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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
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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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Bioconversion of Hemicellulose
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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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Bioconversion of Hemicellulose
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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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Bioconversion of Hemicellulose
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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
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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
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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.
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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
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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
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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.
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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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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.
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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.
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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, 843850,
(2005).
Bisaria, V.S., Ghose, T.K., Biodegradation of cellulosic
materials: Substrates,
microorganisms, enzymes and products, Enzyme Microb. Technol.,
3, 90-104, (1981).
Clarke, J.H., Davidson, K., Rixon, J.E., Halstead, J. R.,
Fransen, M. P., Gilbert, H. J.,
Hazlewood, G. P., A comparison of enzyme-aided bleaching of
softwood paper pulp
using combinations of xylanase, mannanase and -galactosidase,
Appl. Microbiol.
Biotechnol., 53, 661-667, (2000).
Deshpande, V., Keskar, S., Mishra, C., Rao, M., Direct
conversion of
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)--
xylanases and one -xylosidase
from Aspergillus awamori, J. Biotechnol., 27, 249265, (1993).
(cf., de Vries, R.P.,
-
Bioconversion of Hemicellulose
Page 26 of 28
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, 617621 (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, 5363,
(2002).
Rivers, D.B., Emert, G.H., Factors affecting the enzymatic
hydrolysis of municipal solid
waste components, Biotechnol. Bioeng., 31, 278281, (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,
36933700, (2005).
Sehoon, K., Holtzapple, M.T., Lime pretreatment and enzymatic
hydrolysis of corn stover,
Bioresource Technol., 96, 19942006, (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 -Larabinofuranosidases,
endo-1,4--xylanases, and -
xylosidase activities, Biotechnol. Bioeng., 81, 726731,
(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).
-
Bioconversion of Hemicellulose
Page 27 of 28
Szczodrak, J., The enzymatic hydrolysis and fermentation of
pretreated wheat straw to
ethanol, Biotechnol. Bioeng., 32, 771776, (1988).
Vazquez, M.J., Alonso, J.L., Domoinguez, H., Parajo, J.C.,
Xylooligosaccharides:
manufacture and applications, Trends in Food Sci. &
Technol., 11, 387393, (2000).
Yanez, R., Alonso, J.L., Parajo, J.C., Production of
hemicellulosic sugars and glucose from
residual corrugated cardboard, Process Biochem., 39, 15431551,
(2004).
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