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Hydrolysis of cellulose and hemicellulose

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 HYDROLYSIS OF CELLULOSE AND

HEMICELLULOSE

Charles E. Wyman1, Stephen R. Decker2, John W. Brady3, Liisa Viikari4, and Michael E.

Himmel2

1. Thayer School of Engineering, Dartmouth College, Hanover, New Hampshire 03755

2. National Renewable Energy Laboratory, Golden, Colorado, 80401
3. Department of Food Science, Cornell University, Ithaca, New York, 14853
4. VTT Technical Research Centre of Finland, FIN-02044, VTT

1
1 INTRODUCTION 5

2 OVERVIEW OF THE RELEVANT STRUCTURAL FEATURES OF

CELLULOSIC MATERIALS 7

2.1 Cellulose 7

2.1.1 Chemical Composition 8

2.1.2 Structure and Morphology of Cellulose Fibers 8

2.2 Hemicelluloses 14

2.2.1 Structural Functionality of Hemicelluloses 15

2.2.2 Chemical Composition and Sources of Hemicelluloses Subtypes 17

3 HEMICELLULOSE AND CELLULOSE HYDROLYSIS FOR BIOMASS

CONVERSION 23

3.1 Overview Of Conversion Chemistry 23

3.2 Typical Process Steps 24

3.3 Cellulose and hemicellulose hydrolysis reactions 26

4 ACID HYDROLYSIS OF CELLULOSE 28

4.1 System Description and Performance 28

4.2 Kinetic Models 30

5 ACID HYDROLYSIS OF HEMICELLULOSE 35

5.1 System Description and Performance 35

5.2 Kinetic Models 36

6 CELLULASE SYSTEMS FOR CELLULASE HYDROLYSIS 41

6.1 Sources of Cellulase Enzymes 41

6.1.1 Fungal Enzyme Systems 41

6.1.2 Bacterial Enzyme Systems 42

6.2 General Classification of Cellulase Enzymes 44

2
 6.2.1 Endoglucanases 44

6.2.2 Exoglucanases 45

6.2.3 Beta-β-Glucosidase 45

6.3 Cellulase Structure and Function 45

6.3.1 Synergism 45

6.3.2 Glycosyl Hydrolase Diversity 47

6.3.3 Enzyme Adsorption and Non Productive Binding 48

6.3.4 Processive Cellulases 49

7 ENZYMATIC HYDROLYSIS OF CELLULOSE 50

7.1 Experimental Systems 50

7.2 Structural Features Impacting Cellulase Action 53

7.2.1 Crystallinity and Boundary Water Layer 53

7.2.2 Hemicellulose Content 54

7.2.3 Lignin Content 54

7.2.4 Acetyl Content 58

7.3 Kinetic Modelling of enzymatic hydrolysis of cellulose 58

8 PRETREATMENT TO IMPROVE CELLULOSE HYDROLYSIS BY

ENZYMES 60

8.1 Effect of Pretreatment on Enzymatic Hydrolysis 60

8.2 Desirable Pretreatment Attributes 62

8.3 Pretreatment Types 63

8.4 Chemical Pretreatment at Low to Neutral pH 64

8.5 Chemical Pretreatment at High pH 66

9 ENZYMATIC HYDROLYSIS OF HEMICELLULOSE 68

9.1 Sources of Hemicellulases 68

3
 9.2 General Hemicellulose Hydrolysis 69

9.2.1 Xylan Backbone Hydrolysis 70

9.2.2 Mannan Hydrolysis 72

9.2.3 Enzymatic glucan hydrolysis 72

9.2.4 Enzymatic hydrolysis of hemicellulose oligomers 74

9.2.5 Enzymatic debranching of hemicellulose 74

9.3 Action of Hemicellulases on Model Substrates 75

9.4 Hydrolysis of Residual Hemicellulose in the Solid Residue 77

9.4.1 Hydrolysis of Solublized Hemicellulose 78

10 ECONOMICS OF SUGAR PRODUCTION FROM CELLULOSIC BIOMASS 79

10.1 Cellulosic Biomass as a Feedstock 79

10.2 Economics of Hydrolysis Based Technologies 80

10.3 Application to Other Products 84

10.4 Opportunities for Cost Reductions 85

11 BENEFITS AND IMPACTS OF CELLULOSIC BIOMASS CONVERSION 87

11.1 Greenhouse Gas Reductions 87

11.2 Strategic Benefits 89

11.3 Solid Waste Disposal 90

11.4 Economic Benefits 91

11.5 International Fuels Market 91

11.6 Sustainable Production of Organic Fuels and Chemicals 92

12 ACKNOWLEDGMENTS 92

13 REFERENCES 92

4
1 INTRODUCTION

Cellulosic biomass includes agricultural (e.g., corn stover and sugarcane bagasse) and forestry

(e.g., sawdust, thinnings, and mill wastes) residues, portions of municipal solid waste (e.g.,

waste paper), and herbaceous (e.g., switchgrass) and woody (e.g., poplar trees) crops. Such

materials are abundant and competitive in price with petroleum, and cellulosic biomass can

provide a sustainable resource that is truly unique for making organic products. Furthermore,

cellulosic biomass can be produced in many regions of the world that do not have much

petroleum, opening up a new route to manufacturing organic fuels and chemicals.

The structural portion of cellulosic biomass is a composite of cellulose chains joined together

by hydrogen bonding as long cellulose fibers that are in turn held together with hemicellulose

and lignin, allowing growth to large aerial plants that can withstand the weather and resist

attack by organisms and insects. Traditionally, humans have employed the strength offered

by some forms of cellulosic biomass for structural purposes such as construction of homes,

furniture, and baseball bats. Other types of biomass such as grasses can stabilize the soil and

provide protein for feeding of animals. In addition, chemicals can react with biomass at high

temperatures to remove a portion of the hemicellulose and/or lignin to enhance the properties

of the remaining solids for manufacture of paper, particleboard, and other products. However,

the cellulose and hemicellulose portions of biomass, typically representing about 40 to 50%

and 20 to 30% of plants, respectively, are polysaccharides that can be broken down into

sugars through hydrolysis by enzymes or acids, and these sugars can be fermented or

chemically altered to valuable fuels and chemicals. Over the years, such hydrolysis reactions

have been employed to release sugars for making ethanol during periods of war by

approaches such as the Scholler process during the 1940s and for making various products in

controlled economies such as the former Soviet Union. Hydrolysis of the polysaccharides in
5
cellulosic biomass for production of fuels and chemicals offers important strategic,

environmental, and economic advantages, and although the cost has been too historically too

high compared to fossil alternatives, research over the last 2 decades has advanced the

technology to the point that it is becoming economically viable. The challenge is to overcome

the risk of commercializing first-of-a-kind applications and to continue to advance hydrolysis

technologies to become even lower in cost so that fuels and chemicals from cellulosic

resources are competitive without subsidies.

This chapter presents a comprehensive overview of cellulose and hemicellulose hydrolysis. It

begins with a short description of important structural features of cellulosic materials to

provide a context on the demands of hydrolyzing these abundant but recalcitrant

polysaccharides. The next section focuses on applications, process steps, and stoichiometry

for hydrolysis reactions. A discussion of acid hydrolysis approaches and kinetics is then

provided followed by a similar treatment for hemicellulose hydrolysis to give insight into

opportunities and limitations for acid based processes. At this point, the chapter shifts to

enzymatic hydrolysis starting with a short description of different sources of cellulase

enzymes for breaking down cellulose to glucose. This section also includes information on

the different types of enzyme activities involved in converting cellulose to glucose and

features of these components that influence cellulase action. The chapter then examines the

structural characteristics of biomass that influence cellulose hydrolysis by enzymes, types of

cellulose hydrolysis processes, and experimental results for systems for enzymatic conversion

of cellulose, and summarizes some of the factors influencing hydrolysis kinetics. Because

cellulosic biomass must be pretreated to achieve high yields of glucose from cellulose by

enzymes, an overview is provided of leading options for preparing biomass for enzymatic

processing.

6
Many promising pretreatment approaches do not fully hydrolyze hemicellulose to sugar

monomers, and use of enzymes provides an attractive route to making them fermentable.

Hemicellulose hydrolysis is also important in many other applications. Therefore, sources of

such enzymes, how they act, features impacting their action, experimental results with

hemicellulases, and modeling approaches to predict their performance are described. The

economics of hydrolysis-based processes are then summarized including a description of

advances that have reduced costs to be competitive now, identification of opportunities to

reduce costs further, and synergies for production of multiple products from cellulosic

materials. The chapter concludes with a brief description of important benefits of applying

hydrolysis of biomass to generate sugars for conversion to ethanol and other products.

Because covering such a wide spectrum of hydrolysis aspects in a single chapter necessitates

being succinct, numerous references are included to help the reader pursue topics in more

depth, if desired.

2 OVERVIEW OF THE RELEVANT STRUCTURAL FEATURES OF

CELLULOSIC MATERIALS

2.1 Cellulose

As a regular, linear homopolymer, cellulose might reasonably be thought to be structurally

uncomplicated. This perception would not be entirely correct however, since cellulose chains

are organized together into progressively more complex assemblies at increasing size scales.

As a result of this emergent structural complexity and heterogeneity, the structure of cellulose

has been remarkably difficult to unravel for such a nominally simple substance, and it is only

relatively recently that a consensus picture of its organization has begun to emerged. Several

7
recent reviews have surveyed cellulose structures, but this question is still an active topic of

study (1-3).

2.1.1 Chemical Composition

The chemical structure of cellulose, which is a linear polymer of β-(1→4)-linked D-

glucopyranose monomer units, is in fact quite simple. Typically cellulose chains in primary

plant cell walls have DPs (degree of polymerization) in the range from 5,000-7,500 glucose

monomer units, with the DP of cellulose from wood being around 10,000 and around 15,000

for cellulose from cotton (3). The basic repeating unit of cellulose is cellobiose (Figure 1),

the β-(1→4)-linked disaccharide of D-glucopyranose. At ambient temperatures, the relatively

rigid glucose rings are all found in their lowest-energy, 4C1 puckered chair conformation (see

Figure 1), and do not easily make transitions to the other chair conformer or to the various

possible twist-boat forms. With the rings in this conformation, all of the hydrogen-bonding

hydroxyl and hydroxymethyl substituents of the pyranose rings are equatorial, directed around

the periphery of the ring, while all of the hydrophobic aliphatic protons are in axial positions,

pointing either up or down relative to the average plane of the rings. This topological feature

of cellobiose can be seen readily in Figure 1.

2.1.2 Structure and Morphology of Cellulose Fibers

Bulk cellulose has a substantial degree of crystallinity, and its structure has long been the

subject of intense study. A crystalline structure for cellulose was first described by Mark and

Meyer in 1928 (4). Unfortunately, however, disorder and polydispersity in chain lengths

prevent the formation of single crystals, and x-ray diffraction studies of the crystal structure of

cellulose have been limited to fiber diffraction experiments. For this reason, the details of the

crystal structure of native cellulose are still a matter of debate more than 70 years later.
8
However, combined with modeling calculations, such experiments can be used to deduce

plausible conformations consistent with the limited data. All available evidence indicates that

crystalline cellulose chains are in an extended, flat 2-fold helical conformation (described in

detail below), but small variations in this conformation or in the packing of the cellulose

chains within the crystal give rise to a number of crystalline polymorphs, many of which can

be interconverted by various processing treatments (3, 5). Under normal conditions, cellulose

is extremely insoluble in water, which is of course necessary for it to function properly as the

structural framework in plant cell walls.

Seven crystal polymorphs have been identified for cellulose, which are designated as Iα, Iβ,

II, IIII, IIIII, IVI, and IVII, as indicated in the following diagram 1.1 (3).

(1.1)

These polymorphs differ in physical and chemical characteristics such as solubility, density,

melting point, crystal shape, and optical and electrical properties (2, 4). In nature, cellulose

Iα and Iβ are the most abundant crystal forms and hence are referred to as native cellulose.

Initially, X-ray fiber diffraction experiments of different native cellulose sources led to two

models of native cellulose differing in the number and orientation of glucose units in the unit

cell (6). Later experiments by Atalla and VanderHart in 1984 using 1H-NMR indicated that

native cellulose contained two allomorphs which were designated Iα and Iβ (7). This picture

has been confirmed by Wada and coworkers using electron diffraction experiments (8).
9
Cellulose I consists of chains arranged in a parallel fashion such that the (1→4) glycosidic

bonds point in the same direction along the microfibril. The nature of cellulose biosynthesis

requires this parallel packing. Although cellulose I is the enzymatically-synthesized crystal

form for this polymer, it is not the lowest-energy form, and the individual fibers are

kinetically trapped in this arrangement by the crystal lattice after synthesis.

Cellulose Iα is the major allomorph produced by bacterial and fungal sources and is a triclinic

P1 crystal with one cellobiose residue per unit cell (9). The cellulose chains are oriented in a

parallel manner as would be expected for a unit cell with one chain. Cellulose Iα is converted

to the more stable Iβ form through an annealing process at 270°C in various media (3).

Cellulose Iβ is the major crystal form in higher plant species and is monoclinic in nature with

two cellobiose moieties per unit cell. Cellulose Iα and cellulose Iβ are found within the same

microfibril and hence parallel packing of the cellulose chains in cellulose Iβ is the logical

conclusion and is consistent with X-ray diffraction data, analysis with silver staining and

cellobiohydrolase digestion (10-12). There are two possible ways to pack the chains in a two-

parallel-chain unit cell arrangement: parallel up and parallel down. The two chains are

referred to as the corner chain and the origin chain. Differences in the definitions of the unit

cell dimensions have lead to confusion as to the precise packing for cellulose Iβ. Current and

proper convention, as proposed by Sarko and Muggli (6), designates the chain which lies

parallel to the c axis direction as the origin chain and the chain which passes through the

center of the a/b plane, and translated in the c axes direction by a/4 with respect to the origin

chain, as the center chain (11). The result of this designation denotes the chains of a cellulose

Iβ crystal as packing in a parallel up fashion.

Cellulose II is the major polymorph in industrially processed cellulose. Cellulose II can be

formed upon regeneration or mercerization of cellulose I and is also the most

10
thermodynamically stable allomorph. The nature of the cellulose II structure remains to be

definitely agreed upon. For example, arguments continue as to whether cellulose II consists

of parallel or antiparallel chains. Nishimura and Sarko proposed that cellulose II is formed

after crystalline cellulose I is solvated and the amorphous regions re-crystallize into anti-

parallel microfibrils (13). Simon and coworkers proposed that the increase in distance

between chains in the crystal due to swelling is sufficient for the chain to fold at both ends

and then glide along one another to produce the antiparallel arrangement (14). Others claim

cellulose II chains align antiparallel after solvation due to chain exchange between

microfibrils. Kroon-Batenburg and coworkers have suggested that the difference between

cellulose I and cellulose II is simply the rotation of the primary alcohol from the TG

conformer1 to the GT conformer, and that cellulose II is in fact parallel (5). These authors

argue that the unit cell dimensions of GT parallel cellulose II are the same as those for anti-

parallel TG cellulose II, and that it thus is hard to distinguish the packing mode of cellulose II.

(It is interesting to note that neither of these primary alcohol conformers is the lowest-energy

form for free glucose; both NMR and modeling studies find that the GG conformer has the

lowest free energy for the isolated monomer (16-20). Using molecular dynamics techniques,

Marhofer and coworkers calculated Young’s modulus for cellulose II with chains in both the

parallel and antiparallel arrangements (21). Only the parallel arrangement gave good

agreement with experimental results, and they too concluded that cellulose II chains have a

parallel arrangement. Most recently Langan and coworkers produced a cellulose II structure

from neutron fiber diffraction analysis (22). The neutron diffraction data extended to a 1.2 Å

1
tg conformation refers to the rotamer of the primary alcohol group, where the first t (trans) or g (gauche) refers

to the orientation of the C4-C5-C6-O6 torsion with respect to O5 and the second letter designates the orientation

with respect to C4. 15. R. H. Marchessault, and S. Peréz, Conformations of the Hydroxymethyl Group in

Crystalline Aldohexopyranoses, Biopolymers, 18:2369 (1979).

11
resolution from two highly crystalline fiber samples of mercerized flax, and these workers

constructed two models of cellulose II in an anti-parallel arrangement with the primary

alcohols in the GT conformation for the origin chain and in the TG conformation for center

chain. It is important to note that fiber diffraction studies cannot directly determine the

primary alcohol rotamer conformation, which must be deduced from models consistent with

the limited diffraction data. Apparently no workers have attempted to fit cellulose II fiber

diffraction data using a GG model for either parallel or antiparallel chains, and the question of

the structure of cellulose II must be regarded as still unresolved.

The problem of describing the overall conformation of polysaccharide chains can be reduced

to the problem of specifying the rotational torsion angles φ and ψ around each successive

glycosidic linkage, in much the way that the conformation of the backbone of a protein can be

specified by listing the peptide Ramachandran torsional angles (23). For single crystals of

cellobiose the values of these angles are (42˚, -18˚) (24). Because the glycosidic linkage is of

the equatorial-equatorial type (since the O4 group of glucose is equatorial, as is the O1 group

in the β anomer), a broad range of values of φ and ψ are possible which involve no clashes

between two linked rings. For cellulose chains, the linkage torsion angles are approximately

(28˚, -30˚), with small variations in different polymorphs but with all cellulose structures

having essentially the same extended flat ribbon-like conformation in crystals. Figure 2

illustrates the conformation of the cellobiose repeat unit in cellulose, and Figure 3 shows the

conformation of a short hexaose oligomer in this conformation.

There are several very important qualitative features of the crystal conformation of the

individual cellulose chains that should be noted. As can be seen from Figure 3, in the very

extended crystalline conformation each glucose unit is “flipped” by 180˚ with respect to the

previous and subsequent rings, so that the exocyclic primary alcohol groups alternately point

to the right and left of the chain direction. This feature is important for the binding grooves
12
and pockets of cellulase enzymes that must accommodate cellulose chains. Secondly, the

chain is stabilized by strong hydrogen bonds along the direction of the chain, from the

exocyclic O6 primary alcohol hydroxyl group to the O2 secondary alcohol hydroxyl group of

the subsequent residue, and from the O3 hydroxyl group to the O5 ring oxygen of the next

sugar residue. These hydrogen bonds help to maintain and reinforce the flat, linear

conformation of the chain beyond the rigidity that might be expected for this type of linkage.

Importantly, in most models for crystalline cellulose, there are no hydrogen bonds between

chains in different crystal layers. Finally, with all of the aliphatic hydrogen atoms in axial

positions and all of the polar hydroxyl groups in equatorial positions, the top and bottoms of

the cellulose chains are essentially completely hydrophobic, while the sides of the chains are

hydrophilic and capable of hydrogen bonding. This topology is extremely important for the

packing of chains into crystals. In all of the proposed crystal packing schemes, the chains are

stacked with a pairing of hydrophobic faces, and these hydrophobic regions must make some

contribution to the insolubility of cellulose under normal conditions. Furthermore, binding

sites for cellulose segments in proteins must contain hydrophobic surfaces such as tryptophan

or phenylalanine side chains to pair up with these non-polar faces.

In native cellulose found in plant sources, the cellulose chains have extended segments with

the regular, repeating crystal conformation, and a number of independent chains are packed

together into bundles or microfibrils which typically consist of from around 30 to 200

independent chains. In these microfibrils, long sequences of the individual chains are found

in the extended conformation and similar segments from adjacent chains pack together into

highly regular microcrystalline regions. The microfibrils are not entirely crystalline, however,

even in so-called bacterial microcrystalline cellulose samples, as the regular segments of the

individual chain conformations alternate with segments which are irregular in conformation.

This alternation of regular microcrystalline regions with more flexible disordered regions is
13
almost certainly the result of evolutionary selection, since in order to serve effectively as the

principal structural component of plant cell walls, both great strength and flexibility are

needed.

2.2 Hemicelluloses

Hemicelluloses are generally classified according to the main sugar residue in the backbone,

e.g. xylans, mannans, and glucans, with xylans and mannans being the most prevalent.

Depending on the plant species, developmental stage, and tissue type, various subclasses of

hemicellulose may be found, including glucuronoxylans, arabinoxylans, linear mannans,

glucomannans, galactomannans, galactoglucomannans, β-glucans, and xyloglucans. These

different subtypes can be grouped into two general categories based on the hydration of the

fibers. Low hydration polysaccharides include the arabinoxylans, glucuronoxylans,

xyloglucans, and linear mannans. With the exception of the linear mannans, which serve

mainly as a seed storage compound, this class of hemicellulose functions primarily to stabilize

the cell wall through hydrogen-bonding interactions with cellulose and covalent interaction

with lignin. Again, with the exception of the linear mannans, these compounds are water

soluble in their native state, generally due to their branched construction. Standard extraction

protocols using alkali de-esterifies them of some of their sidechains, disrupting the entropy of

the water of solublization enough to cause the partially debranched chains to aggregate

through interchain hydrogen bonding and fall out of solution. Other hemicelluloses, such as

galactoglucomannans, glucomannans, galactomannans, and β-glucans comprise a subset of

highly hydrated hemicelluloses. This class, comprised mainly of hydrocolloids, is used

primarily as an extracellular energy and raw materials storage system and as a water retention

mechanism in seeds. As they have fewer, if any, ester-linked sidechains, alkaline extraction

does not usually render them insoluble and they tend to be heavily hydrated.
14
2.2.1 Structural Functionality of Hemicelluloses

The majority of the polysaccharides found in plant cell walls belong to the cellulose,

hemicellulose, or pectin classes. Cellulose, the dominant structural polysaccharide of plant

cell walls, is a linear β-(1→4)-D-glucopyranoside polymer. Although cellulose functions as

the rigid, load-bearing component of the cell wall, the rigidity of the cellulose microfibril is

strengthened within a matrix of hemicelluloses. Also referred to as cross-linking glycans, the

hemicelluloses are believed to be involved in regulation of wall elongation and modification

Unlike lignin, the hemicelluloses are thought to have a strong effect on the interactions

between cellulose microfibrils and to heavily influence the strength and rigidity of the cross-

linking matrix. This matrix is further complexed and reinforced with the pectins. Pectins are

non-cellulosic acidic cell wall polysaccharides. The pectins vary widely and are divided into

three classes, homogalacturonan, rhamnogalacturonan I, and rhamnogalacturonan II. Pectins

function as a sol-like matrix, providing water and ion retention, support and facilitation of cell

wall modifying enzymes, cell wall porosity, cell-to-cell adhesion, cell expansion, cell

signalling, developmental regulation, and defense.

Hemicelluloses are loosely defined as non-cellulose, non-pectin cell wall

heteropolysaccharides consisting of several polymers, varying in composition of

monosaccharides and glycosidic linkages, substitution pattern and degree of polymerization.

Their chemical composition and structural features vary widely across species, sub-cellular

location, and developmental stages. They have often been reported as chemically associated

or cross-linked to other polysaccharides, proteins or lignin. Studies of bacterial cellulose

produced by Acetobacter xylinum in the presence of various hemicellulose fibers have shown

that these hemicelluloses readily become complexed into the interior and along the surface of

the cellulose microfibril (25-27). The results of this interaction are dependent upon the type

15
of hemicellulose involved. These results have been supported by FT-IR work on intact wood

fibre (28). Probably no chemical bonds exist between cellulose and hemicellulose, but mutual

adhesion is provided by hydrogen bonds and van der Waals forces.

Evidence has been reported for linkages between hemicellulose and lignin in wood and other

plant materials. The side groups of xylan may have an important role in the bonding of lignin

to hemicellulose. Both ester linkages between lignin and methylglucuronic acid residues and

ether bonds from lignin to arabinosyl groups have been reported (29). Low molecular weight

phenolic components, such as ferulic acid and p-coumaric acid, are covalently bound to plant

cell wall polysaccharides of annual plants (30).

Side groups, especially the acetyl substituents, affect the physicochemical properties and

biodegradability of soluble or matrix-bound hemicelluloses. Acetylation has been shown to

increase the solubility of polysaccharides in water, probably by hindering the aggregation of

molecules, and thus making the substrate more accessible to enzymes (31, 32). On the other

hand, the presence of ester-linked non-carbohydrate residues impedes the ability of individual

glycanases to liberate uniform degradation products and therefore decreases the enzymatic

degradability of polysaccharides. Chemical de-acetylation of aspen wood and wheat straw

has been shown to increase their enzymatic degradation. Xylan became 5–7 fold more

digestible which in turn improved significantly the accessibility of cellulose to enzymatic

hydrolysis (33). The effect of acetyl side groups on the enzymatic hydrolysis of

hemicelluloses was first demonstrated by Biely and co-workers (34).

16
2.2.2 Chemical Composition and Sources of Hemicelluloses Subtypes

2.2.2.1 Xylans

The term “xylan” is a catchall for polysaccharides that have a β-(1→4)-D-xylopyranose

backbone with a variety of sidechains. Xylan is the predominant hemicellulose in most plant

cell walls, generally comprising about 1/3 of the total plant biomass (35). Xylan is found

primarily in the secondary cell wall and seems to accumulate on the surface of the cellulose

microfibril as the cellulose synthase complex extrudes the microfibril (36). This deposition

continues as the additional cell wall material is laid down, resulting in heavier xylan

deposition on the cellulose microfibrils in the outermost layers of the secondary cell wall.

Xylans function primarily by forming cross-links between the other cell wall components,

such as cellulose, lignin, other hemicelluloses, and pectin. This interaction is carried out by

hydrogen bonding to the other polysaccharides and by covalent linkages through the

arabinofuranosyl sidechains to the ferulic and coumaric acids found in lignin.

The composition and linkages of the sidechains determines the specific variety of xylan. The

sidechains, usually attached at the O2 and/or O3 positions, include glucuronic acid, 4-O-

methylglucuronic acid, L-arabinofuranose, xylose, and acetyl groups. Removal of these side

chains generally enhances the rate of degradation by endoxylanase enzymes (37). The types

and levels of sidechains are dependent on the particular plant. Due to these differences in

sidechains, xylans from grasses and annuals having an elevated level of α-L-

arabinofuranoside substituents are referred to as arabinoxylans, while hardwood xylans,

highly substituted with 4-O-methyl glucuronic acid, are termed glucuronoxylans. Hardwood

glucuronoxylan, typically comprising 15-30% of the biomass dry weight, has high acetyl and

glucuronic acid moieties. 4-O-methylglucuronic acid is linked to the xylan backbone by α-

(1→2) glycosidic bonds and the acetic acid is esterified at the carbon 2 and/or 3 hydroxyl
17
group. The molar ratio of xylose:glucuronic acid:acetyl residues is about 10:1:7 (38). Side

chains on grasses and other annual plant xylans, also typically 15-30% of the plant cell dry

weight, are mainly arabinofuranose and acetyl groups and are even more heterogeneous than

those from woody tissues. The L-arabinofuranoside branches are linked α-(1→2,3) to the

xylopyranose backbone. The ester-linked acetyl groups are also attached to the C2 or C3

hydroxyl.

In contrast with hardwoods and annual plants, softwood hemicellulose is dominated by

galactoglucomannan (15-20% dry weight), with xylans comprising only 7-10% of the

biomass dry weight. The galactoglucomannans are divided into two subtypes of low and high

galactose content with galactose:glucose:mannose ratios of 0.1:1:4 and 1:1:3 respectively

(38). Softwood xylans, arabino-4-O-methylglucuronoxylans, are not acetylated, but the xylan

backbone is substituted at carbon 2 and 3 with 4-O-methyl-α-D-glucuronic acid and α-L-

arabinofuranosyl residues, respectively. Softwood xylan has a xylose:glucuronic

acid:arabinose ratio of approximately 8:1.6:1 (39) to 10:2:1 (38). Softwood xylan also has a

lower DP of about 100 compared to hardwood xylans with DP around 200 (40)

2.2.2.2 Mannans and mannan derivatives

The term mannan indicates a linear polymer of β-(1→4)-linked mannopyranosyl residues.

The structure, and hence the degradation, of mannan is very analogous to cellulose, both

being linear β-(1→4) linked monosaccharide polymers (41). Mannan, however, is found in

only a few particular plants, notably in the endosperm of the ivory-nut from the Tagua Palm

(Phytelephas macrocarpa) and a few other plants (41). The mannan polymer can be branched

however, with various combination of mainly glucose and galactose residues, giving rise to

much more common glucomannans, galactomannans, and galactoglucomannans.

18
Galactomannan is composed of a polymeric β-(1→4) mannopyranosyl backbone highly

substituted with β-(1→6) linked galactopyranose residues (42, 43). The degree of

substitution varies with source. Areas of galactose-free mannosyl residues can form junction

zones through inter-chain hydrogen bonding where there are six or more unsubstituted

mannose residues. The number of free mannose regions and degree of overall substitution

determines properties such as viscosity and solubility. More highly substituted

galactomannans tend to have high degrees of solubility and form more viscous solutions.

Galactomannans are generally believed to be an extracellular non-amylose form of

carbohydrate storage found predominantly, if not exclusively, in seeds (44). Found mainly in

the legume family, there are two major types of galactomannan, differentiated by the source

and degrees of galactose substitution. Locust bean gum (LBG), derived from the carob tree

(Ceratonia siliqua), contains an average of 2000 sugar residues, with a galactose about every

3.5-mannosyl residues. The other major commercial source is guar gum, from the seed of the

leguminous shrub Cyamopsis tetragonoloba. Guar gum contains more galactose residues

than LBG, having a galactose every 1.5 to 2 mannose units, and is longer than LBG, with

residue counts of around 10,000. The galactomannans form a set of polysaccharides called

gums that include other non-hemicellulosic polysaccharides such as carrageenan (alternating

β-D- and α-D-galactoses from some seaweeds) and xanthan gum (produced by the bacterium

Xanthomonas campestris). Carrageenans are subdivided into λ-, ι-, and κ-carrageenans based

on the sulfonation and 3,6 anhydro bridges present on the individual monomers. The linkages

alter between α-(1→3) and β-(1→4).

Glucomannan is a storage polysaccharide found mainly in the root of the Konjac plant

(Amorphophallous konjac). It consists of a β-(1→4) linked mannopyranose and

glucopyranose backbone in a ration of 1.6:1 (45). The backbone residues are substituted in a

19
β-(1→3) linkage with several sugars and short oligosaccharides, as well as with O-linked

acetyl groups about every 15 residues (46, 47). Hardwoods contain low levels of

glucomannan, with a glucose to mannan ratio of 1:1 to 1:2 (38). Although most grasses do

not contain glucomannan in the mature tissue, ramie has been shown to contain approximately

7.5% glucomannan in the whole plant (48). Other plants found to contain glucomannan

include aspen, lily, dates, western hemlock, lodge pole pine, and spruce (47, 49-52).

Galactoglucomannan is a hemicellulose found mainly in softwoods, although they have also

been described from fern, with fern galactoglucomannan having a lower DP than softwood

galactoglucomannan (53). Structurally, galactoglucomannan is comprised of β-(1→4)-linked

β-D-glucopyranosyl and β-D-mannopyranosyl units which are partially substituted by α-D-

galactopyranosyl and acetyl groups (40). Two types of galactoglucomannans can be

separated; water and alkali soluble fractions, with ratios of mannose:glucose:galactose:acetyl

residues 3:1:1:0.24 for the water soluble fraction and 3:1:0.1:0.24 for the alkali soluble

fraction (54).

2.2.2.3 Glucans

In addition to cellulose, the dominant polysaccharide in plant biomass, two other β-glucans

play important roles in plant cell wall structure and function. β-glucan and xyloglucan are

structurally similar to cellulose, being based on a β-linked glucose backbone. β-glucan

consists of mixtures of β-(1→3) and β-(1→4)-linked glucose residues while xyloglucan is a

straight β-(1→4) glucopyranose polymer with varying degrees of α-(1→6)-linked xylose

residues. Both structures are involved in support and cross-linking of the cellulose matrix

through hydrogen bonding interactions with cellulose, other hemicelluloses, and pectins.

Xyloglucan is believed to be extensively involved in support of the cell wall through

hydrogen bonding interactions with the cellulose (55, 56). These hydrogen bonds are
20
extended to multiple cellulose microfibrils, giving strong support between these cellulose

bundles and forming what is referred to as the cellulose/xyloglucan network (57). Rapid

freezing, deep-etching electron microscopy evidence indicates that the cellulose-xyloglucan

interactions extend beyond surface interactions to include embedding of the xyloglucan into

the cellulose microfibril (56). This is supported by the hydrolysis and extraction work of

Pauly and co-workers (58). Inclusion of xyloglucan chains in the media during cellulose

formation by Acetobacter aceti spp. xylinum has also shown that the interaction of xyloglucan

with cellulose extends into the microfibril(59). This interaction is believed to play a part in

regulating cell wall expansion, mainly through weakening of the rigid structure of the

cellulose microfibrils, allowing flexibility, and modification of these cross-links by specific

enzymes (55, 58).

Xyloglucans are polysaccharide polymers composed of a linear backbone of β-(1→4) linked

glucopyranose moieties with some monomers substituted with xylopyranose in an α-(1→6)

linkage. In this aspect, they are very similar to cellulose. The difference arises due to the

substitution that occurs on some of the glucose residues in the backbone. In xyloglucan, some

of these glucosyl residues are substituted with xylopyranose in an α-(1Æ6) linkage. The two

predominant patterns that occur are based in a repeated tetramer, either three sequential

substituted glucoses followed by a single unsubstituted glucose, or a pair of substituted

glucoses followed by a pair of unsubstituted glucoses. These patterns are referred to as

XXXG and XXGG respectively (60). The xylose side chains can in turn be substituted with

one or more of the following disaccharides: α-(1→2)-L-fucosylpyranose-β-(1→2)-D-

galactopyranose or α-(1→2)-L-galactopyranose-β-(1→2)-D-galactopyranose, with the fucose

residues being found mainly in primary cell wall (57, 61-65). As the linkage goes through a

galactose α-linked to the glucose, these are designated by an L in the pattern designation,

21
such as XXLG, where glucoses 1 and 2 are substituted with xylose, glucose 3 is linked to a

galactose, and glucose 4 is unsubstituted(66). α-(1→2)-L-arabinofuranose has also been

shown to be substituted onto either the main glucose chain or onto the galactose or xylose side

groups (57, 66, 67). In solanaceous plants, such as tobacco, tomato, and potato, the

xyloglucan has a high degree of arabinosyl residues attached to up to 60% of the xylose

sidechains in an O-2 linkage (57). These polymers are referred to as arabinoxyloglucans. It

has been shown that xyloglucans are acetylated through O-linkages to the arabinose or

galactosyl sidechains (57, 64, 67, 68). Despite this side chain substitution, a specific

acetylxyloglucan esterase has not been discovered.

Xyloglucan is found predominantly in dicotyledons, although non-graminaceous

monocotyledons such as onion also contain these polysaccharides (58). In mature wood

from hybrid poplar, xyloglucan comprises approximately 3% of the cell wall dry weight,

reduced from the 6% found in the early primary cell wall (69). In cocoa, xyloglucan makes

up 8% of the dry weight of cell wall material (70), while pea stems contain 21%

xyloglucan(58). Corn coleoptiles (3 days) contain 2.3 and 6.7% xyloglucan in epidermal and

mesophyll cells respectively(71).

β-Glucan is a glucopyranose polymer containing either β-(1→3) or mixed β-(1→3), β- (1→4)

linkages. This polymer is found in plant cell walls under a variety of conditions and in

several specific developmental stages. The ratio of (1→4) to (1→3) linkages varies by

species and gives specific properties to individual β-glucan polymers. In maize coleoptiles,

the mixed β-(1→3), β−(1→4) glucan is deposited early in cell development, comprising

greater than 70% of the cell wall material in the endosperm, and decreases rapidly after day 5

(72). The second type of plant β-Glucan consists of callose, a β-(1→3) linked glucose

polymer found primarily in rapidly growing structures such as pollen tubes and developing

22
seeds (73-77). Additional non-plant β-(1→3) forms include curdlan, produced by the

bacterium Agrobacterium sp. ATCC 31750 (formerly Alcaligenes faecalis subsp.

myxogenes)(78), and krestin, from the fungus Coriola versicolor (79). A subset of the β-

(1→3) glucans include β-(1→6) branch points, and while there are multiple forms of this

subset produced by fungi, the primary plant produced form is laminarin, produced by a brown

algae Laminaria digitata (80).

3 HEMICELLULOSE AND CELLULOSE HYDROLYSIS FOR BIOMASS

CONVERSION

3.1 Overview Of Conversion Chemistry

The cellulose and hemicellulose in lignocellulosic biomass can be hydrolyzed to sugars that

can be microbially fermented into various products such as ethanol or chemically converted

into other products (81-90). In such a case, cellulose is broken down to glucose, the same

sugar released by hydrolysis of starch. Thus, the same fermentation and chemical conversion

processes as now being used to manufacture products such as ethanol, lactic acid, and lysine

from starch glucose can be applied to manufacturing products from the glucose released by

hydrolysis of cellulose. The primary challenge is that the glucose in cellulose is joined by

beta bonds in a crystalline structure that is far more difficult to depolymerize than the alpha

bonds in amorphous starch (91). On the other hand, hemicellulose is an amorphous polymer

that is more easily hydrolyzed into its component sugars than is cellulose. However,

hemicellulose is typically made up of five different sugars - arabinose, galactose, glucose,

mannose, and xylose – as well as other components such as acetic, glucuronic, and ferulic

acids. Native organisms do not efficiently ferment this range of sugars to products, and the

heterogeneity of the mixture can impact product purity for chemical conversion routes. As
23
presented previously, the remaining 20 to 30% or so of cellulosic biomass is often mostly

lignin, a phenyl-propene aromatic compound that cannot be fermented but can be used as a

high energy content boiler fuel. In addition, cellulosics contain protein, minerals, oils, and

other components with the amount varying with species. Ideally, each of these fractions can

be utilized to make fuels, chemicals, food, and feed (92).

3.2 Typical Process Steps

There are many possible routes to hydrolysis of cellulose and hemicellulose to sugars, but we

will focus on a coupled acid and enzymatic hydrolysis approach considered by many to be

most ready for near term applications (81-85, 87-89, 92). Figure 5 presents a simplified

process flow diagram for making ethanol from hemicellulose and cellulose sugars released

during the pretreatment and enzymatic hydrolysis steps, respectively, based on this approach.

The process starts with feedstock transportation to the process facility for possible storage at

the site or immediate feeding to the process. In either event, feedstock is then moved into the

process using suitable conveying equipment coupled with cleaning operations to remove

foreign objects such as rocks and dirt that could jam or otherwise interfere with process

operations. The storage, conveying, and cleaning operations are important steps, and

experience with these operations is vital to insure successful operations. Next, the material is

pressurized for feeding to pretreatment reactors capable of achieving the high solids

concentrations that are vital to minimizing energy (steam) use and maximizing sugar

concentrations, and acid and steam are added to promote the hemicellulose hydrolysis to its

component sugars. It is generally desired to limit acid use to less than about 1% in the

hemicellulose hydrolysis reactors to minimize acid use as well as subsequent neutralization

and disposal costs, with actual conditions determined by tradeoffs among pretreatment time,

temperature, and acid use with some variability in the best conditions among feedstocks. Up
24
to about 80 to 90% of the hemicellulose sugars can be recovered in a an optimized

pretreatment operation (93-95).

Following pretreatment, the reactor contents are usually rapidly cooled by flashing to a lower

pressure to stop sugar degradation reactions and maximize sugar yields. The solids

containing most of the cellulose and lignin in the feed are washed in special equipment to

recover nearly all the sugars released from the hemicellulose in a liquid stream that is then

conditioned with lime to neutralize the pH and remove fermentation inhibitors, and ion

exchange is applied, if necessary, to remove acetic acid and other components that can retard

downstream operations.

Depending on enzyme loading requirements and enzyme production yields, about 4 to 9% of

the washed solids and possibly some liquid from the hemicellulose sugar recovery and

conditioning steps are used to grow an organism such as Trichoderma reesei that can make

enzymes known as cellulase that can be added back to the bulk of the pretreated solids to

hydrolyze cellulose to glucose. Many operations are currently based on adding the cellulase

to the pretreated solids along with the conditioned hemicellulose sugar stream and fermenting

the resultant sugar stream with an organism such as genetically engineered E. coli that

ferments all of the five sugars in hemicellulose and cellulose to ethanol with the high yields

vital to commercial success. In this so-called simultaneous saccharification and co-

fermentation or SSCF process configuration, the glucose released by enzymatic hydrolysis of

cellulose is converted into ethanol as rapidly as it is released. Ethanol is recovered at

appropriate purity from the fermentation broth by a combination of distillation and

dehydration operations. The bottoms from the distillation column contain unreacted

cellulose, lignin, cellulase, fermentative organisms, and other residual ingredients as well as

water, and the solids are recovered and burned to generate all the heat and electricity needed

25
for the conversion process with excess electricity sold for extra revenue. A portion of the

water is recycled to the process while the rest passes to waste water treatment for proper

disposal. Methane gas produced during anaerobic treatment is also fed to the boiler to

generate heat and electricity.

The SSCF configuration is favored to reduce equipment costs and the powerful inhibition of

cellulase activity by glucose and particularly cellobiose. Adding cellulase, pretreated

cellulose, and fermentative organisms in the SSF configuration has been clearly demonstrated

to be favorable for reducing ethanol production costs (82). The low concentration of free

glucose and presence of ethanol also make it more difficult for invading microorganisms to

takeover the fermentation reaction and form undesirable products. Although SSCF has

similar potential to improve fermentation of cellulosic sugars to products other than ethanol,

most of these other products are less volatile than water, and separation of the product from

the more complex fermentation broth at the completion of fermentation is more challenging.

In addition, other combinations of pretreatment and hydrolysis reactions can be applied to

release sugars from cellulose and hemicellulose fractions, as described in other sections of

this chapter.

3.3 Cellulose and hemicellulose hydrolysis reactions

Cellulose is a long chain of glucose molecules covalently joined by beta bonds with many of

the neighboring chains typically linked together through hydrogen bonding in a crystalline

structure (96). Acids or enzymes known as cellulases can catalyze the reaction of water with

the glucose molecules in these chains to release single glucose molecules – monomers – by

the following reaction:

(C 6 H 10 O5 ) n + n H 2 O → n C 6 H 12 O6 (3.1)

26
in which H2O is water, (C6H10O5)n is a chain made up of n glucose molecules that is often

termed glucan, and C6H12O6 is a glucose monomer. Thus, each glucose unit in the long chain

combines with a water molecule, and 180 mass units of glucose are released from 162 mass

units of glucan and 18 mass units of water, an 11.1% mass gain. Oligomers made up of

several glucose molecules may also be released as intermediates in cellulose hydrolysis and

often contain only 2 to perhaps 3 glucose units. Cellulase enzymes are very specific in only

catalyzing addition of water to glucan chains, and the optimum temperature needed for

reaction 3.1 is only about 50oC, virtually eliminating degradation reactions. Thus, only

glucose is formed via enzymatically driven hydrolysis of cellulose, and yields can approach

100%. On the other hand, use of dilute acids (e.g., 1.0% sulfuric acid) to drive reaction 3.1

requires much higher temperatures of about 220oC, and the acid also triggers the breakdown

of glucose to degradation products such as hydroxymethyl furfural, limiting yields.

Concentrated acids (e.g., 75% sulfuric acid) can be used at moderate temperatures to achieve

high yields similar to those for cellulase enzymes.

Hemicellulose can also be hydrolyzed by the addition of water to release individual sugar

chains contained in the longer hemicellulose molecule. The stoichiometry for the reaction of

the hexose sugars galactose, glucose, and mannose that are in hemicellulose is the same as

shown in equation 3.1, and an 11.1% mass gain results for these molecules. On the other

hand, addition of water to the five-carbon sugar molecules arabinan and xylan in the

hemicellulose molecule follows the following stoichiometry:

( C 5 H 8 O4 ) n + H 2 O → n C 5 H 10 O5 (3.2)

with (C5H8O4)n being a chain made up of n arabinose or xylose (pentose) molecules that can

be termed arabinan or xylan, respectively, and C5H10O5 being one of the corresponding

pentose sugars formed by hemicellulose hydrolysis. Based on this reaction, we can see that

27
the molecular weight of the sugar molecule released increases from 132 mass units before

hydrolysis to 150 mass units of pentose sugars formed, a gain of over 13.6%. Hemicellulose

is open to attack at intermediate positions along its long backbone with the release of

oligomers made up of many sugar molecules that can be successively broken down to shorter

chained oligomers before single sugar molecules are formed (97). A suitable cocktail of

enzymes known collectively as hemicellulase can catalyze addition of water to hemicellulose

with high specificity at modest temperatures, thus avoiding sugar degradation and resulting in

high sugar yields. Dilute acids (e.g., sulfuric) can also catalyze hemicellulose hydrolysis to

sugars at temperatures of about 100 to 200oC, but furfural and other degradation products are

formed from the sugars at these temperatures if one targets good yields of hemicellulose

sugars in solution. Nonetheless, degradation of the sugars released can be modest enough to

recover about 80 to 90% of the maximum possible sugars (93-95). On the other hand,

operation without adding acid limits recovery of hemicellulose sugars to about 65% or less,

with most in oligomeric form (98).

4 ACID HYDROLYSIS OF CELLULOSE

4.1 System Description and Performance

When heated to high temperatures with dilute sulfuric acid, long cellulose chains break down

to shorter groups of glucose molecules that then release glucose that can then degrade to

hydroxymethyl furfural (99-109). Generally, the majority of cellulose is in crystalline form,

and harsh reaction conditions (high temperatures, high acid concentrations) are needed to

liberate glucose from these tightly associated chains. Furthermore, it is found that the rate of

cellulose hydrolysis increases faster with increasing temperature than the rate of glucose

degradation does, and as a result, yields increase with temperature and acid concentration,
28
reaching about 70% at 260oC (107). However, pyrolysis and other side reactions become

very important at temperatures above about 220oC, and the amount of tars and other difficult

to handle byproducts formed increases as the temperature is raised above these levels (110).

Such materials can rapidly coat reactor walls and plug lines, limiting the practical temperature

range. In addition, reaction times to maximum glucose yields become very short at high

temperatures, being only about 6 seconds at about 250oC with 1% sulfuric acid, and

implementing commercially viable reactors that can accurately control such short times

presents a major challenge (111).

Various reactors have been tested over the years to release glucose from cellulose with dilute

acid. Older reactors, such as employed during the 1940s, tended to be large vessels that could

be heated through jacketed reactors. More recently, direct steam injection has been applied to

rapidly heat cellulose containing biomass to the high temperatures required (110, 112). Some

of these reactors have been tubular flow systems with steam addition to quickly heat biomass

slurries, but rapid build up of tars limited their use (110). Others have used twin screw

extruders to promote mixing and control vessel residence times (113), but such equipment is

very expensive and not easily scaled to the high throughputs needed for commercial

operations. Overall, yields of about 50 to 60% of the glucose in cellulose can be recovered

from with temperatures of around 220oC in typical co-current or batch reactors (111).

However, recent results with flow of liquid through solids that are compressed throughout the

reaction to minimize void volume show close to theoretical yields of glucose from acid

hydrolysis of cellulose in pretreated yellow poplar wood (114). Unfortunately, no

commercially viable equipment has yet been demonstrated to duplicate this performance.

29
4.2 Kinetic Models

The dilute acid hydrolysis of cellulose is typically pictured as addition of a hydrogen ion to

form a conjugated acid, leading to cleavage of a glycosidic bond as a water molecule is added

to release both glucose or short chains of glucose molecules (glucose oligomers) and an H+

ion. In this sense, the hydrogen ion acts as a catalyst that facilitates hydrolysis without net

consumption of these species. The oligomers can further hydrolyze to form glucose.

Amorphous portions of cellulose and oligomers break down very rapidly to form glucose

monomers while crystalline regions of cellulose react much more slowly. Unfortunately, the

severe conditions needed to hydrolyze cellulose, and particularly crystalline cellulose, also

degrade glucose to products such as hydroxymethyl furfural (HMF), levulinic acid, and

formic acid. Thus, recovery of glucose requires trading off conditions to maximize cellulose

hydrolysis against degradation of glucose (106). An examination of the subsequent glucose

degradation reactions was provided by McKibbons and co-workers (115).

Most cellulose hydrolysis models are based on those developed initially by Saeman in which

dilute acid hydrolysis of Douglas fir in a batch reactor with 0.4% acid was described as a

sequence of consecutive homogeneous first order reactions (103). In this case, cellulose is

hydrolyzed to glucose, which in turn breaks down to degradation products according to the

following series reaction:

k1 k2
Cellulose + Glucose Degradation products (4.1)

Furthermore, because water is present in excess, the rate expressions for each of the steps

represented by the arrows can be described as:

rC = − k1 C (4.2)

30
 rG = k1 C − k 2 G (4.3)

in which rC represents the reaction rate for cellulose, rG is the reaction rate for glucose, C is

the mass of cellulose, G is the mass of glucose, and k1 and k2 are reaction rate constants for

cellulose and glucose, respectively. The rate constants in equation (1) and (2) are typically

assumed to follow an Arrhenius temperature dependence of the form:

k i = k i 0 A mi e − Ei / RT ( 4 .4 )

in which kio is a constant, A is the acid concentration in weight percent, mi has a value close to

1.0, Ei is the activation energy, R is the universal gas constant, and T is the absolute

temperature. For batch systems, the yield of glucose, the focus of many studies, is

determined by incorporating equations 4.2 and 4.3 into time dependent mass balances and

integrating the expressions to arrive at the following relationship:

k1
G = C0 [ ] (e − k 2 t − e − k1 t ) + G0 e − k 2 t (4.5)
k1 − k 2

in which G is the mass of glucose at any time t and C0 and G0 are the initial quantities of

cellulose (expressed as equivalent mass units of glucose) and glucose, respectively.

The rate constants for these equations are usually fit to experimental data using a least squares

method, and more than 10 studies were reported between 1945 and 1990 that applied Saeman

kinetics to follow cellulose hydrolysis data of various cellulosic materials in plug flow or

batch reactors (103, 107, 110-112, 116-122). All models predict that the glucose yield

increases with increasing acid concentration and temperature, but no model predicts yields

greater than 70% of theoretical for the range of conditions examined.

Although these models provide a useful indication of the tradeoffs in converting cellulose to

glucose and help define optimum conditions and operating strategies, various researchers

have tried to enhance the models to more fully describe hydrolysis. For example, Conner and
31
co-workers (123) incorporated reaction of two fractions of cellulose, one more easily

hydrolyzed that the other, as well as reaction of glucose to an array of products, some of

which can reversibly form glucose, according to the following scheme:

Easily hydrolyzed Levoglucosan

cellulose

Glucose (4.6)
Degradation Products

Resistant cellulose Disaccharides Glucosides

This approach improved the fit of experimental data to the model curves.

Because the conversion of oligomers to glucose is 2 to 3 times faster than hydrolysis of

cellulose to soluble oligomers, oligomer formation was not initially recognized (124), but

Abatzoglou and co-workers (122) found oligo-derivatives during hydrolysis of alpha cellulose

in a cascade reactor with 0.2 to 1.0 wt % H2SO4, with particularly significant amounts in the

early stages of hydrolysis. To include this result, the Saeman model (103) was extended:

αk1 βk1 k2
Cellulose Oligoderivatives Degradation Glucose (4.7)
γk1

Based on this mechanism, consideration was given to three possibilities: 1) the

oligoderivatives to glucose reaction is in equilibrium, 2) the oligoderivatives to glucose

reaction is not in equilibrium, and 3) there are no repolymerization reactions (γ= 0). The third

model proved to best represent experimental data. These results led Abatzoglou and co-

workers to suggest using a two-step process for optimal glucose yield in which the reaction of

32
cellulose to oligomer is catalyzed in a first stage followed by the oligomer to glucose reaction

in a second stage under milder conditions (125). This approach would reduce formation of

degradation products.

Bouchard and co-workers (126) applied thermogravimetric analysis, differential scanning

calorimetry, and diffuse reflectance infrared to show that the chemical structure of cellulose

remaining after hydrolysis in a plug flow reactor changed significantly. Using a semi-batch

flowthrough system, Mok and Antal (127) concluded that an acid-catalyzed parasitic pathway

competes with acid-catalyzed hydrolysis to produce cellulose or non-hydrolyzable oligomers

that cannot be hydrolyzed to glucose but that there is no significant chemical change in the

cellulose itself. Based on these studies, the path for reaction of cellulose to non-reactive

modified cellulose and insoluble oligomers that parallels production of reactive soluble

oligosaccharides along with degradation of glucose limit glucose yields to less than 70%, as

suggested by the following reaction pathway:

Modified Cellulose
Insoluble Oligomers
Cellulose Glucose Further Reactions (4.8)

Soluble Oligosaccharides

In addition, this model suggests that high severity processes induce structural rearrangements

that affect the kinetic properties of the cellulose.

The oligomer formation model suggests that cellulose forms shorter chain length species prior

to forming monomeric glucose, as one would expect based on depolymerization models. It is

interesting to note that prior to Saeman reporting his first order kinetic model, Simha in 1941

presented depolymerization models based on random and non random scission of the long

polymer chains to progressively shorter oligomers and monomers (128). For the former, the

33
number and weight average molecular weight both drop rapidly and then tended to slow, with

the average degree of polymerization α given by:

α = 1 − exp ( − k t ) (4.9)

On the other hand, for preferred breaking of the ends of the chain, the weight average

molecular weight decreases much more slowly than the number average value because the

rapid formation of monomers contributes far less to the former. Cellulose hydrolysis was best

described by assuming a reaction behavior intermediate between these extremes with bonds

for molecules at the ends of a chain reacting twice as fast as those in the interior.

Unfortunately, little work has been reported on the application of such depolymerization

models to cellulose hydrolysis subsequent to these studies.

More recently, nearly theoretical yields of glucose from acid hydrolysis of cellulose in

pretreated yellow poplar wood have been reported for flow of liquid through solids that are

compressed by a spring to minimize their volume throughout the reaction in a “shrinking bed

percolation” device (114). The release of glucose varied depending on whether a batch,

percolation, or shrinking bed percolation reactor configuration was employed, with the latter

obtaining the highest yields. While biphasic kinetics were observed for the batch system at

lower hydrolysis severities, the initial hydrolysis rate constant was about five times greater for

the percolation reactor than for batch operation using 0.07% sulfuric acid at 225oC. Overall,

the data suggests that an intermediate step occurs between cellulose hydrolysis and glucose

release to the bulk fluid that is affected by the flow velocity. They also observed a flattening

of the glucose yield curves with increasing acid concentration. Diffusion resistance due to

boundary layer resistances resulting from structured water, viscosity, and re-hydrogen

bonding of released glucose were postulated to cause these changes in performance with flow.

34
5 ACID HYDROLYSIS OF HEMICELLULOSE

5.1 System Description and Performance

In simple terms, acid hydrolysis of hemicellulose is very similar to that for cellulose: acid

catalyzes the breakdown of long hemicellulose chains to form shorter chained oligomers and

then sugar monomers that the acid can degrade. However, because hemicellulose is

amorphous, less severe conditions are required to release hemicellulose sugars. Steam is

often directly injected into the biomass to rapidly heat it up, and the acetic acid contained in

the hemicellulose chains can provide hydrogen ions to promote hydrolysis. Yet, in this case,

hemicellulose sugar yields are limited to about 65% of the maximum possible (98). On the

other hand, adding dilute sulfuric or other acids results in much higher hemicellulose sugars

yields and more digestible cellulose at a relatively low cost (129, 130). Steady progress has

been made in refining the technology to remove hemicellulose with better yields and

improved cellulose digestibility, and the process has been demonstrated to be effective on a

variety of biomass feedstocks (e.g., (93-95, 131-133). For example, yields of about 85 to

90% of the sugars can be recovered from the hemicellulose fraction with temperatures around

160oC, reaction times of about 10 minutes, and acid levels of about 0.7%. In addition, about

85 to over 90% of the remaining solid cellulose can be enzymatically digested to produce

glucose. These high yields are vital to low costs (81-85, 87, 88) and have led many to favor

dilute sulfuric acid hydrolysis for near term applications (89).

Despite often being considered a frontrunner, dilute sulfuric acid hydrolysis is expensive,

representing about one third of the total cost of making ethanol in one study (Lynd et al,

1999). Its corrosive environment mandates use of expensive materials of construction (81-85,

87-89). In addition, reaction degradation products such as furfural and lignin fragments and

35
solublized biomass compounds such as acetic acid must be removed by over-liming, steam

stripping, or other processes before fermentation (134-137). Although lime is least expensive

for acid neutralization and hydrolysate conditioning, the gypsum formed has reverse solubility

characteristics that cause downstream difficulties (81-85, 87-89). Furthermore, even low cost

sulfuric acid and lime impact costs that mount when disposal is included. Additionally, about

a 7-day reaction time with expensive cellulase loadings of up to 20 IFPU/g cellulose is needed

to realize good yields in subsequent enzymatic hydrolysis. Finally, dilute acid pretreatment

can consume considerable process power for size reduction (81-85, 87-89). Thus, it is vital to

optimize dilute acid pretreatment in conjunction with downstream processing to minimize

these costs as much as possible. In line with this, flow through and counter-current flow

reactors have shown many desirable features for pretreatment by hemicellulose hydrolysis

including high yields of hemicellulose sugars and enhanced cellulose digestion with low or no

acid addition (138-141). However, the large volumes of water needed and difficult-to-

implement equipment configurations present important challenges that must be addressed if

the technology is to be applied commercially.

5.2 Kinetic Models

Overend and Chornet combined treatment temperature and time in a single severity parameter

for hemicellulose depolymerization by steam or aqueous pretreatment without acid addition,

defined as (142):

T − 100
RO = t * exp [ ] (5.9)
14.75

in which the time t is usually expressed in minutes and T is the temperature in degrees

Celsius. Xylan removal from hemicellulose solids follows a consistent pattern when plotted

against RO or log RO for a variety of feedstocks and temperatures, allowing one to readily

36
estimate tradeoffs among time and temperature. Although equation 5.9 would only be

expected to describe solids decomposition for first order kinetics, xylose recovery somewhat

coincidentally also tracks well for a wide range of conditions when plotted against RO, and it

has been shown that the log RO is equal to about 3.8 at the point of maximum hemicellulose

sugar yield for a variety of feedstocks and process systems. The severity parameter was

subsequently extended to include the effect of acid addition on hemicellulose solublization

and sugar recovery in a combined severity parameter for dilute acid hydrolysis defined as

(122):

T − 100
CO = [ H + ] t exp [ ] (5.10)
14.75

with [H+] being the hydrogen ion concentration and all other parameters being as defined for

equation 5.9. The severity parameter is similar to a ‘H’ factor relationship used in the context

of Kraft pulping processes (143) and is a valuable tool for both process control and predicting

yields.

Stepwise kinetic models have also been developed to describe hemicellulose hydrolysis, and

most can trace their origins to those applied by Saeman to describe cellulose hydrolysis in

1945 (103). Thus, they assume a homogeneous first order series reaction mechanism.

Furthermore, many of the models incorporate the further assumption that oligomers react so

fast to monomers that oligomers can be omitted from consideration (103, 144-146). For this

two step reaction, hemicellulose is hydrolyzed to xylose that in turn breaks down to

degradation products in a second reaction as follows:

k1 k2
Hemicellulose Xylose Degradation products (5.1)

37
The same rate expressions and integrated forms for batch hydrolysis apply as used to describe

cellulose hydrolysis in Section 4. As before, the rate constants ki are assumed to follow an

Arrhenius temperature relationship of the form

k i = k i 0 A mi e − Ei / RT (5.2)

in which ki0 is the pre-exponential factor, A is the concentration of acid by weight, mi is a

power, and Ei is the activation energy. The maximum yield predicted by this type of model

varies from 83 to 95% (116, 144, 146).

In 1955, Kobayashi and Sakai (147) divided hemicellulose into two arbitrary fractions with

one hydrolyzing more quickly than the other according to two distinct kinetic constants. This

approach was used to account for the decrease in reaction rate with conversion, and most

subsequent models of hemicellulose hydrolysis have been based on this reaction scheme, as

follows (e.g. (115, 124, 147-151):

Fast hydrolyzing kf
hemicellulose
k2
Xylose Degradation products (5.3)

Slow hydrolyzing ks
hemicellulose

The proportion of fast and slow fractions is determined by fitting to kinetic data and are

typically about 65 and 35% respectively, with only slight variations among materials studied.

Although ignored in the models above, oligomer intermediates are frequently observed

experimentally. For example, Kim and Lee measured increases in monomer yields if they

subjected the hydrolysate solution from batch hemicellulose hydrolysis to secondary acid

treatment, showing that dissolved oligomers are present (152). Oligomers are particularly

significant in flow systems such as hydrothermal pretreatment in which oligomers are

38
removed before they have time to form monomers, and the concentration of solublized

material could be more than tripled by re-circulation (153). In a reverse-flow, two

temperature reactor configuration, the fraction of dissolved xylose as monomers can be as low

as 31% (139). Recently, Li and coworkers applied liquid chromatography techniques to

measure a distribution of chain lengths for uncatalyzed hydrolysis of xylan that clearly shows

formation of long chained species followed by their decomposition to shorter chained

oligomers and then monomers (97). A few models include formation of oligomers as

intermediates in the following type of reaction sequence (124):

Fast hemicellulose kf
k1 k2
Oligomers Xylose Degradation products (5.4)

Slow hemicellulose ks

Others have included two groups of oligomers in the pathway for hydrolysis of hemicellulose

without addition of acid to recognize that the initial oligomers are of longer chain length than

those formed later (154). In this case, an intermediate oligomer formation step is added to the

sequence in equation 5.4 as follows:

k1 k2 k3 k4
X OH OL M D (5.5)

in which OH represents oligomers with a higher degree of polymerization and OL represents

those with a lower degree of polymerization. In a subsequent paper, a better fit to data was

reported when a step was added for direct degradation of low degree of polymerization

oligomers to furfural (155).

In addition to considering the nature of the reacting species, hemicellulose models have been

modified in an attempt to more accurately reflect reaction catalysis with several focused on

39
including the effect of neutralizing capacity of the substrate. In one, minerals in the substrate

were reported to neutralize up to 70% of the acid added (124). Procedures were developed to

calculate the molal hydrogen ion concentration, [H+], based on acid addition and the

neutralizing capacity of the substrate, and these values were incorporated into the rate

equation instead of the weight percent acid concentration typically used with all other terms

defined as previously (105):

[ ]
ki = kio * H +
mi
* e ( − Ei / RT ) (5.6)

In a similar manner, the effective acid concentration was determined from the neutralizing

capacity of the substrate and the amount and concentration of the applied acid assuming that

all substrate cations were immediately available and effective to give (123):

[H+] = molality of added acid - molality of the cations (5.7)

Another paper concluded that the weight percent acid concentration do not account for the

nonlinearity of the hydrogen ion concentration with increasing acid addition or consider the

effect of substrate neutralizing capacity and used pH in the rate constants as follows (117):

ki = kio * e ( − Ei / RT − 2.303mi ( pH )) (5.8)

Such an approach could explain the range of yield curves observed when comparing models

using only the applied acid concentration.

Although significant effort has been devoted to describing the kinetics of hemicellulose

hydrolysis, the models do not predict consistent results. For example, the kinetic constants fit

to describe overall hemicellulose dissolution and sugar recovery differ from those determined

for breakdown of pure xylose by acid, and models from different studies project different

hemicellulose removal rates and yields (156). In addition, yields of monomers and oligomers

increase when more water is added for hydrolysis with just water, a finding that is

inconsistent with first order kinetics or the assumption that acetic acid released during
40
hydrolysis catalyzes the reaction (157-161). Pushing liquid through biomass accelerates

removal of hemicellulose with flow rate, again inconsistent with conventional first order

kinetic models that only show an effect of acid concentration or temperature (140, 141, 162-

164). Furthermore, treating the hydrolysis of hemicellulose solids to dissolved sugars as a

homogeneous reaction is counter to the basic nature of this solid/liquid system, and many

questions arise about the models used to date. For example, could the biphasic kinetic really

be attributed to mass transfer limitations that affect release of sugars into solution (165).

Oligomers appear to be important in distinguishing the performance of various process

configurations for breakdown of hemicellulose to sugars and also could help explain the

deviations in model predictions. Could oligomer solubility in the particulate biomass affect

hydrolysis rates? Is the binding of xylan to lignin important? Answering these questions and

clarification of hemicellulose hydrolysis should provide insight that can be valuable to more

confident applications of the technology and also to defining advanced pretreatment

approaches.

6 CELLULASE SYSTEMS FOR CELLULASE HYDROLYSIS

6.1 Sources of Cellulase Enzymes

6.1.1 Fungal Enzyme Systems

More than 100 cellulolytic fungi have been reported to date and this number is continually

increasing (166-168). Aerobic fungi (hyphomycetes, ascomycetes, and basidiomycetes) are

represented in this grouping. Some members of these groups can also modify and degrade

lignin and cellulose in wood samples. The fungal cellulase system was initially interpreted

largely in terms of substantial biochemical and molecular biological developments for the

41
Trichoderma reesei system. In many ways, this system was the developmental archetype

cellulase system. Many reviews have adequately described the 20-plus years of systematic

research conducted at the Army Natick Laboratory on this subject (169). Much of the

subsequent research in this field focused on mutation/selection of better T. reesei strains for

enzyme commercialization, including biomass conversion.

It is also clear that anaerobic as well as aerobic environments produce important and

functionally intriguing fungal cellulase producers (170). Common to the rumen are anaerobic

fungi as well as bacteria that produce enzymes capable of degrading the cell walls of plants

(171). The best studied anaerobic cellulase producers to date are probably Orpinomyces sp.

(172), Piromyces sp. (173), and Neocallimastix sp. (174). The molecular arrangement of the

fungal cellulosome; however, remains unknown, and some predict that it may differ from

bacterial cellulosome in many aspects (175-178).

Today, many researchers are working to better understand the role fungal hydrolytic enzymes

play in the natural process of plant cell wall degradation (179). Aiding in this process is the

application of new tools for direct analysis of biomass, including improved methods for using

electron microscopy (180) and near field scanning optical microscopy (NSOM) as probes of

enzyme action.

6.1.2 Bacterial Enzyme Systems

Bacterial cellulase systems include those produced by aerobic and anaerobic bacteria and

actinomycetes and are the focus of considerable study. It is probable that more diversity in

protein folds and structure/function relationships will eventually be found in the bacterial

paradigm (168). Coughlan and Ljungdahl have extensively reviewed bacterial cellulases

(181), identifying 46 unique bacterial producers of cellulases. More recently, Lynd and

coworkers (182) have reviewed the concept of exploiting C. thermocellum and related
42
microorganisms for "consolidated bioprocessing" (CBP), in which the production of

cellulolytic enzymes, hydrolysis of biomass, and fermentation of resulting sugars to desired

products occur in one step. Perhaps the most studied actinomycete cellulase producer,

Thermobifida fusca, has been shown to harbor cellulases from many glycosyl hydrolase

families, including families 1, 5, 6, 9, 48, and 74 (183-185). It is noteworthy that to date no

bacterial cellulase system includes a cellobiohydrolase from family 7. In general, the

cellulase systems produced by actinomycetes are not associated with cellulosome-like

structures; however, this question is still debated.

In the early 1980s, Bayer and Lamed (186-192) first described a multi-enzyme complex in the

anaerobic, thermophilic, cellulolytic bacterium, Clostridium thermocellum, that was termed

the cellulosome. Using a variety of biochemical approaches, negative staining techniques,

transmission and scanning electron microscopy, the cellulosome in this bacterium was found

to be packaged into protuberance-like cell-surface organelles that are released into the

medium in the latter stages of growth. The molecular mass of the cellulosome complex was

determined to be in the region of several MDa, and isolated cellulosome complexes were

found to be approximately 18 nm in size (192). The composition of a given cellulosome

varies depending on the substrate that apparently regulates enzyme expression. Cellulosomes

can be found on the cell surface and in the extracellular growth medium. The cellulosome has

been described as the principle mechanism by which some anaerobic cellulolytic

microorganisms achieve efficient breakdown of the recalcitrant polysaccharides present in

plant cells. The cellulosome is believed to be more efficient than the free enzyme system

because it “collects” and “positions” enzymes onto the substrate surface. The cellulosome has

been found in numerous cellulolytic, anaerobic microorganisms including clostridia,

ruminococci, and anaerobic fungi (186-191, 193). In the past two decades, molecular

biological approaches (genomics) have been employed to find new cellulosomal genes, an
43
activity that has provided information needed for our collective understanding of the

structure/function relationships of the cellulosome.

Extensive review of the bacterial cellulosome can be found in the literature (194-200). Two

types of subunits have been identified from the bacterial cellulosome complex. Non-catalytic

subunits, called “scaffoldins”, serve to position and organize the other subunits and to attach

the cellulosome to the cell surface and/or to the substrate. The other type of subunit is

represented by the enzymes, which hydrolyze the plant cell wall polysaccharides (201-203).

Based on sequence analysis and directed biochemical evidence, at least three types of protein

domains are involved in the function and assembly of the cellulosome. Catalytic domains

(enzymes) carry out substrate hydrolysis. Binding domains function to provide binding

capacity to the substrate or the cell surface. For example, the carbohydrate-binding module

(CBM) is responsible for mediating the binding of the scaffoldin to carbohydrate substrate

(203). An S-layer homologous (SLH) domain mediates the association of the cellulosome to

the cell-surface (201). Recognition domains, called cohesin and dockerin, are known as

receptor/adaptor pairs. The specific interaction between cohesin and dockerin forms the

positional self-assembly of the cellulosome.

6.2 General Classification of Cellulase Enzymes

6.2.1 Endoglucanases

The "endo-1,4-β-glucanases" or 1,4-β-D-glucan 4-glucanohydrolases (EC 3.2.1.4), which act

randomly on soluble and insoluble 1,4-β-glucan substrates, are commonly measured by

detecting the decrease in viscosity or reducing groups released from carboxymethylcellulose

(CMC) (204).

44
6.2.2 Exoglucanases

The "exo-1,4-β-D-glucanases," include both the 1,4-β-D-glucan glucohydrolases (EC

3.2.1.74), which liberate D-glucose from 1,4-β-D-glucans and hydrolyze D-cellobiose slowly,

and 1,4-β-D-glucan cellobiohydrolase (EC 3.2.1.91), which liberates D-cellobiose from 1,4-β-

glucans. Differentiation of these enzyme classes requires analytical techniques to distinguish

glucose and cellobiose and is usually carried out by HPLC or GC. These enzymes can be

further distinguished by their ability to liberate free sugars from either the reducing or non-

reducing end of the cellulose chain (205). Determination of which preference a given

enzyme has is usually carried out through synergy studies with enzymes of known orientation

(206-208).

6.2.3 Beta-β-Glucosidase

The ”β-D-glucosidases” or β-D-glucoside glucohydrolases (EC 3.2.1.21) act to release

D-glucose units from cellobiose and soluble cellodextrins, as well as an array of glycosides.

Measurement of this activity is carried out either specifically on cellobiose or cello-oligomers

with product analysis by HPLC or GC, or by direct spectrophotometric or fluorometric

analysis of various chromogenic and fluorogenic analogues of cellobiose and cello-oligomers.

6.3 Cellulase Structure and Function

6.3.1 Synergism

Gilligan and Reese (209) first showed that the amount of reducing sugar released from

cellulose by the combined fractions of fungal culture filtrate was greater than the sum of the

amounts released by the individual fractions. Since that initial report, many investigators
45
have used a variety of fungal preparations to demonstrate a synergistic interaction between

homologous exo- and endo-acting cellulase components (165, 169, 209-211). Cross-

synergism between endo- and exo-acting enzymes from filtrates of different aerobic fungi has

also been demonstrated many times (211-214).

Exo-exo synergism was first reported in 1980 by Fägerstam and Pettersson (215). As shown

in this drawing, exo-endo synergism is explained best in terms of providing new sites of

attack for the exoglucanases. These enzymes normally find available cellodextrin "ends" at

the reducing and non-reducing termini of cellulose microfibrils. Random internal cleavage of

surface cellulose chains by endoglucanases provides numerous additional sites for attack by

cellobiohydrolases. Therefore, each hydrolytic event by an endoglucanase yields both a new

reducing and a new non-reducing site. Thus, logical consideration of catalyst efficiency

dictates the presence of exoglucanases specific for reducing termini and non-reducing termini.

Indeed, recent X-ray crystallographic work reported by Teeri and co-workers (216) confirms

that the reducing terminus of a cellodextrin can be shown in proximal orientation to the active

site tunnel; i.e., reducing end in first, of T. reesei CBH I. Claeyssens and coworkers

confirmed from kinetic data that T. reesei CBH II preferred the non-reducing terminus of the

cellulose chain (217).

Baker and co-workers (212) recently reported a standardized, comparative study that

measured glucose release and synergistic effects in the solublization of microcrystalline

cellulose by binary mixtures of 11 fungal and bacterial cellulases (eight endoglucanases and

three exoglucanases). Evaluation of 16 endo/exo pairs revealed that bacterial/fungal hybrid

pairs are very effective in solublizing microcrystalline cellulose. Of nine bacterial/fungal

hybrid pairs studied, six were ranked among the nine most synergistic combinations, and six

bacterial/fungal pairs were also among the top nine pairs in terms of soluble-sugar release.

One hybrid pair (Acidothermus cellulolyticus El and T. reesei cel7A) was ranked first in both
46
synergism and sugar-release. In exo/exo synergism experiments, the performance of

Thermomonaspora fusca E3 confirmed its mode of action as ''CBH II-like'' (i.e., E3 is

synergistic with T. reesei CBH I, but not with T. reesei CBH II).

6.3.2 Glycosyl Hydrolase Diversity

Glycosyl hydrolases have recently been grouped by using protein sequence alignment

algorithms Hydrophobic Cluster Analysis (HCA) by Henrissat and co-workers (218). HCA

relies on the basic rules underlying the folding of globular proteins and uses a bi-dimensional

plot to display the amino acid sequence of a protein depicted as an "unrolled longitudinal cut"

of a cylinder (218). The "helical net" produced by this graphical display allows the full

sequence environment of each amino acid to be examined. Gilkes and co-workers (219)

originally proposed 9 glycosyl hydrolase families, based on the glycosyl hydrolase sequences

available at that time and, in the ensuing five years, these researchers have added substantially

to the original classification list (220, 221). Today, Bairoch (222) has identified 56 glycosyl

hydrolase families. This classification system provides a powerful tool for glycosyl hydrolase

enzyme engineering studies, because many enzymes critical for industrial processes have not

yet been crystallized or subjected to structure analysis. Still, for those working to improve

cellulase function by design, the fact that cellulases and beta-glucosidases are distributed

throughout glycosyl hydrolase families 1, 3, 5, 6, 7, 8, 9, 12, 44, 45, 48, 61, and 74 is

challenging. Since tertiary structure and key residues at active sites are generally better

conserved than amino acid sequence, it is no surprise that structural studies, combined with

sequence comparisons directed at active site residues, have allowed many of the families to be

grouped in clans having a common fold and a common catalytic apparatus (223). Five such

clans recently proposed for the glycosyl hydrolases are GH-A (including families 1, 2, 5, 10,

17, 26, 30, 35, 39, 42, 51, 53); GH-B (families 7 and 16); GH-C (families 11 and 12); GH-D

47
(families 27 and 36); GH-E (families 33 and 34); GE-F (families 43 and 62); GE-G (families

37 and 63); and GE-H (families 13 and 70) (222). Among these, clans GH-A and GH-B

include cellulases. These cellulase Families include members from widely different fold

types, i.e., the TIM-barrel, βαβ-barrel variant (a TIM-barrel-like structure that is imperfectly

superimposable on the TIM-barrel template), β-sandwich, and α-helix circular array. This

diversity in cellulase fold structure must be taken into account when considering the transfer

and application of design strategies between different cellulases.

6.3.3 Enzyme Adsorption and Non Productive Binding

The process of binding to cellulose is an important first step in enzymatic cellulose

degradation, and the study of cellulose binding is of fundamental importance for

understanding the cellulose degradation process. Cellulases that carry either a fused cellulose

binding module (CBM) or a CBM attached by a linker peptide seem highly susceptible to loss

on cellulose. Although the CBM undoubtedly plays a critical role in the hydrolysis of

cellulose for some cellulases, this small, yet highly reactive domain may also lead to general

adsorption and non-productive binding of many enzymes to cellulose (224). The binding of

Cel7 enzymes, such as T. reesei CBH I, and isolated CBMs do not follow a simple Langmuir

adsorption isotherm. This has been explained by the probability that binding sites for the

proteins on the cellulose surface overlap each other; however, the two-domain structure of the

enzymes, where both domains bind, may also play a role (225). Lignocellulose, which

contains lignin and other polysaccharides in addition to cellulose, presents an even more

complex substrate for enzyme action. Clear evidence of a relationship between lignin content

and cellulase digestion of lignocellulosic substrates has been demonstrated (226). Sild and

co-workers recently reported a kinetic and theoretical study of cellobiohydrolase action on

cellulose and proposed that in addition to being a surface peeling process, these enzymes may
48
eventually produce a substrate surface so complex that, unassisted by other cellulases, action

is greatly impeded (227).

6.3.4 Processive Cellulases

Cellulases from glycosyl hydrolase families 6, 7, 9 (and possibly others) are thought to act in

a processive manner on cellulose. That is, these enzymes do not become disengaged with the

single cellodextrin substrate until that chain is fully hydrolyzed, or until the enzyme becomes

denatured or otherwise compromised. They may, in fact, move on the cellulose surface as a

consequence of the hydrolytic cycle. The T. reesei Cel7A was probably the first enzyme in

this group to be studied in depth. Like most family 7 cellobiohydrolases, the T. reesei Cel7A

contains a large catalytic domain connected to a smaller cellulose binding module by an

amino acid linker peptide consisting of about 30 amino acids. Using small angle x-ray

scattering, Receveur and co-workers (228) recently reported the solution conformation of the

entire two domain Ce145 protein from H. insolens as well as the effect of the length and

flexibility of the linker on the spatial arrangement of the constitutive modules. The measured

dimensions of the enzyme show that the linker exhibits an extended conformation leading to a

maximum extension between the two centers of mass of each module corresponding to about

four cellobiose units on a cellulose chain. These results were consistent with a model

proposed by Himmel and co-workers (229) where cellulases can move on the surface of

cellulose with a ”caterpillar-like” linear displacement.

49
7 ENZYMATIC HYDROLYSIS OF CELLULOSE

7.1 Experimental Systems

The initial approach to enzymatically converting cellulose to ethanol involved separate

operations for pretreatment of biomass to open up the structure of biomass for attack of

cellulose by cellulase, production of cellulase enzyme on pretreated biomass, addition of the

cellulase produced to pretreated biomass to release glucose, and glucose fermentation to

ethanol or other products (230). Over time, the title Separate Hydrolysis and Fermentation or

SHF emerged to designate this sequence of operations. Improvements in pretreatment and

cellulase enzymes enhanced the performance of the SHF process by increasing the

accessibility of the substrate to cellulase and improving the balance and level of activity of the

enzyme, respectively, to enhance the performance of the SHF approach (81-83). With regard

to the former, dilute acid pretreatment emerged as a frontrunner for preparing cellulosic

biomass for enzymatic conversion with high yields at a reasonable cost (93-95, 129-133). For

the latter, classical mutation and selection successively improved the enzymes from the

fungus T. reesei discovered during World War II with the evolution from earlier strains such

as QM9414, to improved varieties such as Rut C30 (231, 232), and on to Genencor 150L that

was more effective because of enhanced levels of β-D-glucosidase that convert cellobiose into

glucose (233-237). Unfortunately, glucose and particularly cellobiose are powerful inhibitors

of cellulase action. Although yields can be improved by adding high loadings of cellulase,

this strategy results in excessive enzyme costs. Otherwise, hydrolysis yields have been

historically limited to lower glucose concentrations than considered desirable to achieve high

concentrations of ethanol or other fermentation products that can be recovered more

economically.

50
An alternative strategy known as the simultaneous saccharification and fermentation (SSF)

process adds cellulase and the fermentative organism to the same vessel to rapidly convert

glucose to ethanol as it is released, reducing the accumulation of this powerful inhibitor of

cellulase activity (238, 239). Although the temperature of the SSF process has to be reduced

below the optimum for cellulase alone to accommodate the lower temperature tolerance of

fermentative organisms, particularly at the low sugar and high ethanol concentrations of the

process, SSF performed better in terms of rates, yields, and concentrations of ethanol than

hydrolysis alone at higher temperatures that are optimum for cellulose hydrolysis (82, 234,

237, 240, 241). Nonetheless, cellulase action was still slow, with typical SSF reaction times

of about 5 to 7 days to achieve modest ethanol concentrations.

Following identification of the SSF configuration in the mid 1970s (238, 239), fermentative

organisms were sought that tolerate the combined stresses of higher temperatures (to increase

hydrolysis rates), low glucose levels (due to rapid sugar metabolism by the organism), and

high ethanol concentrations (233-237). However, rapid conversion of cellobiose to glucose

was found to be more important than the fermentation temperature, and the best results were

with a cellulase such as Genencor 150L that is high in β-glucosidase (82, 233, 234). Further

research showed that co-culture of a less ethanol tolerant cellobiose fermenting organism

Brettanomyces clausenii with more ethanol tolerant Saccharomyces cerevisiae yeast enhanced

performance, and later a single yeast, Brettanomyces custerii, was identified that both

ferments cellobiose directly into ethanol and is ethanol tolerant, eliminating the need for the

co-culture system (233, 236). More recently, bacteria have been genetically engineered to

ferment xylose to ethanol and also ferment cellobiose to ethanol either naturally or through

additional genetic modifications, and these organisms provide similar cellobiose utilization

benefits. In addition, additional genetic modifications have been made to these organisms so

they make endoglucanase in addition to fermenting cellobiose (242-246). Such strains can
51
utilize a substantial fraction of amorphous cellulose and could also reduce the need for

externally added enzyme if a mixture rich in cellobiohydrolase is applied.

Ongoing research continues to search for fermentative organisms that are temperature tolerant

and possess other traits that better match operating conditions preferred by cellulase (247-

255). In addition, many have applied SSF with advantage to a wide range of feedstocks

pretreated at various conditions (158, 256-273). Combining SSF with hemicellulose sugar

fermentation has received attention to lower costs (274-282), and SSF has been used to make

products other than ethanol (283-289). SSF has also been applied in fed-batch and continuous

processes for conversion of paper sludge and wood to ethanol (290-292). Although other

approaches are still being considered (293-296), SSF-based technology has been shown to be

a leading candidate for near-term applications and will likely remain so until new cellulases

are developed that work much faster minimal product inhibition at high temperatures (82,

233-237, 297-300).

Continuous and fed-batch processes offer advantages for producing commodity products from

cellulosics as compared to more conventional batch configurations including high cell and

enzyme concentrations over the operating cycle, greatly reduced down time for vessel

turnaround, and lower operating and capital costs for seed fermenters. While these factors are

generally not as significant for making pharmaceuticals, they can have a great potential

impact for producing commodity products. For example, frequent refilling and emptying of

batch fermenters is usually impractical at the >1 million gallon volumes envisioned for such

processes (89). In addition, eliminating seed fermenters lowered the ethanol selling price by

about 12 cents per gallon and the overall cost of processing by about 20 % in a 1996 study

built from a process design used in NREL models (301). Consistent with these observations,

most larger operations in the existing corn ethanol industry are operated in the continuous

mode.
52
While several thousand batch laboratory enzymatic hydrolysis only and SSF studies

operations have been published, as noted above, experimental investigation of continuous and

fed-batch fermentations of cellulosic feedstocks involves added complexity and cost as well

as specialized and usually custom equipment, and only a few fed-batch or continuous

experimental results have been reported (206, 256, 257, 290-292, 302). None considered the

high substrate concentrations or continuous cascade operations projected for commercial use

(89). In a broader context of an extensive review of all types of cellulosic-based systems in

addition to just SSF, only 29 fed-batch and continuous studies were found (303), and only two

used feed concentrations greater than 25 g/L cellulose (290, 291). Thus, far more effort is

needed to develop and understand continuous SSF processes if such systems are to be utilized

commercially.

7.2 Structural Features Impacting Cellulase Action

7.2.1 Crystallinity and Boundary Water Layer

Molecular Mechanics simulations have been used by Brady and coworkers (304) to model the

structuring of water adjacent to two different faces of microcrystalline monoclinic cellulose

Iβ. Strong localization of the adjacent water was found for molecules in the first hydration

layer, due to both hydrogen bonding to the hydroxyl groups of the sugar molecules and also

due to hydrophobic hydration of the extensive regions of hydrophobic surface resulting from

the axial aliphatic hydrogen atoms of the “tops” of the glucose monomer units. Importantly,

significant structuring of the water was found to extend far out into the solution. Thermally-

induced fluctuations in the crystalline surface were observed accompanied in some cases by

penetration of water molecules into the crystal lattice. It is hypothesized that the highly

structured layers of water might present a substantial barrier to the approach of cellulase
53
enzymes toward the (1,0,0) surface in enzyme-catalyzed hydrolysis, and might significantly

inhibit the escape of soluble products in dilute acid hydrolysis, contributing to the slow rates

of hydrolysis observed experimentally. Conversely, the (1,1,0) step face induces much less

structuring, and thus might be more easily approached, resulting in a higher hydrolysis rate on

this face.

7.2.2 Hemicellulose Content

Several attempts have been carried out to obtain a better understanding of the relative

importance of lignin and hemicellulose in the enzymatic hydrolysis of pretreated cellulosic

substrates. Grohmann and co-workers (133) related the improvement in enzyme digestibility

of pretreated wood to the removal of hemicellulose which, according to Grethlein (305)

results in an increase in both the accessible pore volume and the specific surface area. Stone

and Scallan (306) showed that the median pore size is also strongly dependent on the degree

of swelling. Increasing the severity of the pretreatment usually leads to increased

solublization of hemicelluloses in water and improved glucose yields in enzymatic hydrolysis

(307-310).

7.2.3 Lignin Content

The common pretreatment methods do not remove significant amounts of lignin. Formation

of small particles; brown oily substances have been detected both inside and outside exploded

cell walls by photomicrographs (311). Tanahashi suggested that lignin is first depolymerised

and then repolymerised. These results are consistent with the softening and agglomeration of

lignaceous materials and extractives. Donaldson and co-workers (312) found that when these

particles are smeared out by mild alkali, the hydrolysis is greatly reduced. Thus, it appears

that lignin is to a large extent separated from the cellulose although not totally removed from
54
the substrate. This supports that separation of lignin and cellulose, but not complete

delignification, is an important requirement for an effective pretreatment (313).

The generally observed decline of the hydrolysis rate has been suggested to be due to the

exhaustion of the more reactive, amorphous cellulose substrate. Another suggested reason for

this fall-off might be due to enzyme adsorbed to the lignaceous substrate as the hydrolysis

proceeds. It has been shown that the specific rate (rate of hydrolysis per adsorbed enzyme)

decreased significantly as the hydrolysis proceeded. This shows evidence for the non-specific

adsorption of cellulases, obviously on lignin (314). The reasons for the decline of the rate of

hydrolysis is still not fully understood in spite of numerous studies during the last decades

(315-318). Obviously, lignin restricts the hydrolysis by shielding cellulose surfaces or by

adsorbing and inactivating enzymes. It has been proposed that the close association between

lignin and cellulose prevents swelling of the fibres, resulting in limited enzyme accessibility

to the cellulose (319).

Somewhat contradictory results have been obtained on the influence of delignification on

enzymatic digestibility. Ramos and co-workers (320) and Schwald and co-workers (321)

obtained little or no increase in digestibility after extraction of lignin from pretreated wood

substrates. Selective removal of lignin and hemicellulose before steam treatment by

extraction with sodium chlorite and potassium hydroxide, respectively, indicated that short

steaming times were enough to produce in situ hydrolysis of the hemicellulose, resulting in

removal most of the hemicellulose and residual lignin and thus greatly increasing accessibility

to cellulose (321). Attempts to remove some of the remaining lignin by simultaneous

enzymatic delignification and cellulose hydrolysis showed that the enzymatic digestibility

could be slightly improved (322). The approach has not been fully exploited due to the lack

of an efficient enzymatic delignification system.

55
Much of the remaining, water insoluble lignin still contained in the wood structure is

extractable by dilute alkali or by organic solvents. Removal of this alkali insoluble lignin

resulted, however, in only a small increase in the yield of glucose on enzymatic hydrolysis or

even decreased the rate and extent of hydrolysis of steam treated softwood substrates (320,

321). It has been observed that primarily the nature and the redistribution of the guaiacyl

lignins restricted the hydrolysis of cellulose from steam-pretreated softwood. When the

residual substrate remaining after extensive hydrolysis of steam pretreated aspen and

eucalyptus was examined microscopically, it was apparent that this debris was mainly

composed of vessel elements (320). Since the lignin in vessel elements is known to have a

higher guaiacyl to syringyl ratio than other cells found in hardwoods tissues, it is probable

that the distribution of this lignin fraction restricts the swelling of the cellulosic residue and

reduces the surface area available to the enzymes. SEM examination of the ultrastructure of

decayed stumps of red alder indicated that libriform fibres and ray parenchyma cells were

almost totally degraded, whereas vessel elements remained relatively unmodified after

extensive degradation. It appeared that high level of degradation was only observed where

syringyl lignins were predominant and that the lower accessibility of plant vessels was

partially the result of occurrence of the more recalcitrant guaiacyl lignin type in the vessel

walls.

The adsorption capacity of the pretreated wood for the protein in crude cellulase has been

estimated to increase significantly as the temperature of the pretreatment is increased up to

220°C, indicating an enhanced surface area of cellulose. However, it has also been observed

that the adsorption capacity of the lignaceous residue decreases due to increased melting and

agglomeration of lignin (311, 312). This shows that lignin not only shields the cellulose but

also acts a competitive non-specific adsorbent. The hydrophobicity of surface has generally

been regarded as an important factor: the more hydrophobic the surface is the higher the
56
extent of adsorption. All the major T. reesei cellulases have hydrophobic amino acids

exposed on the surface (323). The hydrophobic residues on the surface of the enzyme may

also lead to binding to the hydrophobic surface of lignin. The adsorption of purified

cellulases from T. reesei, CBH I (Cel7A) and EG II (Cel5A) and their catalytic domains on

steam pretreated softwood (SPS) and lignin has been studied using tritium labeled enzymes.

Both CBH I and its catalytic domain exhibited a higher affinity to SPS than EG II or its

catalytic domain. Removal of cellulose binding domain decreased markedly the binding

efficiency (324). Thus, the non-specific unproductive adsorption of cellulases seems to be

one important factor in the decline of hydrolysis rate (314, 317, 325). The rate decline of

enzymatic hydrolysis is also related to enzyme inactivation during hydrolysis. In addition to

end product inhibition, there are reports on soluble enzyme inhibitors. A variety of

substances are formed during the pretreatments. It has been shown that the liquid formed

after pretreatment has an inhibitory effect on the enzymes (137, 326).

Cellulose hydrolysis has been reported to be improved when surfactants are present in the

reaction mixture (327-329). It has been proposed that surfactants prevent the unproductive

binding of cellulases on lignin surfaces. Non-ionic surfactants (such as Tween and Triton)

were found to increase the hydrolysis whereas the charged surfactants decreased hydrolysis.

In the hydrolysis of SPS, the addition of Tween 20 allowed the reduction of the enzyme

loading to about half (330). The positive effect of surfactant addition on hydrolysis of SPS

correlated with a decrease in CEL 7A (CBH I) adsorption. The increase in hydrolysis and

decrease in CEL 7A adsorption observed by Tween 20 was not observed on pure cellulosic

substrates.

57
7.2.4 Acetyl Content

7.3 Kinetic Modelling of enzymatic hydrolysis of cellulose

Kinetic models based on fundamental principles are valuable tools for building confidence in

the ability to predict performance of large scale systems that have never been run

commercially (87). Such models can also clarify cause and effect relationships better than

viewing data alone, giving important insight that can facilitate identifying and overcoming

key bottlenecks. Details of modeling approaches applied to describe the rate of enzymatic

hydrolysis of cellulose would require more space than available here, but some general

concepts important in the development of such models are summarized with more details

available in recent reviews (303, 331).

A key element of hydrolysis reaction models is the attack of cellulose by enzymes to release

glucose. In this regard, endoglucanase and exoglucanase must act on an insoluble substrate,

cellulose and are generally pictured to do so by attaching to the substrate via an adsorption

mechanism to form an enzyme-substrate complex. Furthermore, cellulase enzyme has also

been shown to attach to lignin to form non-productive enzyme-lignin complexes that reduce

the amount of enzyme available to act on cellulose, and cellulase bound to lignin did not

desorb when water was added (325, 332, 333). On the basis of these and other observations,

adsorption models have been developed to relate the concentrations of accessible substrate,

cellulase enzyme, lignin, enzyme-substrate complexes, and enzyme-lignin complexes (325,

332). Langmuir-type adsorption has been shown to adequately represent this relationship in

various studies.

Nutor and Converse (332) calculated a specific hydrolysis parameter defined as the hydrolysis

rate divided by amount of adsorbed enzyme and showed it declined sharply with conversion
58
even when corrected for glucose inhibition effects. Furthermore, the specific rate dropped

with increasing substrate concentration possibly indicating that the effectiveness of the

enzyme is diminished when it is spread to thin over the substrate surface, consistent with the

synergistic action of the key components (334). In addition, the specific rate was almost

independent of total enzyme concentration suggesting that hydrolysis rate is impacted by

substrate conversion and/or deactivation of cellulase over time.

Cellulose is not totally homogeneous, and one would expect that large cellulose enzymes

more readily access some portions than others. Thus, particle reactivity would be expected to

change with conversion for cellulosic substrates as shown in the literature (318, 325, 332).

On this basis, empirical models have been developed to include decreasing cellulose reactivity

with conversion (290, 335, 336). When these are multiplied by a weighting function to

represent the probability that each particle population is still in the reactor for fed-batch and

continuous fermentations, this approach has been shown to improve the ability to describe

fed-batch and continuous SSF processes in which there are particle populations with different

ages and therefore reactivity (290, 336).

Various components in the liquid can also affect cellulose conversion. In particular, glucose

and cellobiose are powerful inhibitors of cellulase action, and this effect must be integrated

into kinetic models (337, 338). For SSF, fermentation of glucose and in some cases other

carbohydrates must be modeled, and considerable literature is available on such systems.

Many yeast kinetic models are unstructured and non-segregated and based on the Monod

equation for substrate limited growth. Structured yeast kinetic models that describe the flux

of individual metabolites, specifically the glycolytic pathway, have also been developed, but

these are generally applied for the purposes of metabolic engineering (339, 340). Additional

terms have been added to the Monod model to describe growth inhibition by ethanol (341,

342) and high sugar concentrations (343-346). Other important factors to consider include the
59
proportionality between growth and substrate consumption as represented by the cell yield

and the decline in cell yield observed at low growth rates (341). Overall models were applied

to describe SSF operations based on considerations of the types described above, although

most were used to predict the performance of batch systems (337, 338, 347-359). As

mentioned before, a model was also developed to integrate the change in reactivity with

substrate age and consider the age distribution of particles that should be considered for

continuous stirred tank systems (290, 336). However, few of the models were applied to

multiple continuous vessels operated in the cascade arrangement expected to be applied

commercially (84, 85, 88, 89). In addition, little experimental data has been produced for

comparison, and no data is available at industrially relevant concentrations or for cascaded

continuous fermenters (303).

8 PRETREATMENT TO IMPROVE CELLULOSE HYDROLYSIS BY ENZYMES

8.1 Effect of Pretreatment on Enzymatic Hydrolysis

Cellulosic biomass must be pretreated to achieve high yields of glucose from cellulose by

enzymes. However, the complex structure of biomass makes it difficult to isolate how

pretreatment influences cellulose digestion, and understanding the relationship of cellulose

digestibility by enzymes to pretreated biomass characteristics is challenging (331). Although

much research is still needed before we can predict a priori how to improve cellulose

digestion by cellulase, enzymatic digestibility of pretreated cellulose has been related to

removal of key constituents including lignin, hemicellulose, and hemicellulose acetylation

(135, 360, 361). Some have shown a direct relationship between cellulose digestion and

hemicellulose removal, and lignin removal was reported not to be needed to achieve good

cellulose conversion (131-133). Yet, complete hemicellulose removal is often not sufficient if
60
the pretreatment temperature is too low, and higher temperatures are needed for hemicellulose

removal to be effective for some substrates than for others, suggesting that more than just

hemicellulose removal is needed (93). Consistent with this idea, several studies have shown

that removing lignin improved cellulose hydrolysis, but differences are reported in the degree

of delignification needed (360-367). Hemicellulose is altered in many lignin removal

techniques, confounding the role of the two components (365, 368-372). Furthermore,

although lignin dissolution remains at less than about 20 to 35% throughout batch operations,

lignin and hemicellulose removal are nearly linearly related if liquid is continually swept

through biomass during hemicellulose hydrolysis, with up to 75% of the lignin removed at

higher flow rates, suggesting that lignin dissolves during hemicellulose hydrolysis but reacts

to form insolubles if left in the system. Evidence has been presented that alteration of

hemicellulose is not needed to affect cellulose digestibility (373-375). Removal of acetyl

groups has also been shown to appreciably improve cellulose digestibility, with some

differences reported in the degree of deacetylation needed (33, 376), but deacetylation has

been concluded to influence hydrolysis rates less than crystallinity or lignin content (360,

362).

Cellulose digestibility has also been related to physical features of the pretreated substrate. In

one approach, enzyme and hydrolysis water penetration were pictured to be impeded by

cellulose crystallinity, but rates were found to drop with increasing crystallinity in some

studies (377-381) and increase in others (305, 311). In addition, even highly crystalline

substrates were hydrolyzed to glucose with high yields (382). An extensive study of model

compounds of varying crystallinity, lignin, and hemicellulose acetylation showed that

hemicellulose acetylation plays a relatively minor role and that crystallinity is less important

than removal of lignin and has a greater impact on rates than yields (360, 362, 377).

Enzymatic consumption of free chain ends generated during pretreatment has been suggested
61
to explain why cellulose digestion slows over time, but measurements of a decline in rate of

hydrolysis with increasing fractional surface coverage by adsorbed enzyme has led others to

conclude that steric hindrance by nearby enzyme molecules, multi-layer adsorption, binding

of enzyme to lignin, and cellulase getting stuck in dead end alleys could play roles (361).

Digestibility has also been explained in terms of the accessibility of cellulose to enzymes, and

hydrolysis rates have been related to pore volume and surface area accessibility (305, 383).

Concerns have been raised about how accurately these features can be measured and whether

the complex structure of cellulases could penetrate such pores effectively (361).

8.2 Desirable Pretreatment Attributes

The following are desirable, although not necessarily completely achievable, attributes for

pretreatment processes (134, 135, 301, 384):

• The cost of chemicals for pretreatment and subsequent neutralization and pre-

fermentation conditioning should be kept low,

• Generation of wastes in pretreatment should be minimized,

• Limited size reduction should be needed prior to pretreatment because biomass

milling is energy intensive and costly,

• Pretreatment should preserve hemicellulose sugars and make them more

accessible for fermentation,

• Fast reactions and/or non-corrosive chemicals are desirable to minimize

pretreatment reactor costs,

• The concentration of hemicellulose sugars from pretreatment should be above

about 10% to keep fermentation vessel sizes reasonable and facilitate

downstream recovery of the fermentation product,

62
 • Pretreatment must promote high product yields in subsequent enzymatic

hydrolysis or fermentation operations with minimal conditioning costs and

little loss of sugars during conditioning,

• Hydrolysate conditioning in preparation for subsequent biological steps should

not form products that present processing or disposal challenges (e.g.,

gypsum).

• Low enzyme loadings should be adequate to realize greater than 90%

digestibility of pretreated cellulose in less than 5 and preferably 3 days, and

• Pretreatment should facilitate recovery of lignin and other constituents for

conversion to valuable co-products and to simplify downstream processing.

8.3 Pretreatment Types

Biological, chemical, and physical methods have been evaluated to prepare cellulosic biomass

for conversion of cellulose and hemicellulose to valuable products. Biological pretreatment

targets using fungi or other microorganisms to selectively remove lignin or other constituents,

thereby improving accessibility to cellulase, and although the low chemical and energy use

anticipated for such approaches is conceptually attractive, no controllable and sufficiently

rapid system has yet been found (375, 385-387). Known physical methods such as

comminution (e.g., milling) and radiation often perform poorly and are costly (388-394). Just

high temperature steam breaks down hemicellulose into sugars and oligomers, but the total

dissolved sugar yields from hemicellulose during pretreatment and subsequently from

cellulose by enzymes are too limited (98, 142, 320, 395, 396). Passing high temperature

water without adding any chemicals (140, 141, 384, 397-404) or with about 0.07% sulfuric

acid (138, 139) through biomass can reduce or eliminate acid use, achieve nearly theoretical

hemicellulose sugar yields, produce much more digestible cellulose, result in liquid
63
hydrolysate that is more compatible with fermentative organisms, lower the cost for materials

of construction, use less neutralization and conditioning chemicals, and cut back on size

reduction requirements (139, 301, 384). Unfortunately, dilute sugar streams and consequently

low ethanol concentrations that increase energy requirements for distillation and pretreatment

result, making current flow-through systems appear to be commercially unattractive. At this

point, the most viable pretreatment options are all based on adding acid or base to lower or

raise the pH, respectively, and the reader is referred to the literature for more information on

biological, physical, and related pretreatment technologies (134, 135, 360, 375, 405-407).

8.4 Chemical Pretreatment at Low to Neutral pH

A leading chemical approach is based on adding about 0.5 to 1.5% sulfuric acid to cellulosic

biomass and heating the mixture to temperatures between approximately 120 and 200oC, with

about 80 to 90% of the hemicellulose recovered as mostly monomeric sugars. Furthermore,

the cellulose left in the solid residue is highly digestible by enzymes (94, 131, 133, 305), and

hemicellulose removal also supports subsequent acid catalyzed cellulose hydrolysis without

sacrificing hemicellulose sugars that would rapidly degrade otherwise (108-110). Because

high yields of sugars from both hemicellulose and cellulose are vital to economic success,

many favor this approach to pretreatment (83, 84, 88, 301, 408), and dilute acid pretreatment

has been the subject of over a decade of research and development (134, 135). However,

dilute acid hemicellulose hydrolysis still has important limitations such as the need to use

exotic and expensive materials of construction to accommodate the very corrosive

environment, costs for neutralizing acid following pretreatment and disposing of the salts

formed, the need to remove toxics released and formed during pretreatment in order to realize

high biological processing yields, and formation of gypsum with problematic reverse

solubility characteristics when neutralized with inexpensive calcium hydroxide (84, 85, 94,
64
131, 133-137). About a 7 day period with somewhat high enzyme dosages is also required to

enzymatically hydrolyze cellulose pretreated by hemicellulose removal by dilute acid, and use

of less enzyme and reaction for shorter times would significantly reduce costs (82, 84, 85).

Most of the lignin is left with the cellulose, and adsorption of cellulase by lignin increases

cellulase loading requirements (314, 409).

Other acids have been evaluated to use in place of sulfuric. For example, hydrochloric acid is

somewhat more effective than sulfuric, but its toxicity and highly corrosive nature make it

more expensive to use (410). Nitric acid performs similarly to sulfuric and is compatible with

less expensive materials of construction (411, 412), but the acid cost is higher. Sulfur dioxide

has been used with good results as well (137, 413), but this chemical presents some safety

concerns with no cost advantage relative to sulfuric acid (414). Addition of carbon dioxide to

generate carbonic acid requires such high pressures as to raise concerns about containment

costs and does not appear to be as effective as sulfuric acid (135, 273, 279, 415). Thus, the

low cost and good performance of sulfuric acid generally favor its use in most current

designs.

An alternative pretreatment under development is based on controlling the pH between 4 and

7 with the goal of reacting hemicellulose to soluble oligomers while minimizing formation of

sugar monomers that can degrade to furfural and other compounds that reduce sugar yields.

This approach reduces the concentration of hydrogen ions formed by breakdown of natural

acids released during pretreatment that catalyze formation and degradation of

monosaccharides (405). Reduction in release of toxic degradation products through this

strategy could also benefit subsequent cellulose hydrolysis by enzymes or ethanol

fermentation. In one application now under investigation, the stillage from a corn ethanol

plant has proven effective in providing buffering capacity for hydrolysis of corn fiber at

controlled pH. In addition, changes in physical structure such as increased pore size and
65
decreased crystallinity are targeted to enhance enzyme penetration and effectiveness (305,

378, 416-418).

8.5 Chemical Pretreatment at High pH

While hydrogen ions catalyze hemicellulose hydrolysis and removal at low pH, operation at a

high pH of over about 10 can solublize and remove lignin and result in improved cellulose

digestibility. Over the years, various alkaline materials such as sodium and potassium

hydroxide have been used for this purpose, but their costs are too high for production of low

value fuels and commodity chemicals (367, 374, 377, 378, 419). Use of solvents in an

organosolv technique has also been considered, but the costs are too high unless cellulose is to

be recovered for high value uses (420-422). On the other hand, ammonia is volatile at

moderate temperatures and can be recovered and reused (423-425). In particular, an ammonia

fiber explosion (AFEX) process has been developed to treat cellulosic biomass with liquid

ammonia at moderate temperatures of about 100oC or less followed by rapid pressure release

to recover the ammonia and open up the structure of the solids. The physical appearance of

AFEX treated material appears similar to that prior to pretreatment, and little change in lignin

or hemicellulose content is found. However, the AFEX process decrystallizes cellulose and

greatly disrupts the cellulose structure, enhancing its susceptibility to cellulase. Although

hemicellulose hydrolysis to monomers is very limited during AFEX, an enzyme mixture rich

in hemicellulase as well as cellulase activity can achieve greater than 90% conversion of

cellulose and hemicellulose to fermentable sugars for a wide variety of lignocellulosic

materials including alfalfa, barley straw, corn residue, wheat straw, rice straw, corn fiber,

sugar cane bagasse, switchgrass, coastal Bermuda grass, and rye grass straw (423, 424, 426-

430). Furthermore, high yields of glucose can be obtained cellulose at low cellulase loadings

of 1 to 10 IU of cellulase/gram of dry cellulosic biomass for most of these materials, but

66
enhanced hemicellulase activity is needed to release residual hemicellulose (424, 427-430).

The hydrolysate is also compatible with fermentative organisms and enzymes without need

for conditioning, and the small amount of ammonia left in the pretreated stream is an effective

nitrogen source for subsequent fermentations (423, 426). The moderate temperatures and low

corrosiveness of ammonia also minimizes materials of construction costs. Although AFEX

was originally developed as a batch process, a new continuous fiber extrusion configuration

called FIBEX reduces treatment time and required ammonia levels significantly while giving

similar hydrolysis results to batch AFEX (431).

Removal of lignin from biomass before biological processing improves cellulose digestibility,

reduces downstream agitation power requirements, provides less sites for non productive

cellulase adsorption, reduces dissolved lignin compounds that are toxic to fermentations,

facilitates cell and enzyme recovery and recycle, and simplifies the distillation step. An

ammonia recycle pretreatment (ARP) approach selectively breaks down lignin in an aqueous

ammonia solution at high temperatures by an ammoniolysis reaction with virtually no effect

on hemicellulose or cellulose content, and the dissolved lignin is removed by circulating

liquid through biomass (372). Furthermore, ARP partially fractionates biomass into three

major fractions: hemicellulose, solid cellulose, and extracted lignin. The treated solids are a

low-lignin cellulosic material with high glucan content that is readily hydrolyzed by enzymes

when ARP is applied herbaceous crops, corn stover, and switchgrass (432). ARP

pretreatment has also been studied in conjunction with additional treatment with hydrogen

peroxide (433) and dilute sulfuric acid (434). Although flow of liquid through biomass could

prove to be too expensive, promising performance was recently observed when liquid

ammonia was mixed with biomass for several days at virtually room temperature, and further

development of this technology could prove very effective.

67
As the least expensive alkali, lime could be cost effective for lignin removal, and recent

studies have shown that lime is feasible for pretreatment at a temperature of about 100oC, a

reaction time of 1 to 2 hours, lime loadings of about 0.1 g Ca(OH)2/g biomass, and water

loadings of 5 to 15 g H2O/g biomass (362, 435, 436). Lime enhanced enzymatic digestibility

of herbaceous biomass by as much as ten times but was less effective on woody biomass

because of its high lignin content. In addition to being inexpensive, lime is safe to handle and

is widely available. Furthermore, lime can be recovered by washing biomass with water

followed by reaction with carbon dioxide to precipitate calcium carbonate for feed to a lime

kiln for recycle. More recently, use of lime has been studied at temperatures between 25 and

55oC and found to remove lignin and some acetyl from biomass. Various solvents such as

ethanol and methanol can also be applied to remove lignin effectively in organosolv

processes, but the costs are too high to be practical for other than recovery of high value

products (369, 370, 421).

9 ENZYMATIC HYDROLYSIS OF HEMICELLULOSE

9.1 Sources of Hemicellulases

Hemicellulases are produced by many species of bacteria and fungi, as well as by several

plants. Many microorganisms produce a multiple pattern of hemicellulases to degrade the

plant materials efficiently. Today, most commercial hemicellulase preparations are produced

by genetically modified Trichoderma or Aspergillus strains. Most microbial hemicellulases

studied are active between pH 4 and 6 and at temperatures below 70°C. More thermophilic

enzymes are of great interest both in hydrolysis and other applications. Xylanases, which are

stable, and function efficiently at high temperatures have been isolated especially from

thermophilic bacteria. Several xylanase genes, encoding proteins active at temperatures from
68
75°C up to 95°C (pH 6–8), have been isolated. The most thermophilic xylanases hitherto

described are produced by species of the extremely thermophilic bacterium Thermotoga

(437). Only few thermophilic mannanases have so far been characterized (438, 439).

9.2 General Hemicellulose Hydrolysis

The complexity of hemicellulose structure requires a high degree of coordination between the

enzymes involved in hemicellulose degradation. Most enzymes have very specific

requirements for tight substrate binding and precise transition state formation, which usually

leads to high catalytic turnover rates. However, even ideal catalytic sites must be "carried to

the substrate" by the macromolecule within which it is housed, and enzymes are large

compared to the polysaccharide oligomers under attack, especially as the particular site of

action may be buried in a heterocrystalline structure of mixed polysaccharides. Of further

complication is that the actions of glycosyl hydrolases often change the chemical environment

of the partially degraded substrate, which in turn affects the actions of other glycosyl

hydrolases. For example, most native hemicelluloses are quite water soluble because, in part,

of the substituents attached to the main chain. These side chains disrupt the water structure

and help to solublize the hemicellulose. Debranching enzymes, which remove these

substituents, may generally decrease substrate solubility when acting alone, and in turn lower

the polysaccharide's susceptibility to endo-acting hydrolases (439, 440).

A major part of the published work on hemicellulases deals with the properties and mode of

action, as well as applications of xylanases (reviewed by e.g. Coughlan and Hazlewood (441),

Sunna and Antranikian (442), and Viikari and co-workers (443). Due to their complex nature,

enzymatic hydrolysis of xylans is intrinsically more complicated than most of the other plant

polysaccharides. Typically, the polymer is debranched, either prior to or in conjunction with

depolymerization of the backbone. As the substituents are removed, xylan can become less
69
soluble and form aggregates that sterically hinder and retard further degradation (443).

Different debranching enzymes are required depending on the specific type of xylan being

hydrolysed. These include arabinofuranosidases, ferulic and coumaric acid esterases, acetyl

and acetyl xylan esterases, glucuronidases, and xylosidases (Figure 6.). Simultaneous

removal of these side chains and cleavage of the polymeric backbone synergistically enhances

the rate of degradation by endoxylanase enzymes (37, 440, 444-449). Thus, a xylan that has

been subjected to acetyl xylan esterase is less susceptible to enzymatic degradation than a

xylan subjected to a mixture of depolymerizing and debranching enzymes (450, 451).

9.2.1 Xylan Backbone Hydrolysis

The endoxylanases (1,4-β-D-xylan xylanohydrolases, EC 3.2.1.8) randomly cleave the main

chain 1,4-β-D-xylosidic linkages and are often quite particular about the type of linkage, type

of sugar, and presence or absence of nearby substituents (451) (see Figures 4 and 6). An

endoxylanase that cleaves β-(1,4) linkages will usually have no effect on β-(1,3) linkages.

Also, an endoxylanase that cleaves main-chain linkages near an O-2 linked arabinose will

have no effect on an open-chain xylan (452). Although there are such specific examples of

endos requiring sidechains for maximal activity (453), the majority of the endo-acting

hemicellulose hydrolases tend to be more active on debranched or partially debranched

hemicellulose, especially in the case of xylanases. The limitation on this increased activity is

probably due to solubility or the polysaccharides, which tend to become more insoluble as the

debranching process continues. Decreasing chain length due to the activity of endo-

hemicellulases mollifies this, allowing the shorter, less substituted fragments to remain

soluble. Exo-acting enzymes, which probably fall into reducing and non-reducing end

specific groups, and oligomer-hydrolysing enzymes, also require debranching as a precursor

to maximal activity. Overall, a balance must be met between removing the branching side
70
chains from the polysaccharide backbone, decreasing the average chain-length, and

hydrolysing the oligomers into free monomers, all while maintaining enough solubility of the

fragments to allow enzyme access. The concerted action of the various hemicellulase enzyme

classes probably accounts for the high synergy observed when the enzymes are used concert

with each other (454).

Most xylanases are rather small proteins (molecular mass around 20 kDa) with a basic

isoelectric point (pI 8 to 10). Xylanases belong to the two principle xylanase groups (glycosyl

hydrolase families 10 and 11) and differ from each other with respect to their catalytic

properties (455). These enzyme families are similar in that they both depolymerize xylan via

the Koshland type, two-step catalysis that leaves products with retained stereochemistry of

anomeric configuration. Family 10 enzymes typically yield lower molecular weight products

(tetramers) than family 11 (pentamers) (455). This is likely due to the difference in binding

sites, with family 10 enzymes having a binding site that recognizes shorter oligosaccharides

than family 11 (456, 457). Although these enzymes are active on native branched xylan,

debranching may increase their activity (455, 458). Xylanases with high Mr/low pI (family

10) seem to exhibit higher catalytic versatility than the low Mr/high pI -xylanases (family 11)

and thus, they are able to more efficiently hydrolyze highly substituted xylans.

Several three dimensional structures of different low molecular mass xylanases belonging to

family 11 have been determined, the first ones from Bacillus pumilus and T. reesei (459-461).

These enzymes have very similar structures; ellipsoidal, well-packed molecules having

diameters between 30 to 45 Å. Most xylanases do not contain a separate substrate-binding

domain. Some bacterial xylanases; however, have been found to contain, either a cellulose

binding domain (462) or a xylan binding domain (463). The first solved crystal structure of

family 10 xylanases was the catalytic domain from Streptomyces lividans, which is about 1.5

times longer than the B. pumilus and T. reesei xylanases (464).

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9.2.2 Mannan Hydrolysis

Endomannanases (1,4-β-D-mannan mannanohydrolase, EC 3.2.1.78) catalyze the random

hydrolysis of β-D-1,4 mannopyranosyl linkages within the main chain of mannans and

various polysaccharides consisting mainly of mannose, such as glucomannans,

galactomannans and galactoglucomannans. Mannanases are generally larger proteins than

xylanases (Mr 30–90 kDa) and have acidic isoelectric points. They seem to comprise a more

heterogeneous group of enzymes than xylanases; i.e. no clear groups based on biochemical

properties have been identified. The mannanase of T. reesei has been found to have a similar

multi-domain structure as several cellulolytic enzymes, i.e. the protein contains a catalytic

core domain, which is separated by a linker from a cellulose-binding domain (465). In

glucomannan and galactomannan, as with xylan, degradation requires both debranching and

depolymerizing enzymes, which work in synergy (449).

9.2.3 Enzymatic glucan hydrolysis

Enzymatic degradation of glucans is not as well understood as some of the other

hemicelluloses. As xyloglucan is believed to be critical in governing the growth and

expansion of plant cell walls, most recent research efforts appear to be focused on

determination of plant enzymes responsible for control and modification of the expanding cell

wall (66, 466-483). Although not as heavily branched as xylans, the xylose and other

substituents on xyloglucan can make enzymatic digestion more complicated than that of

cellulose and β-glucan. Some xyloglucanases require a specific xylose substitution pattern

while others are more general (66). This determination seems to be dependent on the binding

sub-sites in specific endoglucanases, at least in T. reesei endo-acting β-glucanases (484). A

xyloglucanase from Aspergillus niger has been shown to be active against several β-glucans,

72
but having the highest activity against tamarind xyloglucan (485). This, combined with its

lack of synergy with cellulases, indicates enzyme specificity different from traditional

endoglucanases active on cellulose. A similar enzyme has been isolated from A. aculeatus

(486). T. reesei has also been shown to have endoglucanases with activity on xyloglucan

(484). A plant-specific enzyme believed to be responsible for modification of xyloglucan in

the cell wall through endo-hydrolysis and glycosyl transferase activities has also been

demonstrated (468, 479). A recent study on glycanases from Thermomonospore fusca

indicated that some glucan hydrolases are very specific, others have much broader substrate

specificities (487). Cel5A was shown to be active on a wide range of substrates, including β-

glucan, carboxymethyl cellulose, xyloglucan, lichenin, galactomannan, and glucomannan.

Debranching of xyloglucan has been shown to occur via α-fucosidases, which specifically

cleave α-(1→2)-L-fucosides from the galactose side chains (EC 3.2.1.63) bound to the

glucose backbone or are non-specific α-fucosidases (EC 3.2.1.51). As the galactose

substituted onto the glucose is α-(1→2)-linked, this is essentially a fucosylated lactose and

enzymes specific for this side-chain conformation are assayed by activity against 2'-fucosyl-

lactitol but show no activity against p-nitrophenyl-α-L-fucopyranoside (488). There is an

exo-acting enzyme that acts on the non-reducing end of xyloglucan oligomers.

Oligoxyloglucan beta-glycosidase (EC 3.2.1.120) is produced by Aspergillus oryzae and

removes an α-xylo-β-(1→6)-D-glucosyl dimer (isoprimverose) from the non-reducing end.

Because of the differences in the linkages, different enzymes are required to cleave the two

forms of β-glucan (489-494). Enzymatic degradation of β-glucan is accomplished through

glycosyl hydrolase family 12 enzymes (EC 3.2.1.4). Although these endo-acting enzyme are

active on β-(1→4) glycosidic linkages, they are differentiated from other β-(1→4)-acting

enzymes by the distinction of being able to hydrolyze the β-(1→4) linkages in mixed β-

73
(1→3,1→4)-linked polysaccharides. Glucan endo-1,3-beta-D-glucosidase (β-(1→3)

glucanase) (EC 3.2.1.39) is an endo-acting glycosyl hydrolase that acts on β-(1→3) glucan,

but has very limited activity on the mixed linkage β-glucan. Endo-1,3(4)-β-glucanase (β-

(1→3, 1→4) glucanase) (EC 3.2.1.6), is also an endo acting glycosyl hydrolase. There is an

exo-acting glycosyl hydrolase that is active on β-(1→3) glucan. Glucan 1,3-beta-glucosidase

(EC 3.2.1.58) acts by processively releasing glucose from β-(1→3) glucan from the non-

reducing end.

9.2.4 Enzymatic hydrolysis of hemicellulose oligomers

Enzymes needed for further hydrolysis of the short oligomeric compounds produced by endo-

enzymes from hemicelluloses are β-xylosidase (1,4-β-D-xyloside xylohydrolase, EC

3.2.1.37), β-mannosidase (1,4-β-D-mannoside mannohydrolase, EC 3.1.1.25) and β-

glucosidase (EC 3.2.1.21). β-Xylosidases and β-mannosidase catalyze the hydrolysis of xylo-

and manno-oligosaccharides, respectively, by removing successive xylose or mannose

residues from the non-reducing termini. Exoglycanases are generally larger proteins than

endoglycanases, with molecular weights above 100 kDa and they are often built up by two or

more subunits.

9.2.5 Enzymatic debranching of hemicellulose

The side groups connected to xylan and glucomannan main chains are removed by α-

glucuronidase (EC 3.2.1.139), α-arabinosidase (α-L-arabinofuranoside

arabinofuranohydrolase, EC 3.2.1.55) and α-D-galactosidase (α-D-galactoside

galactohydrolase, EC 3.2.1.22). Acetyl and hydroxycinnamic acid substituents bound to

hemicellulose are removed by acetyl xylan esterases (3.1.1.72) and other esterases (see Figure

74
4). Some accessory enzymes, such as an α-arabinosidase and an esterase of Pseudomonas

fluorescens subs. cellulosa and an acetyl xylan esterase of Trichoderma reesei have been

found to contain a multi-domain structure with a separate cellulose binding domain (495,

496). Recently, the three-dimensional structures of acetyl xylan esterases (497, 498) and

feruloyl esterases (499, 500) have been published.

9.3 Action of Hemicellulases on Model Substrates

The enzyme activity is usually determined using isolated substrates, which may or may not be

similar to the actual natural substrate. Especially with hemicelluloses, the isolation of the

substrate from complex raw materials leads often to chemical and structural modification of

the substrate. Some of the side groups may be removed during chemical extraction or the

average degree of polymerization of the hemicellulose may change. The physical and

morphological properties of the isolated substrates in the activity assay are often different

from those in the practical application. Analogously with cellulose, the structure of the

hemicellulose substrate changes during the hydrolysis. Therefore, the kinetic modelling of the

hydrolysis is difficult.

Most of the xylanases characterized act randomly and are able to hydrolyze different types of

xylans, showing differences only in the spectrum of end products. The main products formed

from the hydrolysis of xylans are xylobiose, xylotriose and substituted oligomers of two to

four xylosyl residues. The chain length and the structure of the substituted products depend

on the mode of action of the individual xylanase (455). Some xylanases, however, have

rather strict substrate specificity. A unique xylanase, which requires a glucuronic acid

substituent in the xylan backbone, is produced by Bacillus subtilis (501). Few exoxylanases

75
have also been isolated and characterized. These liberate xylobiose and/or xylotetraose from

the non-reducing end of the xylan chain (502, 503).

The main hydrolysis products of galactomannans and glucomannans by mannanases are

mannobiose, mannotriose and various mixed oligosaccharides. The hydrolysis yield is

dependent on the degree of substitution as well as on the distribution of the substituents (504).

The hydrolysis of glucomannans is also affected by the glucose to mannose ratio. Some

mannanases are able to hydrolyze not only the β-1,4-bond between two mannose units, but

also the bond between the adjacent glucose and mannose units (505, 506). Several

endoglucanases, which hydrolyze mainly cellulose, are also able to cut the linkage between

glucose and mannose in glucomannans and galactoglucomannans.

Side groups, which are still attached to oligosaccharides after the hydrolysis of xylans and

mannans by xylanase or mannanase, respectively, restrict the action of β-xylosidase and β-

mannosidase. The hydrolysis may stop at the substituted sugar unit. The β-xylosidase of T.

reesei and β-mannosidases of A. niger are also able to attack polymeric xylan and mannan,

respectively, liberating xylose and mannose by successive exo-action (507). There are clearly

different types of side-group cleaving enzymes. Some of these accessory enzymes are able to

hydrolyze only substituted short-chain oligomers, which must first be produced by the

backbone depolymerizing endo-enzymes (xylanases and mannanases). Others are capable of

also attacking intact polymeric substrates. Most accessory enzymes of the latter type,

however, prefer oligomeric substrates.

The synergy between different hemicellulolytic enzymes is usually observed by the improved

action of both endoglycanases and accessory enzymes. Clear synergy between xylanases, β-

xylosidase and α-glucuronidase of T. reesei was observed in the hydrolysis of deacetylated

glucuronoxylan (Table 1). Pure xylanase, α-glucuronidase or their combination did not

76
liberate notable amounts of glucuronic acid, even though extensive hydrolysis of the main

chain by xylanolytic activity occurred. However, addition of α-glucuronidase significantly

increased the hydrolysis of xylo-oligomers with the pure xylanase. When the xylanase was

supplemented with β-xylosidase, the improving effect of additional α-glucuronidase on the

yield was even more significant (508). Thus, if the substrate contains substituted xylans, the

enzyme preparations should contain adequate amounts of xylanolytic activities needed.

In the case of acetylated substrates, practically no formation of sugars could be observed

without addition of an acetyl esterase or an acetyl xylan esterase (509). When purified wheat

straw arabinoxylan was used as substrate, the liberation of xylose and arabinose was increased

after the addition of feruloyl esterase and α-arabinosidase (510). Analogous results have been

obtained with acetylated galactoglucomannans (511).

9.4 Hydrolysis of Residual Hemicellulose in the Solid Residue

The pretreatments usually aim at a complete removal of hemicellulose in order to improve the

hydrolysis of cellulose. Thus, the impact of the enzymatic hydrolysis of fibre bound

hemicellulose on cellulose hydrolysis has not been considered important as evaluated by the

number of publications. The hemicellulolytic activity in the cellulase preparations can also be

expected to be high enough to provide the necessary hydrolysis of the residual hemicellulose

in the solid matrix. The enzyme activity pattern of a typical commercial cellulase (Celluclast

from Novozyme), supplemented with β-xylosidase (Novozym 188) is presented in Table 2.

As can be seen, at a frequently used cellulase dosage level of 20 FPU/g substrate, the

corresponding xylanase activity is about 8200 nkat/g substrate. If estimated per gram of xylan

in the substrate, the xylanase dosage becomes even higher, as the residual xylan content in the

substrate is typically far below 10% of the dry weight. The activities of accessory enzymes

77
are fairly low, but their role in the solubilization of matrix bound xylan is expectedly less

significant. The acetyl esterase activity, however, may play a crucial role, if the raw material

is still acetylated.

The hydrolysis of xylans in fibre bound substrates has mostly been studied on cellulose pulps,

however, with the aim of not hydrolyzing cellulose. In these experiments, it has been found

that most accessory enzymes had only minor effects in the hydrolysis of xylans (512).

Although the pulp substrates differ significantly from pretreated lignocellulosic raw materials,

e.g. with respect to the degree of acetylation, these results are probably valid also on other

substrates. It can thus be concluded that if the solid substrate contains deacetylated xylan,

xylanases alone should be able to solublize the xylan to oligomers. However, if the fibre

bound contains acetyl groups, these may restrict the hydrolysis.

9.4.1 Hydrolysis of Solublized Hemicellulose

The pretreatment conditions affect the solublization, recovery and composition of the

solublized hemicelluloses. Depending on the raw material and pretreatment conditions, high

molecular weight fractions, oligosaccharides, monomers or sugar degradation products are

formed. Longer pretreatment times (at around 190°C) and additives, such as SO2, lead

generally to better recovery of monomers. Less severe pretreatment conditions lead to

solublization of xylan and/or formation of oligosaccharides which can be hydrolyzed into

monomers by enzymes (309). The total enzymatic hydrolysis of substituted oligomers needs

the synergistic action of endoenzymes and accessory enzymes. The raw material,

pretreatment conditions and thus the structure of the solubilized hemicellulose oligomers

should be known for the identification of enzymes needed. The solubilized fraction from

steam-pretreated birch wood contained about 10% acetyl groups, which were liberated with a

culture filtrate from T. reesei (513). Synergy between xylanases, β-xylosidase and acetyl
78
esterase of T. reesei was shown to be essential for the production of xylose from steamed

birch xylan. Hydrolysis of the high molecular weight fraction of steamed birch wood

hemicellulose by xylanase alone produced only about 10% of the amount of xylose produced

by the whole set of enzymes (see Table 3).

10 ECONOMICS OF SUGAR PRODUCTION FROM CELLULOSIC BIOMASS

10.1 Cellulosic Biomass as a Feedstock

Cellulosic biomass is a low cost and abundant resource that has the potential to support large-

scale production of fuels and commodity chemicals. To provide a perspective on its cost,

cellulosic biomass at $44/dry metric ton is equivalent to petroleum at about $6/barrel on an

equal mass basis or about $12 to 13/barrel for an equivalent energy content (90). Some types

of cellulosic biomass such as pulp and paper sludge and sugarcane bagasse can be obtained at

virtually no cost in sufficient quantities to support introduction of conversion technologies,

and much more substantial amounts of materials such as herbaceous and woody crops are

projected to be available at less than about $44/dry ton (301, 514). Thus, it is apparent that

the cost of the resource is not a dominant concern but that the primary barrier is the cost of

converting cellulosic materials into valuable products. In the case of biological conversion

operations, the cost of hydrolyzing hemicellulose and cellulose in biomass into fermentable

sugars represents the primary cost barrier to widespread commercial use (301).

Another view of the economics of converting cellulosic biomass into fuels and chemicals can

be gained by considering the basics of the conversion process. If we focus on release of

sugars from the cellulose and hemicellulose portions for fermentation to ethanol as an

example, about 100 gallons of ethanol can be derived from a dry ton of cellulosic biomass for

mature technology. Thus, a feedstock costing about $40/dry ton would contribute about $0.40
79
to the cost of one gallon of ethanol. Then if we assume that the feedstock represents about

two thirds of the total cash and annualized capital cost of product as is typical for many

commodity products, the overall cost of making ethanol would be about $0.60/gal. These

values change with the carbohydrate content, yield of sugars and ethanol, and assumed capital

costs (515), but the overall concept suggests that ethanol could be produced from cellulosics

at low costs for mature technology. The challenge is to improve the technology sufficiently to

realize these costs.

10.2 Economics of Hydrolysis Based Technologies

Capital and operating costs have been estimated for biological conversion of cellulosic

biomass to ethanol and some chemicals via hydrolysis in a number of studies over the last

several years (82-84, 87-89, 92, 516-518). To support such analyses, serious efforts were

devoted to estimating performance through experimental research and analysis with support

from various engineering firms and vendors with experience in the field, and these studies

provide useful insight into cost trends for producing sugar intermediates as well as ethanol

and coproducts derived from these sugars. However, it is important to note that no such

processes are yet in operation, and important cost and performance assumptions for cost

projections have not been verified at a commercial scale. Furthermore, many firms

experienced in design, construction, and operation of such processes are reluctant to disclose

information that may be released into the public domain, and the results are specific to

particular process configurations, technologies, and data. In addition, biomass processing

costs tend to be site specific and will change with location. The financial structure for the

project in terms of such factors as use of debt, return on investment requirements, and tax

deductions will have a major impact on capital recovery costs as well. Also, published

studies often assume low interest rates with extended time scales for payment as may apply to
80
mature technology, but higher rates of return will be demanded to finance implementation of

first-of-a-kind processes. Ultimately, costs only become real when the process is fully

operational. Consequently, published technoeconomic evaluations should not be viewed as

providing absolute measures of process economics but as giving useful insight that can help

select promising paths for processing biomass, measure technical progress, and define

research opportunities with potential impact.

Initially, a wide range of acid and enzymatically catalyzed options were considered for

hydrolysis of cellulose and hemicellulose to sugars for production of ethanol and other

products. Many of the concentrated acid catalyzed approaches were projected to require

costly acid recovery operations, and several present significant safety and environmental

hazards (519). Dilute acid processes were projected to be more cost effective but suffered

from low yields (108). Although cellulase enzymes were expensive and did not achieve

optimal performance initially, nearly theoretical yields are conceptually feasible, and it was

believed that the technology could be improved through emerging tools in biotechnology (81,

87, 92). In light of this opportunity and reduced federal research budgets, research was

focused on improving enzymatically catalyzed conversion technologies (92).

At first, enzymatic processes were built around a sequential enzymatic hydrolysis and

fermentation approach in which cellulose was first enzymatically hydrolyzed to glucose

followed by fermentation to ethanol in a separate operation, and the selling price was

projected to be about $3.60/gallon for this configuration in 1979 with use of a fungal strain

known as QM9414 for cellulase production (81). Changing to cellulase from an improved

strain known as Rut C30 with better balance in enzyme activity components and lower end

product inhibition 3 years later dropped the projected cost to about $2.66/gallon. Next,

cellulase developed by Genencor Corporation and designated as 150L improved hydrolysis

further, and the projected cost dropped to about $2.25/gallon in 1985. By taking advantage of
81
reduced end-product inhibition for the simultaneous saccharification and fermentation (SSF)

configuration with this same 150L enzyme, the estimated cost of ethanol dropped to about

$1.78/gallon for the year 1986. At this point, the decision was made to keep the feed rate to

the processing facility constant rather than maintaining a constant ethanol capacity, and the

cost became only about $1.65/gallon through retention of economies of scale (81). An

important limitation of the technology to this point was that the five carbon sugars arabinose

and xylose could not be fermented to ethanol with high yields by known organisms, and

incorporation of a newly discovered genetically engineered organism that could use all sugars

dropped the total cash and capital recovery costs to only $1.22/gallon even though the capital

recovery factor was increased to somewhat better reflect private capital requirements for

mature technology (84, 85, 92). These values have been updated in two recent studies that

show how sensitive the cost projections are to key technical and economic parameters (88,

89). Several companies now are striving to commercialize the technology but face major

obstacles because of the high capital costs coupled with the risk of implementing first-of-a-

kind technology.

Overall, historical improvements in projected costs for converting cellulosic biomass to

ethanol can be grouped into two broad categories: overcoming the recalcitrance of cellulosic

biomass and overcoming the diversity of its sugars. The first category includes advances in

pretreatment that improved the recovery of sugars from hemicellulose while also opening up

the structure of cellulose to promote access by cellulase enzymes, and pretreatment with

dilute sulfuric acid realized both of these goals. Improvements in cellulase enzymes from the

original QM9414 to Genencor 150L also enhanced yields of glucose from cellulose while

reducing cellulase loadings. Although end-product inhibition limited glucose concentrations

and yields, application of the integrated SSF approach allowed improvements in yields while

reducing enzyme demands. Other changes such as use of cellobiose fermenting organisms in
82
SSF and supplementation with beta-glucosidase also enhanced performance. Addition of the

broth from cellulase production into the SSF fermentation improved results by taking

advantage of enzyme activity otherwise lost with the fungal bodies and converting substrate

left after enzyme production to ethanol (238, 520). Although these advances are all vital to

realizing competitive product costs, overcoming the diversity of biomass sugars was essential

if yields are to be sufficient to achieve low costs. In this case, insertion of two genes from the

bacteria Zymomonas mobilis into E. coli and other bacteria that naturally take up all

hemicellulose sugars resulted in the nearly theoretical yields of ethanol needed (242, 521,

522).

Over the years, relatively little research has been funded on improving cellulase production

for hydrolysis of pretreated cellulose, and some uncertainty and even controversy surrounds

its cost. Early studies projected that cellulase costs were important but not a major single

factor (84, 301). However, more recent investigations have projected greater impact for

cellulase. For example, the cost of making cellulase from pretreated biomass with technology

viewed as being currently viable was projected for a process with a feed of 2,000 dry metric

tons per day of cellulosic biomass (516). For a feedstock costing $25/dry ton, the overall cost

of enzyme was projected to be about $0.32/gallon of ethanol produced, with the capital costs

estimated at $0.126/gallon, electricity at $0.107/gallon, and fixed costs, feedstock, nutrients,

and other raw materials costing about $0.085/gallon. However, a number of key assumptions

had to be made about the performance of the enzyme production and cellulose hydrolysis

process that have important implications for the cost projections. More recently, companies

experienced in commercial cellulase development and production have been contracted to

reduce the cost of making cellulase for large scale application to hydrolyzing cellulose to

glucose for fermentation to ethanol and other products.

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10.3 Application to Other Products

Although the majority of technoeconomic analyses of hydrolysis-based conversion processes

have focused on manufacture of ethanol from cellulosic biomass, most of the operations used

for making ethanol are applicable for producing sugars that could be fermented to several

chemicals, and a few studies identified possible products to make from such sugars (523,

524). Furthermore, criteria were developed and applied to find economically promising

products, and more detailed process designs were used to estimate costs for some of the better

possibilities (518, 525). In general terms, most of these require fermentation yield

improvements to be economically competitive with fossil based products, although making

succinic acid and a few other products appear promising now. In addition, lactic acid, 1-3

propanediol, and other new products being introduced for corn glucose fermentation could

benefit from cellulosic biomass sugars.

Another study examined making both ethanol and chemicals from hemicellulose and cellulose

hydrolysis sugars in a single cellulosic refinery, and important economic synergies were

projected when fuels and chemicals were produced in the same facility (517). In particular,

economies of scale associated with using large equipment were projected to be significant and

appeared to outweigh increases in feedstock transportation costs with facility size, similar to

findings by others (89, 526). Markets for many chemicals that could be made from

fermentation of hemicellulose and cellulose sugars were too small to support such a large

facility, while converting these sugars to ethanol for use as a fuel would require many such

large operations (518, 527). Thus, the low cost sugars that would result from operating a

large scale ethanol facility make production of higher margin chemicals more profitable than

they would be otherwise, and making both fuel and chemicals resulted in lower costs for

either one than making just one in a dedicated facility for the same rate of return on capital.

84
Furthermore, burning lignin and other components not fermented to products could produce

all the heat and electricity needed to run the conversion process, and electricity in excess of

that needed for the process could be sold at a low price for generation of baseload power,

further enhancing the synergies of the cellulosic biomass refinery.

10.4 Opportunities for Cost Reductions

Because cellulase is costly, sensitivity studies were used to estimate how technology

improvements could impact cellulase costs in one investigation (516). Raising cellulase

productivity on mixtures of hemicellulose sugars and pretreated cellulose from currently

assumed values of about 75 to 200 FPU/(L-hr) lowered enzyme costs rapidly to about

$0.20/gallon of ethanol by reducing both equipment and power demands, two of the largest

contributors to costs. However, batch cellulase production that entailed filling, operating,

draining, and cleaning cycles was assumed, and only the operating portion of the total cycle is

reduced as productivity was enhanced. Thus, the benefits of increases in productivity

diminished rapidly if the productivity was raised above about 200 FPU/L-hr, implying that a

continuous enzyme production system would be needed to realize additional benefits.

Increasing cellulase specific activity from 600 FPU/gram of protein also dramatically reduced

costs initially, but the improvements dropped off rapidly beyond about 1,600 FPU/gram

because of the power law dependence of equipment costs on scale of operation. Cellulase

costs drop nearly linearly with reductions in enzyme loading during hydrolysis, suggesting

that pretreatments that make cellulose more easily hydrolyzed to glucose by enzymes would

significantly reduce processing costs as well. Because of the dominance of capital, aeration,

and other costs, changing feedstock costs had little effect on enzyme economics.

Another study was undertaken with the goal of determining if an advanced technology

scenario could be defined that could reduce ethanol production costs enough to compete with
85
conventional fuels in an open market (301). In particular, advanced pretreatment technology

based on hemicellulose hydrolysis and use of a single organism for cellulase production,

cellulose hydrolysis, cellulose sugar fermentation, and hemicellulose sugar fermentation were

substituted for previously assumed sequential process operations. Complete material and

energy balances were applied for one scenario based on advanced technology that was

believed to be achievable through analogy with similar commercial systems and for another

scenario that assumed the best performance conceivable. Next, operating and capital costs

were estimated. From this, the overall costs of making cellulosic ethanol including both cash

costs and capital recovery charges were calculated to be about $0.50/gallon and $0.34/gallon

for the advanced and best technology cases, respectively. Such low costs would be

competitive with gasoline for use of ethanol as a pure fuel without subsidies.

The advanced pretreatment technology assumed that biomass could be pretreated with high

hemicellulose yields without acids or milling to fine particles and that the hydrolysate would

be fermentable without preconditioning based on observations for liquid hot water

pretreatment (384). Such a pretreatment is also expected to require lower cost materials of

construction. It was further assumed that use of the consolidated biological processing (CBP)

for cellulose hydrolysis and sugar fermentation could realize cellulose hydrolysis yields of

92%, fermentation yields to ethanol of 90%, and an ethanol concentration of 5% by weight in

a fermentation time of 36 hours. In addition, CBP was conducted in continuous fermenters to

eliminate costly seed vessels. The processing facility was also assumed to process about 2.74

million dry tons per year of feedstock costing $38.60/delivered dry ton to achieve some

economies of scale. Overall, the key drivers in reducing costs were simplification of both the

pretreatment and biological conversion steps with the potential of achieving such low costs

limited if the process configuration was not changed from process designs currently projected

for near term commercial use.

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It is also important to note that although concerns are sometimes raised about high-energy use

and costs for ethanol purification, modern distillation and dehydration technology is able to

recover ethanol in high purity at reasonable costs (88, 301). As a result, even dramatic

improvements in this area would have little impact on costs, and research funds are better

spent on advancing technology for hydrolyzing cellulose and hemicellulose to sugars, that is,

on overcoming the recalcitrance of cellulosic biomass.

11 BENEFITS AND IMPACTS OF CELLULOSIC BIOMASS CONVERSION

11.1 Greenhouse Gas Reductions

Perhaps the most unique and powerful attribute of converting cellulosic biomass to fuels and

chemicals is very low net greenhouse gas emissions, particularly when compared to other

options for making organic transportation fuels and chemicals (87, 528-531). For ethanol

production in particular, non-fermentable and unconverted solids and wastes left after making

targeted products can be burned to provide all the heat and electricity to run the process, and

no fossil fuel is required to operate the conversion plant for mature technology (84, 85, 89).

Similar results are expected for making many chemicals from biomass provided process heat

and power requirements are not excessive. Additionally, low levels of fertilizer and

cultivation are needed for many lignocellulosic crops, thereby keeping energy inputs for

growing biomass low (514, 528). The result is that most of the carbon dioxide released

during ethanol manufacture and utilization in a cradle-to-grave or lifecycle analysis is

recaptured when new biomass is grown to replace that harvested, and the net release of carbon

dioxide is less than 10% of that for gasoline (530-533). Use of renewable resources in other

portions of the ethanol production cycle (e.g., fueling farm machinery with ethanol instead of

diesel fuel) would further reduce carbon dioxide release. Furthermore, if electricity exported
87
from the facility is assumed to displace generation by coal or other fossil fuels, essentially no

net carbon dioxide is released into the atmosphere (534).

Because the transportation sector emits about one third of all carbon dioxide, the dominant

greenhouse gas, in the United States and the United States is responsible for about 25% of

world greenhouse gas emissions, making ethanol from cellulosic biomass can have a large

benefit if applied on a large scale (535). In addition, ethanol is a versatile liquid fuel,

currently produced from corn and other starch crops, and is widely accepted by vehicle

manufacturers and users for blending with over 10% of the gasoline consumed in the United

States. Furthermore, light duty vehicles are also manufactured that use blends containing up

to 85% ethanol with gasoline. Thus, cellulosic ethanol could be rapidly integrated into the

vehicle fleet and would not require extended timeframes to realize its benefits.

Although fossil fuel use and greenhouse gas emissions for production of chemicals from

cellulosic biomass have not been examined as extensively, many such processes would be

expected to also reduce greenhouse gas emissions. First, the fossil energy requirements to

plant, grow, harvest, and transport cellulosic biomass is the same whether ethanol and

chemicals are produced. For anaerobic fermentations to chemicals, the amount of energy to

convert biomass into sugars and ferment those sugars should also be similar with some minor

differences expected in the amount of energy to stir fermenters due to possible differences in

residence times and fluid viscosity, a relatively minor impact. However, unlike ethanol, many

chemicals are lower in volatility than water, and more energy may be required to recover

some chemicals from the fermentation broth. Nonetheless, for many processes, the

requirements should still be largely covered by heat and power from lignin combustion,

minimizing if not eliminating the need for fossil inputs. Finally, many chemical products,

such as succinic acid, have a virtually one-to-one weight yield from biomass sugars, without

release of CO2, while almost half the weight of the sugars is released as CO2 during sugar
88
fermentation to ethanol. If the chemical is used quickly and ultimately burned or decomposes

in landfills, the CO2 balance is very similar to that for cellulosic ethanol except for whatever

generally small differences result from product recovery. On the other hand, if the chemical

is made into plastics or other products with long lifetimes, CO2 is actually sequestered,

effectively drawing CO2 out of the atmosphere when we grow plants and converting that CO2

into forms that have extended lifetimes. Nonetheless, low energy input processes such as

anaerobic fermentations must be employed because some aerobic fermentations result in

greater fossil fuel use than needed to make their petrochemical counterparts (536).

11.2 Strategic Benefits

Despite much political clamour to reduce petroleum use during several energy crises in which

petroleum supplies were tightened and prices ballooned, petroleum imports have grown to

two thirds of U.S. petroleum use, the largest fraction ever, making the country vulnerable to

supply disruptions and price hikes. In addition, petroleum remains the largest and still

growing source of energy in the United States, providing almost 40% of about 100 quads of

total energy (quadrillion BTUs or 1015 BTU) (535). While other energy sectors are well-

diversified, over 96% of transportation energy is from petroleum, and the transportation sector

uses about two thirds of all petroleum consumed (535). Many chemicals also are derived

from petroleum. Today, an interruption in oil supplies or increase in petroleum costs would

badly damage or even cripple transportation and organic chemicals production.

Although there is controversy in estimating biomass availability, use of petroleum for making

chemicals is far less than for fuels, and there is almost certainly more than enough cellulosic

biomass to support manufacturing all the chemicals that could be derived from this resource

(86), and many studies estimate that enough cellulosic biomass could be available from

wastes and dedicated energy crops to make a significant dent in the huge amount of gasoline
89
consumed in the United States (514, 528). Furthermore, improved efficiency in fuel

utilization could dramatically reduce biomass demands, making it even more likely that all

light duty vehicles could be powered by cellulosic ethanol. Many types of cellulosic biomass

such as alfalfa and switchgrass contain significant levels of protein, and over half the

agricultural crop land in the United States is used to grow animal feed (427, 428, 537, 538).

Development of technology for recovery of animal feed quality protein from cellulosic

biomass would allow land to be used to support both fuel and feed production, greatly

enhancing fuel production potential. Overall, more efficient transportation coupled with more

complete capitalization on the ingredients in biomass crops should make it possible to meet

both fuel and feed needs from biomass. Use of cellulosic biomass has been called the “New

Petroleum” because of its tremendous, although still untapped, potential (539).

11.3 Solid Waste Disposal

Disposal of wastes is a mounting problem as the population continues to grow and we seek to

maintain and improve the environment. For example, farmers must reduce the burning of rice

straw after a harvest in northern California and less waste wood can be burned in British

Columbia to improve air quality. Suppression of natural forest fires has resulted in dense

forests that cause more damage to the soil and mature trees because hotter fires result when

they finally rage beyond control, and many are seeking to thin the forests to restore them to

their natural plant density. Converting such waste biomass into fuels and chemicals through

hydrolysis of cellulose and hemicellulose would provide a valuable solution to these growing

problems while also generating valuable products. However, solid waste disposal via

hydrolysis to sugars for fermentation and reaction to valuable products is under appreciated

and deserves far more attention. In addition, many chemicals that can be made from biomass

are biodegradable, reducing the accumulation of solid wastes and facilitating waste treatment
90
for liquids and gases. Thus, production of such materials could provide an economically

attractive path to solid waste disposal.

11.4 Economic Benefits

Conversion of cellulosic biomass to fuels and chemicals requires ready access to large

amounts of raw material, and such facilities must be located in rural areas to keep raw

material transportation costs as low as possible. Thus, relatively high-paying, skilled,

manufacturing jobs for well-trained chemists, biologists, operators, and maintenance and

supervisory personnel are created in rural America. In addition, new jobs must be developed

on farms to plant, grow, and harvest cellulosic biomass, and opportunities emerge for trucking

and railroad employment to ship biomass, chemicals, nutrients, and other supplies to the plant

and to transport product to markets. Additional jobs are also generated in local communities

through increased demand for housing, schools, stores, etc.

11.5 International Fuels Market

Energy demand is growing rapidly in developing countries (540, 541), and increased mobility

will become more important as well, driving up the demand for transportation fuels. Given

that transportation is so dependent on petroleum now, meeting this growing demand will be

challenging unless new fuel sources are developed. Producing fuels from domestic sources of

biomass would also reduce trade deficits for both developed and developing countries with

limited petroleum resources and help grow their economies.

91
11.6 Sustainable Production of Organic Fuels and Chemicals

When one lines up all sustainable resources and compares them to human needs, biomass

provides the only known route to produce organic fuels and chemicals sustainably (528).

Furthermore, liquid fuels provide many important attributes for transportation including ease

of fueling and storage and relatively high energy density. Thus, fermenting of sugars released

by hydrolysis of cellulose and hemicellulose to ethanol or other liquid fuels can provide a

unique and powerful path to making fuels for transportation on an ongoing basis. It can also

help diversify a transportation sector that is almost totally dependent (>96%) on one source,

petroleum, for energy. In addition, most organic chemicals also come from petroleum and

other fossil resources (535), and making these products from biomass would help stabilize the

price and availability of organic chemicals. Although sugar and starch now supply

fermentation sugars for manufacture of several products, hydrolysis of cellulosic biomass can

provide abundant, low cost sugars that could support even larger scale uses provided

processing costs are reduced.

12 ACKNOWLEDGMENTS

The authors would like to thank the Thayer School of Engineering at Dartmouth College, the

National Renewable Energy Laboratory, and the Office of the Biomass Program of the United

States Department of Energy for making preparation of this chapter possible.

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156
Table 1

Enzymes Hydrolysis products, % of d.w.

Methyl glucuronic Xylo-oligomers

Reducing sugars
acid (X1-X5)

XYL 0.25 14.5 13.0

α-GLU 0.12 0.0 0.1

XYL + α-GLU 0.50 18.8 21.1

β-X 0.12 1.4 1.6

XYL + β-X 0.62 20.2 14.9

β-X + α-GLU 1.12 5.8 5.3

XYL + β-X + α-GLU 4.88 33.5 28.8

157
Table 2

Cellulase and hemicellulase activities Activity

FPU* 20 FPU/g

HEC * 6100 nkat/g

Mannanase 1200 nkat/g

Xylanase 8200 nkat/g

β-glucosidase 950 nkat/g

β-mannosidase 15 nkat/g

β-xylosidase 100 nkat/g

α-arabinosidase 160 nkat/g

α-galactosidase 140 nkat/g

158
Table 3.

Enzymes Hydrolysis products, % of d.w.

Acetic acid Xylose Xylobiose

XYL 4.5 6 6.0

β-X 0.5 6 0

AE 1.2 0 0

XYL + β-X 5.9 25 1.0

XYL + AE 8.5 8 16.0

β-XYL + AE 3.2 16.0 0

XYL +β-X + AE 9.4 42.0 1.0

XYL + β-X + AE + α-GLU 12.0 50.0 3.0

159
Figure 1.

160
Figure 2.

161
Figure 3.

162
A.

Ph
⇒
MeGlcA Ara Ac Ara
Ö  ⇒ x 
Xyl — Xyl — Xyl — Xyl —Xyl — Xyl —Xyl — Xyl — Xyl

→
Xyl — Xyl

Î
→ Endoxylanase Ö α-Glucuronidase ⇒ Esterase
Î β-Xylosidase x α-Arabinosidase

B.

Gal Ac Ac
Î ⇒ ⇒
Glc — Man — Man — Glc — Man — Man — Man
→

Man — Man Glc — Man

x

→ Endomannanase Ö β-Glucosidase Ö
⇒ Esterase
Î α-Galactosidase x β-Mannosidase

Figure 4.

163
 Ethanol

Biomass Conveying Pretreatment & SSCF Ethanol

feedstock & cleaning conditioning fermentation recovery

Cellulase
production

Steam and Steam &

power electricity
Treated water
generation for process
for recycle or
disposal and export

Ash

Figure 5

164
 CH3 CH3 2 CH3

1
H O O H H O O H H O O

H H O H H O H
HO H O H O
OH H OH OH H
H OH H OH H
H H H
O O OH
H H H O H H O
O
H H H H O H
O
OH H O 3
OH H
H O O H
H 4 O O
H H OH
OH
HO H
H OH 5

OH
CH3
O
O
H3C
OH

H OH
O O
CH3 H
1 OH H
2
H O
H O O H H H O
3
H OH
H H O H H O H
HO O O
OH H H OH
H H OH H H H
H OH OH H
O H OH
H O H H O H H
O O
H H OH H H H

CH3
6
7
H O O H
H H O
HO OH
OH H
H H
H OH
O
H O H

H H OH

Figure 6

165
Table 1. Synergy of different xylanolytic enzymes purified from T. reesei in the hydrolysis

of deacetylated glucuronoxylan. Abbreviations: α-GLU, α-glucuronidase 740

nkat/g; β-X, β-xylosidase 1000 nkat/g; XYL, xylanase pI 5.5 10 000 nkat/g,

hydrolysis 40°C for 24 hours (508).

Table 2. Cellulase and hemicellulase activities in Celluclast supplemented with β-

glucosidase (Novozym 188; *β-glucosidase activity excluded), calculated at a FPU

dosage of 20 FPU/gram of substrate.

Table 3. Hydrolysis of steamed birchwood hemicellulose fraction by different xylanolytic

enzymes from T. reesei. Abbreviations: XYL, xylanase 12 000 nkat/g, β-X, β-

xylosidase 500 nkat/g; AE, acetyl esterase 500 nkat/g; α-GLU, α-glucuronidase 20

nkat/g; hydrolysis 45°C for 24 hours (513). This substrate from the steaming

process was a technical preparation containing about 70–80% xylans, the remainder

consisting of extractives, tannins, and eventually pectic substances and solublized

lignin. In addition to the synergism needed for the complete breakdown of the

xylan, other enzymes may also be needed, depending on the raw material.

166
 Figure 1. Cellobiose, the basic repeating unit of cellulose.

Figure 2. Flat, ribbon like conformation of cellobiose as found in the linear cellulose chain.

Figure 3. Cellohexaose showing the planar conformation and 180o flipping of the cellobiose

residues in linear cellulose.

Figure 4. Enzymes participating in the hydrolysis of xylans (A) and glucomannans (B): Ac,

acetyl; Ph, phenolic groups; Ara, arabinose; MeGlcA, methyl glucuronic acid; Xyl,

xylose; Gal, galactose; Glc, glucose; Man, mannose

Figure 5. A simplified process flow diagram for hydrolysis of hemicellulose and cellulose to

sugars for fermentation to ethanol based on dilute acid pretreatment, enzymatic

hydrolysis of cellulose, and fermentation of both cellulose and hemicellulose to

ethanol in the same vessel as for cellulose hydrolysis by the SSCF operation.

Figure 6. Enzyme activities in depolymerization of arabinoxylan. The structure is a

generalized diagram of arabinoglucuronoxylan. Enzyme activities are 1)

endoxylanase, 2 acetyl xylan esterase, 3) α-L-arabinofuranosidase, 4) α-D-

glucuronidase, 5) ferulic acid esterase, 6) acetyl esterase, and 7) β-xylosidase

167

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