharide bonds are not entirely cleaved

during alkali pulping and require accessory enzymes to help cleave these linkages,

improving pulp digestibility (Buchert, 1992). Studies with steam exploded wheat

straw have found synergistic interactions between crude cellulases and accessory

enzymes (xylanase and esterases) (Tabka et al., 2006). However, it would be

difficult to study the interaction of glycosyl hydrolases on pretreated

lignocellulosics using crude enzyme mixtures. Purification of individual enzyme

components to high purity has helped elucidate the role of cellulase and

hemicellulase synergy during hydrolysis of acid and ammonia pretreated biomass

(Selig et al., 2008b; Gao et al., 2010).

2.3 Types of chemical pretreatment

Pretreatments can be classified based on the nature of cell wall disruption (i.e.

physical or chemical) during the process into four categories (Sun and Cheng,



2002; Mosier et al., 2005b; da Costa Sousa et al., 2009); (a) physical, (b) solvent

fractionation, (c) chemical and, (d) biological-based pretreatments.

2.3.1 Physical pretreatment

Physical pretreatments disrupt the lignocellulose structure with little or no

chemical modifications to the individual cell wall components. Some typical

methods for physical pretreatment include biomass particle size reduction

through communition, including dry, wet, vibratory and compression-based ball

milling (Millett et al., 1975; Sidiras and Koukios, 1989; Tassinari et al., 1980;

Alvo and Belkacemi, 1997). Physical treatments help enhance enzyme

digestibility by increasing the cell wall accessible surface area to volume ratio.

In some cases, extensive ball milling results in decrystallization of cellulose and

reduction in degree of polymerization (Fan et al., 1981). Particle size reduction

alone is not a sufficient pretreatment to significantly enhance the rate of

enzymatic hydrolysis. The cost of size reduction also increases exponentially as

the desired particle size decreases, making the process economically unfeasible

in a commercial scenario (McMillan, 1994). However, some sort of minimal

particle size reduction is necessary prior to most thermochemical pretreatments

to improve material handling during processing.

2.3.2 Solvent fractionation-based pretreatment

Solvent fractionation using various solvents that can selectively solubilize

cellulose, hemicellulose and lignin can also be used to pretreat lignocellulosic

biomass. Cellulosic solvents like hydrazine, dimethyl sulfoxide (DMSO) and

concentrated mineral acids can disrupt hydrogen bonding between cellulose

microfibrils to solubilize crystalline cellulose (Heinze and Koschella, 2005).

Solvent fractionation-based pretreatments may be further classified into the

following three categories, based on the solvent system used.

Organosolv process

Historically, the organosolv process was investigated largely from the perspec-

tive of paper production (via pulping) from hardwoods. Organosolv process

includes extracting lignin from lignocellulosic biomass using organic solvents

like aromatic alcohols (phenols) or aliphatic alcohols (e.g. ethylene glycol,

methanol, ethanol, butanol, glycerol) typically with an acidic catalyst (Sidiras

and Koukios, 2004; Pan et al., 2006; 2007; Sun and Chen, 2008). The effect of

organosolv-based process parameters (e.g., solvent composition, temperature,

liquid to solid loading) on fractionation of the major components in woody

biomass has been extensively studied over the years (X Zhao et al., 2009).

Typical ethanol-based organosolv pretreatment parameters have the following



ranges; temperatures (90±120 ëC for grasses and 155±220 ëC for hard/soft

woods), reaction times (25±100min), acid catalyst loading (0.5±2%), and

ethanol concentrations (25±75% v/v). Compared to aliphatic alcohols, aromatic

alcohols were found to be more effective in solubilizing lignin from different

feedstocks (Lee et al., 1987). In addition to alcohols, various amines have been

used to delignify lignocellulosic biomass, but amines also tend to solubilize

substantial amounts of carbohydrates in addition to lignin. An ethanol-based

organosolv process has been commercialized by Lignol Innovation Corporation

(Vancouver, Canada) to separate lignin, hemicellulose and cellulose fractions

from woody biomass (Arato et al., 2005). The insoluble cellulose fraction is

subjected to enzymatic hydrolysis to produce fermentable sugars, while the

liquor from the organosolv step can be further processed to recover lignin,

furfural, xylose, acetic acid and lipophylic extractive-based fractions. Ethanol

used in the process is distilled and reused. The lignin fraction recovered can be

used as an additive binder among other applications. In the case of poplar, about

88% of the total cellulose is recoverable as monomeric glucose after 100 hours

of enzymatic hydrolysis of the residual solid fraction.

Fractionation using phosphoric acid

Fractionation of lignocelluloses to amorphous cellulose, hemicellulose, lignin

and acetic acid using concentrated phosphoric acid, acetone and water-based

mixtures is novel solvent fractionation-based pretreatment (Zhang et al., 2007).

Phosphoric acid-based pretreatments may be carried out at moderate reaction

conditions (i.e. 50 ëC, 1 bar), which minimizes acid catalyzed degradation of

polysaccharides. The process gave 98% and 89% recovery yield for glucose and

xylose, respectively, upon fractionation of corn stover. The selective fractiona-

tion of cell wall components is possible due to significant difference in solubility

of cellulose, hemicellulose and lignin in phosphoric acid-acetone-water

mixtures. The organic solvents are easily recoverable due to difference in

volatility with respect to phosphoric acid (Zhang et al., 2006a; 2007; Moxyley et

al., 2008). Despite several advantages associated with this pretreatment method,

which include enhanced rate of amorphous cellulose hydrolysis and low pre-

treatment utility costs (e.g. 50 ëC vs. 100±200 ëC for other thermochemical

treatments); there are several technical challenges (e.g., solvent cost and

recovery, xylose recovery and fermentation) that need to be addressed to make

this process commercially viable. Other issues include phosphoric acid/sugar

separation, acid recovery, and acid re-concentration.

Ionic liquid-based fractionation

Ionic liquids (IL) are non-volatile solvents, under atmospheric conditions, com-

posed exclusively of ions held together by coulombic forces. The first reported



IL that could solubilize cellulose was back in 1934 using molten N-ethyl-

pyridinium chloride, in the presence of nitrogen-containing bases (Graenacher,

1934). However, the application of IL as lignocellulose pretreatment catalyst has

gained momentum in recent years (Swatloski et al., 2002). Cellulose-dissolving

IL usually contains anions of chloride, formate, acetate or alkyl phosphonate,

since these ions form strong hydrogen bonds with cellulose. During a typical IL

pretreatment of lignocellulosics, the biomass added to ionic liquids (ratio of

1 : 10±15, wt/wt) is heated (50±150 ëC) to solubilize the cellulosic, hemi-

cellulosic and lignin components (Singh et al., 2009). The un-dissolved residue

is filtered from solution and anti-solvents (e.g., water/methanol/ethanol) are

added to the solution to recover the solubilized cellulose (as amorphous

cellulose). The mass recovery of lignin and hemicellulose is currently unknown

for corn stover (Singh et al., 2009). IL can be recovered from the anti-solvent by

flash distillation and re-used in the process (Joglekar et al., 2007; Rinaldi et al.,

2008, Dadi et al., 2006; 2007). Typically, ionic liquids that incorporate anions

are found to be more effective in solubilizing cellulose, due to stronger hydrogen

bonding between the anion and cellulose hydroxyl groups. On the other hand, IL

with non-coordinating anions and long-chain substitution are poor cellulose

solvents. IL with chloride anions appear to be the most effective solvents, while

IL with aromatic side chains require higher temperatures to solubilize cellulose,

due to their higher melting points and viscosities (H Zhao et al., 2009).

While most of the reports on IL-based pretreatments are on processed

cellulose (e.g., Avicel, Sigmacell), there are recent publications that have

pretreated woody biomass (Kilpelainen, 2007; Lee et al., 2008). The solubiliza-

tion efficiency of hard woods in IL was found to vary in the following order;

ball-milled wood powder > sawdust > thermomechanical pulp > wood chip.

Wood flour solubilization was tested using [Emim]+[CH3COO]± (1-ethyl-3-

methylimidazolium acetate)-based IL as a function of pretreatment time and

temperature. Increasing residence time and IL treatment temperature caused a

significant decrease in cellulose crystallinity; while improving lignin solubility

(20±60% solubilized) and enhancing regenerated biomass enzymatic digesti-

bility. Greater than 90% glucan digestibility was achieved using commercial

enzymes after 24 hours of hydrolysis. There are several concerns that need to be

addressed to facilitate commercialization of IL-based pretreatments. These

include cost of ionic liquids, recovery of IL after pretreatment, hemicellulose

recovery yield and enzymatic/microbial compatibility of IL (Turner et al., 2003).

2.3.3 Chemical pretreatment

Acidic-based pretreatments

Most acidic-based pretreatments (e.g., dilute acid, steam explosion and liquid

hot water treatment) have very similar chemistries but vary in thermochemical



severity. The acidic catalyst is either supplemented externally (as mineral acids)

or is formed due to hydrolysis of hemicellulose acetyl linkages (forming acetic

acid) and degradation of polysaccharides/lignin to short-chain aliphatic acids

and phenolic acids. Water itself behaves as an acid at high temperatures

typically employed in most pretreatments (Weil et al., 1998). The acid is

responsible for the hydrolytic cleavage and removal of hemicellulose and lignin,

hence improving accessibility of residual cellulose to glycosyl hydrolases. The

extent of removal of hemicellulose and lignin varies considerably between

various acidic pretreatments depending on the severity of pretreatment. The

severity of acidic pretreatments can be quantified using a simple relationship

which helps couple the effect of time (t), temperature (T) and acidity (pH) into a

single fudge factor known as the `combined severity factor (S)' (Chum et al.,

1990).

log�S� � log�R0� ÿ pH

R0 � t � exp�T ÿ 100�14:75

� �Higher acid pretreatment severities, referring to longer residence times,

higher temperatures and lower pH values, result in extensive hydrolysis of

hemicellulose to monomeric sugars. However, higher pretreatment severity also

results in the degradation of lignin and hemicellulose derivatives to compounds

like furfural, 5-hydroxymethylfurfural, levulinic acid, phenolic acids/aldehydes

and other aliphatic acids that can severely inhibit downstream biological

processing (Klinke et al., 2004). Most acidic-based pretreatments look to

optimize severity by trading-off between maximizing cellulose enzymatic

digestibility, hemicellulose acid catalyzed hydrolysis to xylose and minimizing

formation of biological inhibitors.

Dilute-acid pretreatment (using either sulfuric, hydrochloric or nitric acid)

was one of the first pretreatment methods implemented to pretreat ligno-

cellulosic biomass in order to produce ethanol (Ruttan, 1909). Traditionally,

dilute acid-based hydrolysis of lignocellulosic biomass has been used to

manufacture furfural through hydrolysis and dehydration of hemicelluloses

(Zeitsch, 2000). The remaining cellulosic fraction was typically hydrolyzed to

monomeric glucose using concentrated acid (10±30%) prior to fermentation

(Harris and Begliner, 1946). However, with the development of genetically

modified microbes that can co-ferment glucose and xylose it became necessary

to prevent extensive degradation of hemicellulose to furfural to increase ethanol

yields. This has resulted in the development of dilute-acid-based pretreatments,

typically using sulfuric acid, carried out at lower acid concentrations (0.05±5%)

and temperatures (160±220 ëC) (Kim et al., 2001; Torget et al., 1992). Dilute-

acid pretreatment can be carried out in a stationary batch mode or flow-through

continuous mode (Lloyd and Wyman, 2005; Lee et al., 1999).



Steam explosion is one of the most widely implemented pretreatment

methodologies to pretreat several different types of lignocellulosic biomass (e.g.,

agricultural residues, hardwoods and softwoods). Steam explosion is typically

carried out by rapidly heating lignocellulosic biomass with high pressure

saturated steam with/without external chemical addition (typically acids) for

certain duration of time (ranging from a few seconds to minutes), at a fixed

temperature (160±290 ëC) prior to explosively releasing the pressure (Ballesteros

et al., 2006; Ballesteros et al., 2000; Chandra et al., 2007). There have been

several reported variants of the steam explosion process. These variants

typically incorporate pre-impregnation with an acid (like sulfuric acid, sulfur

dioxide) or alkali (like sodium hydroxide, ammonia) prior to steam explosion

(Chen et al., 2005; Playne, 1984; Brownell and Saddler, 1984; Hsu, 1996).

Liquid hot water pretreatment (also known as hydrothermal or aquasolv

treatment) is a low severity acid-based pretreatment that uses water at high

pressures (>5MPa) to maintain the liquid state at elevated temperatures (160±

230 ëC) to pretreat lignocellulosics (Bobleter et al., 1981; Mosier et al., 2005b;

Weil et al., 1998). The liquid water is contacted with the biomass in three

possible modes; co-current, counter-current and flow-through (Mosier et al.,

2005a,b,c). Recent variants to the liquid hot water pretreatment process allows

for better pH control (ranging from pH 4±7) that limits non-specific degradation

of polysaccharides (Mosier et al., 2005b).

Carbonic acid, formed by dissolved carbon dioxide in water, has also been

used as a pretreatment catalyst (van Walsum et al., 2007). In addition to mineral

acids (like sulphuric acid, nitric acid), organic acids (carbonic, acetic, succinic,

fumaric, maleic, citric acid) have been used (at 50 mM concentration) as

pretreatment catalysts (Mosier et al., 2001; 2002). It was reported that maleic

and fumaric acids possess superior selectivity for the production of fermentable

sugars from cellulose than sulfuric acid (Kootstra et al., 2009). Both sulfuric and

maleic/fumaric acid have the ability to hydrolyze �-(1-4)-glycosidic linkages.

But, at 150 ëC sulfuric acid degrades glucose/arabinose to undesirable by-

products, while maleic/fumaric acid does not. For example, corn stover treated

at high solids loading (150±200 g/liter) by sulfuric acid results in 30% degrada-

tion of xylose. Maleic acid gave ~95% monomeric xylose yield with trace

amounts of furfural, along with 90% glucose yield after enzymatic hydrolysis of

the xylan depleted substrate (15 FPU cellulase/g glucan). The resulting uncon-

ditioned hydrolyzate was fermented by recombinant S. cerevisiae giving 87%

theoretical ethanol yield. Since organic acids are weaker acids than mineral

acids, protons are only partially dissociated under comparable reaction condi-

tions. This could be one possible reason for the selective catalytic activity of

organic acids compared to mineral acids. One of the major concerns is the

recovery of organic acids. Suitable economic analyses have to be carried out in

order to determine the feasibility of using di-carboxylic acids vs. mineral acids

within a cellulosic biorefinery (Lu and Mosier, 2007; 2008).



Alkali-based pretreatments

Alkali-based pretreatments typically differ in the type and thermodynamic state

of the catalyst-solvent systems employed. Alkalis used for pretreatment typically

include calcium hydroxide, ammonia and sodium hydroxide. Most alkali

pretreatments help lower recalcitrance of lignocellulosics through saponification

(or ammonolysis in the presence of liquid ammonia) of hemicellulose acetyl and

lignin-carbohydrate complex linkages (Chang and Holtzapple, 2000; Laureano-

Perez et al., 2005; Weimer et al., 1986). The extraction of lignin and hemi-

cellulose from the biomass during most alkaline pretreatments also helps reduce

non-specific binding of enzymes during cellulose enzymatic hydrolysis (Kim

and Lee, 2005a).

Ammonia fiber expansion (AFEX) is a unique alkaline pretreatment method

that uses concentrated liquid ammonia-water mixtures to pretreat lignocellu-

losics (Dale et al., 1996; Dale and Moreira, 1982). AFEX is a low temperature

process (60±140 ëC) that is carried out by adding liquid ammonia (0.3±2 kg

ammonia per kg dry weight biomass) to pre-wet biomass (0.05±1 kg water per

kg dry weight biomass) in a pressurized reactor that is cooked for 5±45 minutes

before explosively releasing the ammonia. AFEX is a dry to dry process, unlike

most other pretreatments, resulting in no separate liquid stream at the end of the

pretreatment. The volatility of ammonia allows for easy recovery and reuse

within a continuous process (Sendich et al., 2008). AFEX results in de-acetyla-

tion and cleavage of lignin-carbohydrate complexes through base catalyzed

hydrolysis (Laureano-Perez et al., 2006). The extent of ammonolysis of ester

linkages within the cell wall after AFEX is poorly understood (Chou, 1986;

Chundawat, 2009; Weimer et al., 1986). Liquid ammonia is also thought to de-

crystallize cellulose (typically via formation of a more swollen crystal structure

known as cellulose IIII from native cellulose I�) as shown by X-ray diffraction

studies (Lewin and Roldan, 1971; Wada et al., 2006). Interestingly, cellulose III

has been shown to have 3±5-fold higher rate of enzymatic hydrolysis compared

to native cellulose I (isolated from Cladophora algae cell walls). The extent of

cellulose III formation during AFEX (and related ammonia-based pretreatments)

treatment of lignocellulosic biomass is being currently investigated (Chundawat,

2009). On-going research also indicates significant modification of the cell wall

ultra and macro-structure during AFEX that results in enhanced enzymatic

accessibility (unpublished data). An improved fundamental understanding of the

mechanism of ammonia-based pretreatments would allow novel process

modifications to further enhance the overall rate of enzymatic hydrolysis and

fermentation of plant cell walls.

There have been several variants of the ammonia treatment process that have

been reported in the literature; such as supercritical ammonia treatment, ammonia

recycle percolation (ARP), soaking in aqueous ammonia (SAA) and ammonia-

peroxide pretreatment (Weimer et al., 1986; Kim and Lee, 1996; 2005a; 2005b;



Kim et al., 2003; Hennessey et al., 2007). Most of these pretreatments vary in the

thermodynamic state of ammonia-water mixtures for varying ammonia

concentrations. ARP pretreatment is carried out in a flow-through, recycle mode

by percolating ammoniacal solutions (5±15% concentration) through a column

reactor packed with biomass under high pressure (2±3MPa) and temperatures

(160±180 ëC). On the other hand, calcium hydroxide or lime-based pretreatments

are typically carried out at 50±120 ëC, for 1 hr to 4 weeks using 0.05±0.5 g

Ca(OH)2/g biomass and 2±10 g water/g biomass. Typically, lime treatment is also

conducted in the presence of air or pressurized oxygen to facilitate lignin

degradation and removal (Kim and Holtzapple et al., 2005; 2006). Oxidative

decomposition of lignin in poplar was shown to be facilitated by high pressure

oxygen supplementation during conventional lime pretreatments (Sierra, et al.,

2009). A significant amount of lime is consumed in the process at elevated

temperatures. However, the recovery of the solubilized catalyst in the liquor is

possible (after neutralization to calcium carbonate) via integration to a suitable

lime kiln technology (Kaar and Holtzapple, 2000). Unlike AFEX, there is a

significant extraction of lignin and hemicellulose in other alkaline pretreatments

like ARP and lime-based pretreatments.

Oxidative pretreatments

Ozone has also been used an oxidizing agent to break down lignin within

lignocellulosic biomass to help increase enzymatic digestibility (Puri, 1983;

Quesada et al., 1999; Garcia-Cubero et al., 2009). Traditionally, ozone had been

used to bleach pulps replacing chlorine as the oxidant (Roncero et al., 2003).

Ozonolysis, typically conducted at atmospheric conditions, has been used in

combination with other alkaline pretreatments to extract lignin from forage

residues and hence enhance enzymatic digestibility (Akin and Morrison, 1988;

Ben-Ghedalia and Miron, 1981; Morrison et al., 1991). Ozone reacts selectively

with lignin-based aromatics producing several types of degradation products

(primarily aromatic aldehydes and aliphatic organic acids) that have been found to

inhibit biological processing of ozone treated biomass (Quesada et al., 1997).

Ozonolysis-based intermediate degradation products (like free radicals) are

responsible for unwanted side-reactions, like polysaccharide degradation

(JablonskyÂ et al., 2004; Ragnar et al., 1999; Shatalov and Pereira, 2007). There

is a lack of ozonolysis pretreatment-based data for various agricultural residues

(Silverstein et al., 2007). The possible inhibitory effects of various ozonolysis

degradation products are currently poorly understood. However, the biggest hurdle

for an economically feasible ozonolysis-based pretreatment is the cost of ozone.

Alkaline wet oxidation is another form of oxidative pretreatment carried out

typically under alkaline conditions at high temperatures (170±220 ëC) using

pressurized air/oxygen or hydrogen peroxide as the oxidant (Martin and

Thomsen, 2007; Varga et al., 2003; McGinnis et al., 1983). Alkalis typically



employed for wet oxidation include sodium carbonate (0.02±0.05 g Na2CO3/g

dry biomass) and calcium hydroxide to help solubilize the hemicellulose and

lignin fraction more effectively (Klinke et al., 2002; Martin and Thomsen, 2007;

Sierra et al., 2009). The advantage of an alkaline medium during treatment at

elevated temperatures is to minimize formation of various furan-based

degradation products.

2.3.4 Biological pretreatments

Most chemical pretreatments are carried out at high temperature/pressure-based

reaction conditions, and thus require significant capital investment (Eggeman

and Elander, 2005). High thermochemical severity also results in the formation

of several degradation products that are inhibitory to downstream biological

processing (i.e. enzymatic hydrolysis and fermentation). Contrary to chemical-

based pretreatments, biological pretreatments (using enzymes or microbes as

pretreatment catalysts) would consume lesser energy since they are carried out

under milder reaction conditions (Lee, 1997; Keller et al., 2003; Lee et al.,

2007). Interestingly, termites enhance digestibility of lignocellulosic biomass to

gut microbiota via exposure to low temperature based oxidative alkaline

pretreatment like conditions within the hind-gut (Brune, 1998). However, most

industrially relevant biological treatment processes are slow (reaction time

ranges between several hours to months) and have low-throughput. Biological

pretreatments are typically carried out by inoculating the biomass with fungal

spores (e.g., white rot basidiomycetes and even certain actinomycetes) or

externally supplementing accessory enzymes (e.g., ferulic acid esterases and

hemicellulases). White-rot fungi have been found to be effective to degrade

lignin while minimizing polysaccharide consumption (Sun and Cheng, 2002;

Kerem et al., 1992). During microbial pretreatments, a substantial amount of

delignification can take place, possibly at the expense of polysaccharide con-

sumption depending on the microbial source (Kerem et al., 1992). White-rot

microbes typically secrete lignin peroxidases, along with various types of

glycosyl hydrolases, that cleave the C±C lignin backbone in the presence of

hydrogen peroxide. Other enzymes involved in aerobically catalyzed lignin

degradation include Mn-dependent peroxidases, laccases (monophenol oxidase)

and superoxide dismutase (Leonowicz et al., 1999). Details about the lignin

degrading microbial systems have been summarized elsewhere (Lee, 1997;

Leonowicz et al., 1999). In recent years, the use of ferulic acid esterases and

other accessory enzymes as catalysts to cleave LCC linkages within plant cell

walls to help reduce thermochemical severity during subsequent pretreatment

and/or to maximize recovery of valuable phenolic by-products have been

explored (Anderson et al., 2005; Akin et al., 2006).

Several articles on microbial pretreatments using solid state fermentation (SSF)

have been published for both grasses and hard woods (MendoncËa et al., 2008; Lee



et al., 2008). The effect of substrate moisture content, inorganic salt concentration,

culture time on lignin degradation, solids recovery, and availability of carbohydrate

on the biological pretreatment process have been explored. The solid state

cultivation of cotton stalks using Phanerochaete chrysosporium, at 75% moisture

content without salts was the preferable pretreatment condition resulting in 28%

lignin degradation, 71% solids recovery and 42% availability of carbohydrates over

a period of 14 days (Shi et al., 2008). Another approach is to use minimally treated

mushroom spent straw (MSS) as a feedstock for downstream thermochemical and

biological processing (Balan et al., 2008). When MSS was used without any

pretreatment, the glucan digestibility was around 40%, using standard commercial

cellulases (15 FPU/g glucan); and 20% for untreated rice straw. Further

thermochemical pretreatment is necessary for getting higher sugar yields from

MSS. By using microbial pretreated biomass as a feedstock for thermochemical

pretreatments; it might be possible to lower processing costs by reducing

pretreatment severity and minimizing chemical usage while obtaining higher

overall hydrolysis yields. Unfortunately, there have been no detailed economic

studies comparing the feasibility of scaling-up and integrating biological-based

pretreatments within a conventional cellulosic biorefinery.

2.4 Comparing effectiveness of leadingpretreatments on corn stover and poplar

A Consortium for Applied Fundamentals and Innovation (CAFI) for improving

understanding and comparing leading pretreatment technologies was formed in

2000. The CAFI project has allowed a systematic comparison of pretreatments

on common feedstocks (e.g., corn stover, poplar, switchgrass) using comparable

standard methodologies. Some of the leading pretreatments that are currently

part of the CAFI project include dilute acid, steam explosion (with sulfur

dioxide), controlled pH liquid hot water, ammonia fiber expansion (AFEX),

ammonia recycle percolation (ARP) and lime-based pretreatments.

Table 2.1 summarizes the optimum pretreatment conditions; inclusive of

reaction time, catalyst and water loading, for each pretreatment that resulted in

optimal enzymatic hydrolysis yields for corn stover and poplar (Adapted from

OÈ hgren et al., 2005; 2007; Kumar et al., 2009; Wyman et al., 2005; 2006). Most

acidic pretreatments (except ARP) typically required high temperatures (170±

200 ëC) to effectively pretreat corn stover. However, for lignin-rich poplar all

pretreatments required much higher temperatures (>150 ëC) and chemical load-

ing for maximizing enzymatic digestibility. AFEX employs lower temperatures

(90±160 ëC) and water loadings (0.6±3.2 g/g dry biomass) compared to other

pretreatments. Ongoing process improvements have further allowed significant

reduction in ammonia usage during AFEX (Sendich et al., 2008).

Some of the common effects of CAFI-based pretreatments on the physico-

chemical properties of corn stover and poplar are shown in Table 2.2. AFEX and



Table 2.1 Optimum pretreatment conditions for several leading CAFI-based thermochemical pretreatments that maximize enzymaticdigestibility of pretreated corn stover and poplar (data for poplar shown in parentheses)

Pretreatment Temperature Reaction Catalyst Catalyst Water Specific notestype (ëC) time loading loading

(min) (g/g dry (g/g drybiomass) biomass)

Dilute acid 160 (190) 20 (1.1) Sulfuric acid 0.015 (0.02) 3 (3.3) In batch mode using a parr reactorSulfur dioxide 190 (190) 5 (5) Sulfur dioxide 0.03 (0.03) 4 (4) Soaked overnight in 3% acid solution prior to

treatmentControlled pH/ 190 (200) 15 (10) ± ± 5.25 (5.67) In flowthroughmode (treated poplar washedhot water with hot water)

AFEX 90 (180) 5 (10) Ammonia 1 (2) 0.6 (2.3) Liquid ammonia added to moist biomass priorto heating reactor

ARP 170 (185) 10 (27.5) Ammonia 0.5 (0.55) 2.8 (3.2) Flowthrough mode using 5ml/min ofammoniacal solution, 15%w/w

Lime 55 (160) 4 weeks Calcium 0.07 (0.2) 10 (1.6) w/wo purging with air (purged with oxygen(120) hydroxide at 200psi, 39% solids)

Adapted fromÚhgren et al. (2005, 2007);Wyman et al. (2005, 2009). Note: lime pretreatment for poplar was performed in the presence of pressurized O2.

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Table 2.2 Prominent physicochemical effects of various leading thermochemical pretreatments on corn stover/poplar (data for poplarshown in parentheses). The cellulose crystallinity index for both untreated corn stover and poplar is 50 units each. The degree ofpolymerization for untreated corn stover and poplar cellulose is 7000 and 3500, respectively. The percent acetyl removed value for AFEXcorn stover (marked by *) is lower than expected based on ongoing investigations (unpublished data; Chundawat, 2009)

Pretreatment type Cellulose % % % Cellulose % acetylcrystalinity cellulose hemicellulose lignin degree of groups

index removed removed removed polymerization removed

Dilute acid 53 (51) 5±10 (10±15) 70±75 (90±95) 18 (nd) 2700 (500) 55 (90)Sulfur dioxide-based steam nd (56) 3±5 (1±5) 40 (90±95) 40±50 (nd) 3000 (650) 55 (80±85)explosion

Controlled pH liquid hot water 45 (54) 5±10 (1±5) 40 (55±60) nd (nd) 5600 (1800) 55 (70±75)Ammonia fiber expansion (AFEX) 36 (48) 0 0 0 6600 (2700) 30±35* (70±80)Ammonia recycle percolation 26 (50) 1±5 (5±10) 50±60 (30±35) 75±85 (40) 4600 (3200) 85±90 (85)(ARP)

Lime 56 (55) 1±3 (1±3) 30±35 (3±5) 55±60 (50) 3200 (1600) 90±95 (95)

Adapted fromKim andHoltzapple (2005, 2006); Kim and Lee (2005a); Laureano-Perez et al. (2005); LloydandWyman (2005);Mosier et al. (2005a); Úhgren et al.(2005, 2007); Teymouri et al. (2005);Wyman et al. (2009); and Kumar et al. (2009). nd = not determined or unknown. Note: lime pretreatment for poplar wasperformed in the presence of pressurized O2.

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ARP are seen to cause a significant reduction in the crystallinity of corn stover

compared to other pretreatments. This is probably due to modification of

cellulose I to III during AFEX or due to extraction of lignin and hemicellulose

during ARP (Lewin and Roldan, 1971; Kim and Lee, 2005a). However,

measurement of crystallinity by XRD (X-ray diffraction) is compounded by

presence of residual amorphous lignin and hemicellulose (Park et al., 2009).

Acidic pretreatments that typically hydrolyze and extract the amorphous

cellulose and hemicellulose components result in increasing the crystallinity as

measured via XRD. The degree of polymerization of cellulose is reduced

significantly after most pretreatments except AFEX (Kumar et al., 2009). Unlike

other pretreatments, AFEX is a dry-to-dry process with no secondary liquid

stream being generated at the end of the treatment. Most pretreatments (except

AFEX) result in significant removal of cellulose (1±10%), hemicellulose (30±

95%) and lignin (20±85%). The solubilized monomeric and oligomeric sugars in

the liquid stream for acidic-based pretreatments are sensitive to thermally

induced decomposition that results in the formation of furans and other

degradation products (Chen et al., 2006). Almost all pretreatments resulted in

extensive deacetylation of hemicellulose ranging between 30 and 95% (basis is

theoretical acetyl content).

The effect of thermochemical pretreatments on corn stover/poplar glucan

and xylan hydrolysis yields at the end of pretreatment (Stage A) and enzymatic

hydrolysis (Stage B) (using 15 FPU/g glucan cellulase loading) is shown in

Table 2.3 (Adapted from Wyman et al., 2005; 2009). Most pretreatments per-

form reasonably well on corn stover with AFEX resulting in the highest total

glucan (96%) and xylan conversion (91%). A significant amount of

hemicellulose is solubilized as oligomeric sugars (20±60%) for most treatments

in the liquid stream after pretreatment. The sugar oligomers must be

hydrolyzed to monomeric xylose through acid or enzyme catalyzed hydrolysis.

Unlike other treatments, AFEX retains all the hemicellulose in the biomass at

the end of pretreatment which is hydrolyzed by hemicellulases during

enzymatic hydrolysis resulting in much higher monomeric sugar yields.

However, for poplar most acidic pretreatments perform better than alkaline

pretreatments except oxidative lime treatment. Steam explosion catalyzed by

sulfur dioxide resulted in higher glucan yields compared to dilute sulfuric acid

treatment for poplar. Unlike corn stover, a significant amount of hemicellulose

was hydrolyzed to monomeric sugars for poplar during acidic-based

pretreatments. This is probably due to the higher pretreatment severity

employed for poplar compared to corn stover. Lime pretreatment was the only

alkaline pretreatment that performed well on poplar. This is likely because

pressurized oxygen was used during lime pretreatment that resulted in a more

effective oxidatively catalyzed delignification of the cell wall compared to

other alkaline treatments. Supplementation of xylanases was found to play a

critical role in the hydrolysis of alkali pretreated poplar giving much higher



Table 2.3 Effect of leading thermochemical pretreatments on corn stover (I) and poplar (II) glucan and xylan percent conversions (based oncomposition of untreated biomass) at the end of pretreatment (Stage A) and enzymatic hydrolysis (Stage B) (enzymatic hydrolysisconducted at 15FPU/g glucan cellulase loading)

%Glucan conversion for pretreated corn stoverPretreatment Stage A Enzymatic Stage B Total glucan conversion (A+B)

Pretreatment type Monomeric Oligomeric Monomeric Oligomeric Monomeric Oligomeric Total

Dilute acid 6 0 86 0 92 0 92Controlled pH hot water 0 5 85 0 86 5 91AFEX 0 0 96 0 96 0 96ARP 0 0 90 0 90 0 90Lime 0 1 92 1 92 1 94

%Xylan conversion for pretreated corn stoverPretreatment Stage A* Enzymatic Stage B* Total xylan conversion (A*+B*)

Monomeric Oligomeric Monomeric Oligomeric Monomeric Oligomeric Total

Dilute acid 82 2 8 0 91 2 93Controlled pH hot water 2 55 24 0 26 55 81AFEX 0 0 77 14 77 14 91ARP 0 47 41 0 41 47 88Lime 1 23 52 0 52 23 76

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Table 2.3 Continued

%Glucan conversion for pretreated poplarPretreatment Stage A Enzymatic Stage B Total glucan conversion (A+B)


SO2 steam explosion 3 0 97 0 100 0 100Dilute acid (Sunds) 24 0 63 0 87 0 87Controlled pH hot water 0 2 54 0 54 2 56AFEX 0 0 53 0 53 0 53ARP 0 1 49 0 49 1 49Lime 0 0 90 5 90 5 96

%Xylan conversion for pretreated poplarPretreatment Stage A* Enzymatic Stage B* Total xylan conversion (A*+B*)


SO2 steam explosion 54 20 9 0 64 20 84Dilute acid (Sunds) 63 0 9 0 72 0 72Controlled pH hot water 4 54 38 0 42 54 96AFEX 0 0 52 0 52 0 52ARP 0 37 31 1 31 38 69Lime 0 5 65 8 65 12 78

Adapted fromWyman et al. (2005, 2009). Note: lime pretreatment for poplar was performed in the presence of pressurized O2.

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glucan and xylan conversions than has been reported in Table 2.3 (Kumar and

Wyman, 2009a; 2009b).

Comparison of glucose and xylose fermentability (by Saccharomyces

cerevisiae LNH-ST 424A) of enzymatic hydrolyzates from various pretreat-

ments for poplar results in varying ethanol metabolic yields (ranging from 85 to

100%) depending on the type of pretreatment (Lu et al., 2009). However, a more

rigorous comparison between various leading pretreatment technologies is

required to compare fermentability of the liquid extract (this is typically

ignored) and solid fractions resulting from the pretreatment with and without

detoxification at industrially relevant conditions (e.g., high solids loading to

produce >4% w/w ethanol concentration). Recent findings comparing AFEX

and dilute acid pretreated corn stover have shown that some amount of

detoxification might be necessary for acidic-based pretreatments to prevent

microbial inhibition (Lau et al., 2008b). While AFEX treated corn stover was

found to be easily fermentable with no necessity for substrate water washing,

detoxification or external nutrient supplementation (Lau et al., 2008a; 2008b;

Lau and Dale, 2009). There is a significantly higher amount of potentially

inhibitory degradation products (e.g., organic acids and phenolics) formed

during dilute acid pretreatment compared to AFEX (Chundawat et al., 2007;

2008b). Without any detoxification, E. coli KO11 was unable to grow on acid

treated corn stover hydrolyzate while Saccharomyces cerevisiae LNH-ST 424A

performed poorly compared to growth on AFEX stover hydrolyzate (Lau et al.,

2008b).

2.5 Characteristics of an ideal pretreatment

2.5.1 Factors affecting viability of any pretreatmenttechnology

The efficacy of any pretreatment technology should not be evaluated exclusively

based on the enzymatic digestibility of the pretreated solid biomass fraction

(typically ignoring the liquid fraction). Some authors defend that this evaluation

should be performed using a holistic perspective spanning technological,

economical and environmental factors (da Costa Sousa et al., 2009). In this

context, there are two important decisions to make before analyzing the efficacy

of any pretreatment: 1) decide what factors are important to take into con-

sideration, and 2) decide where to place the mass/energy boundaries to evaluate

the role of each factor on the system. The factors that are important to take into

consideration should not only comprise overall mass and energy balances for the

process (inclusive of product yields, concentration and reaction rates), but

should also encompass secondary factors that contribute to the economic and

environmental impacts of the pretreatment. These secondary factors need to be

analyzed within the boundaries of the study, which should be placed at the



radius of influence of the pretreatment, and possibly beyond the realm of

biorefinery activity. From Figs 2.2, 2.3 and 2.4 it is possible to see how

pretreatment can influence both pre- and post-processing steps such as, biomass

type, harvesting conditions, milling, enzyme type and amount requirement,

hydrolyzate preconditioning, microbial fermentation, by-product utilization,

waste residue handling, and ethanol recovery. This makes pretreatment the

central unit operation in a biorefinery with all-pervasive technological and

economic implications on other processing steps.

This kind of a cradle-to-grave assessment comparing pretreatments has been

largely absent, until recently. The CAFI project was the first attempt towards

this goal, allowing a systematic comparison of pretreatments (e.g., dilute-acid,

AFEX) on common feedstocks (e.g., corn stover, poplar) using consistent

analytical methods (e.g., same batch of commercial enzyme formulations). The

technical and economic performance of all CAFI pretreatments for corn stover

and poplar has been published in recent years (Wyman et al., 2005; 2009).

However, CAFI considered only a few of the pretreatment technologies

available for their study (dilute acid, liquid hot water, AFEX, ARP and lime-

based pretreatments).

In a crude attempt to have a general idea about the state-of-the-art of the most

important pretreatment technologies available today, Tables 2.4 and 2.5 were

compiled using information available in the literature. These tables list some of

the important factors to take into consideration when comparing pretreatment

technologies. In this case, it is important to note that the pretreatment processes

that were involved in CAFI project show data that have a comparable basis.

However, this is not always true for other pretreatment technologies, since

experiments were carried out using different feedstocks, enzyme mixtures,

enzyme loadings and microbial fermentation conditions. The goal was to give a

general perspective of the requirements of each pretreatment and study their

effect on downstream biological processing.

From Table 2.4, it is possible to find useful information about each type of

pretreatment, such as the type of chemicals and the typical quantities used in the

process, hazard information about these chemicals, their cost (ICIS, 2006),

chemical recovery requirements, process conditions (temperature, pressure,

residence time). Another aspect that should be taken into consideration is the

`generality' of the pretreatment. Generality of a pretreatment refers to how the

pretreatment performs on a variety of feedstocks, such as hardwoods, softwoods

and grasses, in the dry or wet form. This is especially important when

operational flexibility of a biorefinery is required in the presence of variable

feedstocks.

The effect of pretreatment method on enzymatic hydrolysis performance can

be also analyzed from the typical glucan and xylan conversions (to monomeric

and oligomeric sugars), enzyme loading and hydrolysis time. The pretreatment

conditions and enzymatic hydrolysis performance are dependent on the type of



2.2 Material and energy flow balance for a generic pretreatment-based biorefinery unit operations from `cradle' (e.g., biomass cultivation)to `grave' (e.g., lignin residue and/or ethanol combustion).

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2.3 Detailed mass balance for generic unit operations involved in a second generation lignocellulosic biorefinery incorporating thermo-chemical pretreatment, enzymatic hydrolysis and microbial fermentation.

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2.4 Pretreatment is the center of the biorefinery `universe'. The figure depicts the strong inter-dependence between thermochemicalpretreatment with other upstream/downstream operations and process-product economic viability.

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Table 2.4 Important pretreatment-related parameters (e.g., chemical usage, hazards, cost of chemical, catalyst recovery, effluent waste, pressure/temperature/reaction time) for biological and thermochemical pretreatments

Pretreatment Pretreatment-related parameterscategorya

Chemical Chemical Chemical Chemicals Chemical recovery % Solid Temperature Pressure Residence Referencesusage/ hazardsb costc processes Catalyst effluent (ëC) (psi) timeMg $/Mg recovery

biomass

FractionationOrganosolv (++) C2H5OH, 4:1 C2H5OH H:2, F:3, $1300 (C2H5OH), Precipitation/Filtration/ 97% Yes 90±220 200±300 25±100 min ICIS, 2006; Pan et al., 2006;

H2O (+ acid 2:1 H2O R:0 $95 (H2SO4) Distillation +Waste water Sidiras and Koukios, 2004;or base) treatment/Water recovery Sun and Chen, 2008; X Zhao

et al., 2009

Phosphoric acid H3PO4, 13.5:1 H3PO4, H:2, F:0, $450 (H3PO4) Distillation/Flash N/A Yes 50 ± 60±80 min ICIS, 2006; H. Li et al., 2009;(+++) H2O, 19:1 CH3COCH3 R:0/H:1, $1390 (CH3COCH3) Separation +Waste water Zhang et al., 2007

CH3COCH3 24:1 H2O F:3, R:0 treatment/Water recovery

Ionic liquids (++) Ionic 10:1 Ionic N/A $45000 (Ionic Active carbon adsorption + N/A Yes 100±150 ± 0.5±2 hours Dadi et al., 2007; ICIS, 2006;liquids, liquid liquid)d Diethyl ether washing + Q. Li et al., 2009; WasserscheidH2O, Distillation +Chloroform/ and Haumann, 2006

CH3OH, methanol washing +C2H5OH activated alumina

adsorption + Distillation +Waste water treatment/Water recovery

ChemicalDilute-acid (+++) H2O, 0.03:1 H2SO4/ H:3, F:0, $95 (H2SO4) Acid neutralization (e.g. N/R Yes 160±220 30±220 2±30 min Eggeman and Elander, 2005;

H2SO4 4:1 H2O R:2, O:W with lime) +Waste water ICIS, 2006; Lu et al., 2009;treatment/Water recovery Mohagheghi et al., 1992; Schell

et al., 2003; Wyman et al., 2005

Steam explosion H2O, 0.005:1 H:3, F:0, $95 (H2SO4) Acid neutralization (e.g. N/R Yes 160±290 200±350 5±15 min ICIS, 2006; Li and Chen, 2008;(+++) H2SO4 H2SO4/ R:2, O:W/ $230 (SO2) with lime) +Waste water H. Li et al., 2009; Varga et al.,

or SO2 0.03:1 SO2 H:2, F:0, treatment/Water recovery 2004; Wang et al., 2009R:0

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Liquid hot H2O 6:1 H2O H:0, F:0, $0.50 (H2O)e Waste water treatment/ N/R Yes 160±230 350±400 15±20 min Ballesteros et al., 2002;water (++) R:0 Water recovery Eggeman and Elander, 2005;

ICIS, 2006; Laser et al., 2002;Lu et al., 2009; Mosier et al.,2005a

AFEX (++) H2O, NH3 1:1 NH3/ H:3, F:1, $280 (NH3) Distillation/Condensation/ 97±99% No 60±140 200±600 5±15 min Alizadeh et al., 2005; Eggeman0.6:1 H2O R:0 Water Quenching/ and Elander, 2005; ICIS, 2006;

Compression/ Lau et al., 2008b; Lu et al., 2009;Water recovery Sendich et al., 2008

ARP (++) H2O, NH3 0.47:1 NH3/ H:3, F:1, $280 (NH3) Distillation/Condensation/ N/A No 160±180 300±400 10±20 min Eggeman and Elander, 2005;2.7:1 H2O R:0 Water Quenching/ ICIS, 2006; Kim et al., 2003;

Compression/ Kim and Lee, 2005a; Lu et al.,Water recovery 2009

Lime (++) Ca(OH)2, 0.75:1 H:1, F:0, $180 (Ca(OH)2) Neutralize base with CO2/ N/A Yes 25±150 0±200 1±8 weeks Eggeman and Elander, 2005;air/O2 Ca(OH)2/ R:1 Regenerate with lime kiln ICIS, 2006; Kim and Holtzapple,

(optional) 10:1 H2O technology/Water recovery 2005; Lu et al., 2009

Ozonolysis H2O, O3 0.027:1 O3/ Not rated N/Af Recompression of the non- N/A No 25 ± 2±3 hours Garcia-Cubero et al., 2009;(+++) 0.3:1 H2O by NFPA reacted ozone to the feed Quesada et al., 1999;

line/Water recovery Sun and Cheng, 2002

Alkaline wet H2O, O2 1.2MPa O2, H:3, F:0, $820 (H2O2) Base neutralization/ N/A No 170±220 45±175 15 min ICIS, 2006; Klinke et al., 2003;oxidation (++) H2O2, 0.03:1 R:0, O:OX/ $495 (Na2CO3) Waste water treatment/ Martin et al., 2008; Varga et al.,

Na2CO3 Na2CO3, H:1, F:1, Water recovery 200315:1 H2O R:2

BiologicalFungi or N/R N/R N/A N/A Waste water treatment/ N/R No 20±25 ± 14±23 days Balan et al., 2008; Taniguchibacteria (+) Water recovery et al., 2005; Watanabe, 2007

Notes:a Generality of pretreatment: High (+++), Medium (++), Low (+).b Based on NFPA standards: H, Health; F, Flammability; R, Reactivity; O, Other hazard information (0 ±No special hazard, 4 ± Severe Hazard,W ±Reactivity withwater, OX ±Oxidizer).c 2006 price by ICIS.d Current best case scenario projection for a generic ionic liquid.e Price of water typically ranges between $0.25 and $1/Mg.f Ozonewas considered to be produced in-house. Cost of production not available.g N/A, N/R andMg stand for Not available, Not required andMegagram (equivalent to1metric tonne), respectively.

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Table 2.5 Enzymatic hydrolysis-fermentation related parameter ranges (e.g., glucan and xylan hydrolysis yields) comprising various cellulosicsubstrates (i.e., grasses/straws and hardwoods/softwoods) and overall process economics for various biological and thermochemicalpretreatments

Pretreatment Enzymatic hydrolysis-related parametersb Fermentation-related parametersb Economicsa Referencescategory

Total Total Cellulase Hydrolysis Final Ethanol Washing Detoxification Totalglucose xylose loading/ time ethanol yieldc require- and nutrient fixedyield yield per g (hrs) concen- (%) ment supple- capital(%) (%) glucan tration mentation $/gal

(%)

FractionationOrganosolv 85±100 N/A 20 FPU 48 3.7 99.5 (G) Yes Yes N/A ICIS, 2006; Pan et al., 2006; Sidiras and

Koukios, 2004; Sun and Chen, 2008;X Zhao et al., 2009

Phosphoric acid >90 80 25 FPU 24 4.75 93 (G) Yes Yes N/A ICIS, 2006; H. Li et al., 2009; Zhanget al., 2007

Ionic liquids 55±97 N/A N/A 6±24 N/A 86 (G) Yes Yes N/A Dadi et al., 2007; ICIS, 2006; Q. Li et al.,2009;Wasserscheid and Haumann,2006

ChemicalDilute acid 85±95 70±95 15 FPU 72 5.7 86 (G) Yes Yes 1.48 X Eggeman and Elander, 2005; ICIS, 2006;

Lu et al., 2009; Mohagheghi et al., 1992;Schell et al., 2003;Wyman et al., 2005,2009

Steam explosion 85±100 85±95 25 FPU 72 4.7 86 (G, X) Yes Yes N/A ICIS, 2006; Li and Chen, 2008; H. Li etal., 2009; Varga et al., 2004; Wang et al.,2009;Wyman et al., 2009; Lu et al., 2009

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Liquid hot water 55±90 80±95 20 FPU 24 2.9 82.7 (G, X) No Yes 1.82 X Ballesteros et al., 2002; Eggeman andElander, 2005; ICIS, 2006; Laser et al.,2002; Lu et al., 2009;Mosier et al.,2005b; Wyman et al., 2005, 2009

AFEX 55±100 55±95 15 FPU 72±168 4 88.5 (G, X) No No 1.48 X Alizadeh et al., 2005; Eggeman andElander, 2005; ICIS, 2006; Lau et al.,2008a,b; Lu et al., 2009; Sendich et al.,2008;Wyman et al., 2005, 2009

ARP 50±90 70±90 15 FPU 12±72 2 98.6 (G,X) Yes No 1.82 X Eggeman and Elander, 2005; ICIS, 2006;Kim et al., 2003; Kim and Lee, 2005a; Luet al., 2009; Wyman et al., 2005, 2009

Lime (with O2) >90 80 15 FPU 72±168 4 100 (G, X) Yes No 1.33 X Eggeman and Elander, 2005; ICIS, 2006;Kim and Holtzapple, 2005; Lu et al.,2009;Wyman et al., 2005, 2009

Ozonolysis 80±90 N/A 29 FPU 48 N/A N/A N/A N/A N/A Garcia-Cubero et al., 2009; Quesada etal., 1999; Sun and Cheng, 2002

Alkaline wet 70±80 50±55 73 FPU 24 5.2 83 (G) Yes No N/A ICIS, 2006; Klinke et al., 2003;oxidation Martin et al., 2008; Varga et al., 2003

BiologicalFungi or bacteria 40 N/A 15 FPU 72 N/A N/A Yes No N/A Balan et al., 2008; Taniguchi et al., 2005;

Watanabe, 2007

Notes:Lime pretreatment for poplar was performed in the presence of O2.a Ideal fixed capital given by X = $2.51/gal of ethanol.b Enzymatichydrolysis/fermentationdata is for corn stover, poplar andother relatedagricultural residues/energycrops. Data for CAFI pretreatments is basedoncorn stoverand/or poplar.c Ethanolmetabolic yield is either based on initial glucose (G), xylose (X) or both sugars (G/X).

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feedstock used. In Table 2.5, the data shown for the various pretreatments can

correspond to different types of feedstock (e.g., corn stover, poplar and other

lignocellulosics) and may not be always comparable. However, the data refer

mostly to the best case scenario for each pretreatment.

Fermentation is also affected by the choice of pretreatment, since the type of

chemical modifications occurring in the plant cell wall can dictate the formation

of potential inhibitors to microbial growth. The detoxification step is usually

done by increasing the pH of the hydrolyzate to 9±11 at high temperature

(~90 ëC) (Saha et al., 2005) using lime. This process will produce an insoluble

waste residue (comprising various lignin and furan-based inhibitory

compounds) that needs to be considered for the environmental and economical

evaluation of the complete system. In addition to this fact, the extractive nature

of some pretreatments promotes the removal of several micronutrients which

could be used by microbes during fermentation (Lau and Dale, 2009). For this

reason, in some processes it will be necessary to supplement the fermentation

media with additional nutrients, which also impact the overall economics of the

process.

To further extend this analysis, it would be necessary to expand the

boundaries of these studies to pre- and post-processing steps within a ligno-

cellulosic biorefinery. This would help assess both economical and environ-

mental impacts caused by the biorefinery for different pretreatment

technologies. To complete this goal, it is necessary to integrate land manage-

ment, feedstock processing, waste handling, distribution chains, animal feed

production, etc., when evaluating the biorefinery process. In this context, life

cycle analysis (LCA) is considered to be a powerful tool that could be used to

help evaluate and design this system in a sustainable fashion (Kim and Dale,

2005; 2006).

2.5.2 Mass and energy balances

It is critical to carry out detailed material and energy balances over the entire

biorefinery related operations (as shown in Figs 2.2 and 2.3) to estimate the

process economic and environmental costs. Economic analysis would help

determine high-impact process parameters that would significantly influence the

feasibility of the operation. The interplay of various parameters like type of

feedstock (prairie grass vs. hardwood), extent of biomass milling, total

hemicellulase loading, hexose/pentose co-fermentation and co-product (residual

protein) recovery with the pretreatment options can be better explored from this

holistic perspective. For example, depending on the type of feedstock (corn

stover vs. poplar) the severity of thermochemical pretreatment and total enzyme

requirement would vary drastically. The type of pretreatment used (dilute acid

vs. AFEX) would also influence detoxification and microbial fermentation

strategies. It will be possible to estimate production costs for ethanol when



producing useful co-products such as animal feed using low severity

pretreatments like AFEX (Bals et al., 2007; Carolan et al., 2007). The strategies

for biomass collection, handling and transportation would also likely be

influenced by the type of pretreatment technology. It would be easier to predict

the actual economic potential of various biorefinery scenarios using detailed

material and energy balances integrated with financial models (Eggeman and

Elander, 2005; Sendich et al., 2008; Wooley et al., 1999).

2.5.3 Product yield, reaction rate, product concentration

The three important criteria that are typically used to compare various

pretreatments are overall product yield (per unit mass of feedstock), rate of

reaction and final product concentration. These parameters are typically found to

most significantly influence the final minimum ethanol selling price (MESP) for

all economic models (McAloon et al., 2000; Eggeman and Elander, 2005). In

general, these factors will influence the equipment size and fixed cost invest-

ments for a biorefinery. More specifically, high yield is important to minimize

feedstock use, which is considered to be the highest variable cost in a biorefinery

(Aden et al., 2002). The minimization of feedstock usage is also necessary to

maintain sustainability of the bioeconomy (compare to `bottom of the barrel'

processing arguments in petroleum refineries), providing as much product (e.g.

bioethanol) per acre of land as possible.

To compare effectiveness of various pretreatments in terms of product yield,

reaction rates and product concentration, it is important to have identical

experimental setups. Most of the time, it is impossible to perform a fair

comparison between pretreatment technologies, since the information available

in the literature, comes from completely different experimental bases.

Monomeric sugar yields during enzymatic hydrolysis are closely dependent

on total solids loading, enzyme activity and inhibition. The solids to liquid ratio

employed during pretreatment would affect the total solids loading employed

during hydrolysis. Most pretreatments employ large quantities of water (5±10 kg

water per kg biomass) that result in slurries at the end of pretreatment (which is

typically washed and separated from the solid fraction that is used for hydrolysis

experiments). Depending on the severity of acid-based treatments the lignin and

hemicellulose are selectively solubilized from the solid glucan rich fraction.

Most of the time, the solubilized hemicellulose is not easily fermentable to

ethanol due to presence of inhibitors such as furfural and 5-hydroxymethyl-

furfural (Klinke et al., 2004), which results in lower overall ethanol yield.

However, this situation opens the possibility for utilization of this stream for

other applications, such as chemical conversion of sugars into valuable products

(Zhao et al., 2007).



2.5.4 Economic and environmental feasibility

It would also be critical to conduct life cycle analysis (LCA) around various

biorefinery scenarios taking in consideration the utilization of different feed-

stocks, agricultural practices, and technologies to produce biofuels and useful

co-products (Kim and Dale, 2005). Certain feedstocks have higher biomass

yields (per hectare) and might need lesser water and fertilizer inputs, like

Miscanthus. However, the severity of thermochemical pretreatment necessary to

achieve equivalent ethanol yields might negate the advantage of using mis-

canthus over corn stover as the biorefinery feedstock (Murnen et al., 2007). It

therefore becomes pertinent to evaluate the non-tangible benefits of using

certain feedstocks and technologies in a biorefinery, which have traditionally

been ignored.

It is also useful to evaluate biorefinery operations from a `bottom-of-the-

barrel' processing outlook. Some parameters that contribute to the overall

environmental and economical impact have to do with waste generation and

disposal. For this reason, technologies that are able to utilize all lignocellulosic

feedstock components should ideally be favored. Currently, 20±30% of the

lignin from processed biomass is being burnt to generate energy. However, with

further improvements in lignin utilization chemistry and technology it might be

possible to recover useful by-products as feedstock for the petroleum-based

polymer industry (Shevchenko et al., 1999; Lora and Glasser, 2002). In addition,

processes that minimize water and chemical utilization should be favored, since

it is important to minimize wastewater treatment and disposal of the non-

recycled chemicals for economical and environmental reasons. The quantity and

nature of the chemical used during pretreatment will also dictate the investment

in safety equipment as well as handling and disposal infrastructures. The use of

different pretreatment chemicals will naturally generate variable environmental

and economical issues to handle.

Finally, one other interesting factor that should be considered for evaluating

pretreatments is the potential of treated biomass to be used as animal feed

(Weimer et al., 2003; Carolan et al., 2007). Most of the land in the United States

is used to produce animal feed and thus the use of pretreated lignocellulosic

biomass to feed ruminant animals can contribute to an important reduction of

land use for animal feed production (Carolan et al., 2007). From this point of

view, more land will be available to produce lignocellulosic biomass for

biofuels, with minimal impact on the food chain.

2.6 Conclusions

Most studies on pretreatments have looked to optimize pretreatment parameters

through maximization of hydrolysis (and/or fermentation) product yields. It is

important to determine the chemical and ultra-structural modifications



incorporated within the cell wall during pretreatment to identify the key limiting

factors that result in recalcitrance of native lignocellulosic biomass to bio-

processing. Further advances in this field would help in the development of novel

pretreatment technologies that are cost effective and environmentally friendly.

Another area of research that has lagged behind in this field of research has

been in trying to predict the rate of enzymatic hydrolysis for pretreated

lignocellulosic biomass (Laureano-Perez et al., 2005). Both substrate and

enzyme related factors would be expected to contribute to the saccharification

process. The rate of hydrolysis of a heterogenous, insoluble lignocellulosic

substrate can be classified into two phases primarily: (a) the initial rate of

hydrolysis that typically is dependent on the gross enzyme accessibility of the

substrates. This initial rate of hydrolysis can be well correlated to the porosity of

the biomass depending on the type of pretreatment (Grethlein, 1985); (b) the

terminal rate of hydrolysis is dependent on several factors ranging from

cellulose degree of polymerization, hemicellulose side-chain branching, non-

productive protein binding to lignin and enzyme inhibition by pretreatment and

hydrolysis degradation products. There is a need to coherently incorporate these

parameters for both phases of hydrolysis when developing saccharification

kinetic models (Zhou et al., 2009).

Consolidated bioprocessing (CBP) is a novel processing strategy for

lignocellulosic biomass which is looking to consolidate the four biologically-

mediated steps; of cellulase production, cellulose hydrolysis, hexose and pentose

fermentation into a single processing step. This would help significantly reduce

processing costs for a cellulosic biorefinery. There is no native microbial system

that is currently able to perform CBP. Efforts are currently underway to develop a

suitable CBP microbe. It would be of interest to study the effect of various

pretreatment methodologies on the performance of CBP-based microbial systems.

Cleavage of LCC linkages and extraction of hemicellulose-lignin residues

seem to be the most important ultra-structural modification that takes place

during most chemical pretreatments. However, in order to further enhance the

rate of enzymatic hydrolysis of cell walls it would be necessary to modify the

crystal structure of cellulose as well. The solid crystalline morphology of

cellulose seems to be the ultimate rate-limiting step for efficient hydrolysis of

plant cell walls (Zhou et al. 2009). Pretreatments that can further reduce the

crystallinity of cellulose (either through formation of cellulose III or via

conversion to amorphous cellulose), in a cost-effective manner, would have a

significant advantage over existing pretreatment technologies.

2.7 Acknowledgements

The authors would like to thank CAFI team members for providing necessary

data and making important suggestions during the course of preparation of this

chapter. Some of the data for Tables 2.1, 2.2 and 2.3 were adapted from



previously published CAFI related paper by Kim and Holtzapple, 2005; 2006;

Kim and Lee, 2005a; Laureano-Perez et al., 2005; Lloyd and Wyman, 2005;

Mosier et al., 2005a; Teymouri et al., 2005; Wyman et al., 2009; Kumar et al.,

2009; Kumar and Wyman, 2009a,b. This work was partly funded by DOE Great

Lakes Bioenergy Research Center (www.greatlakesbioenergy.org) supported by

the US Department of Energy, Office of Science, Office of Biological and

Environmental Research, through Cooperative Agreement DE-FC02-

07ER64494 between The Board of Regents of the University of Wisconsin

System and the US Department of Energy.

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