Chapter – 1

General introduction

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1.0 Introduction At present we are confronted with two problems of fundamental importance. First

in the present world, accumulation of lignocellulose in large quantities in places where

agricultural residues present a disposal problem that results not only in deterioration of the

environment but also in loss of potentially valuable material that can be used in paper

manufacture, bio-fuel production, animal feed, composting, humus and among others. The

primary component of lignocellulosic biomass consists of cellulose, hemicelluloses and

lignin in an average ratio of 4:3:3 (Brauns et al., 1949). These are the most abundant

organic compounds on earth and have the potential to be a renewable source for energy

and chemicals. The estimated global annual production of biomass is 1x10 tons,

sequestering 2x10 J of energy (Smeets et al., 2007). The exact percentage of these three

components varies from source to source (Smeets et al., 1979). Second the world fossil

fuel resources are depleating and the nuclear energy production is facing many significant

questions which still have not been clarified. In view of rising prices of crude oil due to

increasing fuel demands, the need for alternative sources of bio-energy is expected to

increase sharply in the coming years. Among potential alternative bio-energy resources,

lignocelluloses have been identified as the prime source of bio-fuels and other chemicals.

Plant biomass is abundant and potential renewable energy source in nature, which

is produced by photosynthesis. Lignocellulosic residues from wood, grass, agricultural and

forestry wastes as well as municipal solid wastes are particularly abundant in nature and

have a potential for bioconversion. Several novel markets have identified importance of the

lignocellulosic residues recently. The use of fungi in low cost bioremediation projects

might be attractive given their highly efficient lignocellulose hydrolysis enzyme

machinery. The major components of lignocellulosic biomass are cellulose (C6 sugars),

hemicellulose (C5 sugars) and lignins (polyphenols). Glucose produced from cellulose can

be used as feedstock for ethanol production, while C5 sugars, from the hemicellulose, can

be used to produce furfural or ethanol. Lignin can be a potential source of a wide range of

phenolic compounds (Klass, 1998).

Microbial enzymes are a fast growing field in biotechnology. The global market of

industrial enzymes was closed to a billion dollars in 1990 and crossed the $2.0 billion mark

in 2005 (Krishna 2005). The market has been estimated at $3.3 billion in 2010 and is

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expected to reach $4.4 billion by 2015. The production cost and the yields of enzymes are

considered the major problems in commercial exploitation. Enzymes can carry out their

myriads of biochemical reactions under ambient conditions, which make their use eco-

friendly and often the best alternative to polluting chemical technologies. They are specific

in function speed up reaction by providing alternative pathway of lower activation energy

without being consumed. Currently, the chemical additives are being replaced by the

enzymes in numerous food applications. The use of enzymes has become a need of the

time, because they promote effects similar to those of chemical additives with the

advantage of being considered as safe natural additives (Penstone 1996). Enzymatic

treatment provides the same level of output as it is achieved through conventional methods

that use harsh chemicals.

Ethanol production from lignocellulosic biomass through the biological route

seems very attractive and sustainable due to several reasons among which the renewable

and ubiquitous nature of biomass and its non-competitiveness with food crops are the

major ones. Lignocellulosic biomass accounts for 50% of all the biomass available in the

world asserting its potential as a feasible raw material for ethanol production. There are

number of reports on the uses of these renewable sources (Lynd et al., 1991). Since last

decade, there has been increasing research interest in the value of bio-sourced materials

recovered from residual biomass and agro-wastes (Fitzpatrick et al., 2010).Ethanol

production from lignocelluloses is mainly categorized into 4 steps:(1) Pretreatment process

which can reduce the lignin content and render cellulose and hemicelluloses content for

enzymatic hydrolysis.(2) Convert enzymatic hydrolysis to breakdown polysaccharide to

simple sugars.(3) Conversions of sugars (C6 and C5) for ethanol production through

microbes. (4) Production of ethanol from sugars. The Figure 1.1 illustrates the step by step

procedure for ethanol production from lignocelluloses.

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Fig.1.1. Generalized schematic representation of lignocellulosic materials bio-

conversion into ethanol. Adapted from Asgher et al., (2014)

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1.1 Plant cell wall architecture

Plant cell wall is a dynamic network highly organized which changes throughout

the life of the cell. The new primary cell wall is born in the cell during cell division and

rapidly increases in surface area during cell expansion. The middle lamella forms the

interface between the primary walls of neighboring cells. Finally, at differentiation, many

cells elaborate with the primary wall a secondary cell wall (either lignified or unlignified),

building a complex structure uniquely suited to the function of the cell. Contiguous cells

are bound together by deposition of lignin in the middle lamella (Ornston et al., 1987). The

highest concentration of lignin is observed in the middle lamella and the primary wall

(Saka and Goering, 1985). The secondary wall contains most of the cell lignin as it forms

the greatest volume of cell wall (Harris, 1990). The biological activity of some molecules

derived from the primary cell wall polysaccharides.

1.1.1 Principle components of cell wall

Plant cell wall is a highly specialized network formed by a heterogeneous mixture

of cellulose, hemicelluloses and lignin. In nature, the above substances grow and decay

during the year. It has been estimated that around 7.5x1010

tonnes of cellulose are

consumed and regenerated every year (Kirk-Otmer, 2001). It is thereby the most abundant

organic compound in the world. Cellulose and hemicelluloses polymers bring rigidity to

the wall and pectin provides fluidity throw the gelatinous polysaccharides matrix.

Cellulose and hemicelluloses are embedded in the amorphous pectin polymers and

stabilized by proteins and phenolic compounds. Hemicelluloses bind to the surface of

cellulose network preventing direct contact among microfibrils, and pectin is linked to

hemicelluloses forming a gel phase. The major component of lignocellulosic materials is

cellulose, followed by hemicellulose and lignin.

1.1.2 Cellulose

Cellulose is the β-1, 4-polyacetal of cellobiose (4-O-β-D-glucopyranosyl-D-

glucose). Cellulose is more commonly considered as a polymer of glucose because

cellobiose consists of two molecules of glucose. Many properties of cellulose depend on its

degree of polymerization (DP), i.e. the number of glucose units that make up one polymer

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molecule. The DP of cellulose can extend to a value of 17000, even though more

commonly a number of 800-10000 units is encountered (Kirk-Otmer, 2001).

Cellulose chains aggregate together to form microfibrils, which are highly

crystalline and insoluble structures, each one about 3 nm in diameter, chemically stable

and resistant to enzymatic attack. Cellulose microfibrils comprise the core of the plant cell

wall; one third of the total mass of wall is cellulose. Cellulose is found in both the

crystalline and the non-crystalline structure. The coalescence of several polymer chains

leads to the formation of microfibrils, which in turn are united to form fibres. In this way

cellulose can obtain a crystalline structure. Figure 1.2 illustrates structure as well as the

placement of cellulose in the cell wall.

Fig.1.2: Formation of micro and macrofibrils (fibers) of cellulose and their position

in the wall

The β-1, 4-linkages in a cellulose chain and chemical formula of cellulose is

(C6H

10O

5). Cellulose consists of linear macromolecular chains of glucose, linked by β-1, 4-

glucosidic bonds between the number one and number four carbon atoms of the adjacent

glucose units (Figure 1.3). Partial hydrolysis of cellulose produces a range of

oligosaccharides including cellobiose, cellotriose, and cellotetraose (Feller et al., 1986).

Cellulose is a relatively hygroscopic material absorbing 8-14% water under normal

atmospheric conditions (20°C, 60% relative humidity). Nevertheless, it is insoluble in

water, where it swells. Cellulose is also insoluble in dilute acid solutions at low

temperature. The solubility of the polymer is strongly related to the degree of hydrolysis

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achieved. As a result, factors that affect the hydrolysis rate of cellulose also affect its

solubility that takes place, however, with the molecule being in a different form than the

native one.At higher temperatures it becomes soluble, as the energy provided is enough to

break the hydrogen bonds that hold the crystalline structure of the molecule. Cellulose is

also soluble in concentrated acids, but severe degradation of the polymer by hydrolysis is

caused. In alkaline solutions extensive swelling of cellulose takes place as well as

dissolution of the low molecular weight fractions of the polymer (Krassig and Schurz,

2002). Solvents of cellulose that have been applied in industrial or laboratory practice

include uncommon and complex systems, such as cupriethylenediamine (cuen) hydroxide

or the cadmium complex Cadoxen. Additionally, aqueous salt solutions, such as zinc

chloride, dissolve limited amounts of cellulose (Kirk-Otmer, 2001).Cellulose does not melt

with temperature, but its decompositions starts at 180°C (Thermo wood handbook, 2003).

1.1.3 Hemicellulose

Hemicellulose is the second abundant renewable biomass in nature, and xylan is the

major hemicellulose component and approximately amounts to 20-35% of plant cell wall

dry weight (Jiang et al., 2010).Hemicelluloses have the property of being soluble in dilute

alkali. They are usually classified according to the sugar residues present xylan, mannans,

arabinans and galactans. Most hemicelluloses do not occur as homo-polysaccharides but as

hetero-ploysaccarides, containing different types of sugar in the backbone chain and in the

side chain or appendages. These may be D-xylose, L-arabinose, D-mannose acid, O-acetyl

groups or feruloyl and coumaryl ester linked via L-arabinose residues to the backbones

(Timell et al., 1965). The most common type of polymers that belongs to the

hemicellulose family of polysaccharides is xylan. As shown in Figure 1.3, the molecule of

a xylan involves 1-β-4 linkages of xylopyranosyl units with α-(4-O)-methyl-D-

glucuronopyranosyl units attached to anhydroxylose units. The result is a branched

polymer chain that is mainly composed of five carbon sugar monomers, xylose, and to a

lesser extent six carbon sugar monomers such as glucose.

Different plants may contain the same basic xylan structure but different

arrangements with other sugar residues, especially L-arabinose, D-glucuronic acid and 4-

methyl ether may occure (Wilkie, 1979). Arabinose is normally the next most plentiful

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component in hemicellulose. Minor components, including mannose, galactose, and uronic

acids may also be present. Some hemicelluloses contain both glucomannans and

galactoglucomannans. The glucomannans contain D-glucose and D-mannose units in a

ratio of 30:70. The galactoglucomannans contain D-galactose, D-glucose, and D-mannans

units in a ratio of 2:10:30 (Klass, 1998; Paster, 2003). Hemicellulose is an insoluble in

water at low temperature. However, its hydrolysis starts at a temperature lower than that of

cellulose, which renders it soluble at elevated temperatures (Thermwoodhandbook, 2003).

Hemicellulose utilization is a considered difficult because the branched structure of

hemicelluloses slows enzymatic hydrolysis.

1.1.4 Xylans

Schulze, (1891) introduced the name hemicellulose for the fractions isolated or

extracted from plant materials with dilute alkali. The principal monomers present in most

of the hemicelluloses are D-xylose, D-mannose, D-galactose and L-arabinose.

Composition and distribution of the substitutions is wide by variable according to the plant

cell species. Xylans usually contain many arabinose residues attached to the backbone

which are known as arabinoxylans and glucuronoarabinoxylans; high amounts of

arabinoxylans are present in the endosperm of cereals. Hemicelluloses include xylan,

mannan, galactan and arabinan as the main heteropolymers. Xylan contains D-xylose as its

monomeric unit and traces of L-arabinose. The galactan contains D-galactosyl units

whereas mannan is made up of D-mannosyl units. The arabinan is composed of L-

arabinose.The substrates for xylanases are polysaccharides composed of β-1, 4 linked

xylopyranose units. They are highly branched and in firm association with other polymers

shows in Figure 1.3 (The structure of xylan and hemicelluloses is same). Acid hydrolysis of

xylan is a rapid process for production of xylose but a number of toxic and undesirable

substances are also produced (Ferreira-Filho et al., 1994). Xylan contains of D-xylose units

and is linked from the number one to the number four carbon of each residue and

composed of chains of 1-4 linked β-D-xylopyranose residues. Xylan is the major type of

hemicellulose and its hydrolysis depends on two classes of enzymes. The Endoxylanase

(EC 3.2.1.8) cleave the xylan backbone into smaller parts like oligosaccharides that are

further degraded to xylose by xylosidases (EC 3.2.1.37). During the last few decades, great

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interest has been developed in xylan and its hydrolytic enzymatic complex, for

application in bread production, supplement in animal feed, preparation of drinks, textiles,

bleaching of cellulose pulp, ethanol and xylitol production.

The structure of xylans found in cell walls of plants can differ greatly depending

on their origin and different structures attached to the xylan backbone. Although most of

the xylans have branched structures, however some linear polysaccharides have been

isolated (De Vries et al., 2001). Lignin is bound to xylans by an ester linkage to 4-O-

methyl-D-glucuronic acid residues. These linkages in lignocellulose can be disrupted by

using various pretreatment methods that expose most of the polysaccharide components to

enzymatic hydrolysis (Chang et al., 2001). Selective hydrolysis of xylan has been observed

when purified and crude enzyme was applied in which cellulases were inhibited (Mora et

al., 1986).

Fig.1.3. Diagrammatic illustration of the framework of lignocelluloses: Cellulose,

Hemicellulose and Lignin (Adopted from Menon and Rao, 2012).

1.1.5 Lignin

Lignin is a complex phenolic polymer that provides an embedding material for the

cellulosic polymers of the secondary cell walls. Lignins are always associated with

hemicellulose, not only in intimate physical mixture, but also anchored to the latter by

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actual covalent bonds (Sarkanen and Ludwic, 1971). It is an amorphous three-dimensional

polymer with phenylpropane units as the predominant building blocks. More specifically,

p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol are the ones most commonly

encountered. Dividing higher plants into two categories, hardwood (angiosperm) and

softwood (gymnosperm), it has been identified that lignin from softwood is made up of

more than 90% of coniferyl alcohol with the remaining being mainly p-coumaryl alcohol

units. Contrary to softwoods, lignin contained in hardwood is made up of varying ratios of

coniferyl and sinapyl alcohol type of units (Kirk-Otmer, 2001). From the nutritional point

of view, lignin has always been blamed as an important barrier to polysaccharide

utilization (Van Soest, 1994). Lignin resists the growth of microorganisms and stores more

solar energy than either cellulose or hemicellulose (Hu, 2002).

The structure of lignin that is depicted in Figure 1.3 is complex, disordered, random

and consists mainly of ether linked aromatic ring structures, which adds elasticity to the

cellulose and hemicellulose matrices (Paster, 2003). Lignin is mainly composed of

phenylpropane or C9 units linked together by different types of bonds, including alkyl-aryl,

alkyl-alkyl, and aryl-aryl ether bonds. The molecular weight of lignins may be 100 kDa or

more.

Solvents that have been identified to significantly dissolve lignin include low

molecular alcohols, dioxane, acetone, pyridine, and dimethyl sulfoxide. Furthermore, it has

been observed that at elevated temperatures, thermal softening of lignin takes place, which

allows depolymeristation reactions of acidic or alkaline nature to accelerate (Connor et al.,

2007). In addition, phenolic acids are structural components of the lignin core in plant cell

wall (Shimada, et al., 1971). The concentration of phenolics in grass cell walls varies from

8 to 28 mg/g and is less than 3 mg/g in legumes (Eraso and Hartley, 1990). Ester bonds are

labile to alkali treatment and as phenolic acids are ester-linked to both lignin and

hemicellulose they can be released by alkali treatment (Hartley et al., 1985). The effect of

phenolic acids on rumen microbial fermentation has been extensively studied. Chesson et

al., (1982) reported that p-coumaric and ferulic acids were the most toxic phenolic acids to

rumen cellulolytic bacteria. P-coumaric acid has an inhibitory effect on colonization of

fibres by fungi (Akin and Rigsby, 1985) and cellulolytic bacteria (Akin et al., 1988).

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1.2 Chemical interaction between components

There are four main types of bonds identified in the lignocellulose complex. Those

are ether type of bonds, ester bonds, carbon-to-carbon bonds and hydrogen bonds. These

four bonds are the main types of bonds that provide linkages within the individual

components of lignocellulose (intrapolymer linkages), and connect the different

components to form the complex (interpolymer linkages). The position and bonding

function of the latter linkages is summarized in Table 1.1 (Faulon et al., 1994).

Table 1.1 Overview of linkages between the lignocelluloses polymers.

Bonds within different components (intrapolymer linkages)

Ether bond Lignin, (hemi) cellulose Carbon to carbon Lignin Hydrogen bond Cellulose Ester bond Hemicellulose Bonds connecting different components (interpolymer linkages) Ether bond Cellulose-Lignin; Hemicellulose-lignin Ester bond Hemicellulose-lignin Hydrogen bond Cellulose-hemicellulose; Hemicellulose-Lignin

Cellulose-Lignin

1.2.1 Functional groups and chemical properties of lignocellulose components

From the aspect of producing sugar monomers from lignocellulose and ultimately

ethanol or other chemical building blocks, the functional groups that are of interest are:

1. Functional groups that are involved in the hydrolysis of the polysaccharides to their

monomers and the possible subsequent degradation reactions of these monomers (e.g.

to furfural).

2. Functional groups that are involved in the (partial) depolymerisation of lignin (into

fragments or phenolic compounds) so that the cellulose fraction becomes more

accessible for enzymes.

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Table 1.2 Functional groups in components of lignocelluloses

Functional Group Lignin Cellulose Hemicellulose

Aromatic ring ×

Hydroxyl group ×

Carbon to carbon linkage ×

Ether (glucosidic) linkage × × ×

Ester bond ×

Hydrogen bond* × ×

*The hydrogen bond is not a functional group, as its reaction does not lead to chemical

change of the molecule. However, it changes the solubility of the molecule, though and it

is therefore important for the breakdown of lignocelluloses (Table 2.2)

1.3 Xylanase market scenario

The increasing of lignocellulosic material in the environmental leads to pollution.

However, these renewable carbon sources can be utilized for the production of xylanases

by several companies for use on many industries like pulp, poultry, textile, paper krafting

and bleaching, feed etc.,

The DSM Nutritional product enzyme Ronozyme WX is an outstanding xylanase

designed to meet the requirements of modern poultry and pig feeding. Characterized by

exceptional heat stability, a broad pH range and a high, consistent level of xylanase

activity, Ronozyme WX can be used as the sole NSP (non-starch polysaccharides) enzyme

in diets based on wheat, rye and triticale or these cereals in combination with barley and

cereal by-products. Ronozyme WX is derived from Thermomyceslanuginosus spp., an

organism that thrives in hot and dry conditions. This benefit is passed on, ensuring that the

enzyme can remain active even at high temperatures. Ronozyme WX is an endoxylanase

which hydrolyses arabinoxylans and xylans in feeds. Plant polysaccharides play an

important role in feedstuffs. They can function as a major nutritional component in the diet

(e.g. starch) but a number of NSP are detrimental to non-ruminants such as poultry and

pigs. Poultry and pigs are not capable of digesting these NSP's due to a lack of the relevant

enzymes in their digestive tracts (DSM Nutritional products, 2011).

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ALKX (L/P) wide range xylanase enzyme was a xylan hydrolyzing xylanase

enzyme with heat and pH stability derived from a genetically modified strain of

Aspergillus. Xylans are polysaccharides composed of β-1, 4- linked xylopyranose units.

They are one of the major constituents of plant cell walls and account for more than 30 %

of the dry weight of terrestrial plants. The enzyme falls into the category of endo- β-1, 4-

xylanase which is of prime importance for hydrolysis of xylan polymers in cereal grains

and lignocellulosic biomass. The enzyme breaks xylan to short chain xylo-oligosaccharides

of varying length. (Enzyme-India, 2011)

The optimal usage level of the enzyme in viscosity reduction depends on

processing parameters such as type of Raw material, Processing time, pH, Temperature

and the quality composition. A typical Product code-ALKX (L/P) dosage rate for viscosity

reduction during liquefaction of starch is 0.02-0.04 % w/w of the material and degrading

xylan in lignocellulosic biomass is 0.15-0.6 % w/w of the material. Kaypeeyes biotech pvt

ltd manufacture multizyme is a cocktail of different enzymes for improving the digestion

& bioavailability of nutrition

Varuna biocell private limited manufacture the xylacid, used in the bleaching

process. In pulping, almost 90% of the wood lignin is solubilized in the cooking liquor.

The remaining lignin is primarily responsible for the brown color of the unbleached pulp

and papers. This residual lignin is progressively removed from the pulp in the bleaching

process. The main part of the hemicellulose bound lignin is located in the outer layers of

the fibers. The concept of xylanase bleaching of pulp is based on the partial hydrolysis of

xylan, which is the main component of the hemicellulose. The enzymatic action increases

the extractability of lignin from the pulp in the subsequent bleaching process using

chlorine and oxygen.

Xylacid has been screened to have negligible cellulase activity (FPU). Xylacid is

active over a wide range of pH and temperature i.e. from 5-9 pH and from 40 to 70°C

respectively. Advanced Enzyme Technologies ltd manufactures the acid xylanases for

degradation of soluble NSP thereby improving the nutrients in GI Tract which is most

effective for high fibre diet.

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1.4. Xylanase distribution

Over 100 years ago the role of enzymes in the breakdown of xylan was observed

by Bastawde (1992). The xylan hydrolyzing enzymes are ubiquitous and diverse by nature

(Collins et al., 2005). A number of different sources, including bacteria (Battan et al.,

2006), fungi (Kuhad et al., 1998), actinomycetes (Ball et al., 1989), and yeast (Liu et al.,

1998) have been reported to produce xylanase. In the last decade, a number of

hyperthermophilic archaea have been isolated that are able to grow around the boiling

point of water (Niehaus et al., 1999). Xylanases also can be found in marine algae,

protozoans, crustaceans, insects, snails and seeds of land plants (Sunna and Antranikian

1997). Various bacterial and fungal species viz. Thermomonospora sp, Bacillus, sp,

Melanocarpus albomyces, Chaetomium thermophilum, Nonomuraea flexuosa,

Streptomyces sp., Dictyoglomus sp., Thermotogales sp., Thermoactinomyces thalophilus,

Thermoascus aurantiacus, Fusarium proliferatum, Clostridium abusonum, Arthrobacter

etc. are reported to produce alkalo-thermophilic xylanases (Khandeparkar et al., 2006;

George et al., 2001). Bacteria just like many industrial enzymes fascinated the researchers

for alkaline thermostable endo-1, 4-β-xylanase producing trait. Bacillus SSP-34 produced

higher levels of endo-1, 4-β-xylanase activity under optimum nitrogen condition. Bacillus

SSP-34 produced an endo-1, 4-β-xylanase with activity of 506 IU/ml in the optimized

medium (Subramaniyan et al., 2002). Earlier, Ratto (1992) reported endo-1, 4-β-xylanase

with an activity of 400 IU/ml from Bacillus circulans. It had optimum activity at pH 7 and

40% of the activity was retained at pH 9.2. However, the culture supernatant also showed

low levels of celluloytic activities with 1.38 IU/ml of endoglucanase (CMCase EC 3.2.1.4)

and 0.05 U/ml of cellobiohydrolases. Bacillus stearothermophilus strain T6 has been

reported to produce cellulase free endo-1, 4-β-xylanase. It actually had slight cellulolytic

activity of 0.021 IU/ml. Mathrani et al., (1992) reported endo-1, 4-β-xylanases from

Dictyoglomus sp. having optimum activities at pH 5.5 and 90ºC. It merits the significant

pH stability at a range of pH values 5.5 to 9.0.

Xylanase genes have been isolated from microorganisms of various genera and

expressed in Escherichia coli. In bacteria xylanases are not only produced at lower activity

levels than in fungi, but are also restricted to the intracellular or periplasmic fractions.

Furthermore, enzymes expressed in bacteria are not subjected to post-translation

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modifications, such as glycosylation. Among microbial sources, filamentous fungi are

especially interesting as they secrete these enzymes into the medium and their xylanase

levels are very much higher than those found in yeasts and bacteria

1.5 The xylanolytic complex

Xylanases catalyze the hydrolysis of xylans. These enzymes are produced mainly

by microorganisms and take a part in the breakdown of plant cell walls, along with other

enzymes that hydrolyze polysaccharides, and also digest xylan during the germination of

some seeds (e.g. in the malting of barley grain). In addition to the production of a variety

of xylanolytic enzymes, many microorganisms produce multiple xylanases (Gilbert et al.,

1993). The heterogeneity and complexity of xylan has resulted in an abundance of diverse

xylanases with varying specificities, primary sequences and folds, and hence has lead to

limitations with the classification of these enzymes by substrate specificity alone (Wong et

al.,1988).

The complex structure of xylan needs different enzymes for its complete

hydrolysis. Endo-1, 4-β-xylanases (1, 4-β-D-xylanohydrolase, E.C.3.2.1.8) depolymerise

xylan by the random hydrolysis of xylan backbone and 1, 4-β-D-xylosidases (1, 4-β-D-xylan

xylohydrolase (E.C.3.2.1.37) split off small oligosaccharides. The side groups present in xylan

are liberated by α-L-arabinofuranosidase, α-D-glucuronidase, galactosidase and acetyl xylan

esterase.

1.5.1 Endo-1, 4-β-xylanases (1, 4-β-D-xylanohydrolase, E.C.3.2.1.8)

These enzymes act cleaves the glycosidic bonds in the xylan backbone, bringing

about a reduction in the degree of polymerization of the substrate. Xylan is not attacked

randomly, but the bonds selected for hydrolysis depend on the nature of the substrate

molecule, i.e. on the chain length, the degree of branching, and the presence of

substituent’s (Li et al., 2000). Initially, the main hydrolysis products are β-D-

xylopyranosyl oligomers, but at a later stage, small molecules such as mono, di and tri

saccharides of β-D-xylopyranosyl may be produced. The hydrolysis of xylan by an

endoxylanase may be written as follows:

H (C5H804)n OH + H2O → H (C5H8O4) n-p OH+ H(C5H804) p OH

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This equation shows the stochiometry of a single hydrolytic event in a xylan

molecule; such a reaction may occur at many points in the chain. The endoxylanases have

been classified in several ways. Based on Wong et al., (1988) they are grouped into nine

groups:

a) Non-arabinose liberating endoxylanases: This class of enzymes cannot act on

arabinosyl initiated branch points at β-(1→4) linkages and produce only xylobiose and

xylose as the major end products. These enzymes can breakdown oligosaccharides as

small as xylobiose.

b) Non-arabinose liberating endoxylanases: These are unable to cleave at α-(1→2) and

α-(1→3) branch points produce mainly xylooligosaccharides larger than xylobiose.

These xylanases are unable to cleave xylotriose and xylobiose.

c) Arabinose liberating endoxylanases: These enzymes cleave the xylan chain at the

branch points and produce mainly xylobiose, xylose and arabinose.

d) Arabinose liberating endoxylanases: These enzymes can produce hydrolyzation of

branch points and produce intermediate size oligosaccharides and arabinose.

e) Exo-1, 4-β-xylanases (1,4-β-D-xylanxylanohydrolase): These enzymes remove the

single xylose units from non-reducing end of the xylan chain.

f) β-xylosidase or xylobiase: These enzymes hydrolyze disaccharides like xylobiose and

the higher xylooligosachharides with decreasing specific activity.

g) α-L-arabinofuranosidase (E.C.3.2.1.55): These enzymes cleave side chain α-1,3

arabinofuranose from the xylan main chain.

h) α-(4-O-methyl)-D-glucuronidase (E.C.3.2.1.-): These can remove D-glucuronysyl or

4-O methyl glucuronosyl residues from xylan.

i) Acetyl xylan esterase: (E.C.3.1.1.6): These enzymes liberate acetyl groups from the

xylan backbone.

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Owing to its heterogeneous structure, xylan degradation needs not just one enzyme,

but an enzyme complex. The components of this system that have been most extensively

studied are the endoxylanases and the β-xylosidases. Ferulic acid esterase, p-coumaric acid

esterase, acetylxylan esterase and α-glucuronidase were discovered only at the end of the

1980s, probably because of the difficulty of obtaining suitable substrates. These enzymes

are present in fungi and bacteria, but until now few have been purified and analysed in

terms of their physical and chemical properties.

1.6 Rate limiting factors in enzymatic hydrolysis

The rate of conversion of cellulose fibres to individual, easily hydrolysable shorter

chains is dependent on some factors. For enzymatic hydrolysis of natural cellulose, as cited

by Lynd et al., (2002), a number of determining factors of hydrolysis rate have been

postulated, as follows.

1.6.1 Crystallinity

This is generally regarded as a key factor influencing cellulose hydrolysis at both

enzymatic and microbial levels. The highly crystalline regions of cellulose chains are

recalcitrant to hydrolysis, as a result of their tightly packed nature which prevents

accessibility of the enzymes. The more of these regions present the slower the rate of

hydrolysis. Pretreatments of biomass that reduce crystallinity usually enhance the

hydrolysis of cellulose by fungal cellulases, but some pretreatments effective in enhancing

hydrolysis have been reported to increase crystallinity (Focher et al.,1981).

1.6.2 Degree of polymerization: The longer the cellulose chain, the lower the rate of

hydrolysis (Walker et al., 1990).

1.6.3 Particle size

Within any given cellulose sample, there is a great measure of unevenness of the

size and shape of individual particles, which thus affects the rate of hydrolysis.

Chapter-1

17

1.6.4 Pore volume

The pore structure of cellulosic materials must be able to accommodate particles of

the size of a cellulolytic enzyme. The greater the availability, the more the enzymes that

are adsorbed (Mosier et al., 1999). The pore structure is a much more important

determinant of hydrolysis in natural biomass materials than in purified celluloses, which

have relatively smooth surfaces and lower porosity.

1.6.5 Accessible surface area

Since cellulose hydrolysis is a surface phenomenon, available surface area is a

potential determinant of hydrolytic rate. The available surface area is a more important

determinant of rate of hydrolysis or utilization than is crystallinity. Most cellulose chains

are hidden within the microfibrils, which prevents exposure to enzymes and thus limiting

the rate of hydrolysis. It has been shown that the pore structure of cellulosic materials can

accommodate particles of the size of a cellulolytic enzyme, and good correlation has been

observed between total surface area (Stone et al., 1968) and the rate of substrate

hydrolysis. Gama et al., (1994), however, applying a modified solute exclusion technique

to five different celluloses, have reported that cellulolytic enzymes do not penetrate the

pore structure of purified celluloses.

The shortage of fossil fuels and the arising need to find alternative sources for

renewable energy and fuels, there is a renewal of interest in the bioconversion of

lignocellulosic biomass using cellulases, xylanases and other enzymes.

1.7 Pretreatment of lignocellulosic biomass

Pretreatment is a crucial process step for the biochemical conversion of

lignocellulosic biomass into e.g. bioethanol. It is required to alter the structure of cellulosic

biomass to make cellulose more accessible to the enzymes that convert the carbohydrate

polymers into fermentable sugars (Mosier et al., 2005). Pretreatment has been recognised

as one of the most expensive processing steps in cellulosic biomass to fermentable sugars

conversion and several recent review articles provide a general overview of the field

(Hendriks and Zeeman, 2008). Pretreatment involves the alteration of biomass so that

(enzymatic) hydrolysis of cellulose and hemicellulose can be achieved more rapidly and

Chapter-1

18

with greater yields. Possible goals include the removal of lignin and disruption of the

crystalline structure of cellulose.

1.7.1 Mechanical pretreatment

a) Milling

Reduction of particle size is often needed to make material handling easier and to

increase surface/volume ratio. This can be done by chipping, milling or grinding.

Mechanical pretreatment is usually carried out before a following processing step, and the

desired particle size is dependent on these subsequent steps. For mechanical pretreatment

factors like capital costs, operating costs, scale-up possibilities and depreciation of

equipment are very important.

b) Ultrasonic pretreatment

The method of ultrasonication for lignocellulosic biomass is investigated at

laboratory scale. It is a well known technique for treatment of sludge from waste water

treatment plants. The experiments show the effect of the pretreatment of pure cellulose on

its enzymatic hydrolysis, using a model compound (Carboxyl methyl cellulose, CMC). The

issue of lignocellulose pretreatment is not addressed in the experiments. The experimental

results showed that when a suspension of cellulose is provided with energy by irradiation,

the reaction rate of the subsequent enzymatic hydrolysis is increased by approximately

200% (Imai et al., 2004). The mode of action is not investigated. Presumably, it is the

hydrogen bonds of the cellulose crystalline structure that break if treated with enough

energy. However, the energy provided in this case is 130 kJ/g CMC, which is significantly

higher than the energy of the hydrogen bond 0.12 kJ/g (Bochek, 2003).

1.7.2 Chemical pretreatment: To this group belong the pretreatments that are purely

initiated by chemical reactions for disruption of the biomass structure.

a) Liquid hot water

Liquid hot water (LHW) processes are biomass pretreatments with water at high

temperature and pressure. Other terms are hydrothermolysis, hydrothermal pretreatment,

Chapter-1

19

aqueous fractionation, aquasolv (Mosier et al., 2005). Aquasolv by hot compressed water

contacts water with biomass for up to 15 min at temperatures of 200-230 °C. Between 40

and 60% of the total biomass is dissolved in the process, with 4-22% of the cellulose, 35-

60% of the lignin and all of the hemicellulose being removed. Over 90% of the

hemicellulose is recovered as monomeric sugars when acid was used to hydrolyze the

resulting liquid. In addition, acetic acid is formed during the treatment and acts as a

catalyst for polysaccharide hydrolysis. The results in the formation of monomeric sugars

that may further decompose to furfural (inhibitor of fermentation). Variability in results

was related to the biomass type with high lignin solubilization impeding recovery of

hemicellulose sugars (Mok and Antal, 1992).

b) Weak acid hydrolysis

Dilute acid treatment is one of the most effective pretreatment methods for

lignocellulosic biomass. In general there are two types of weak acid hydrolysis:

1. High temperature and continuous flow process for low-solids loading (T> 160°C, 5-10

wt% substrate concentration).

2. Low temperature and batch process for high-solids loading (T≤160°C, 10-40% substrate

concentration).

c) Strong acid hydrolysis

Concentrated strong acids such as H2SO

4 and HCl have been widely used for

treating lignocellulosic materials because they are powerful agents for cellulose hydrolysis

(Sun and Cheng, 2002), and no enzymes are needed subsequent to the acid hydrolysis.

Advantages of concentrated acid hydrolysis are the flexibility in terms of feedstock choice,

high monomeric sugar yield as well as mild temperature conditions that are needed.

Drawbacks of using concentrated acids are corrosive nature of the reaction and the need to

recycle acids in order to lower cost. To date, several companies are in the process of

commercialising strong acid hydrolysis of lignocellulosic biomass for microbial

fermentation purposes (BlueFire Ethanol, 2010; Biosulfurol, 2010).

Chapter-1

20

d) Alkaline hydrolysis

The major effect of alkaline pretreatment is the removal of lignin from the biomass,

thus improving the reactivity of the remaining polysaccharides. In addition, alkali

pretreatments remove acetyl and the various uronic acid substitutions on hemicellulose that

lower the accessibility of the enzyme to the hemicellulose and cellulose surface (Chang

and Holtzapple, 2000). It is reported that alkaline hydrolysis mechanism is based on

saponification of intermolecular ester bonds crosslinking xylan hemicelluloses and other

components such as lignin (Sun and Cheng, 2002).

e) Calcium hydroxide or sodium hydroxide

By using these components salts are formed that may be incorporated in the

biomass and need to be removed or recycled (González et al., 1986). Process conditions

are relatively mild but reaction times can be long. These mild conditions prevent

condensation of lignin, resulting in high lignin solubility, especially for biomass with low

lignin content such as softwood and grasses. Due to the mild conditions, degradation of

sugars to furfural, HMF and organic acids is limited. The addition of air or oxygen to the

reaction mixture greatly improves the delignification, especially highly lignified materials

(Chang and Holtzapple, 2000).

f) Ammonia

Pretreatment of biomass with aqueous ammonia at elevated temperatures reduces

lignin content and removes some hemicellulose while decrystallising cellulose. Ammonia

pretreatment techniques include the ammonia fibre explosion-method (AFEX), ammonia

recycle percolation (ARP) and soaking in aqueous ammonia (SAA). With ARP the

biomass is pretreated with aqueous ammonia in a flow-through column reactor. The liquid

flows at high temperature through the reactor column, which has been previously packed

with biomass. To prevent flash evaporation the reactor system must be slightly pressurized

(e.g. 2.3 MPa) (Kim and Lee, 2005). After reaction the solid fraction, rich in cellulose and

hemicellulose, is separated from the liquid. This liquid fraction is sent into a steam-heated

evaporator for ammonia recovery and lignin and other sugar separation. Ammonia is then

recycled to the reactor inlet whereas the separated fraction is sent into a crystallizer. After

Chapter-1

21

crystallization a washing step is carried out in order to extract the sugars that have been

retained in the solid matrix. Soaking in aqueous ammonia (SAA) at low temperature

removes efficiently the lignin in the raw material by minimizing the interaction with

hemicellulose. As a result an increase of surface area and pore size is achieved. Thus,

retained hemicellulose and cellulose can be hydrolyzed to fermentable sugars by most

commercial xylanase and cellulase mixtures. Kim et al., 2008 evaluated SAA as a

pretreatment method of biomass (Kim et al., 2008). However, biomass pretreatment

economics are also strongly influenced by total sugar yields achieved.

1.7.3 Combined chemical and mechanical pretreatment: These methods combine

mechanical and chemical action. To this group belong the following pretreatments:

a) Steam explosion

Steam explosion (uncatalysed or catalysed) is one of the most applied pretreatment

processes owing to its low use of chemicals and limited energy consumption. With this

method high-pressure saturated steam is injected into a batch or continuous reactor filled

with biomass. During the steam injection, the temperature rises to 160-260 ºC.

Subsequently, pressure is suddenly reduced and the biomass undergoes an explosive

decompression with hemicellulose degradation and lignin matrix disruption as result.

b) Ammonia fiber explosion (AFEX)

In the AFEX process, biomass is treated with liquid ammonia at high temperature

and pressure (Teymouri et al., 2005). After a few seconds, pressure is swiftly reduced. A

typical AFEX process is carried out with 1-2 kg ammonia/kg dry biomass at 90 °C during

30 min. It reduces the lignin content and removes some hemicellulose while decrystallising

cellulose. The cost of ammonia and especially of ammonia recovery drives the cost of the

pre-treatment (Holtzapple et al., 1991 and 1994), although ammonia is easily recovered

due to its volatility.

Chapter-1

22

c) CO2 explosion

This method is similar to steam and ammonia fibre explosion; high pressure CO2

is

injected into the batch reactor and then liberated by an explosive decompression. It is

believed that CO2

reacts to carbonic acid (carbon dioxide in water), thereby improving the

hydrolysis rate. Yields of CO2

explosion are lower than those obtained with steam or

ammonia explosion, but they are higher than those reached with enzymatic hydrolysis

without pretreatment (Sun and Cheng, 2002).

d) Mechanical/alkaline pretreatment

Combined mechanical/alkaline pretreatment consists of a continuous mechanical

pretreatment (e.g. milling, extrusion, refining) of lignocellulosic biomass with the aid of an

alkali. The resulting fractions consist of a soluble fraction (containing lignin, hemicellulose

and inorganic components) and a cellulose-enriched solid fraction. As opposed to the acid-

catalysed methods, the general principle behind alkaline pretreatment methods is the

removal of lignin whereas cellulose and part of the hemicelluloses remain in the solid

fraction. The solid fraction is submitted to enzymatic hydrolysis for the production of C6

and C5 sugars and this pretreatment method is especially suitable in combination with

fermentation routes in which both C6 and C5 sugars can be converted to products. By

performing extrusion and chemical pretreatment in one step the accessibility of cellulose

for enzymes is improved, resulting in higher delignification values and improved

enzymatic hydrolysis. In addition, the moderate operation temperatures of this process

prevent the formation of degradation and oxidation products.

1.7.4 Biological pretreatment

In this group of pretreatments microorganisms such as white, brown and soft rot-

fungi are employed to degrade hemicellulose and lignin. Advantages of biological

pretreatments are low energy requirement and mild operation conditions. Nevertheless, the

rate of biological hydrolysis is usually very low, so this pretreatment requires long

residence times (Cardona and Sanchez, 2007; Sun and Cheng, 2002; Tengerdy and

Szakacs, 2003).

Chapter-1

23

1.8 Applications of xylanases

Xylanolytic enzymes from microorganism have attracted a great deal of attention in

the last decade, particularly because of their biotechnological potential in various industrial

processes (Bajpai et al., 1999), such as food, feed, and pulp and paper industries.

Xylanases have shown an immense potential for increasing the production of several

useful products in a most economical way. Commercial xylanases are industrially

produced in countries such as Japan, Finland, Germany, Republic of Ireland, Denmark,

Canada and the USA. The microorganisms used to obtain these enzymes are Aspergillus

niger, Trichoderma sp. and Humicola insolens. Nevertheless, commercial xylanases can

also be obtained from bacteria. Xylanase began to be used in the 1980s: initially in the

preparation of animal feed and later in the food, textile and paper industries. Currently,

xylanase and cellulase, together with pectinases, account for 20% of the world enzyme

market.

a) Bio-ethanol production

The process of ethanol production from lignocellulosic biomass includes de-

lignification of plant biomass and hydrolysis of cellulose and hemicellulose to

monosaccharides (Beg et al., 2001). The hydrolysis process can be performed by treatment

with acids at high temperatures or by enzyme action. The acidic hydrolysis requires a

significant energy consumption and acid-resisting equipment which makes the process

more expensive. However, enzymatic hydrolysis does not have these disadvantages.

Because of the complex composition of lignocellulosic biomass, the synergistic action of

several enzymes viz. endoglucanases, (EC 3.2.1.4), β-glucosidases (EC 3.2.1.21), endo-1,

4-β-xylanases (EC 3.2.1.8) and β- xylosidases (EC 3.2.1.37) is required for complete

hydrolysis (deVries and Visser 2001). In some cases, endo-1, 4-β xylanase has been

reported to be a bifunctional enzyme having endo-1, 4-β-xylanase as well as cellulase

activity. Bi-functionality of endo-1,4-β-xylanase could result in more efficient and cheaper

saccharification process of the agricultural residues, municipal and industrial wastes used

for bio-ethanol production as it can degrade both cellulose and xylan residues.

Saccharification of the cellulose and hemicellulose in biomass results in sugar-rich liquid

streams useful for the production of a variety of value-added products including ethanol,

Chapter-1

24

furfural, and various functional biopolymers (Fuller et al., 1995). An increased possibility

of fermentation of both hexoses and pentoses sugars present in lignocelluloses into

methanol has also been reported (Senn and Pieper 2001).

b) Textiles

The xylanolytic complex can be used in the textile industry to process plant fibres,

such as hessian or linen. For this purpose, the xylanase should be free of cellulolytic

enzymes. One process consists of incubating dried ramee (China grass) stems with

xylanase to liberate the long cellulose fibres intact. After using this method, there is no

need to use the strong bleaching step, since the lignin does not undergo oxidation, which

would lead to darkening of the fibres (Prade 1995; Brühlmann et al., 2000; Csiszar et al.,

2001). Relatively little research has been done on the enzymatic preparation of textile

fibres, and yet this appears to be a promising market demanding the development of new

techniques.

c) Xylanases in animal feed

The use of enzymes in the production of feed is an important sector of agribusiness,

with an annual world production exceeding 600 million tons and a turnover of >50 billion

dollars. Xylanases are used in animal feed along with glucanases, pectinases, cellulases,

proteases, amylases, phytase, galactosidases and lipases. These enzymes break down

arabinoxylans in the ingredients of the feed, reducing the viscosity of the raw material

(Twomey et al., 2003). The arabinoxylan found in the cell walls of grains has an anti-

nutrient effect on poultry. When such components are present in soluble form, they may

raise the viscosity of the ingested feed, interfering with the mobility and absorption of

other components. If xylanase is added to feed containing maize and sorghum, both of

which are low viscosity foods, it may improve the digestion of nutrients in the initial part

of the digestive tract, resulting in a better use of energy.

d) Pharmaceutical and chemical applications

Xylanase and xylan are little used in the pharmaceutical industry. Xylanases are

sometimes added in combination with a complex of enzymes (hemicellulases, proteases

Chapter-1

25

and others) as a dietary supplement or to treat poor digestion, but few medicinal products

can be found with this formulation. Hydrolytic products of xylan, such as β-D-

xylopyranosyl residues, can be converted into combustible liquids (ethanol), solvents and

artificial low-calorie sweeteners. The first steps are the delignification of hemicelluloses

material rich in xylan, followed by hydrolysis by xylanases and hemicellulases, to produce

sugars such as β-Dxylopyranosyl units. Next, the products are fermented, mainly by yeasts

(Pichia stipitis and Candida shehatae) to produce xylitol or ethanol (Shapack et al., 1987;

Screenath and Jeffries 2000). Among the sugars used in the production of ethyl alcohol, β-

D-xylopyranosyl residues represent between 5 and 20%. Xylitol is a polyalcohol with a

sweetening power comparable to that of sucrose (Parajo et al., 1998). It is a non-cariogenic

sweetener, suitable for diabetic and obese individuals and recommended for the prevention

of osteoporosis and respiratory infections, lipid metabolism disorder, kidney and parenteral

lesions. A variety of commercial products containing xylitol, such as chewing gum, can be

found on the market.

e) Fruit juice and beer clarification

Endo-1,4-β-xylanase helps in increasing juice yield from fruits or vegetables and

also in the maceration process. Besides, it reduces the viscosity of the fruit juice improving

its filterability (Biely, 1985). Endo-1, 4-β-xylanase also improves extraction of more

fermentable sugar from barley and therefore useful for making beer, as well as processing

the spent barley for animal feed. In addition, added endo-1, 4-β-xylanase reduces the

viscosity of the brewing liquid, improving its filterability (Garg et al., 2010).

f) Baking

Enzymes play a central role in baking technology. Xylanase has been reported to

have used in bread making (Beg et al., 2001). It has been described that enzymatic

hydrolysis of non-starch polysaccharides leads to improvement of rheological properties of

dough, bread specific volume, and crumb firmness (Martinez-Anaya and Jimenez 1997).

Endo-1,4-β-xylanase (xylanase) randomly attacks arabino-xylan backbone to cause a

decrease in the degree of polymerization, hence leaving a strong impact on arabino-xylan

structure and function (Courtin and Delcour 2002; Qi Si and Drost-Lustenberger 2002). At

Chapter-1

26

the optimum doses, xylanase improves dough machinability, dough stability, oven spring,

loaf volume, crumb structure, and shelf-life (Hamer 1995; Poutanen 1997). As a

consequence of the hydrolytic action of xylanase, free sugars such as pentoses could be

released, which might be used by microbes for fermentation. The non-starch

polysaccharides in the endosperm cell wall of wheat constitute up to 75% of the dry matter

weight (Mares and Stone 1973). There are 3-4% (w/w) pentosans in normal wheat flour

which are partially soluble (Qi Si and Drost-Lustenberger 2002). Several endo-1, 4-β-

xylanases from bacterial and fungal sources have been used in baking industries (Pariza

and Johnson 2001).

g) Paper Industry

Chlorinated phenolic compounds as well as polychlorinated biphenyls, produced

during conventional pulp bleaching being toxic and highly resistant to biodegradation,

form one of the major sources of environmental pollution.

h) Kraft Process

Removal of residual lignin from Kraft pulp is physically and chemically restricted

by hemicelluloses. Lignin has been reported to link with hemicelluloses (Puls 1997;

Karlsson et al., 2001) and there are reports regarding the isolation of lignin carbohydrate

complexes from the kraft pulp (Iverson and Wannstrom 1986) The most common pulping

process is the kraft process or sulphate process where cooking of wood chips is carried out

in a solution of Na2S/NaOH at about 170°C for two hours resulting in the degradation and

solubilisation of lignin. The resulting pulp has a characteristic brown colour which is

primarily due to the presence of residual lignin and lignin derivatives. The intensity of pulp

colour is a function of the amount and chemical state of the remaining lignin. To obtain

pulp of very high brightness and brightness stability, all the lignin must be removed from

the pulp. For that, chemical pulping is more effective than mechanical pulping. However,

there is the formation of residual lignin which has to be removed by bleaching process.

The residual lignin in chemical pulp is dark in colour because it has been extensively

oxidized and modified in the cooking process. This residual lignin is difficult to be

removed due to its covalent binding to the hemicellulose and perhaps to cellulose fibres.

Chapter-1

27

The bleaching of the pulp can be regarded as a purification process involving the

destruction, alteration or solubilization of the lignin, coloured organic matters and other

undesirable residues on the fibres (Madlala et al., 2001).

i) Bio-bleaching

Bleaching of chemical pulp to a higher brightness without complete removal of

lignin has not been successful so far. Conventionally chlorine is used for bleaching.

Chlorination of pulp does not show any decolourising effect, and in fact, the colour of the

pulp may increase with chlorination and it is the oxidative mechanism which aids the pulp

bleaching (Loras 1980). At low pH the main reaction of chlorine is chlorination rather than

oxidation. Thus chlorine selectively chlorinates and degrades lignin compounds rather than

the carbohydrates (e.g. hemicelluloses-xylan) moieties in the unbleached pulp. The

dominant role of chlorine in bleaching is to convert the residual lignin in the pulp to water

or alkali soluble products. The effluents that are produced during the bleaching process,

especially those following the chlorination and the first extraction stages are the major

contributors to waste water pollution from the pulp paper industry. (Subramaniyan and

Prema, 2000). During the Kraft process part of the xylan is relocated on the fibre surfaces.

Considerable amount of xylan is present in the fibres after pulping process. Enzymatic

hydrolysis of the reprecipitated and relocated xylans on the surface of the fibres apparently

renders the structure of the fibre more permeable. The increased permeability allows the

passage of lignin or lignin-carbohydrate molecules in higher amounts and of high

molecular masses in the subsequent chemical reactions.

j) Degumming

Xylanase system with pectinolytic enzyme system can be used for the degumming

of bast fibers (Fu et al., 2008) such as flax, hamp, jute and ramie. Xylanase-pectinase

combination can also be used in the debarking process, the first step in wood processing

(Wong and Saddler, 1997). Pectinases are believed to play a major role in removal of

binding materials from plant tissues, but xylanase may also be involved in this process.

Chapter-1

28

k) Pulp fiber morphology

After comparing SEM micrographs of soft wood sulphate pulp with that of the

same pulp after xylanase prebleaching and alkali extraction, Pekarovicova et al., (1986)

found that there is no marked change in the shape of fibre after xylanase prebleaching.

However, flattening of the fibre arise after alkaline extraction, confirming that the lignin

extraction from the cell wall results in its collapse. Another report on application of

xylanases for bagasse sulfite pulp pre-treatment also confirmned the formation of ‘peels’

and ‘cracks’ of fibre surfaces. Perhaps this can be explained as resulting from the digestion

of the readsorbed linear xylan from the pulp fibre surface. Surface modification and the

subsequent pentration of surface layers aid the easy removal of chromophoric compounds

by mild oxidising agents.

l) Improving silage

It is known that endo-1, 4-β-xylanase and cellulase treatment of forages produces

better quality silage that improves the subsequent rate of plant cell wall digestion by

ruminants. There is a considerable amount of sugar sequestered in the xylan of plant

biomass. As a result of endo-1, 4-β-xylanase treatment, there is increased nutritive sugar

that is useful for digestion in cow and other ruminants. It is also known that endo-1,4-β-

xylanase also produces compounds which are the nutritive source for many ruminal

microflora (Garg et al., 2010).

1.9. Research objectives

The huge amount of residual plant biomass considered as waste can potentially be

used to produce various value-added products like bio-fuels, enzymes, animal feeds,

chemicals. Wheat straw, rice straw, corncobs, tobacco stalk and on, are rich in lingo-

cellulose and are considered as potential feed stocks for the industrial utilization. Since

they are usually left to rot or burned in the field after harvesting, utilization of these

materials for industrial purpose not only solve the proper disposal of the wastes, but also

provide an additional income to the farmers and generate employment. Corncobs are

reported to be an excellent substrate for growth of various industrially important bacteria,

fungi and yeast for the production of pharmaceutically and nutraceutically important

Chapter-1

29

enzymes. Corn cobs have been recently identified as an ideal source of renewable energy

due to their high carbohydrate concentration coupled with a high density which reduces the

overall cost of ethanol production.

The chemical properties and physical characteristics of corn cobs make for a

feedstock suitable for several methods of energy generation. A group of studies (Clark and

Lathrop, 1953; Foley, 1978) found that corn cobs contain 32.3-45.6 % cellulose, 39.8 %

hemicelluloses mostly composed of pentosan, and 6.7-13.9% lignin. Cellulose is a

polysaccharide of glucose units that serve as the main structural component of the cob’s

cell walls. Hemicellulose is a less complex polysaccharide that can more easily be broken

down to simpler monosaccharides, simple sugars. Lignin is a complex, non-carbohydrate,

structural component which binds to cellulose and stiffens plant cell walls. Generally xylan

constitutes D-xylopyranose units in the backbone linked through 1→4 glycosidic bonds.

The majority of D-xylans have other sugars in side chains, such as 4-O-methyl-D-

glucuronic acid, O-acetyl-L-arabinose, L-arabinose, and D-glucuronic acid. Concerning,

specifically, the xylan from corn cobs, it has been demonstrated that such polymer presents

a chemical composition of 4-O-methyl-D-glucuronic acid, L-arabinose and D-xylose in the

proportion of 2:7:19, respectively (Ebringerova and Hromadkova, 1999; Garcia et al.,

2000; Karucákova et al., 1994; Silva et al., 1998; Whistler and Smart, 1953). Corn cob is a

renewable, widespread and cheap lignocellulosic material largely available in India.

Annually approximately 204 million dry metric tons of corn residue are returned to the

ground as waste byproduct in corn grain production (Perlack et al., 2005). While corn

stover, the above ground corn plant excluding corn kernels, has much potential in use as a

biomass feedstock (Graham et al., 2007), there are concerns associated with its removal

from the ground. Corn cob is a renewable, widespread and cheap lignocellulosic material

largely available in India.

Cobs, leaves and stalks are important residues of corn processing and consumption.

For every 1 kg of dry corn grains produced, about 0.15 kg of cobs, 0.22 kg of leaves and

0.50 kg of stalks are produced (Sokhansanj et al., 2002; 2010 (USDA, 2011). This results

the production of about 130.13, 190.85 and 433.76 million tonnes of cobs, leaves and

stalks in 2011, respectively. Currently, these residues have a number of limited

applications including: (a) use of corn cobs as building material and activated carbon

Chapter-1

30

(Pinto et al., 2012; Cao et al., 2006), (b) use of corn leaves as a feedstock for fermentable

sugars and supplemental fiber source for paper pulp (Shinners and Binversie, 2007; Su et

al., 2006) and (c) use of corn stalks as livestock feed and biofertilizer (Chen et al., 2010a;

Li et al., 2007). Currently, these residues have a number of limited applications including:

(a) use of corn cobs as building material and activated carbon (Pinto et al., 2012; Cao et

al., 2006), (b) use of corn leaves as a feedstock for fermentable sugars and supplemental

fiber source for paper pulp (Shinners and Binversie, 2007; Su et al., 2006) and (c) use of

corn stalks as livestock feed and biofertilizer (Chen et al., 2010a; Li et al., 2007). About

690,668,292 tones maize is produced annually in the world. India produces 21,060,000

tones, is the sixth largest producer and it reaches 24 mn, tones of corn cobs production in

2013 (In Andhra Pradesh cultivation of corn is high).

After juice extraction from fruits generates a lignocellulosic waste material like

mango peel, banana peel, citrus peel, grape pomace and pine apple, it is formed from the

skins, seeds are called zero value-added substrates and it is constitutes about 15 to 18% of

the original fruit. These are having high amount of carbon source and degraded in the soil

by microorganisms. Basically India is an agricultural country that’s why the lignocellulose

is available in abundance, these residues are considered good substrates for enzymes

production. Especially mangos and other fruits are available in our surroundings like

chittoor district, Andhra Pradesh.

Based on these assumptions the work carried out in this thesis is focused on the

optimization, production and characterization of xylanase enzyme from corn cobs by using

fungi, and its application for the production of ethanol from pretreated corn cobs. The

present work deals with the following objectives.

1) Isolation, molecular characterization and screening of fungal strains for the production

of xylanolytic enzymes from agricultural substrates through solid state (SSF) and

submerged (SmF) fermentations.

2) Novel supplementations of fruit processing waste for optimization of xylanase

production by Trichoderma koeningi isolate using response surface methodology

(RSM).

Chapter-1

31

3) Strain improvement through UV and chemical mutagenesis for enhanced xylanase

production based on molasses or Jaggery supplementation in SSF and their optimization

through RSM.

4) Purification and characterization of cellulase-free xylanase of Trichoderma koeninigi.

5) Comparative study on simultaneous saccharification and fermentation of

lignocellulosic agricultural wastes to bio-ethanol using yeast strains.

6) Effect of enzymes supplementation on corn-wheat based poultry feed and its

digestibility in broilers.