bioconversion of hemicellulose

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Table of Contents

1. Introduction .......................................................................................................................... 1

1.1. Plant cell wall .................................................................................................................. 1

2. Chemistry of Cellulose ......................................................................................................... 3

2.1. Introduction ..................................................................................................................... 3

2.2. Structure of Cellulose ..................................................................................................... 3

3. Chemistry of Hemicellulose ................................................................................................ 6

3.1. Introduction ..................................................................................................................... 6

3.2. Holocellulose .................................................................................................................. 7

3.3. Structure of Hemicellulose ............................................................................................. 7

3.3.1. Hardwood Hemicelluloses ....................................................................................... 8

3.3.2. Softwood Hemicelluloses ........................................................................................ 9

3.4. Composition of Hemicellulose in various feedstocks ................................................... 10

3.4.1. Xylans & Mannans ................................................................................................ 10

4. Hydrolysis of Hemicellulose .............................................................................................. 11

5. Bioconversion of Hemicellulose ........................................................................................ 12

5.1. Introduction ................................................................................................................... 12

5.2. Bioconversion by Enzymatic Hydrolysis ...................................................................... 13

5.2.1. Hemicellulase ......................................................................................................... 13

5.3. Bioconversion by Microbial Organisms ....................................................................... 15

5.3.1. Introduction ............................................................................................................ 15

5.3.2. Fermentation by Fungi ........................................................................................... 15

5.3.3. Fermentation by Bacteria ....................................................................................... 18

5.4. Synergic activities between enzymes............................................................................ 18

6. Effect of pre-treatment ...................................................................................................... 20

7. Comparison of various methods ....................................................................................... 22

8. Conclusion .......................................................................................................................... 23

9. Reference ............................................................................................................................ 25

LIST OF SYMBOLS & NOTATIONS USED .................................................................... 27

Bioconversion of Hemicellulose

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1. Introduction

1.1. Plant cell wall

Lignocellulose, the major component of biomass, makes up about half of the matter produced

by photosynthesis. It consists of three types of polymers cellulose, hemicellulose, and lignin

that are strongly intermeshed and chemically bonded by non-covalent forces and by

covalent cross-linkages. Lignocelluloses in nature derive from wood, grass, agricultural

residues, forestry wastes and municipal solid wastes.

The major component of lignocellulose materials is cellulose, along with lignin and

hemicellulose. Cellulose and hemicellulose are macromolecules from different sugars;

whereas lignin is an aromatic polymer synthesized from phenylpropanoid precursors. The

composition and percentages of these polymers vary from one plant species to another.

Moreover, the composition within a single plant varies with age, stage of growth, and other

conditions. Long cells enveloped by a characteristic cellular wall form wood. This wall is a

complex structure that acts at the same time as plant skin and backbone.

Cellulose makes up about 45% of the dry weight of wood. This lineal polymer is composed

of D-glucose subunits linked by -1,4 glucosidic bonds forming cellobiose molecules. These

form long chains (called elemental fibrils) linked together by hydrogen bonds and van der

Waals forces. Hemicellulose and lignin cover microfibrils (which are formed by elemental

fibrils). The orientation of microfibrils is different in the different wall levels. These

microfibrils group together and constitute cellulose fiber. Cellulose can appear in crystalline

form, called crystalline cellulose. In addition, there is a small percentage of non-organized

cellulose chains, which form amorphous cellulose.

Hemicellulose is a complex carbohydrate heteropolymer and makes up 2530% of total wood

dry weight. It is a polysaccharide with a lower molecular weight than cellulose. It consists of

D-xylose, D-mannose, D-galactose, D-glucose, L-arabinose, 4-O-methyl-D-glucuronic acid,

D-galacturonic and D-glucuronic acids. Sugars are linked together by -1, 4- and

occasionally -1,3-glucosidic bonds. The principal component of hardwood hemicellulose is

glucuronoxylan, whereas glucomannan is predominant in softwood.


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Lignin is the most abundant aromatic polymer in nature. It is present in the cellular cell wall,

conferring structural support, impermeability, and resistance against microbial attack and

oxidative stress. Structurally, lignin is an amorphous heteropolymer, non-water soluble and

optically inactive; it consists of phenylpropane units joined together by different types of

linkages. The polymer is synthesized by the generation of free radicals, which are released in

the peroxidase-mediated dehydrogenation of three phenyl propionic alcohols: coniferyl

alcohol (guaiacyl propanol), coumaryl alcohol (p-hydroxyphenylpropanol), and sinapyl

alcohol (syringyl propanol). Coniferyl alcohol is the principal component of softwood

lignins, whereas guaiacyl and syringyl alcohols are the main constituents of hardwood

lignins. The final result of this polymerization is a heterogeneous structure whose basic units

are linked by C-C and aryl-ether linkages, with aryl-glycerol -arylether being the

predominant structure.

Figure 1.1: Configuration of plant cell wall

The biological degradation of cellulose, hemicellulose, and lignin has attracted the interest of

microbiologists and biotechnologists for many years. The diversity of cellulosic and

hemicellulosic substrates has contributed to the difficulties found in enzymatic studies. Fungi

are the best-known microorganisms capable of degrading these polymers. Because the


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substrates are insoluble, both bacterial and fungal degradation have to occur exocellularly,

either in association with the outer cell envelope layer or extracellularly. The most important

type of extracellular enzymatic system is the hydrolytic system, which produces hydrolases

like cellulose & hemicellulase and is responsible for cellulose and hemicellulose degradation.

But before studying the bioconversion of these polymers, one has to know about the

constituting monomers, their structure and the linkages between these monomers

2. Chemistry of Cellulose

2.1. Introduction

The chemistry of cellulose can be dated back to 1838 when it was believed that the cell

wall of plants is not made of one uniform chemical substance but peculiar to each species.

But the subsequent works, which involved the extraction of samples from the plants under

more severe conditions, proved that the fibrous tissue of all young plant cells consists of

one uniform chemical substance: a carbohydrate comprised of glucose residues and

isomeric with starch (C, 44.4%; H, 6.2%), which was named as CELLULOSE.

In spite of opposition, where it was believed that in the plant, the cellulose, lignin, pectin,

and fatty material merged into one another by insensible chemical gradations, the concept

of cellulose as the carbohydrate portion of the cell wall derived exclusively from glucose

was finally accepted.

2.2. Structure of Cellulose

Although the presence of Cellulose in cell wall was confirmed in the early 1850s, it took

seventy more years in defining the proper structure of cellulose. Thanks to the knowledge

gained through the development of several related sciences, such as, advances made in the

chemistry of the simple sugars, in x-ray diffraction, and in colloid chemistry, the structure

of cellulose as a linear macromolecule consisting of anhydroglucose units finally came to

the picture by early 1920s.


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The contribution of Methylation chemistry was significant to this development. A simple

methylation reaction involves replacement of OH group by an OCH3 group and by

detecting this OCH3 group, we could confirm the position of OH group. Thus, it was

shown that the hydroxyl groups occupied the 2nd

, 3rd

, and 6th

position of each

anhydroglucose unit. But this study does not give any proof of cellulose as a linear

macromolecule. This absence of proof led to another theory commonly called the

Association theory or Micellar theory, which was based on the idea that cellulose was a

colloidal substance and therefore, consisted of aggregates (or micelles) of smaller

molecules rather than a single, long, linear macromolecule.

After 1927, however, evidence favoring the linear macromolecular chain structure began to

accumulate. The fact that no reproducible or conclusive evidence in favor of the Association

theory had been obtained, together with the new experimental results definitely in agreement

with the macromolecular concept, established the latter concept on a firm basis, and it

became almost universally accepted after 1932. Thus structure of cellulose evolved as a

linear macromolecule consisting of a large number of hexose units linked together by main-

valence glucosidic links.

Figure 2.1: Structure of linear anhydropyranose units

In this cellulose chain shown above in the figure, the glucose units are in 6-membered

rings, called pyranoses. They are joined by single oxygen atoms between the C-1 of one

pyranose ring and the C-4 of the next ring. Since a molecule of water is lost when an

alcohol and a hemiacetal react to form an acetal, the glucose units in the cellulose polymer

are referred to as anhydroglucose units


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The next debate which came up after establishing the above structure was that whether

cellulose consist exclusively of Glucose units or any other monomer is also involved. For

this purpose, Cotton, the purest form of cellulose was subjected to hydrolysis under various

conditions by different workers. By using chromatographic techniques, the products upon

hydrolysis were separated and chemical composition was determined. The conclusion of

these studies firmly confirmed that a very pure form of cellulose, such as ramie, yields only

glucose upon hydrolysis.

Yet, one particularly curious fact could not be satisfactorily explained. Since cellulose is

made of identical monomer, it was expected that its solubility in alkali solution would be

same under identical conditions. But, the experimental data often pointed significant

variations in alkali solubility with different samples of cellulose. Those experiments also

showed that some cellulose samples had higher viscosities than other samples. This led to the

conclusion that, since cellulose had been shown to consist of linear chains of glucose units,

the different in physical properties meant that different cellulose chains were made up of

different numbers of glucose units.

Thus, the number of monomers n, participating in the formation of cellulose may differ from

each cellulose chain. However, all these monomers are linked through an identical glucosidic

bond, particularly a - glucosidic bond. But there is a shadow of doubt about the uniformity

of the linkages, owing to the fact that the large size of the cellulose molecule shadows the

small number of non-glucosidic bonds which appears here and there. Many investigators had

attempted to clear these doubts, but until now, the controversy has not been settled

conclusively.

Another curious fact pertaining to cellulose is about their end groups. The two terminal

glucose residues in a cellulose chain not only differ from the glucose residues forming the

chain itself, but also differ from each other. One contains a reducing hemiacetal group and is,

therefore, known as the reducing end group, whereas the other contains an extra secondary

hydroxyl group and is known as the non-reducing end group. These end groups are present in

native cellulose, and they are also obtained during strictly hydrolytic cleavage where; for


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each glucosidic bond cut, two new end groups---one of each type will appear. For this reason,

the determination of end groups has been used as a means of measuring the molecular weight

of cellulose as well as for following the course of hydrolytic degradation. Degradation by

other than hydrolytic means (e.g., oxidative), however, may result in the formation of entirely

different types of end groups.

3. Chemistry of Hemicellulose

3.1. Introduction

The cell wall of all plants contains an important group of hetero-polysaccharides, which is

termed as Hemicellulose. Hemicelluloses are usually defined as plant cell components of

branched-chain heteropolysaccharides containing hexosans and pentosans and are easily

hydrolyzed to give simple sugars and some acetic acid. The term Hemicellulose has also been

used to include all the polysaccharide components of the cell wall other than cellulose.

Hemicelluloses are easily soluble by dilute alkali solution. Based on the solubility in alkaline

solutions, Hemicellulose may be separated into two basic fractions, hemicellulose A and B.

However, there are no other clear distinctions between the two types except that

hemicellulose B usually contains a higher proportion of uronic acid than hemicellulose A. It

is worth noting here that most of the hemicelluloses are water insoluble prior to the treatment

of cell wall with strong chemicals.

Some typical examples of hemicellulose are Galactoglucomannans, Arabinoglucuronoxylan,

Arabinogalactan, Glucuronoxylan, Glucomannan etc. All the hemicelluloses are essentially

polymers having certain degree of polymerization (DP) formed by certain monomers. It is

reported that DP of short-chained hetero polymers of hemicellulose is usually less than 200.

Some important monomers which constitutes these hemicelluloses are,

Pentosans

1. D-Xylose

2. D-arabinose

Hexosans

1. D-glucose

2. D-mannose


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Other monomers which appear less frequently are L-Rhamnose, D-Glucuronic acid, D-

Galacturonic acid, 4-O-methyl-D-Glucuronic acid.

Generally speaking, hemicelluloses are extensively branched. It is even found to be linked

with other lignocellulosic materials such as Cellulose and Lignin. The hemicelluloses which

are closely linked with cellulose are called as Cellulosans and those hemicelluloses which are

closely linked with lignin are called as Polyuronides.

3.2. Holocellulose

As mentioned earlier, cellulose, hemicellulose and lignin are termed as lignocellulosic

material of wood. But, when reference is made only to the polysaccharide fraction of the

wood, it is termed as Holocellulose. Holocellulose includes only cellulose and hemicellulose

and the lignin part, which is an aromatic polymer, is excluded.

So, essentially, preparation of holocellulose starts with the removal of lignin from wood.

Wood can be delignified by treating it with chlorine followed by alcoholic ethanolamine.

Lignins property of getting dissolved in chlorine dioxide can also be made to our advantage

by treating wood with the mixture of acetic acid and sodium chloride (the mixture gives

chlorine oxide).

In both the methods, the residue obtained is holocellulose, which is the delignified form of

wood. Holocellulose is an excellent raw material for hemicellulose isolation and synthesis.

3.3. Structure of Hemicellulose

Xylan is the most abundant of all the hemicelluloses. The basic skeleton of the xylans found

in the tissue of all land plants is a linear backbone of 1,4'--anhydro-D-xylopyranose units

linked. The most common monomer found attached to the xylan chain is D-Xylose. The

xylan framework is always found modified in nature. There are many possibilities by which a

Xylan chain might have been modified. Some of the most common frameworks are discussed

here.


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3.3.1. Hardwood Hemicelluloses

Glucuronoxylan

The backbone consists of -D-xylopyranose units, linked by (1 4) bonds. Most of the

xylose residues contain an acetyl group at C-2 or C-3 (about seven acetyl residues per ten

xylose units). These acetyl groups are easily cleaved by alkali, and the acetate formed during

kraft pulping of wood mainly originates from these groups. The xylose units in the xylan

chain additionally carry (1 2) linked 4-O-methyl--D-glucuronic acid residues, on the

average about one uronic acid per ten xylose residues.

Figure 3.1: Structure of Glucuronoxylan

The xylosidic bonds between the xylose units are easily hydrolyzed by acids, whereas the

linkages between the uronic acid groups and xylose are very resistant and they are slowly

hydrolyzed to acetic acid.

Glucomannan

Besides xylan, hardwoods contain 2 -5% of a glucomannan, which is composed of -D-

glucopyranose and -D-mannopyranose units linked by (1 4)-bonds.

Figure 3.2: Structure of Glucomannan

The glucose:mannose ratio varies between 1:2 and 1: 1, depending on the wood species. The

mannosidic bonds between the mannose units are more rapidly hydrolyzed by acid than the

corresponding glucosidic bonds, and glucomannan is easily depolymerized under acidic

conditions.


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3.3.2. Softwood Hemicelluloses

Galactoglucomannans

Galactoglucomannans are the principal hemicelluloses in softwoods (about 20%). Their

backbone is a linear or possibly slightly branched chain built up by (1 4)-linked -D-

glucopyranose and -D-mannopyranose units. The -D-galactopyranose residue is linked as a

single-unit side chain to the framework by (1 6)-bonds. An important structural feature is

Figure 3.3: Structure of Galactoglucomannan

that the C-2 and C-3 positions in mannose and glucose units are partially substituted by acetyl

groups, on the average one group per 3 -4 hexose units. Galactoglucomannans are easily

depolymerized by acids and especially so the bond between galactose and the main chain.

The acetyl groups are much more easily cleaved by alkali than by acid.

Arabinoglucuronoxylan

In addition to galactoglucomannans, softwoods contain an arabinoglucuronoxylan (5 -10%).

It is composed of a framework containing (14)-linked -D-xylopyranose units which are

partially substituted at C-2 by 4-0-methyl--D-glucuronic acid groups, on the average two

residues per ten xylose units. In addition, the framework contains a- L-arabinofuranose units,

on the average 1.3 residues per ten xylose units. Because of their furanosidic structure, the

arabinose side chains are easily hydrolyzed by acids. Both the arabinose and uronic acid

substituents stabilize the xylan chain against alkali-catalyzed degradation.

Figure 3.4: Structure of Arabinoglucuronoxylan


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Arabinogalactan

The heartwood of larches contains exceptionally large amounts of water-soluble

arabinogalactan, which is only a minor constituent in other wood species. Its backbone is

built up (1 3)-linked -D-galactopyranose units. Almost every unit carries a branch

Figure 3.5: Structure of Arabinogalactan

attached to position 6, largely (1 6)-linked -D-galactopyranose residues but also L-

arabinose. There are also a few glucuronic acid residues present in the molecule. The highly

branched structure is responsible for the low viscosity and high solubility in water of this

polysaccharide.

3.4. Composition of Hemicellulose in various feedstocks

Hemicellulose carbohydrates constitute 30-40% dry matter of lignocellulosic materials. But

within the hemicellulose group, the sugars present differ as the source varies. The amount of

hemicellulose varies widely, depending on plant materials, type of tissue, stage of growth,

growth environment, physiological conditions, storage, and method of extraction.

Considerable differences also exist in the hemicellulose content and composition between the

stem, branches, roots, and bark.

3.4.1. Xylans & Mannans

Hardwoods (Angiosperms) and softwoods (Gymnosperms) yield different sugars when

subjected to hydrolysis. The distinction arises from the fact that hardwood is mostly

dominated by monomer xylose whereas softwood has the predominant presence of mannose.

Thus hemicelluloses are classified on this basis also. On hydrolysis, if a hemicellulose gives

more of the xyloses, then those xylose yielding hemicelluloses are referred as Xylans or

Pentosans. On the other hand, if a hemicellulose yields mostly mannose on hydrolysis, then


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they are called as Mannans. Thus, most of the hardwoods such as birch are Xylans and most

of the softwoods such as spruce, pine, etc are Mannans.

Table 3.1 Composition of Hemicellulose in various agricultural residues

Agricultural residues % of total sugars

Xylose Arabinose Glucose Others*

Wheat Straw 57.9 9.1 28.1 5

Soybean 59.9 6.6 6.1 27.4

Sunflower 60.6 2.2 32.6 4.6

Flax straw 64.6 12.8 1.2 21.4

Sweet clover hays 49.3 21.9 8.9 9.9

Peanut hulls 46.3 5 46.6 2.1

Sugar cane bagasse 59.5 14.5 26 -

*Others comprises Mannose & Galactose

4. Hydrolysis of Hemicellulose

Hemicelluloses are hydrolyzed to give simple sugars. On hydrolysis, different hemicelluloses

yield different sugars by which they are formed and some amount of acetic acid. This

hydrolysis of hemicellulose can be done by either chemical or biological method. A wide

range of microorganisms produce different types of hemicellulases in response to the

different types of hemicellulose in their environments.

The total number of hemicellulases and the role of each enzyme are not clear. In

combination, hemicellulase enzymes can hydrolyze hemicellulose to its constituent sugars.

On the other hand, much progress has been made in understanding of chemical hydrolysis of

hemicellulose. During acid hydrolysis of hemicellulose, pentosans and pentoses are degraded

rapidly to furfural and condensation by-products. In order to prevent the decomposition of

sugars, especially pentoses, a more dilute acid, a shorter reaction time, a lower temperature,

and the rapid removal of hydrolytic agents are required. Thus, an efficient process has been

developed recently to hydrolyze hemicellulose by dilute acids at moderate temperature and

atmospheric pressure. Many acids are known to be good hydrolytic agents. The common


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method of acid hydrolysis uses dilute acid. One of the earliest commercial hydrolysis

processes was a dilute sulfuric acid process carried out at a relatively low temperature for a

prolonged period of time. Recently, a great deal of research has examined the dilute acid

hydrolysis of woods and agricultural residues to produce sugars.

It is worth noting here that, chemical hydrolysis of hemicelluloses is much easier to

accomplish than the hydrolysis of cellulose due to the heterogeneous structure and

composition of hemicellulose and its low degree of polymerization.

One important observation made during the hydrolysis of holocellulose is that formation of

Galacturonic acid. Usually, when Polygalacturonides is hydrolyzed, it gives galacturonic

acid. So this confirms presence of Polygalacturonides in holocellulose. These

Polygalacturonides, whose presence is of so small quantity that it is not easily separable from

the hemicellulose, is not included in hemicellulose family but rather called as Pectic

materials.

5. Bioconversion of Hemicellulose

5.1. Introduction

Hemicellulose, as stated earlier, comprises up to 25 to 40 % of all biomass and it is one of the

major constituents of plant materials. There are many potential uses for hemicellulose and

hemicellulose derived carbohydrates. They can be converted by microorganisms to various

products, such as methane, organic acids, sugar alcohols, solvents, animal feed, and ethanol.

The relevance of this idea of converting hemicellulose into useful products is linked to the

availability of biomass energy sources containing hemicellulose. It is estimated that

hemicellulose, composed principally of pentosans such as xylan, represents 20 to 40% of

most lignocellulosic agricultural residues and in India alone 150 million metric tons (for

United States, this figure comes out to be 71 million metric tons) of collectible surplus crop

residues produced annually contain millions of tons of D-xylose sugar residues in the

hemicellulose fraction. Clearly, the utilization of the xylose component of cellulosic biomass

is an important factor in the overall economics of biomass conversion into value-added

products.


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Of the many products available from hemicellulose-derived carbohydrates, ethanol has

received the most attention. This interest in ethanol production focuses on its potential use for

blending with petroleum to make "gasohol". In addition to its use as a fuel or petroleum

supplement, ethanol is also a versatile chemical feedstock, and many chemical products are

derived from ethanol. The other chemical sectors which find the application of hemicellulose

are the food industry and paper and pulp industry. The interest of paper and pulp industry in

the hemicellulose bioconversion arises from the significance importance given nowadays to

biopulping, biobleaching.

A number of biological processes have been investigated for the conversion of cellulose,

starch, and sugars to fuels and chemicals, while little progress has been made toward the

conversion of hemicellulose and hemicellulose-derived pentoses. This sluggishness in growth

of technology of hemicellulose conversion stems from the fact that unlike cellulose which is

made of single monomer, hemicellulose contains many monomers and to find the pathway of

breaking this complex branched chain into monomeric sugars which can then be fermented to

value added products, indeed, introduces the bigger complexity into the picture. But much

has been developed in the recent years which can lead the right path towards the overall

viability of the biomass program.

5.2. Bioconversion by Enzymatic Hydrolysis

5.2.1. Hemicellulase

The ability of enzymes to degrade the carbohydrates into value added products is the basis of

the biomass program, since biomass is the single largest source of carbohydrates which are

fermententable by microorganisms. But these carbohydrates are not available in the free

form. These carbohydrate monomers are present in the extensively polymerized nature and

hence it is essential to break this polymer chain to its constituent monomer units. And each of

these polymers is degraded by a variety of enzymes which produce a battery of enzymes that

work synergically. One such group of complex enzymes which do this job of breaking the

hemicellulose polymer chain into its constituent sugars like D-xylose, D-mannose and L-

arabinose etc are called as Hemicellulase.

Hemicellulases are classified according to the substrates they act upon, by the bonds they

cleave and by their patterns of product formation, but greater variety exists among the endo-


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xylanases and -glucosidases than is reflected in this simple classification system. One

notable distinction is made between endo-1, 4--xylanase and xylan 1, 4--xylosidase. While

the former produces oligosaccharides from random cleavage of xylan, the latter acts

principally on xylan oligosaccharides producing xylose. Some endo-xylanases appear to have

greater specificity for straight chain substrates, and others appear to be able to accommodate

more frequent side chains or branching. Some authors have also described enzymes that

remove acetyl, arabinose and 4-O-methyl glucuronic acid side chains from xylan backbones.

Hemicellulases are usually characterized by their action on defined substrates. In practice,

however, most native substrates are relatively complex and bear little similarity to the

substrate which is obtained after isolation. Native substrates (and especially xylans) are often

in acetylated or in esterified form. And the most common method for hemicellulose recovery,

which is solubilization in alkali, readily removes all ester linkages. Similarly, when the acetyl

groups are removed, hydrogen bonding leads to xylan precipitation and this deactylation

generally increases susceptibility of the substrate to enzyme attack.

Another type of classification which has aroused the interest among researchers is the

thermophilic hemicellulases, which are thermally stable. Ristoph and Humphrey et. al. (1985)

described a thermo-stable xylanase, which is stable for approximately 1 month at 55 C and

could withstand up to 80 C in a 10 min assay.

The use of xylanases in bleaching pulps has stimulated the search for enzymes with alkaline

pH optima. Most xylanases from fungi have pH optima between 4.5 and 5.5. Xylanases from

actinobacteria are active at pH 6.07.0. However, xylanases active at alkaline pH have been

described by many in literature. Alkaline pH activity could be important for certain

applications related to enzymatic treatments of kraft pulps.

As xylan is the main carbohydrate found in hemicellulose, it is naturally understandable that

the xylanase is most studied hemicellulase in the literature. The complete degradation of

xylan requires the cooperative action of a variety of hydrolytic enzymes. An important

distinction should be made between endo-1, 4--xylanase and xylan 1, 4--xylosidase. While

the former generates oligosaccharides from the cleavage of xylan, the latter works on xylan

oligosaccharides, producing xylose. Endo-xylanases are much more common than -

xylosidases, but the latter are necessary in order to produce xylose. Most -xylosidases are


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cell bound, and the enzymes are large relative to endo-xylanases. Should the desired product

upon xylan bioconversion be an oligosaccharide of lower DP (compared to xylan chain)

rather than the xylose monomer, then the enzyme activity with low exo-xylanase (-

xylosidase) activity is desired.

Glucuronoxylan (O-acetyl-4-O-methylglucuronxylan), one of the most common

hemicellulose present in the hardwoods, requires four different enzymes to degrade it, such as

endo-1, 4--xylanase (endoxylanase), acetyl esterase, -glucuronidase and -xylosidase.

Similarly, bioconversion of galactoglucomannan, one of the common hemicellulose in

softwood, starts with rupture of the polymer by endomannases, then acetylglucomannan

esterases remove acetylgroups and -galactosidases eliminate galactose residues. Finally, -

mannosidase and -glycosidase break down the endomannases generated oligomers -1, 4

bonds.

5.3. Bioconversion by Microbial Organisms

5.3.1. Introduction

When it comes to biological methods of converting the substrates, the first thought goes to

the so called biocatalysts alias enzymes such as cellulase, hemicellulase etc. These

hemicellulases are produced by growing the microorganisms such as bacteria, fungi upon the

xylan, mannan substrates. The biggest constraint brought by this method is the isolation and

purification of hemicellulase from the culture from which it is grown. Instead of going

through this process of purification and isolation, the idea of feeding the substrates directly to

the microorganisms seems to be more attractive. This process of production of hemicellulase,

enzymatic hydrolysis of hemicellulose and fermentation of all sugars in one single step is

called as Consolidated Bioprocessing.

5.3.2. Fermentation by Fungi

The organisms of the fungal lineage include mushrooms, rusts, smuts, puffballs, truffles,

morels, molds, and yeasts, as well as many less well-known organisms. About 70,000 species

of fungi have been described; however, some estimates of total numbers suggest that 1.5

million species may exist.


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As the sister group of animals and part of the eukaryotic crown group that radiated about a

billion years ago, the fungi constitute an independent group equal in rank to that of plants and

animals. Rather than requiring a stomach to accomplish digestion, fungi live in their own

food supply and simply grow into new food as the local environment becomes nutrient

depleted. It is with their intention they export hydrolytic enzymes that break down

biopolymers, which can be absorbed as their own nutrition. We take advantage of this

characteristic of fungi to breakdown the biopolymer of our interest. So, once we find the

suitable fungi which will release the hemicellulase to breakdown the hemicellulose chain to

feed on it, we can use such fungi for the purpose of bioconversion of hemicellulose chain.

The genus Aspergillus is one such group of filamentous fungi with a large number of species

which can conveniently degrade the plant cell wall polysaccharides to simple sugars. Many

sub-groups within this genus have been classified but most important for industrial

applications are the eight members of the group of black aspergilli. The black aspergilli have

a number of characteristics which make them ideal organisms for industrial applications.

Moreover, the wide range of enzymes produced by Aspergillus for the degradation of plant

cell wall polysaccharides is of major importance to the bioprocessing industry.

As stated earlier, biodegradation of the xylan backbone depends on two classes of enzymes

such as Endoxylanases and -xylosidases. Both classes of enzymes, as well as their encoding

genes, have been characterized from many organisms. Various endoxylanases have been

identified in Aspergillus. Although variation in those endoxylanases is detected in terms of

molecular mass and optimal pH, the major difference between the enzymes is in their

specificity toward the xylan polymer. Some enzymes cut randomly between unsubstituted

xylose residues whereas the activity of other endoxylanases strongly depends on the

substituents on the xylose residues neighboring the attacked residues.

In several aspergilli, three different endoxylanases have been identified. The best-studied

Aspergillus endoxylanases, with respect to substrate specificity, are the three enzymes from

A.awamori. Knap et. al. (1994) found that A.awamori endoxylanase I is unable to remove

one unsubstituted xylose residue adjacent to singly substituted xylose residues or two

unsubstituted xylose residues adjacent to doubly substituted xylose residues. Similarly,

Kormelink et. al. (1993) reported that A.awamori endoxylanase III was not able to remove


Page 17 of 28

two unsubstituted xylose residues adjacent to singly or doubly substituted xylose residues

toward the reducing end.

Also, variation in the DP of the product is observed for the same aspergilli with different side

chains. For instance, hydrolysis of a glucuronoxylan by an endoxylanase from A.niger

resulted mainly in xylobiose, xylotriose, and xylose, but hydrolysis of an arabinoxylan by the

same enzyme resulted mainly in oligosaccharides with a degree of polymerization of more

than 3. This suggests that the action of this endoxylanase is reduced by the presence of

arabinose residues on the xylan backbone.

The other important enzyme for degradation of xylan, -Xylosidases, has also been identified

in several aspergilli. These enzymes are highly specific for small unsubstituted xylose

oligosaccharides (degree of polymerization of up to 4) and their action results in the

production of xylose. Although this activity is of major importance for the complete

degradation of xylan, absence of the enzyme does not interfere with the induction of the

xylanolytic system. The ability of an A.awamori -xylosidase to release xylose from

xylooligosaccharides was studied to determine its substrate specificity. This enzyme was able

to release xylose from the non-reducing end of branched oligosaccharides only when two

contiguous unsubstituted xylose residues were present adjacent to singly or doubly

substituted xylose residues.

Apart from xylan, galactoglucomannan is abundant in softwood hemicellulose. The

degradation of the galactoglucomannan backbone depends on the action of -mannosidases

and -endomannanases, generally referred to as -mannanases. Both of these enzymes are

commonly produced by aspergilli. Being a true endohydrolases, the enzyme -mannanases

hydrolyze the backbone of galactoglucomannans and releases predominantly mannobiose and

mannotriose from mannan.

The ability of -mannanases to degrade the mannan backbone depends on several factors,

such as the number and distribution of the substituents on the backbone and the ratio of

glucose to mannose. It is most active if the glucomannan backbone is less substituted. This is

evident from the observation that the presence of galactose residues on the mannan backbone

significantly hinders the activity of -mannanase. But this effect is small if the galactose


Page 18 of 28

residues in the vicinity of the cleavage point are all on the same side of the main chain. It has

been shown that A.niger -mannanase binds to four mannose residues during catalysis.

The other enzyme which degrades mannan chain is the -mannosidases (exo-acting

enzymes), which releases mannose from the non-reducing end of manno-oligosaccharides.

The substrate specificity of A.niger -mannosidase has recently been studied by Ademark et

al (1999). They found that the enzyme is able to completely release terminal mannose

residues when one or more adjacent unsubstituted mannose residues are present. The

presence of a galactose-substituted mannose residue adjacent to the terminal mannose residue

reduces the activity of -mannosidase to 18 to 43%, compared to unsubstituted substrates.

5.3.3. Fermentation by Bacteria

Bacteria, like all other living organisms, require nutrients for growth. Essential nutrients

supply bacteria with an energy source and elements for macromolecular biosynthesis. Of

various forms of energy sources available, bacteria use inorganic chemicals (e.g., soil

bacteria), a light source (phototrophs), and organic compounds (heterotrophs).

It was shown over fifty years ago that suspensions of mixed ruminal bacteria are capable of

degrading xylans to xylose, arabinose, xylobiose, xylotriose, xylotetraose, xylopentose, and a

series of higher oligosaccharides . By using media containing xylan as the only added

carbohydrate source, active xylan-fermenting bacterial strains were isolated which conformed

to the description of Butyrivibrio fibrisolvens. Thereafter, number of other ruminal bacteria,

including Bacteroides ruminicola, Bacteroides succinogenes, Bacillus fermus, Bacillus

pumilus were found capable of extensively hydrolyzing and/or fermenting a wide variety of

xylans.

5.4. Synergic activities between enzymes

In native substrates, binding of the polymers are so complex and heterogeneous, as in the

case of hemicelluloses, where different constituents are linked by different types of bonds. So

it is essential that efficient degradation of polysaccharides requires cooperative or synergistic

interactions between the enzymes responsible for cleaving the different linkages. their

bioconversion demands several enzymes. In literature, many reports have been published

relating to synergic activities between enzymes, which itself demonstrates that synergy is, in

fact, a general phenomenon.


Page 19 of 28

The mechanism of how synergic activities between enzymes enhance efficiency of

bioconversion in hemicelluloses can be best explained by considering the research work of

Sorensen et. al. (2003). They investigated the individual and combined efficiency of

commercial, cellulytic and hemicellulytic enzyme preparations, Celluclast 1.5 L and Ultraflo

L, in catalyzing the liberation of arabinose and xylose from water-soluble wheat

arabinoxylan. The 50:50 mixtures of this enzyme preparation showed no synergistic

cooperation in arabinose release, but a synergistic interaction in xylose release was found

between Ultraflo L and Celluclast 1.5 L. This happens due to the fact that the partial removal

of arabinosyl residues from the substrate by -L-arabinofuranosidase which is present in

Ultraflo L makes the attack by the endo-1,4--xylanase present in both the enzymes more

specific to release xylobiose, xylotriose from the partially shaved xylan backbone. Finally,

the complete hydrolysis of xylobiose, xylotriose, and shortchain xylooligosaccharides to

xylose happens by the activity of -xylosidase present in Celluclast 1.5 L. Thus the early

release of arabinose facilitates the clear pathway for other enzymes to attack specifically.

Synergistic action has also been observed (Kormelink et. al., 1993) between many enzymes

from Aspergillus such as endoxylanase, -xylosidase, arabinofuranohydrolase, acetylxylan

esterase. Synergy has also been observed between these enzymes and some of the other

xylanolytic enzymes. Both endoxylanase and -xylosidase positively influenced the release

of 4-O-methylglucuronic acid from birchwood xylan by A.tubingensis -glucuronidase.

Recent studies revealed that synergistic interactions in the degradation of xylan not only are

present between mainchain-cleaving enzymes and accessory enzymes but also occur among

accessory enzymes and that nearly all accessory enzymes positively influence the activity of

the main-chain-cleaving enzymes. A strong synergistic effect has been observed for the role

of A. niger acetylxylan esterase in the hydrolysis of steamed birchwood xylan by three

endoxylanases from A. niger. The addition of acetylxylan esterase resulted in an increase in

the release of xylose and short xylooligosaccharides by a factor of 1.9 to 4.4, 6.8 to 14.7, and

2.5 to 16.3 for endoxylanase I, II, and III, respectively, depending on the incubation time.

The synergic activities between enzymes have been well explored to enhance Kraft Pulping

process. The paper manufactures by the kraft method requires whitening by a multistage

bleaching sequence. Such a bleaching process essentially involves delignification from the

softwood paper pulp by means of extensive use of chlorine and chlorine oxide. The search for


Page 20 of 28

methods which reduce the requirement for elemental chlorine during bleaching has been

prompted by the severe environmental damage caused by the presence of toxic and highly

refractive chlorinated organic byproducts in bleaching effluents. One enzymatic approach to

aid chemical bleaching of kraft pulps relies on the removal of hemicelluloses that may trap

residual lignin within the fiber matrix.

The softwood we are taking about is dominated by xylans & mannans and hence the

biological degradation of arabinoxylans and galactoglucomannans, relies on activity of

enzymes such as xylanases, mannanases and a side-chain removing accessory enzyme -

galactosidase. Due to the relative locations of different substrates within the pulpwood, the

use of two or more hemicellulases signicantly improve pulp bleachability (which can be

observed by decrease in kappa number), as a result of synergistic interactions. If Mannanase

and xylanase are used in this context, Mannan hydrolysis has been shown to further enhance

xylanase-aided pulp bleaching, due to improved xylanase accessibility to residual matrix

xylan, which may in turn be mediated by improved mannanase accessibility to

galactomannan and galactoglucomannan in the pulp matrix, through dispersal of

reprecipitated xylan by the xylanase.

6. Effect of pre-treatment

The hemicellulose substrates or, in fact, any of the lignocellulosic substrates occurring in the

biomass are in highly complex form and webbed with one another and thus the possibility of

enzyme finding and then attacking its substrate is very lean, as the enzyme has to satisfy

several barriers such as particle size, surface area accessible to enzymatic hydrolysis and

lignin content. This reduces the conversion rate of substrates and yield obtained whence is

very low. In order to improve the overall efficiency, the enzymes need to be facilitated with

proper orientation to attack their substrates. This can be done by partially breaking down the

biomass using a suitable pretreatment method.

Adrados et. al. (2005) studied the effect of pre-treatment in the release of hemicellulose

sugars from native hemicelluloses of wheat bran. Xylan is the major constituent of wheat

bran followed by arabinan and glucan. Enzymatic hydrolysis of this wheat bran with and

without pretreatment was conducted using commercial enzymes Celluclast and Ultraflo. Acid

hydrolysis by Sulfuric acid is the pretreatment method used by them. While the enzymatic


Page 21 of 28

hydrolysis alone fetched a yield of only 22% and acid hydrolysis alone gave yield of 50.4%,

the combined method, in which acid hydrolysis is followed by enzymatic hydrolysis, the

process is much more cleaner and released the 53% of sugars (Table 6.1). Thus, in order to

recover maximum amount of sugars from hemicellulose, pretreatment is essential.

Table 6.1: Maximum yields following the various kinds of hydrolysis methods investigated

Hydrolysis methods* Arabinose Xylose Glucose Total

Enzymatic hydrolysis

PT: 170 C, 20 min + EH

AH: 1% H2SO4, 130C, 40 min

PT: 0.2% H2SO4, 160C, 20min + EH

3.8

8.1

17.3

13.3

13.4

19.4

31.3

23.3

4.8

17.7

1.8

16.4

22.0

45.2

50.4

53.0

*Enzymatic hydrolysis (EH), pretreatment (PT), and acid hydrolysis (AH).

Having said this, industrially most important pretreatment methods available are,

Steam explosion

Lime pretreatment

Acid/Alkali pretreatments

The high pressure steam modifies the plant cell wall structure, yielding the partially

hydrolyzed hemicelluloses and a water-insoluble fraction composed of cellulose, residual

hemicellulose and a chemically modified lignin that can be further extracted by mild alkali or

oxidizing agent like alkaline hydrogen peroxide. As the higher temperatures prevail

throughout the pretreatment process, degradation of sugars happens which is a major

drawback of steam pretreatment. The product formed upon the degradation acts as the

inhibitor for the further microbial growth.

Biomass can also be pretreated with lime in the presence of water. Lime pretreatment

efficiently removes the 85% (Kim et. al., 2005) of initial lignin present in the biomass. Lime

pretreatment can be conducted either in oxidative or non-oxidative manner. During the 16

weeks lime pretreatment, non-oxidative delignification removed up to 43.6%, 46.3%, 48.4%,

and 47.7% of the initial lignin at 25, 35, 45, and 55C, respectively. However, oxidative

delignification removed up to 57.8%, 66.2%, 80.9%, and 87.5% of the initial lignin at 25, 35,

45, and 55C, respectively during the same period.


Page 22 of 28

In acid pretreatment methods, the lignocellulosic fraction is suspended in an acidified

aqueous medium that is maintained under pressure at an elevated temperature. This operation,

if not properly controlled may lead to formation of toxics which has to separated before

subjecting the partially hydrolyzed biomass for enzymatic hydrolysis. This presents a

particular problem in large scale operations where such a purification step would not

converge to be economical.

In alkali pretreatment, biomass is treated with alkali such as NaOH, NH4OH etc. Native

substrates (and especially xylans) are often acetylated or otherwise. This pretreatment, where

we these substrates are solubilized in alkali, readily removes all ester linkages and

deacetylation takes place which increases the generally increases susceptibility of the

substrate to enzyme attack.

Thus, each pretreatment has own pros and cons and hence there should be some trade-off to

be made when selecting the appropriate pretreatment of biomass before subjecting it to

enzymatic hydrolysis.

7. Comparison of various methods

The various methods available for the conversion of hemicellulose are

Acid Hydrolysis

Biological methods

Enzymatic Hydrolysis

Direct Microbial conversion Consolidated Bioprocessing.

The primary factors one has to consider before finalizing a conversion methods are maturity

in technology, toxic chemicals produced, selectivity and yield, cost, capacity etc


Page 23 of 28

Table 7.1: Comparison of various conversion methods

Methods

Factors

Chemical methods,

example: Acid Hydrolysis

Biological methods

Enzymatic Hydrolysis DMC*

Technology

Toxicity

Selectivity

Cost

Capacity

Matured

High

Low

Medium

High

Comparatively less

Less

High

High

Medium

Infant stage

Less

High

High

Medium

*Direct Microbial Conversion

8. Conclusion

1. It has been shown in the various studies that the pre-treatment prior to bioconversion of

hemicellulose chain is effective. So one has to decide what kind of pre-treatment can be

given to the particular feed stock we are interested in using as substrate. Economics plays

an important role in this step, as purpose of the pretreatment is to facilitate the

downstream process of enzymatic hydrolysis. Many separation stages involved in this

pretreatment step may nullify the effect of the same economically.

2. The composition and source from which the feed stock is obtained is important issue in

making further decisions related to process as each type of plant and wood chips contains

hemicellulose sugars in different compositions and structure of the backbone chain may

have different levels of substitution. Hence, composition of feedstock must be determined

beforehand in the laboratory.

3. Once we are able to pinpoint a particular sugar which forms the backbone chain, then we

can go for the deciding hemicellulases required for the bioconversion. The optimum

enzyme and substrate concentration should be decided in the laboratory by varying

substrate concentration for each enzyme concentration. Also the optimum pH and stable

temperature should be decided. The hemicellulase selected should be dynamic with

substrate such that it can act upon the feedstock even if the composition and source of

feedstock varies to certain extent.


Page 24 of 28

4. The decision of whether the commercial enzymes should be employed or should the

enzymes be isolated and purified from the suitable organism should be best driven by the

capacity of the plant and availability of separation technology and source of

microorganisms from which the required enzymes should be extracted. The in-house

enzyme production becomes essential if the capacity of the plant is very large.

5. It has been found that the synergic activities between hemicellulases can be made use of

in achieving substantially higher yield of sugars. For instance, if it is known that the

feedstock contains xylan backbone chain often substituted with arabinose units, then -L-

arabinofuranosidase should be synergically used with xylanases so that former breaks the

branched arabinose units from the backbone facilitating the latter to break the main

backbone chain to xylose units.

6. On comparing the various pathways available to convert the hemicellulose

polysaccharides into their corresponding sugars, it has been found that although acid

hydrolysis is matured on the basis of technology it is quite a laggard when it comes to

toxicity and selectivity. Even if we find some way to dispose the toxic materials in

relatively safe manner, the selectivity achieved from this process will not be

commercially viable in near future even if it is acceptable in the current scenario. On both

these account, that is toxicity and selectivity, biological methods are far superior to the

acid hydrolysis.

7. Enzymatic hydrolysis method is promising one where the complex enzyme

hemicellulases are used to degrade the polymer chain. This method brings much more

selectivity and produces almost no toxic chemicals. But cost of commercial enzymes is

very high (for example, 100ml of Celluclast costs $17 USD and 100ml of Ultraflo costs

$4) and proposition of in-house production of enzyme requires equally resource

demanding isolation and purification techniques.

8. The Direct microbial conversion (DMC) method involves the microorganisms like fungi,

yeast and bacteria to convert lignocellulosic materials to the corresponding sugars and

then to value-added products. Their ability to grow on the carbon substrates is tapped to

efficiently degrade the polymer chains without the necessity of intermediate steps like

hemicellulase isolation and purification.


Page 25 of 28

9. Over the surface, DMC seems to be a convenient technology than others but the most

important and demanding part is in finding organisms that can perform all of the required

functions robustly on a variety of feedstocks after mild pretreatments. Thus the discovery

of the fermenting organisms that produce hemicellulase in sufficient quantities to

completely hydrolyze the hemicellulolytic biomass is of primary importance and lowering

the cost of producing these organisms is of secondary importance. If the required

technological advances can be achieved through genetic engineering followed by cost

reductions through improved practice, then consolidated bioprocessing can be the best

solution for the increasing global energy demands.

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LIST OF SYMBOLS & NOTATIONS USED

Xyl xylopyranose unit

R acetyl group

Me methyl group

Glu A glucuronic acid unit

Glu glucopyranose unit

Man mannopyranose unit

Gal galactopyranose unit

Ara f arabinofuranose unit

Ara arabinopyranose unit

bioconversion of hemicellulose

Documents

bioconversion of hemicellulose

hydrolysis of hemicellulose

chemistry of hemicellulose

structure of hemicellulose

composition of hemicellulose

chemistry of cellulose

structure of cellulose

various feedstocks