Biomass Fundamentals Modules 12: Cellulose & Hemicelluloses A capstone course for BioSUCCEED: Bioproducts Sustainability: a University Cooperative Center of Excellence in EDucation The USDA Higher Education Challenge Grants program gratefully acknowledged for support

This course would not be possible without support from: USDA Higher Education Challenge (HEC) Grants Program

Cellulose  -D-anhydroglucopyranose units linked by (1,4)-glycosidic bonds

Cellulose  -D-anhydroglucopyranose units linked by (1,4)-glycosidic bonds

Why the Cellulose Story is so Convoluted? Polymorphism or allotropy refers to the existence of more than one crystalline forms differing in physical and chemical properties. Four major polymorphs of cellulose have been reported –Cellulose I, –Cellulose II, –Cellulose III, –Cellulose IV

Early Models of Cellulose Morphology Fringed Micellar Structure Micellar Structure Before Polymers After Polymers Blocks of material mixed with amorphous material Alignment of chains followed by folding; entropically not very favourable

More Recent Models of Cellulose Morphology Microfibrils exist as discrete crystalline regions of >600Å, separated by less ordered amorphous domains. No chain folding

What is Happening During Acid Hydrolysis? The non-ordered domains of the long Microfibrils are degraded leaving behind the discrete crystallites

Packing of the Cellulose Microfibrils in Cell Wall Tc R R R R

or Tc R R R R R R

First Reported Unit-Cell for Cellulose Meyer-Mark-Misch- Chains run anti-Parallel within the unit cell. Meyer et al. (1929)

Gardner & Blackwell Chains run Parallel within the unit cell. Gardner and Blackwell (1974) The unit cell parameters were almost double that of the Meyer-Mark and Misch unit cell. a = Å, b = 15.72Å, c = 10.38Å (fiber axis), angle β = 97 o.

In summary for Native Cellulose I Blackwell Unit-cell Parallel chain orientation Accepted today Meyer-Misch Unit-cell Anti-parallel chain orientation

How does one go about unequivocally confirming chain orientation? Nature has provided a way to do so. The fact that cellulose has two distinct ends a reducing end group (aldehyde) and a non-reducing end group allows for some creative chemistry to be done

Staining of the Chain Ends R NR Hieta et al., Biopolymers, 23, 1807,1984

Elegant Proof of Chain Orientation by selective staining of the reducing end- groups Kuga et al. (1984) and Chanzy For parallel model one expects the black dots to preferentially be on one end of microfibrils.

Cellulose I Intra-molecular Hydrogen-Bonding The intra-molecular hydrogen-bonds are responsible for the stiff and rigid nature of the cellulose molecule. Due to the equatorial orientation of the hydroxyl groups and its linear structure, cellulose molecules have a strong tendency to form intra- and inter-molecular hydrogen-bonds.

Cellulose I Intra-molecular Hydrogen-Bonding Two kinds of such bonds form within the same chain: C3-OH with endocyclic oxygen C6 –OH (primary ) with the C2-OH

Cellulose I Inter-molecular Hydrogen-Bonding However, one kind of H-bond forms between neighboring chains C3 OH and C6 OH

Cellulose nanofiber bundles 6 assembly proteins (rosette) which produce cellulose nanofibers C. Haigler & L. Blanton Cellulose : Nature Working Across a Length Scale >10 10 ! ~28nm 1 /4

Cellulose II, Another from of Cellulose Created from From cellulose I –Via Mercerization % NaOH –And Regeneration - precipitated from solution Two-chain unit-cell –Anti-parallel chain orientation Of Lower crystallinity than Cellulose I

Mercerization Cellulose I Cellulose II

Why the Transformation? Mercerization; Heterogeneous alkali swelling Several mechanisms proposed –Conformational change “Bent” cellulose I to “twisted & bent” cellulose II –Recrystallization of cellulose II on cellulose I “shish-kebab” structure –Chain-folding –Progressive ‘shifting’ of sheets or chains

Why the Tansformation contd. Since the C 6 hydroxyl group is involved in two secondary valence interactions, it is precluded from interacting with molecules in neighboring planes (above and below). Therefore cellulose has a sheet-like structure with only weak van der Waals forces holding the sheets together. These sheets fall apart during mercerization New orientations and H-bonds may now form Along different planes C2 OH may now H- bond

Cellulose II Hydrogen-bonding Plane 1 Plane 2 Plane 3 New H bonds between the C2 OH groups in neighbouring planes can set in after mercerization.

Current Microscopic Model of Cellulose

Structural Models of a Typical Fiber

Hemicelluloses Structurally, hemicelluloses are co-polymers of two or more sugars and sugar acids –glucose, mannose, galactose, xylose, arabinose and 4-O-methylglucuronic acid They are of low DP with short branching chains, making them amorphous heteropolysaccharides

These are Heteropolysaccharides Supporting material in cell walls that vary from plant to plant and from one plant part to another. In woody plants, there are two basic types D glucomannans D glucuronoxylans The composition and amount of each is species dependent Softwood vs Hardwood Hemicelluloses

Softwood Hemicelluloses (major) galactoglucomannans The principle hemicellulose of softwoods is the galactoglucomannans (~ 20% of woody material) Alternating Glucose & Mannose along the main chain; Galactose branches off; Random acetates at C5 & C3 of main chain Note β-1-4 links along the main chain Note: Galactose C4 OH is axial Mannose C2 OH is axial

Softwood Hemicelluloses (major) galactoglucomannans The principle hemicellulose of softwoods is the galactoglucomannans (~ 20% of woody material) They are subdivided as : High Galactose content: Galactose 1/Glucose 1/Mannose/4 Low Galactose content Galactose 0.1/Glucose 1/Mannose/3

Softwood Hemicelluloses (minor) rabinoglucuronoxylans The minor hemicellulose of softwoods is the Arabinoglucuronoxylans (~5-10% of wood) Xylanose linked along the main chain; arabinose and glucuronic acid branches off Note β-1-4 links along the main chain Note: Xylan has no C6 primary OH Note: Arabinose is a Furan; 5 member ring sugar structure with a C4 primary OH

Harwood Hemicelluloses (major) glucuronoxylans The principle hemicellulose of hardwoods is the glucuronoxylans (15-30% of woody material) Note β-1-4 links along the main chain

Hardwood Hemicelluloses (minor) glucomannans The minor hemicellulose of hardwoods is the glucomannans (2-5% of wood) Note β-1-4 links along the main chain