Xyloglucans are components of the primary walls of most seed plants examined so far. Fucogalactoxyloglucans are the commonest structural type and occur in the primary walls of coniferous gymnosperms, eudicotyledons and in the non-commelinid monocotyledons, at least in onion (Allium cepa) and garlic (A. sativa) (3). These xyloglucans have a re­peating backbone subunit consisting of four (1^4)-linked (3-D-Glcp residues, with the three Glcp residues nearest the non-reducing end each bearing a single (1^6)-linked

Cellulose [(1—>4)-p-Glucan]

^4)-P-d-GIcp-(1^4)-P-d-GIcp-(1^4)-P-d-GIcp-(1->4)-P-d-GIcp-(1^ (1^3,1—>4)-p-Glucan

^3)-p — D-Glcp-(1 ^4)-P-d -Glcp-(1 ^4)-p — d — Glcp-(1 -^3)-P-d — Glcp-(1 ^ Heteroglucan [Fucogalactoxyloglucan]

a-D-xylopyranosyl (D-Xylp) residue (Figure 4.1). About half of the subunits have a L-fucopyranosyl (L-Fucp)-D-galactopyranosyl (D-Galp) side chain attached to the Xylp residue closest to the reducing end; about one half of the subunits with this side chain also have a Galp residue attached to the adjacent Xylp residue (3). In the eudicotyledon to — bacco/tomato family (Solanaceae) and related families, the xyloglucans are arabinoxyloglu — cans that contain L-arabinofuranosyl (L-Araf) but no L-fucopyranosyl (L-Fucp) residues (3, 20, 21). Fucose is also usually absent from the xyloglucans of the grasses; their xyloglucans also contain less xylose and much less galactose than the fucogalactoxyloglucans (3, 22, 23).

Acetyl groups also occur on xyloglucans, for example, in fucogalactoxyloglucans, the D-Galp residues may have an acetyl group attached to C(O)6 (24). In the Nicotianaplumbaginifolia (Solanaceae) XG, acetyl groups are found on 44% of the C(O)6 of the Glcp backbone residues not substituted with Xylp residues and at C(O)5 of 15% of the terminal Araf residues (25).

4.2.1.2.2 HETEROXYLANS (e. g, O-acetyl-(4-O-methylglucurono)xylans) Heteroxylans are the predominant non-cellulosic polysaccharides in many types of cell walls. They all have a basic backbone chain of (3-D-Xylp residues, linked through (1^4)- glycosidic linkages, and substituted by various monosaccharide and oligosaccharide side chains (2, 3, 26). Heteroxylans are the major non-cellulosic polysaccharides in the ligni­fied secondary walls of all eudicotyledons, including the hardwoods where they constitute 10-35% of the wood, and possibly the lignified secondary walls of all non — commelinid monocotyledons. In all these walls, the heteroxylans have 4- O-methyl-a-D- glucopyranosyluronic acid (4- O-methyl-a-D-GlcpA) residues attached to about every tenth Xylp residue (4-O-methylglucuronoxylans) (Figure 4.1) primarily to the C(O)2 and to a lesser extent the C(O)3 position of the Xylp residues. Acetyl groups are esterified to the Xylp residues; mostly at the C(O)2, but a small proportion at C(O)3, with ~3.5—7 acetyl groups per ten Xylp residues (27). However, variation in the structures of 4- O-methylglucuronoxylans occurs. For example, in the heteroxylan from the wood of the eudicotyledon Tasmanian blue gum (Eucalyptus globulus), some of the 4- O-methylglucuronic acid residues are substituted at C(O)2 with a-D-Galp (28).

In softwoods, heteroxylans are usually the second most abundant non-cellulosic polysac­charides, making up ~7—15% of the wood (27). As in hardwoods, these heteroxylans have 4- O-methyl-a-D-GlcpA residues attached predominantly at the C(O)2 position (about one residue every 5—6 Xylp residues), but they also have a-L-Araf residues attached mainly at the C(O)3 position of Xylp residues (about one residue in every 8 Xylp residues) (Figure 4.1) and are usually referred to as arabino(4- O-methylglucurono)xylans. Softwood heteroxylans are not acetylated.

In the vegetative organs of grasses, heteroxylans with similar structures to gym — nosperm arabino(4- O-methylglucurono)xylans are usually the most abundant non — cellulosic polysaccharides in both the primary and lignified secondary walls. These polysac­charides have single a-L-Araf and a-D-GlcAp (or its 4- O-methyl derivative) residues linked at C(O)3 and C(O)2, respectively, to the xylose residues of the (1^4)-S-D-Xylp backbone (Figure 4.1) and have traditionally been referred to as glucuronoarabinoxylans (GAXs) (3, 18, 29—31). Oligosaccharide side chains also occur that contain a number of monosac­charides including the following: L-Araf D-Xylp, D-Galp, and L-Galp. Ferulic acid (FA), small amounts of p-coumaric (pCA) and sometimes sinapic acid (SA), are esterified by their carboxyl groups to the C(O)5 hydroxyl of some of the arabinosyl residues, including those occurring singly and in oligosaccharides (32, 33). In GAXs from the walls of peren­nial ryegrass, and barley and wheat straw, alkali labile substituents are attached at C(O)2 and C(O)3 on 50% of the Xylp residues of the main chain. Acetyl groups could account for 50—70% of the substitutions (34). In GAXs from the walls of bamboo (Phyllostachys edulis) shoots, acetyl groups are on the Arafresidues (35). The acetyl substituents on wheat bran xylans are more labile than the hydroxycinnamic acid (HCA) esters, but no detectable bound acetate remained after extraction with 1 M KOH (36). GAXs in lignified secondary walls have backbones with a lower overall degree of substitution than GAXs in primary walls.

GAXs with similar structures to those of the grasses are also major non-cellulosic polysac­charides of the primary walls of all commelinid monocotyledons except for the palms (Arecaceae) (3, 37-40). They are also probably the major non-cellulosic polysaccharides of lignified secondary walls of all commelinid monocotyledons (2, 3). Small proportions of heteroxylans have been reported in the primary cell walls of eudicotyledons. In the walls of sycamore suspension-cultured cells, GAXs have side chains all linked at C(O)2 to the xylose residues of the (1^4)-S-D-Xylp backbone (41). In white clover (Trifolium repens), 75% of the Xylp main chain residues carry acetyl substituents (34).

4.2.1.2.3 HETEROMANNANS (galactoglucomannans and glucomannans) Galactoglucomannans are found particularly in the lignified secondary walls of coniferous gymnosperms (softwoods), where they are usually the predominant non-cellulosic polysac­charides, comprising ~ 12-18% of the wood (2, 3, 42, 43). They have a linear chain of (1^4)-linked р-D-mannopyranosyl (Manp) and (3-D-Glcp residues, randomly arranged, with a-D-Galp residues attached by (1^6)-linkages to both the Manp and Glcp residues (27) (Figure 4.1). The ratio of Glc:Man:Gal varies from 1:3-4:0.1-1.0. In galactogluco­mannans from spruce wood, more of the D-Galp residues are attached to Manp than Glcp residues (44). In this species, O-acetyl groups are attached mostly to some of the Manp residues at the C(O)2 and C(O)3 positions (43, 45). However, in the galactoglucomannans from wood of the Parana pine (Araucaria angustifolia), O-acetyl groups are reported to be attached at the C(O)3 position of 15.6% of Manp and 6.4% of Glcp residues (23).

Glucomannans are usually the second most abundant non-cellulosic polysaccharides in the walls of hardwoods, making up ~2-5% of the wood (27). They have backbone structures similar to the galactoglucomannans of softwoods, but lack Galp residues and usually a ratio of Glc:Man of ~1:2 although those from birch (Betula spp.) have a ratio of ~ 1:1. O-Acetylated glucomannans have been isolated from aspen (Populus tremula) and birch wood and, as with the galactoglucomannnans of the softwood spruce, the acetyl groups are attached to the C(O)2 and C(O)3 positions of some of the Manp residues (46).

Galactoglucomannans also occur in the primary cell walls of most seed plants (angiosperms and gymnosperms), but usually only in small proportions. Structural char­acterizations of galactoglucomannans obtained either from primary walls of various eu — dicotyledon suspension-cultured cells or from the extracellular culture medium have been made (47-50). Unlike the heteromannans from secondary walls, they contain about equal proportions of Glcp, Manp, and Galp, have alternating (3-D-Glcp and (3-D-Manp residues in the backbone, and either single a-D-Galp residues or (3-D-Galp-(1^2)-a-D — Galp residues attached at C(O)6 of the Manp residues. A similar galactoglucomannan but with a Glc:Man:Gal ratio of 1:1:0.5 has been characterized from primary walls of kiwifruit (Actinidia deliciosa) outer pericarp (51).

4.2.1.2.4 PECTIC POLYSACCHARIDES (pectins)

The pectic polysaccharides (pectins) are a family of complex polysaccharides that occur in the primary cell walls of all seed plants, where they are major components except in the walls of the grasses and other commelinid monocotyledons other than the palms (Arecaceae). The polysaccharides have a block or domain structure with four commonly occurring domains: homogalacturonan (HG), rhamnogalacturonan I (RG-I), rhamnogalacturonan II (RG-II), and xylogalacturonan (XGA) (52-54). Homogalacturonan (Figure 4.2), which is usually the most abundant domain, is composed of linear chains of a-D-galacturonic acid (a-D-GalAp)

Homogalacturonan

—>4)-a — D-GalpA-(1 —>4)-a — D-GalpA-(1 —>4)-a-D — GalpA-(1 —>4)-a- D-GalpA-(1 Rhamnogalacturonan I

—>2)-a-L-Rhap-(1—>4)-a-D-GalpA-(1—>2)-a-L-Rhap-(1-+4)-a-D-GalpA-(1—>

4

t

(1 ->5)-a-arabinan, (1 ->4)-p-galactan, arabino-4-galactan or arabino-3,6-galactan

(1—>5)-a-Arabinan

a — L-Araf

4

2

—>5)-a-L-Araf-(1-»5)-a-L-Araf-(1—>5)-a-L-Araf-(1^5)-a-L-Araf-(1—»

2 3

t t

a-L-Araf a-L-Araf

(1->4)-p-Galactan

^4)-p-D-Galp-(1^4)-p-D-Galp-(1—>4)-p-D-Galp-(1—>4)-p-D-Galp-(1- Arabino-4-galactan

a-L-Araf

4

5

a-L-Araf

I

3

->4)-p-D-Galp-(1^4)-p-D-Galp-(1->4)-P-D-Galp-(1->4)-p-D-Galp-(1

Arabino-3,6-galactan

P-D-Galp

4

6

P-D-Galp a-L-Araf-(1—>3)-p-D-Galp

4 4

6 6

-^3)-p-D-Galp-(1^3)-p-D-Galp-(1^3)-P-D-Galp-(1^3)-P-D-Galp-(1

6

t

p-D-Galp

6

T

p-D-Galp

Figure 4.2 Structures of pectic polysaccharides. Homogalacturonan, rhamnogalacturonan I (RG-I), and polysaccharide side chains found on RG-I: (1 ^5)-a-arabinan, (1 ^4)-|3-galactan, arabino-4-galactan, and arabino-3,6-galactan.

residues that maybe methyl-esterified and acetylated to varying extents. The RG-I domain, which is usually also an abundant domain, consists of alternating a-D-GalAp and a-L-Rhap residues (Figure 4.2). The a-D-GalAp residues of RG-I may be acetylated on C(O)2 or C(O)3, or both, but it is not known if these residues maybe methyl esterified (52, 53, 55). There is evidence that the HGA and XGA domains in pectin of the eudicotyledon apple (Malus domestica) are connected linearly with RG-I domains (56). Usually 20-80% of the Rhap residues in RG-I domains bear polysaccharide or oligosaccharide side chains rich in Araf and Galp residues, and include (1^5)-a-arabinans, (1^4)-p-galactans, arabino-4- galactans (57) and small proportions of arabino-3,6-galactans (Figure 4.2). Arabinans are composed of linear chains of a-L-Arafresidues joined by (1^5)-linkages, with side chains of single or multiple a-L-Araf residues attached through C(O)2, C(O)3 or both (Figure 4.2). Galactans consist mostly of linear (1^4)-p-D-galactan chains, and arabino-4-galactans, have a (1^4)-p-D-galactan backbone with side chains of a-L-Araf residues (Figure 4.2). Arabino-3,6-galactans, similar in structure to those described in the next section may also occur as side chains (55).

Ferulic acid has been shown to be ester-linked to Araf and Galp residues of RG-I in the primary walls of spinach (Spinacia oleracea), sugarbeet (Beta vulgaris), and amaranth (Amaranthus caudatus), all members of the Amaranthaceae (Caryophyllales) (58). Ferulic acid also occurs ester-linked to the primary walls in related “core” families of the Caryophyl­lales and is probably also linked to the side chains of RG-I (3, 59). In addition, a linear (1^4)-p-D-galactan, chemically identical to the galactan side chain on RG-I, occurs in the lignified secondary walls of compression wood of gymnosperms, although it is not known if it forms part of an RG-I (60). In the bast fibers of flax (Linum usitatissimum), both short (2-3 residues) and long (up to 28 residues) (1^4)-p-D-galactan chains are linked to RG-I domains (4).

A xylogalacturonan (XGA) domain occurs particularly in the walls of cells that have sep­arated, or are about to separate, from adjacent cells, for example, in the root cap, and has single S-D-Xylp residues attached to C(O)3 of the a-D-GalAp residues of an HG backbone (61). In contrast, the RG-II domain occurs ubiquitously, but only in low concentrations. RG-II is a highly complex, low-molecular mass (~5—10 kDa) domain that contains the fol­lowing different monosaccharides: the hexoses D-Glcp, D-and L-Galp; the hexuronic acids D-GlcpA and D-GalpA; the pentoses L-Araf L-Arap and 2Me-D-Xylp; the branched pentose D-Apif the deoxyhexoses L-Fucp, L-Rhap, and 2Me-L-Fucp; and the rare sugars L-aceric acid, 2-keto-3-deoxy-D-lyxo-heptulosaric acid, and 2-keto-3-deoxy-D-manno-octulosonic acid. At least seven galacturonic acid residues form a backbone to which are attached four structurally different side chains: A, B, C, and D. A contains 2Me-D-Xylp and is an oc — tasaccharide; B contains 2Me-L-Fucp and, depending on species, is a heptasaccharide, an octasaccharide or a nonasaccharide; C and D are disaccharides, C contains 2-keto-3-deoxy — D-manno-octulosonic acid and D contains 2-keto-3-deoxy-D-lyxo-heptulosaric acid. Except for the differences in the B-side chain, the structure of RG-II is highly conserved and occurs as a dimer cross-linked by 1:2 borate—diol esters between (3-D-Apif residues on adjacent RG-II domains (62).

4.21.26 ARABINO-3,6-GALACTANS

The arabino-3,6-galactans are water-soluble polysaccharides that occur in large proportions as deposits in the lumen of the tracheids in the wood of the coniferous gymnosperm larch (Larix spp.), and in smaller proportions in other softwoods (27, 63). They are composed of a backbone of (1^3)-linked p-D-Galp residues with a side chain attached at the C(O)6 position on nearly every residue. Many of the side chains are single p-D-Galp or a-L-Ara/ residues or the disaccharides p-L-Arap-(1^3)-a-L-Ara/-(1^ or p-D-Galp-(1^6)-p-D — Galp-(1^. In some species of larch, longer, branched side chains also occur (Figure 4.2) (64). Arabino-3,6-galactans are present in spruce, pine (Pinus sylvestris), and larch (Larix sibirica) heartwoods (65-67) and carry GlcAp. Structurally similar arabino-3,6-galactans occur as covalently bound forms, as minor side chains of RG-I (see Section 4.2.1.2.6) and on hydroxyproline-rich proteins in the ubiquitous arabino-3,6-galactan-proteins(AGPs) found associated with plasma membranes and also in exudate gums of Acacia spp. (27, 68-70).

Maize (Zea mays L.) is a monocotyledonous plant whose anatomical structure has been well studied. It resembles other grasses in the arrangement of tissues in the stem, leaf, and

Biomass Recalcitrance: Deconstructing the Plant Cell Wall for Bioenergy. Edited by Michael. E. Himmel © 2008 Blackwell Publishing Ltd. ISBN: 978-1-405-16360-6

Zea mays stem cell types

Primary cell walls

Parenchyma

Phloem

Air spaces

Intercellular air space Lacuna

Secondary cell walls

Vessel (protoxylem) Vessel (metaxylem) Bundle sheath Sclerenchyma (fibers) Epidermis

Figure 3.1 Transverse section of maize stem showing cell types.

root. The stems of monocots generally have epidermis, scattered vascular bundles, and parenchyma pith (Figure 3.1).

The biorefinery feedstock supply system encompasses all the unit operations necessary to move biomass feedstocks from the land to the biorefinery (29). An overview of the feedstock supply system is depicted in Figure 2.3. Biomass production is the beginning of the feedstock

supply chain. It involves producing biomass feedstocks to the point of harvest. Production addresses important factors such as selection of feedstock type, land use issues, policy issues, and agronomic practices that drive biomass yield rates and directly affect harvest and collection operations.

Harvesting and collection encompasses all operations associated with getting the biomass from its source to the storage or queuing location. In addition to obvious operations such as cutting (or combining, swathing, or logging) and hauling, this often includes some form of densification such as baling, bundling, or chipping to facilitate handling and storage. Storage and queuing are essential operations in the feedstock supply system. They are used to deal with seasonal harvest times, variable yields, and delivery schedules. The objective of a storage system is to provide the lowest-cost method (including cost incurred from losses) of holding the biomass material in a stable, unaltered form (i. e., neither quality improvements nor reductions) until it is called for by the biorefinery.

Prior to conversion, the feedstock must be preprocessed to physically transform it into the format required by the biorefinery. Preprocessing can be as simple as grinding and preparing the biomass for increased bulk density or improved conversion efficiency, or it can be as complex as improving feedstock quality through fractionation, tissue separation, and blending.

Transportation generally consists of moving the biomass from the storage location to the biorefinery via truck, rail, barge, or pipeline. The system used directly affects how the feedstock is handled and fed into the conversion process. Transporting and handling methods are driven by the format and bulk density of the material; this makes them highly dependent on each other and on all the other operations in the feedstock supply chain.

Whereas plant cell walls have been measured by electron microscopy (EM) (13, 32-36), the drawback of EM techniques lies in the sample preparation and imaging processes commonly used, which involve chemical extraction, dehydration, and embedding. Because biomass samples go through both chemical pretreatment and enzymatic hydrolysis, methods must be developed to minimize the disruption of sample preparation, and conduct imaging under physiological conditions. Recently developed imaging methods, such as AFM, nonlinear optical microscopy, and single molecule methods, are capable of imaging the cell wall at nanoscale resolution without extensive sample preparation. These techniques can also be applied to directly map the macromolecular structures of cell walls, and to visualize their degrading enzymes simultaneously.