Downloaded from FaaDoOEngineers.com 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
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 β-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 25–30% 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 β-1, 4- and occasionally β-1,3-glucosidic bonds. The principal component of hardwood hemicellulose is glucuronoxylan, whereas glucomannan is predominant in softwood.
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Bioconversion of Hemicellulose 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 β-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 Page 2 of 28
Bioconversion of Hemicellulose 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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Bioconversion of Hemicellulose 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 mainvalence 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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Bioconversion of Hemicellulose
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 β- 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 Page 5 of 28
Bioconversion of Hemicellulose 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 Page 6 of 28
Bioconversion of Hemicellulose Other monomers which appear less frequently are L-Rhamnose, D-Glucuronic acid, DGalacturonic 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. Lignin’s 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'-β-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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Bioconversion of Hemicellulose
3.3.1. Hardwood Hemicelluloses Glucuronoxylan The backbone consists of β-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-α-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 β-Dglucopyranose and β-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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Bioconversion of Hemicellulose
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 β-Dglucopyranose and β-D-mannopyranose units. The α-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 β-D-xylopyranose units which are partially substituted at C-2 by 4-0-methyl-α-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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Bioconversion of Hemicellulose
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 β-D-galactopyranose units. Almost every unit carries a branch
Figure 3.5: Structure of Arabinogalactan
attached to position 6, largely (1 6)-linked β-D-galactopyranose residues but also Larabinose. 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 Page 10 of 28
Bioconversion of Hemicellulose 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 Page 11 of 28
Bioconversion of Hemicellulose 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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Bioconversion of Hemicellulose 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 Larabinose 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 endoPage 13 of 28
Bioconversion of Hemicellulose xylanases and β-glucosidases than is reflected in this simple classification system. One notable distinction is made between endo-1, 4-β-xylanase and xylan 1, 4-β-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 6.0–7.0. 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-β-xylanase and xylan 1, 4-β-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 βxylosidases, but the latter are necessary in order to produce xylose. Most β-xylosidases are Page 14 of 28
Bioconversion of Hemicellulose 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 (βxylosidase) 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-β-xylanase (endoxylanase), acetyl esterase, α-glucuronidase and β-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 α-galactosidases eliminate galactose residues. Finally, βmannosidase and β-glycosidase break down the endomannases generated oligomers β-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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Bioconversion of Hemicellulose 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 β-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
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Bioconversion of Hemicellulose 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, β-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 β-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 β-mannosidases and β-endomannanases, generally referred to as β-mannanases. Both of these enzymes are commonly produced by aspergilli. Being a true endohydrolases, the enzyme β-mannanases hydrolyze the backbone of galactoglucomannans and releases predominantly mannobiose and mannotriose from mannan. The ability of β-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 β-mannanase. But this effect is small if the galactose
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Bioconversion of Hemicellulose 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 β-mannanase binds to four mannose residues during catalysis. The other enzyme which degrades mannan chain is the β-mannosidases (exo-acting enzymes), which releases mannose from the non-reducing end of manno-oligosaccharides. The substrate specificity of A.niger β-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 β-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 18 of 28
Bioconversion of Hemicellulose
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 β-L-arabinofuranosidase which is present in Ultraflo L makes the attack by the endo-1,4-β-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 β-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, β-xylosidase, arabinofuranohydrolase, acetylxylan esterase. Synergy has also been observed between these enzymes and some of the other xylanolytic enzymes. Both endoxylanase and β-xylosidase positively influenced the release of 4-O-methylglucuronic acid from birchwood xylan by A.tubingensis β-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 19 of 28
Bioconversion of Hemicellulose 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 αgalactosidase. 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 20 of 28
Bioconversion of Hemicellulose 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
3.8
13.4
4.8
22.0
PT: 170 C, 20 min + EH
8.1
19.4
17.7
45.2
AH: 1% H2SO4, 130°C, 40 min
17.3
31.3
1.8
50.4
PT: 0.2% H2SO4, 160°C, 20min + EH
13.3
23.3
16.4
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 55°C, 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 55°C, respectively during the same period. Page 21 of 28
Bioconversion of Hemicellulose
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
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Bioconversion of Hemicellulose Table 7.1: Comparison of various conversion methods
Methods Factors
Biological methods
Chemical methods, example: Acid Hydrolysis
Enzymatic Hydrolysis
DMC*
Technology
Matured
Comparatively less
Infant stage
Toxicity
High
Less
Less
Selectivity
Low
High
High
Cost
Medium
High
High
Capacity
High
Medium
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.
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Bioconversion of Hemicellulose 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 β-Larabinofuranosidase 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 24 of 28
Bioconversion of Hemicellulose
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.
9. Reference Adrados, B.P., Choteborska, P., Galbe, M., Zacchi, G., “Ethanol production from non-starch carbohydrates of wheat bran”, Bioresource Technol., 96, 843–850, (2005). Bisaria,
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cellulose/hemicellulose to ethanol by Neurospora crassa ”, Enzyme Microb. Technol., 8, 149-152, (1986). de Vries, R.P., Visser, J., “Aspergillus Enzymes Involved in Degradation of Plant Cell Wall Polysaccharides”, Microb. Mole. Biol. Reviews, 65, 497-522, (2001). Gongs, Ch.S., Chen, L.F., Tsao, G.T., Flickinger, M.C., “Conversions of Hemicellulose Carbohydrates”, Adv. in Biochem. Engg. Biotechnol., 20, 93-118, (1981). Knap, I. H., Carsten, M., Halkier, T., Kofod., L.V., International patent WO 94/23022. (cf., de Vries, R.P., Visser, J., “Aspergillus Enzymes Involved in Degradation of Plant Cell Wall Polysaccharides”, Microb. Mole. Biol. Reviews, 65, 497-522, (2001).) Kormelink, F. J. M., Searle-van Leeuwen, M. J. F., Wood, T.M., Voragen, A.G.J., “Purification and characterization of three endo-(1,4)-β- xylanases and one β-xylosidase from Aspergillus awamori”, J. Biotechnol., 27, 249–265, (1993). (cf., de Vries, R.P., Page 25 of 28
Bioconversion of Hemicellulose Visser, J., “Aspergillus Enzymes Involved in Degradation of Plant Cell Wall Polysaccharides”, Microb. Mole. Biol. Reviews, 65, 497-522, (2001).) Lynd, L.R., Weimer, P.J., van Zyl, W.H., Pretorius, I.S., “Microbial Cellulose Utilization: Fundamentals and Biotechnology”, Microb. Mole. Biol. Reviews, 66, 507-577, (2002). Lin, K.W., Patterson, J.A., Ladisch, M.R., “Anaerobic fermentation: microbes from ruminants”, Enzyme Microb. Technol., 7, 98-107, (1985). Norman, A.G., Chapter 4 & 9 in Wise, “Wood Chemistry”, Reinhold Publications, New York, p71-73, 1944. Nunes, A. P., Pourquie, J., “Steam explosion Pretreatment and enzymatic hydrolysis of Eucalyptus wood”, Bioresource Technol., 57, 107-110, (1996). Pellerin, P., Gosselin, M., Lepoutre, J.P., Samain, E., Debeire, P., “Enzymatic production of Oligosaccharides from Corncob Xylan”, Enzyme Microb. Technol., 13, 617–621 (1991). Perez, J., Munoz-Dorado, J., De la Rubia, T., Martinez, J., “Biodegradation and biological treatments of cellulose, hemicellulose and lignin: an overview”, Int. Microbiol., 5, 53–63, (2002). Rivers, D.B., Emert, G.H., “Factors affecting the enzymatic hydrolysis of municipal solid waste components”, Biotechnol. Bioeng., 31, 278–281, (1988). Rydholm, S., “Pulping Process”, John Wiley & Sons, New York, p157-165, 1967. Rosario Freixoa, M., Maria Norberta de Pinho, “Enzymatic hydrolysis of beechwood xylan in a membrane reactor”, Desalination, 149, 237-242, (2002). Saha, B.C., Iten, L.B., Cotta, M.A., Wu, Y.V., “Dilute acid pretreatment, enzymatic saccharification and fermentation of wheat straw to ethanol”, Process Biochem., 40, 3693–3700, (2005). Sehoon, K., Holtzapple, M.T., “Lime pretreatment and enzymatic hydrolysis of corn stover”, Bioresource Technol., 96, 1994–2006, (2005). Sjostrom E., “Wood Chemistry – Fundamentals & Applications”, Academic Press, Florida, p60-66, 1981. Sorensen, H.R., Meyer, A.S., Pederson, S., “Enzymatic hydrolysis of water soluble wheat arabinoxylan. 1. Synergy between α-Larabinofuranosidases, endo-1,4-β-xylanases, and βxylosidase activities”, Biotechnol. Bioeng., 81, 726–731, (2003). Stephenson, J., “Pulp & Paper Manufacturing”, McGraw Hill, New York, Vol 4, p71-73, 1950. Suurnakki, A., Tenkanen, M., Buchert, J., Viikari, L., “Hemicellulases in the Bleaching of Chemical Pulps”, Adv. in Biochem. Engg. Biotechnol., 57, 261-288, (1997). Page 26 of 28
Bioconversion of Hemicellulose Szczodrak, J., “The enzymatic hydrolysis and fermentation of pretreated wheat straw to ethanol”, Biotechnol. Bioeng., 32, 771–776, (1988). Vazquez, M.J., Alonso, J.L., Domoinguez, H., Parajo, J.C., “Xylooligosaccharides: manufacture and applications”, Trends in Food Sci. & Technol., 11, 387–393, (2000). Yanez, R., Alonso, J.L., Parajo, J.C., “Production of hemicellulosic sugars and glucose from residual corrugated cardboard”, Process Biochem., 39, 1543–1551, (2004).
LIST OF SYMBOLS & NOTATIONS USED Xyl ρ xylopyranose unit R acetyl group Me methyl group Glu ρA glucuronic acid unit Glu ρ glucopyranose unit Man ρ mannopyranose unit Gal ρ galactopyranose unit Ara f arabinofuranose unit Ara ρ arabinopyranose unit
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