bioprocessing of textiles (2014)

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Bioprocessing of textiles



Bioprocessing of textiles

Dr. C. Vigneswaran

Dr. M. Ananthasubramanian

Dr. P. Kandhavadivu

WOODHEAD PUBLISHING INDIA PVT LTD

New Delhi



Published by Woodhead Publishing India Pvt. Ltd.

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First published 2014, Woodhead Publishing India Pvt. Ltd.

© Woodhead Publishing India Pvt. Ltd., 2014

This book contains information obtained from authentic and highly regarded

sources. Reprinted material is quoted with permission. Reasonable efforts have

been made to publish reliable data and information, but the authors and the

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Contents

Preface ix

Acknowledgement xi

1. Enzyme technology 1

1.1 Introduction 1

1.2 Enzymes and their classications 2

1.3 Enzyme structure and catalysis 3

1.4 Enzyme kinetics and their reactions 12

1.5 Present and future trends in biotechnology 15

1.6 References 17

2. Industrial enzymes 23

2.1 Introduction 23

2.2 Enzyme-manufacturing process 24

2.3 Application of enzymes in detergents, paper, leather 31

and food industries2.4 Drawback in conventional textile processing 33

2.5 Enzymes in bioprocessing of textiles 34

2.6 Applications of enzymes in textile industry 37

2.7 Features of enzyme application in textile processing 45

2.8 References 47

3. Bioprocessing of natural bres 53

3.1 Introduction 53

3.2 Bioprocessing of cotton and their characteristics 54

3.3 Bioprocessing of jute and their characteristics 87



3.4 Bioprocessing of ax and their characteristics 112

3.5 Bioprocessing of wool and their characteristics 125 3.6 Bioprocessing of silk and their characteristics 148

3.7 References 161

4. Bioprocessing of synthetic bres 189

4.2 Bioprocessing of polyester and their characteristics 190

4.3 Bioprocessing of polyamide and their characteristics 214

4.4 Bioprocessing of regenerated cellulosic and their 220

characteristics

4.5 Biodegradability of plastics 232

4.6 References 240

5. Enzymes in textile efuents 251

5.1 Introduction 251

5.2 Textile processing operations 252

5.3 Textile efuent characteristics 258 5.4 Present technology in treating waste efuents 260

5.5 Treatment of textile efuents by various methods 265

5.6 Difculties of operation of efuent plants 271

5.7 Advanced technology in efuent treatment practices 271

5.8 Role of enzymes in decolouration 276

5.9 Prospects and future research 288

5.10 References 289

6. Safety and precaution in handling enzymes 299


6.3 Enzyme safety program 301

6.4 Safe handling of enzymes 305

6.5 Symptoms of enzyme exposure 306

6.6 Practical aspects – handling and safety 309 6.7 First-aid treatment 311

6.8 Safety in enzyme therapy 312

6.9 Routes of exposure and possible controls 312

vi Bioprocessing of textiles



6.10 Medical monitoring program for enzyme workers 313

6.11 Safety measures 314 6.12 References 315

7. Bioprocessing of organic cotton textiles 319


7.2 Organic cotton 320

7.3 Biodesizing of organic cotton fabrics with alphas 323

amylase

7.4 Bioscouring of organic cotton with alkaline pectinase 333

7.5 Bioscouring of organic cotton fabric using protease 341

enzyme

7.6 Bioscouring of organic cotton fabric using lipase enzyme 346

7.6 Binary enzyme treatment on bioscouring of organic 352

cotton fabric

7.7 Bioscouring of organic cotton fabrics through specic 355

mixed enzymatic system7.8 Sonication and aerodynamic principles – enzymatic 364

activity

7.9 Biopreparation of organic cotton fabric 370

7.10 References 395

8. Biotechnology and biomaterials for hygienic and health 398

care textiles


8.2 Medical textiles 399

8.3 Modern wound dressings 402

8.4 Enzymes in medical applications 408

8.5 Advanced biopolymer materials 417

8.7 Future trends in medical textiles 426

8.8 References 426

Index 435

Contents vii



Preface

The book mainly deals with the basic fundamentals of enzyme technology

and their applications in textile processing of both natural and synthetic bresfor enhancing their functional characteristics. With increasing awareness

about the environmental concern and usage of eco-friendly agents, today

the researchers and scientists are nding ways to treat the textile materials

without use of chemicals. To fulll the space many researchers are developing

the enzyme-based products for industrial applications to enhance the product

functional properties and uses.

The book is broadly divided into eight chapters namely enzyme technology,

industrial enzymes, bioprocessing of natural bres, synthetic bres, enzymesin textile efuents, safety and precaution in handling enzymes, bioprocessing

of organic cotton and enzyme processing for hygienic and health care textiles.

Processing of cotton fabrics in single bath and continuous operations of

desizing, scouring and bleaching processes and their efuent loads have been

discussed both enzyme- and chemical-based wet processing. This book also

describes the future scope of enzyme technology for hygienic and healthcare

textile product development and safety aspects of handling enzymes in the

textile industries.

This book would be very useful to undergraduate and postgraduatestudents of textile technology, fashion technology, textiles chemistry and textile

processing; researchers working in academic and industrial R&D, and colleges

and universities offering textile technology and biotechnology programs. In

short, this book presents a refreshingly original approach in emphasizing the

interface between enzyme technology and textile materials apart from dealing

with diverse methods and techniques used in industrial practices.

We hope that it would provide guidelines to researchers, scientists and

industrialists as well as students a useful source of information in the importanteld of textile processing through enzyme technology.

Dr. C. Vigneswaran

Dr. M. Ananthasubramanian

Dr. P. Kandhavadivu



Acknowledgement

We owe our gratitude to the people who have contributed to this eld of

Science and Technology enabling us to write this book. We immensely thankthe Principal, Management and Heads of our Departments, PSG College of

Technology for providing us the congenial atmosphere, facilities and the

required support for writing the book. We thank our family members for their

moral support and encouragement. We sincerely thank Woodhead Publishing

India for providing us to bring our knowledge and experience in printable

form.

Dr. C. Vigneswaran

Dr. M. AnanthasubramanianDr. P. Kandhavadivu



Abstract: Enzyme technology – a sub-eld of biotechnology is a developing eldin manufacturing in bulk and high added value in various industrial end uses

both in process and product developments. Enzymes are of globular proteinsand have three-dimensional structures of multi-subunit proteins. This chapterdiscusses the use of enzymes and their classications based on functions andapplications for industrial purposes. The chapter also discusses the mechanismof enzyme such as lock-and-key mechanism with substrate and briefs the rateof reactions such as temperature, pH, concentration and inhibitors. Today theapplications of enzymes in industrial uses are very wide opportunity and willpave the new spectrum in forthcoming days. Modern biotechnology is onetool that can help meet the challenge this growth poses and also contribute toeco-friendly environment and safe to health. The chapter then discusses thepresent and future trends in biotechnology and has applications in many elds

including organic synthesis, clinical analysis, textile processes and nishing,pharmaceuticals, detergents, food production and fermentation.

Keywords: biotechnology, enzyme, enzyme structure, enzyme kinetics, enzymeinhibition, textile process

1.1 Introduction

Enzymes vital to existence of life also plays important role in many aspects of

life. Mankind has used enzymes for many years without understanding what

they were or how they work (Robert 1989). Over the generations, science

has unlocked the mystery of enzymes and has applied this knowledge to

make better use of them in many numbers of applications (Gram et al 2001).

Enzymes play crucial roles in producing our food, on our clothes, even in

producing fuel for our automobiles. Enzymes are also important in reducing

both energy consumption and environmental pollution (Hayavadana and

Renuka 2003).

Enzymes are proteins, which act as a catalyst in many chemical reactions

present in almost all life forms (Hoffmann 1954; Minton 2001). Any substancethat increases the rate of chemical reaction without them changed overall in

the process is a catalyst. These catalysts act on substrates converting them into

product. Each type of enzyme chemically interacts with only one particular

substance or type of substance, termed a substrate (Warshel 1991). For

1

Enzyme technology



2 Bioprocessing of textiles

carryout biochemical reactions, enzymes are responsible in microorganisms,

plants, animals, and human beings. Enzymes are essential for all metabolic

processes, but are not alive. Although like all other proteins, enzymes are

composed of amino acids (Chen et al 1992), they differ in function and they

have the unique ability to facilitate in the biochemical reactions without

undergoing any change themselves (Smith 1997). This catalytic capability

is what makes enzymes unique (Warshel et al 2006). Enzymes are natural

protein molecules that act as highly efcient catalysts in biochemical

reactions, that is, they help a chemical reaction take place quickly and

efciently (Berg et al 2002). Enzymes not only work efciently and rapidly,

they are also biodegradable. Normally enzymes are highly efcient andincrease the rate of reaction in biochemical processes that otherwise proceed

very slowly, or in some cases, not at all. Most of the enzymes increase the

rate of reaction by factors of 103 to 1016 relatives to the rate of uncatalysed

reaction (Bugg 2004).

Enzymes play a diversied role in many aspects of everyday life including

aiding in digestion, the production of food and several industrial applications

(Colwell and Rita 2002). Enzymes are nature’s catalysts (Garcia-Viloca et

al 2004). Mankind has used for thousands of years to carry out chemical

reactions for product manufacturing such as cheese, beer, and wine. Bread andyogurt also owe their avor and also texture to a range of enzyme producing

organisms that domesticated many years ago.

1.2 Enzymes and their classications

Enzymes are active compounds characterized by their function as well as

by their molecular structure (Jun Ogawa and Sakavu Shimizu 1999; Wu-

Kuang Yeh 2010). Generally, enzymes names end with “ase” with exception

for some of the originally studied enzymes such as pepsin, rennin, and

trypsin (Cech 2000; Lilley 2005). The Enzyme Commission has classied

the 1,500 different enzymes into six types by their mechanism of action.

The EC number provides systematic method for classifying enzymes. It

takes a form of letters “EC” followed by four numbers separated by periods

(Nomenclature committee 1979). The rst number broadly classies the

enzyme based on the total reaction catalysed (Cech 1993). The second

number is the enzyme subclass (Table 1.1). The third indicates the sub-

subclass. It should be noted that the subclass and sub-subclass designationmeans different things in different classes (Enzyme Technical Association

2001). The last number is the serial number. Eg: EC 1.1.1.1 means alcohol

dehdrogenase.



Enzyme technology 3

Table 1.1 Classication of enzymes

Enzymeclassication

(EC Number)

Functions Examples

Oxidoreductases (1) Catalyzes the transfer of electrons

from one molecule (the reductant,

also called the hydrogen or electron

donor) to another (the oxidant, also

called the hydrogen or electron

acceptor). This group of enzymes

usually utilizes NADP or NAD as

cofactors.

Catalases

Glucose oxidases

Laccases

Transferases (2) Transfer a functional group (e.g. a

methyl or phosphate group)

Fructosyltransferases

Glucosyltransferases

Hydrolases (3) Catalyze the hydrolysis of various

bonds

Amylases, Cellulases

Lipases, Proteases

Lyases (4) Cleave various bonds by means

other than hydrolysis and oxidation

Pectate lyases

Alpha-acetolactate

decarboxylases

Isomerases (5) Catalyze isomerization changes

within a single molecule

Glucose isomerases

Ligases (6) Join two molecules with covalent

bonds

1.3 Enzyme structure and catalysis

Most enzymes are pure proteins. These proteins are long linear chains of

amino acids fold to produce a three-dimensional product (Annsen 1973;

Fersht 1999). Protein structure is a various levels of organization of protein

molecules (Rodnina and Wintermeyer 2001). A linear sequence of aminoacids (Polypeptide chain) is a primary structure (Smith 1994). The secondary

structure refers to regular local substructure (Alpha helix, beta sheets). The

three dimensional structure of single protein molecule is called a tertiary

structure (Buchholz and Poulson 2000; Tornroth-Horseeld and Neutze

2008). Quaternary structure is three dimensional structures of a multi-subunit

protein and having subunits t together (Fig. 1.1)

Enzymes are of globular protein type i.e., relatively spherical shapes

having complex tertiary and. sometimes quaternary structures (Annsen

1973). A protein contains all its natural structural elements (polypeptide) and

possesses biological activity is a native. When a protein has been unfolded,

it no longer possesses biological activity even though the backbone and the

amino acid groups remain intact (Jaeger and Eggert 2004). Unfolding also




causes subunit dissociation if there are no intersubunit covalent links between

them unfolded, inactive proteins are denatured (Vasella et al 2002).

Fig. 1.1 Three-dimensional structure of a multi-subunit protein

Many enzymes are larger than the substrates they act on and only few

amino acids of the enzyme are involved directly in the catalysis (Eisenmesser

et al. 2002, 2005). Each specic amino acid sequence produces a specic



Enzyme technology 5

structure having unique properties. Individual protein chains in amino groups

may form group together to derive a protein complex (Radzicka and Wolfenden

1995; Dunaway 2008). Depending on the type of enzyme, denaturation may

be reversible or irreversible. Enzymes can be denatured that is, unfolded and

inactivated by heating or chemical denaturants, which may disrupt the three-

dimensional structure of the protein in polypeptide linkages.

The enzymes consist of a protein (apoenzyme) and a non protein

component (cofactor). If the attachment between the co factor and apoenzyme

is firm then cofactor is termed prosthetic group (Jencks 1987; Xue et al 2006).

The loosely bound cofactor is called coenzyme.

Enzymes carry out catalysis of a chemical reaction. For example ifX+Y=Z

The molecule X and Y should collide in correct orientation and should

possess enough energy for the chemical bonds to alter (Wagner Arthur 1975).

As X and Y come near, the electron clouds around them create repulsion.

An initial input energy, called Activation energy overcomes this repulsion for

them to come near and cause bond rearrangement (Bergermeyer 1974; Page

and Williams 1987). This leads to the transition state where the bonds are

stretched to their limit that leads to the formation of the product.

Enzymes can act in several ways that lowers the free energy • Lowering the activation energy by creating an environment in which

the transition state is stabilized

• Lowering the energy of the transition state, but without distorting

the substrate, by creating an environment with the opposite charge

distribution to that of the transition state.

• Providing an alternative pathway. For example, temporarily reacting

with the substrate to form an intermediate ES complex, this would be

impossible in the absence of the enzyme.• Reducing the reaction entropy change by bringing substrates together

in the correct orientation to react.

• Increases in temperatures speed up reactions. Thus, temperature

increases help the enzyme function and develop the end product even

faster. However, if heated too much, the enzyme’s shape deteriorates

and the enzyme becomes denatured. Some enzymes like thermo

labile enzymes work best at low temperatures.

For more than 100 years, the behaviour of enzymes had been explained

by the “lock-and-key” mechanism developed by pioneering German chemistEmil Fischer (1907) (Fig. 1.2). Fischer thought that the chemicals undergoing

a biological reaction t precisely into enzymes like a key into a lock. But

Koshland’s work suggested that this view was too rigid that enzymes

sometimes had to change their shape to accommodate the chemicals and that




this shape change could be part of the catalytic reaction (Fig. 1.3). He called

it the “induced t theory,” but in the late 1950s traditional journals weren’t

interested in publishing his rst paper about it.

Fig. 1.2 Lock-and-key mechanism of enzyme

Fig. 1.3 Active site and substrate reaction of enzyme

1.3.1 Rate of enzyme reactions

Energy reaction processes that generate energy are termed ‘exergonic’

reactions. Reactions that require energy to initiate the reaction with substrate

are known as ‘endergonic’ reactions. Mainly the rate of enzyme reaction is

based on temperature, pH, and substrate concentration.



Enzyme technology 7

1.3.1.1 Temperature

Generally enzymes works at 40°C, but there are some enzymes working at –10°C and some other enzymes working at 90°C are available in nature.

Increasing temperature in the reaction that increases the kinetic energy in

and between molecules possess (Daniel et al 2010). In a uid, this means

that there are more random collisions between molecules (Bruins et al 2003).

Since enzymes catalyse reactions are randomly colliding with substrate

molecules, increasing temperature increases the rate of reaction, forming

more product. While increasing temperature that also increases the vibrational

energy between molecules, specically in this case enzyme molecules, which

puts strain on the bonds that hold them together (Walsh 1979). As temperature

increases, more bonds, especially the weaker hydrogen and ionic bonds, will

break as a result of this strain (Flomenbom et al 2005). Breaking bonds within

the enzyme will cause the active site to change shape. This change in shape

means that the active site is less complementary to the shape of the substrate,

so that it is less likely to catalyse the reaction. Eventually, the enzyme

will become denatured and will no longer function (Illanes et al 1999). As

temperature increases, more enzymes molecules‘ active sites‘ shapes will be

less complementary to the shape of their substrate, and more enzymes will bedenatured. This will decrease the rate of reaction (Fig. 1.4).

Fig. 1.4 Effect of temperature on enzyme activity

1.3.1.2 pH

The pH of the solution plays a signicant role in enzyme activity (Neet 1995).

Figure 1.5 shows the optimum pH selection for better rate of enzyme activity




in the substrate and enzyme activity in the solution. Most enzymes work at a

pH range between 7- 8 though some enzymes have optimum activity at pH 1.

Fig. 1.5 Effect of pH on enzyme activity

The pH affects the charge of the amino acids at the active site, so the

properties of the active site change and the substrate can no longer bind. For

example a carboxyl acid R groups will be uncharged a low pH (COOH), but

charged at high pH (COO – ) (Kopelman 1988; Todd and Gomez 2001).

1.3.1.3 Enzyme concentration

The reaction rate increases with the increase in enzyme concentration, (more

enzyme molecules more active sites), As a result more enzyme-substrate

complexes form (Olsson et al 2006). However, this too will only have an

effect up to a certain concentration (Fig. 1.6), where the enzyme concentration

is no longer the limiting factor (Henri 1902).

Fig. 1.6 Effect of enzyme concentration on rate of reaction



Enzyme technology 9

1.3.1.4 Substrate concentration

If the substrate concentration increases the rate of enzyme reaction increases because more substrate molecules interact with active sites of enzymes (Hunter

1995). Hence more substrate enzyme complex forms (Walsh 1979). Once the

enzymes are saturated with the substrates, addition of substrate makes no

difference in the reaction rate (Koshland 1958). At higher concentrations the

enzyme molecules become saturated with substrate (Fig. 1.7), and there are

few free active sites, so adding more substrate doesn’t make much difference

(though it will increase the rate of E-S collisions).

Fig. 1.7 Effect of substrate concentration on rate of reaction

1.3.1.5 Covalent modifcation

In biological systems enzymes may be in inactive state which will be modied

to active state (or vice versa) by another enzyme either by adding or deleting

a phosphate or methyl group (Irwin 1993).

1.3.1.6 Inhibitors

Inhibitors inhibit the activity of enzymes, reducing the rate of their reactions.

They are found naturally, but are also used articially as drugs, pesticides and

research tools (Changeux and Edelstein 2005)

1.3.2 Allosteric interactions (feedback inhibition)

The activity of enzymes is controlled by certain molecules binding to a

specic regulatory (or allosteric) site on the enzyme known as binding site,

distinct from the active site (Walsh 1979). Different molecules can either

inhibit or activate the enzyme, allowing sophisticated control of the rate of




biochemical reactions. Only a few enzymes can do this, and they are often

at the start of a long biochemical pathway (Tousignant and Pelletier 2004).

They are generally activated by the substrate of the pathway and inhibited

by the product of the pathway, thus only turning the pathway on when it is

needed. This process is known as ‘feedback inhibition’ (Cowan 1997). This

allosteric interaction may allow an enzyme to be temporarily inactivated in

the biochemical reactions (Fig. 1.8). The binding of an allosteric effector

changes the shape of the enzyme in binding site, inactivating it temporarily

while the effector is still bound (Jacques et al. 1963). This kind of mechanism

is commonly employed in feedback inhibition of biochemical activators.

The action of an allosteric inhibitor on the enzyme reaction may be negativecontrol for product formation in the particular biochemical reactions (Tuček

and Proška 1995; Nina et al. 2008).

Fig. 1.8 Action of Allosteric inhibitor on the enzyme reaction

1.3.3 Enzymes: Organic catalysts

Enzymes normally allow many chemical reactions to occur within the

homeostasis constraints of a living system because enzymes act as organic

catalysts in biochemical reaction (Bennett and Frieden 1969). Many enzymes

function by lowering the activation energy of biochemical reactions. By bringing

the reactants closer together, chemical bonds in reaction may be weakened andreactions will proceed faster than without the catalyst. Enzymes can act rapidly,

as in the case of carbonic anhydrase, which causes the chemicals to react 107

times faster than without the enzyme present (Pfeiffer 1954). Carbonic anhydrase

speeds up the transfer of carbon dioxide from cells to the blood. Enzymes are



Enzyme technology 11

substrate specic and enzyme which has peptidase (which breaks peptide bonds

in proteins) will not work on starch (Fig. 1.9). The arrangement of molecules on

the enzyme produces an area known as the ‘active site’ within which the specic

substrate(s) will “fit” and it recognizes connes and orients the substrate in a

particular direction in the catalysis system.

Fig. 1.9 Space lling model of an enzyme with binding site

1.3.4 Factors affecting enzyme activity

The basic enzyme kinetic theory is important in enzyme analysis to understand

the basic enzymatic mechanism. Several factors affect the rate at which

enzymatic reactions proceed - temperature, pH, enzyme concentration, substrate

concentration, and the presence of any inhibitors or activators (Martinek 1969).

Fig. 1.10 Saturation curve for an enzyme reaction showing the relation between the

substrate concentration (S) and rate (v).




1.3.5 Specicity of enzymes

One of the properties of enzymes that makes them as important as diagnosticand research tools is the specicity of its nature they exhibit relative to the

reactions they catalyze (Holum 1968). A few enzymes exhibit absolute

specicity; that is, they will catalyze only one particular reaction (Harrow and

Mazur 1958). Other enzymes will be specic for a particular type of chemical

bond or functional group. In general, there are four distinct types of specicity:

• Absolute specicity - the enzyme will catalyze only one reaction.

• Group specicity - the enzyme will act only on molecules that have

specic functional groups, such as amino, phosphate and methyl

groups.• Linkage specicity - the enzyme will act on a particular type of

chemical bond regardless of the rest of the molecular structure.

• Stereochemical specicity - the enzyme will act on a particular steric

or optical isomer.

1.4 Enzyme kinetics and their reactions

Many enzyme reactions may be modeled by the reaction scheme. If E, S, and

P represent the enzyme, substrate and product, respectively, and ES representan enzyme–substrate complex.

E + S — E + P [1.1]

The assumption is that the equilibrium between S and ES is established

rapidly, so that the second reaction is the one mainly determining the rate

d[P]/dt of appearance of the product P. This reaction will follow a rst-order

rate law, i.e.:

d[P]/dt = – kcat [ES] [1.2]

With a rate constant kcat called the catalytic constant or the turnover

number. At any given conditions and at given initial concentrations [E]

and [S] of enzyme and substrate, respectively, the rate of appearance of P

will typically decrease over time. The rate observed during conversion

of the rst few percent of the substrate is called the initial rate V. In 1913,

Leonor Michaelis and Maud Menten showed that the above model leads

to the following relation between the initial rate V and the initial substrate

concentration [S] at any given enzyme concentration (Michaelis and

Menten 1913).

V = max

M

V [S]K [S]+

[1.3]

Where K M

is a constant called the Michaelis constant and Vmax is

a constant dependent on the enzyme concentration. This dependence of V




on [S] leads to the characteristic curve shape (Fig. 1.10). At low substrate

concentrations the initial rate is, with good approximation, proportional to [S],

and at high values of [S] (substrate saturation) it approaches the limit value

Vmax

, aptly called the maximum rate. The calculations further show that Vmax

= k cat

[E]. The Michaelis constant is independent of the enzyme concentration,

and it can be seen from the formula above that KM can be found as the

substrate concentration for which V = Vmax

/2. In general, for a given enzyme,

different substrates and different sets of conditions (temperature, pH) will

give different values of kcat and KM and thus different initial rates will be

measured under otherwise identical conditions. This means in practice that

each enzyme has an optimum range of pH and temperature for its activity witha given substrate (Agarwal et al 2004). The presence or absence of cofactors

and inhibitors may also inuence the observed kinetics (Schnell et al 2006).

Enzyme activity is usually determined using a rate assay and expressed in

activity U/ml units. The substrate concentration, pH, and temperature are kept

constant during these assay procedures. Standardized assay methods are used

for commercial enzyme preparations.

1.4.1 Enzyme inhibition

An enzyme inhibitor is a molecule which binds to enzymes and decreases their

activity during biochemical reaction. Drug products are most manufactured

with enzyme inhibitors by blocking an enzyme’s activity known as pathogen

or correct a metabolic imbalance. Enzyme reaction rates can be decreased by

various types of enzyme inhibitors.

1.4.1.1 Competitive inhibition

In competitive inhibition, the inhibitor and substrate compete for the enzyme(cannot bind at the same time). Competitive inhibitors strongly resemble the

real substrate of the enzyme (Villa et al 2000). For example, methotrexate

is a competitive inhibitor of the enzyme dihydrofolate reductase, which

catalyzes the reduction of dihydrofolate to tetrahydrofolate. The binding

of the inhibitor need not be to the substrate binding site, if binding of the

inhibitor changes the conformation of the enzyme to prevent substrate binding

and vice versa (Ferguson et al 2002). In competitive inhibition the maximal

velocity of the reaction is not changed, but higher substrate concentrations arerequired to reach a given velocity, increasing the apparent Km. Competitive

inhibitors bind reversibly to the enzyme, preventing the binding of substrate

(Ellis 2001). On the other hand, binding of substrate prevents binding of the

inhibitor. Substrate and inhibitor compete for the enzyme reaction (Fig. 1.11).




Fig. 1.11 Enzyme reactions and inhibition

1.4.1.2 Uncompetitive inhibition

In uncompetitive inhibition the inhibitor can not bind to the free enzyme,

but only to the ES-complex. The EIS-complex thus formed is enzymatically

inactive. This type of inhibition is rare, but may occur in multimeric enzymes

(Hassan and Richter 2002).

1.4.1.3 Non-competitive inhibition

Non-competitive inhibitors can bind to the enzyme at the binding site at thesame time as the substrate, but not to the active site (Agarwal et al 2002).

Both the EI and EIS complexes are enzymatically inactive (Briggs and

Haldane 1925). The inhibitor cannot be driven from the enzyme by higher

substrate concentration (in contrast to competitive inhibition), the apparent

Vmax

changes. But because the substrate can still bind to the enzyme, the K m

stays the same.

1.4.1.4 Mixed inhibitionThis type of inhibition resembles the non-competitive, except that the

EIS-complex has residual enzymatic activity (Ellis 2001). This type of inhibitor

does not follow Michaelis-Menten equation (Savageau 1995; Vincent et al




2008). In many organisms inhibitors may act as part of a feedback mechanism

(Yang and Bahar 2005). If an enzyme produces too much of one substance

in the organism, that substance may act as an inhibitor for the enzyme at the

beginning of the pathway that produces it, causing production of the substance

to slow down or stop when there is sufcient amount (Schnell and Turner

2004; Agarwal 2005). This is a form of negative feedback. Enzymes which

are subject to this form of regulation are often multimeric and have allosteric

binding sites for regulatory substances (Passonneau 1993).

1.5 Present and future trends in biotechnology

During the twentieth century humankind has harnessed microorganisms to

produce useful biochemical including antibiotics, vitamins, amino acids,

avors and colors, as well as specic proteins (Carlier 2001). Some of these

proteins have important medical uses such as insulin, human growth hormone

and blood factors like erythropoietin. In fact, manufacturers have developed

a series of proven, safe, microbial hosts for use in the production of several

enzymes (Warke and Chandratre 2003). Further, enzymes that have not been

readily available in adequate quantity can be produced using technology.

This in turn has opened up important applications benecial to humankind.Additionally, modern techniques are leading to the development of tailored

enzymes with optimized functional properties specic for their intended use.

An example of this is the modication in specic proteases so that they work

more efciently in the alkaline environment of detergent formulations. As a

result, less of the modied protease is needed to deliver equivalent cleaning

power, while using fewer resources during the manufacturing process.

The microbial cell, a bacterium, yeast, or mold, is the key instrument

in many enzyme production processes. To optimize the microbial strains for

production of the desired enzyme, the strain’s genetic properties are often

modied either through natural evolution or through classical breeding and

selection techniques; these classical techniques have been used for decades

to improve microbial production strains. The precise methods of genetic

modication have been developed. The methods, sometimes termed genetic

engineering, are based on processes occurring in nature - the transfer of genes

between different cells.

Scientists to transfer genetic material between cells from the same or

different species, microorganisms such as yeasts, molds and bacteria withnew or improved properties for industrial applications can be developed. In

nature, genetic modication has been occurring since life began. Such genetic

changes are generally random, with a natural selection process favoring

the changes best adapted for survival. Using this process, animal, plant and




microbial breeders have likewise selected individuals within a species with

desired characteristics for further propagation.

Using the tools of modern biotechnology, modications can now be

made more precisely and with much less chance of developing unwanted

secondary changes that could potentially have undesired effects. In nature

and in our production systems, microbes do not express only single enzymes.

Rather, each microbial cell has the genetic nature to produce many different

enzymes. Frequently, only one of these enzyme activities is needed for a

specic application and the “side activities” are removed or substantially

reduced during the recovery process. Often, these side activities are unwanted

and may even be detrimental to the nal use. Additionally, scientists are nowable to discover and/or evolve enzymes that will catalyze pure compounds

for applications including textile wet processing such as enzymatic desizing

with alpha amylase, bioscouring with pectinase, protease, lipase and cellulase

enzymes, binary and mixed enzymatic system, and biopolishing with cellulase

enzymes, lipase enzyme for improving hydrophilic nature of polyester bre

both greatly reducing unwanted byproduct production as well as making the

target product potentially safer and more effective (Buschie-Diller et al 1994).

Modern biotechnology is one tool that can help meet the challenge

this growth poses and also contributed to (a) ecofriendly environment (b)safety and health, (c) reduced water demand in manufacturing processes,

(e) reduced industrial waste and (f) aided in pollution remediation. Enzymes

produced using modern biotechnology contributes to this effort by assuring

the availability of safe, pure enzymes that replace harsh chemical processes

(reducing energy consumption and environmental burden). Modern tools of

biotechnology, enzymes from nature can be accessed which are sufciently

robust to be useful at extremes of pH and temperature and thus hold great

promise for replacing certain chemical processes with much cleaner protein-catalyzed processes (Gubitz and Cavaco-Paulo 2001). Just as exciting, these

new enzymes can make the dream of converting waste biomass to useful

energy an economic reality. Overall, the use of modern biotechnology for

enzyme production can have a major impact on improving the cost and quality

of products at the same time working towards sustainability.

Enzymes have applications in many elds, including organic synthesis,

clinical analysis, textile processes, and nishing, pharmaceuticals, detergents,

food production and fermentation. The application of enzymes to organic

synthesis is currently attracting more and more attention. The discovery ofnew microbial enzymes through extensive and persistent screening will open

new, simple routes for synthetic processes and consequently, new ways to

solve environmental problems (Calafell et al 2005).




Research on enzyme systems for textile processing and nishing has

mainly focused on amylases and cellulases. However, recent biotechnology

and genetic engineering advances have opened opportunities for successful

applications of other enzyme systems, such as lipases, xylanases, laccases,

proteases and pectinases (Emilla Csiszar et al 1998). Today, enzymes can be

customized for specic target areas; for example, enzymatic degumming of

silk, bioscouring of cotton textiles and antifelting and softening of wool. The

basic mechanisms involving enzyme systems and interactions with textile

substrates are likely noticed. Using several enzyme systems and application

conditions, few researchers are being involved in studying the bre/enzyme

interactions and the compatibility of enzymes in combination (Gisela Buschle-Diller, and S. Haig Zeronian 1998).

Biotechnology offers an increasing potential for the production of goods to

meet various human needs. In enzyme technology, a subeld of biotechnology,

new processes have been and are being developed to manufacture both bulk

and high value added products utilizing enzymes as biocatalysts (Tzanko

et al 2002). Enzymes are also used to provide services, as in washing and

environmental processes, or for analytical and diagnostic purposes. The

driving force in the development of enzyme technology, both in academia and

industry, has been and will continue to be:

• The development of new and better products, processes and services

to meet these needs; and/or

• The improvement of processes to produce existing products from

new raw materials as biomass.

Enzymes from nature can be accessed which are sufciently robust to

be useful at extremes of pH and temperature and thus hold great promise

for replacing certain chemical processes with much cleaner protein-catalyzed

processes (Csiszar et al 2001). These new enzymes can make the dream of

converting waste biomass to useful energy an economic reality. Overall, the

use of modern biotechnology for enzyme production can have a major impact

on improving the cost and quality of products at the same time working

towards sustainability.

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Abstract: This chapter discusses the manufacturing process of enzymes dealswith the fermentation process in which enzymatic processes usually carried

out with organism media production, Sterilization, Inoculation and down steamprocessing. Application of enzymes in detergents, paper, leather and foodindustries are outlined with commercial enzymes such as cellulase and proteases.The chapter then discusses the drawback of conventional textile wet processingand application of various types of enzymes in replacement of chemicals in wetprocessing treatments in textiles. This includes desizing, scouring, mercerization,bleaching and washing. For all these steps, the chemicals used are quite toxic.The various textile wet processing such as bio-singeing, bio-desizing, bio-scouring, integrated bio-desizing and bio-scouring, bio-bleaching, peroxidekillers, enzyme effect on color, bio-polishing, bio-carbonizing, degumming ofsilk, textile auxiliaries, decolorization of dye water efuent, nishing of cotton

knits and denim washing have been outlined with use of enzymes such as alphaamylase, pectinase, protease, lipase, and cellulase.

Keywords: Enzyme manufacturing, textile wet processing, biodesizing,bioscouring, biobleaching, denim wash

2.1 Introduction

Today environmental consciousness is one of the major importances in the

textile wet processing industries being concern. Textile industry contributes

to one of the major industrial pollution problems facing the country and the

pollution causing chemicals such as lime, sodium sulphide, salt, solvents,

synthetic pigments, etc., are arise mainly from the desizing, scouring,

bleaching and dyeing processes of textile wet processing (Cavaco-Paulo

and Gubitz 2003). In order to overcome the hazards caused by the chemical

efuents, use of enzymes as a viable alternative has been resorted to in

preparatory dyeing operations such as desizing, scouring, and bleaching

treatments (Karmakar 1998). This review focuses on the use of microbial

enzymes as an alternate technology to the conventional methods, andhighlights the importance of these enzymes in minimizing the pollution load.

Environmental pollution has been a major irritant to industrial development.

This chapter also discusses the advantages and limitations of bioprocessing

techniques in the various textile wet processing such as desizing, scouring,

2

Industrial enzymes




bleaching, biopolishing, bio-singeing and bio-decolouration in the dyeing

efuents for cellulosic and non-cellulosic materials (Abadulla 2000;

Vigneswaran and Keerthivasan 2008).

2.2 Enzyme-manufacturing process

Enzyme molecules are far too complex to synthesize by purely chemical

means, and so the only way of making them is to use living organisms. The

manufacturing process of enzymes deals with the fermentation process in

which enzymatic processes usually carried out at mild conditions (Anto et al.

2006). Furthermore, enzymatic processes have led to a range of new products

and processes. Enzymes are applied in various areas of application, the most

important ones are technical use, manufacturing of food and feed stuff,

cosmetics, medicinal products and as tools for research and development

(Whitehurst and Law 2001).

The major sources of industrial enzymes are from microbes.

Microorganisms produce enzymes inside their cells (intracellular enzymes) and

may also secrete enzymes for action outside the cell (extracellular enzymes).

The microorganisms selected are usually cultured in large fermentation

chambers (known as fermenters) under controlled conditions to maximizeenzyme production (Hema Anto et al. 2006). Manufacturing process comprise

large-scale fermentation to yield high volumes of microbes (Chandrika 1999).

Enzymes are either accumulating inside the cells or are secreted into the

media of the fermentation tanks. In subsequent steps the disrupted cells are (or

the media including the enzymes) subjected to further purication processes

using variety of chemical, mechanical and thermal techniques (concentration,

precipitation, extraction, centrifugation, ltration, chromatography). The

resulting enzyme concentrate is then formulated to the nal ready-to-sell

product by adding stabilizers, standardizing agents, preservatives and salts.

The nal enzymes preparations are usually commercially marketed in granular

or liquid forms (Pandey et al. 1999).

2.2.1 Large scale fermentation of enzymes

Use of an aerobic submerged culture in a stirred tank reactor is the typical

industrial process for enzyme production involving a microorganism that

produces an industrial enzyme. Figure 2.1 shows a owchart of a typical production process and described the role of the organism in enzyme

fermentation, the media as the raw material, the requirement of sterile

environments for enzyme production and the fermentation process itself.




Type Microorganisms Enzymes Neurospora crassa Trysinase

Penicillium funiculosum Dextranase

P. notatum Glucose oxidase

Rhizopus sp. Lipase

Saccharomyces cerevisiae Invertase

S. fragilis Invertase

Trichoderma reesei Cellulase

T. viride Cellulase

2.2.1.2 Media

Microorganisms require food for their growth, called culture media. The

media should have sources for carbon, nitrogen, various nutrient salts, or

certain trace elements. Sugars are the main sources for microbial processes

for both carbon and energy (Loew 1900). Raw materials like molasses, sulte

liquor, unrened sugar, grape juice and starch materials from cereals are

being used. Besides, Nitrogen, phosphorus and potassium are added in the

form of inorganic compounds such as phosphates, ammonium compounds,or potassium chloride. Some low-price nutrients include soy meal, sh meal,

cotton seed; low-quality protein materials such as casein or its hydrolysate,

millet, stillage, and especially corn steep liquor are also used as low-price

nutrients (Tzanko Tzanov 2003). This complex media also contain trace

elements and growth promoters. Downstream processing (purifying the

product) should be kept in mind while using the complex media.

2.2.1.3 SterilizationPure culture is the population of single species of cells. Mostly pure

cultures are used in the production of enzymes. Contamination (unwanted

microorganisms) will spoil the media and overall productivity of the product.

Hence aseptic conditions should be deployed for enzyme production (Pandey

1992). This is done by sterilization. It is a process that removes or kills all

forms of microbial life. The raw materials are subjected to high temperatures

for a dened period of time to kill all microbial forms. Most solid substrates

are sterilized in rooms with elevated temperatures where as liquid media aresterilized in situ (in the reaction vessels itself). The temperature normally used

is above 100°C and it is decided by factors including the raw material used,

pH, microbial load. Air used in aerobic process is usually ltered through

glass wool lters, sintered materials, or membranes of appropriate design.

Contd...



Industrial enzymes 27

2.2.1.4 Inoculation

Introducing the microorganism into the medium is called inoculation. Afterinoculation with the cells, they multiply exponentially. Microbes are cultured

initially in small scale and later called upto inoculate in huge cultures (Robinson

et al. 2001; Banarjee and Bhattacharyya 2003). The starter culture is kept deep

frozen (–70 to –90°C) for preservation. The bacterial cells will be thermally

activated before inoculation. In fungal inoculates proper wetting of the spores

is achieved by adding small amounts of surfactants to the broth. If inoculation

by spare suspensions is not optimal, mycelial pellets can be used for start-up.

2.2.1.5 Fermentation

The fermenters used for enzyme production ranges from 20 to 200 m3 in

volume. The use up of oxygen by microbes in the aerobic process necessitates

supply of oxygen to the culture medium in the fermenter. Enzyme synthesis

rate and microbial growth rate relationships are highly complex (Flickinger

and Drew 1998). The total enzyme synthesis rate depends both on growth

rate and biomass concentration. There are many interdependent factors and

hence be optimized in a pilot plant and further scaled to production size.Cultivation of microbes in fermenter can be carried out either by batch, batch

fed or continuous process (Mala et al. 2007). In batch process, all substrates

are added in the beginning and hence growth rate cannot be controlled by

dosed feeding (Wang et al. 1974). In fed batch, low initial biomass is used

to maintain the desired growth rate. In continuous process excess biomass

is continuously removed so that synthesis rate and biomass concentration is

optimal. However fed batch process is preferred for enzyme production as it

produces higher concentrations of enzymes.

2.2.2 Downstream processing

Enzyme collection concentration and purication from the fermented media is

an important step for both intracellular and extracellular enzymes (Fig. 2.2).

For extracellular enzymes, the enzyme should be concentrated, separated and

puried from spent media. Intracellular enzymes are obtained by breaking the

cell and further separated and puried. The degree of purity of enzymes ranges

from raw enzymes to highly puried forms and depending on its application.

High product concentrations in the supernatant or inside the cells and efcient

purication are therefore important aspects in the overall economy of enzyme

manufacture. Often enzymes may be puried several hundred-fold but the yield

of the enzyme may be very poor, frequently below 10% of the activity of the

original material (Murado et al. 1996; Pandey et al. 1999). In contrast, industrial




enzymes will be puried as little as possible, only other enzymes and material

likely to interfere with the process which the enzyme is to catalyse, will be

removed. Unnecessary purication will be avoided as each additional stage is

costly in terms of equipment, manpower and loss of enzyme activity. As a result,

some commercial enzyme preparations consist essentially of concentrated

fermentation broth, plus additives to stabilize the enzyme’s activity. However,

the content of the required enzyme should be as high as possible (e. g. 10% w/w

of the protein) in order to ease the downstream processing task.

2.2.2.1 Cell disruption

The intracellular enzymes are obtained by disruption of the cells of the microbe.

This could either achieved by mechanical or non mechanical means. The

mechanical disruption of cells is carried out by rupturing cell by shear forces

and simultaneous decompression through high-pressure homogenization

(Durand et al. 1993). Chemical, thermal or enzymatic lyses are the preferred

methods for non mechanical disruption. The drying of microorganisms and the

preparation of acetone powders are standard procedures in which the structure

of the cell wall is altered to permit subsequent extraction of the cell contents.

Animal organs Plant material

Fermentation

Microorganisms

Extracellular

Enzymes

Intracellular

Enzymes

DisruptionExtraction

Grinding

Filtration

Concentration

Purication

Drying

Enyzme

concentrate

Fig. 2.2 Flowchart of the downstream processing of enzymes




2.2.2.2 Separation of solid matter

After cell disruption, the next step is separation of extracellular or intracellularenzymes from cells or cellular fragments, respectively. This operation is rather

difcult because of the small size of bacterial cells and the slight difference

between the density of the cells and that of the fermentation medium (Pandey

et al. 2000). Continuous ltration is used in industry. Large cells, e.g. yeast

cells, can be removed by decantation. Today, efcient centrifuges have been

developed to separate cells and cellular fragments in a continuous process.

Residual plant and organ matter can be separated with simpler centrifuges or

lters. Besides ltration and centrifugation, extraction and occulation are

also applied.

2.2.2.3 Concentration

The concentration of enzymes in the processed media is often very low.

Hence the volume of the starting material must be decreased by concentration

without inactivating the enzyme. Only mild concentration procedures

which do not inactivate enzymes can be employed (Pandey 2003). These

procedures include thermal methods, precipitation, and to an increasingextent, membrane ltration. Enzymes are thermolabile. Hence heat treatment

should be done for a short time. Precipitation of enzymes by salts (e.g.

Ammonium sulfate), polymers (Polyethylene glycols), organic solvents

(ethanol, acetone) and isoelectric points. In processing enzymes, cross-

ow ltration is used to harvest cells, whereas ultraltration is employed

for concentrating and desalting. The desalting of enzyme solutions can be

carried out conveniently by dialtration. The small salt molecules are driven

through a membrane with the water molecules and permeate is continuously

replaced by fresh water.

2.2.2.4 Purication

Partially puried enzyme preparations is sufcient for many industrial

applications, however for analytical purpose highly puried enzymes are

used. Special procedures employed for enzyme purication are crystallization,

electrophoresis, and chromatography. Crystallization and electrophoresis

are not relevant for large scale purications (Pettier and Beckord 1945).

Chromatography, in contrast, is of fundamental importance to enzyme

purication. Molecules are separated according to their physical properties

(size, shape, charge, hydrophobic interactions), chemical properties (covalent

binding), or biological properties (biospecic afnity).




found growth in use of new enzymes and development of new enzyme

properties in enzyme manufacturing. Enzyme expression is dramatically

increased by the use of strong expression or multi-copy systems at gene level.

New enzymes not accessible before can be cloned into and produced from

a well known host organism. Thereby, enzymes from almost any source in

nature become accessible, including exotic sources such as extremozymes,

exhibiting unusual properties such as extreme thermostability (Alat 2001).

They are also an excellent illustration of how different industry structural and

market considerations can affect the uptake of enzyme technology.

2.3 Application of enzymes in detergents, paper,leather and food industries

2.3.1 Detergents

The major applications of enzymes are in the detergent industry (Showell and

Baas 1998). They work efciently in removing dirt and other stains in the

cloth particularly at low temperatures used during washing. Mainly proteases,

lipases, amylases are used in detergents (Obendorf et al. 2003). The most

widely used detergent enzymes are hydrolases, which remove soils formedfrom proteins, lipids, and polysaccharides (Fujii et al. 1986). Cellulase is a

type of hydrolase that provides fabric care through selective reactions not

previously possible when washing clothes. Cellulases clean indirectly by

hydrolyzing glycosidic bonds. Many detergent brands are based on a blend

of two, three, or even four different enzymes (Tyndall 1996; Ee et al. 1997).

2.3.2 Paper

Trees turning to white paper involve a lot of chemical processing. Organicsubstances with chlorine are one of the major toxic products of bleaching

pulp (for whitening the paper). It also produces a range of chlorinated organic

compounds in waste water deteriorating the ecosystem. Hence enzymes are

now the popular alternative to be used in the paper industry. Use of amylases

for modication of starch coating (Bernfeld 1955) and xylanases to reduce

the consumption of bleach chemicals were the most well known applications.

Lipases used for pitch control, esterases used for stickiness removal, amylases

and cellulases for deinking have become an integral part of the solutions used

in the pulp and paper industries today (De Maria et al. 2007). The enzymatic

treatment opens up the pulp matrix allowing better penetration of the

bleaching agents and better extraction or washout of lignin and the associated

dark brown compounds.




2.3.6 Enzymes for scientic and analytical use

Enzymes can be used as chemicals to determine the concentration of substrates,measure the catalytic activity of enzymes present in biological samples and serve

as labels in immunoassays to determine the concentrations of enzymatically

inert substances. For instance, enzymes are routinely used in determination of

glucose (glucose oxidase, horse-radish peroxidase), urea (urease, glutamate

dehydrogenase), and triglycerides (lipase, carboxylesterase, glycerol kinases

etc.) in clinical diagnosis (Winkler et al. 1990). Carbohydrates, organics acids,

alcohols and other food ingredients are routinely determined in food analysis

using enzymes (El-Shafei and Rezkallah 1997). Research and development

in life science is often using genetic engineering techniques which in turn are

largely depending on various types of DNA-modifying enzymes: restriction

endonucleases, ligases, and polymerases etc.

2.4 Drawback in conventional textile processing

In the conventional textile wet processing, the grey cotton fabric has to undergo

a series of chemical treatments before it turns into a nished fabric. This

includes desizing, scouring, mercerization, bleaching and washing. For allthese steps, the chemicals used are quite toxic. During fabric manufacture, the

non-cellulosic and foreign constituents are removed partially or completely in

the various pre and post operations; the extent of removal of these constituents

decides the characteristics of the nal textile fabric (Shukla and Jaipura 2004).

Besides chemical treatment, certain enzymatic treatments are also necessary

to get optimum results. Chemical and chemical-based industries are the prime

targets of the environmentalists for their crusade against pollution, and textile

industry has also not been left out of the reckoning. The generation of pollution

is signicantly high in the preparatory and dyeing operations compared to the post dyeing operations (Menzes and Desai 2004). In fact, one third of the

pollution caused by the textile industries results from the wastes generated

during desizing operations. The wastes from the dyeing houses are let out

into the drains which in turn empty into the main sewerage causing hazard to

those who use this water. Many dyeing houses have been forced to close down

because of their noncompliance with the standards laid down. In a short span

of time, Indian textile industry has faced serious challenges such as German

ban on pentachlorophenate, certain azo dyes, formaldehyde, etc. on one hand,

and court order for compliance with environmental regulations (Vigneswaran

et al. 2011). The attention of dyeing units is focused towards revamping the

processing methods, recovery systems, and efuent treatment techniques

to make textile processing eco-friendly. Intensive efforts are being directed




towards using a viable alternative technology for pre and post processes using

enzymes. This could be one of the ways of solving the industrial pollution

problems resulting from textile waste water efuents and sustainable planet.

2.5 Enzymes in bioprocessing of textiles

Today enzymes have become an integral part of the textile processing. Though

enzyme in desizing application was established decades ago, only in recent

years the application has widened with new products introduced (Gubitz and

Cavaco-Paulo 2001). With the increase in awareness and regulation about

environment concerns, enzymes are the obvious choice because enzymes

are biodegradable and they work under mild conditions saving the precious

energy (Lowe 1992). Enzymes being biocatalysts and very specic are used in

small amounts and have a direct consequence of lesser packing material used,

the transportation impact is lower. Enzymes are responsible for many essential

biochemical reactions in microorganisms, plants, animals, and human beings

(Vigneswaran et al. 2011). Enzymes are essential for all metabolic processes,

but are not alive. Although like all other proteins, enzymes are composed

of amino acids, they differ in function in that they have the unique ability

to facilitate biochemical reactions without undergoing change themselves.This catalytic capability is what makes enzymes unique (Gupta et al. 2004).

Enzymes are categorized according to the compounds they act upon. Some

of the most common include; protease which break down proteins, cellulase

which break down cellulose, lipase which split fats (lipids) into glycerol and

fatty acids, and amylase which break down starch into simple sugars (Egmond

and Van Bemmel 1997). Table 2.2 and 2.3 show the major types of hydrolase

and oxidoreductase enzymes used in the textile wet processing industries.

Table 2.2 Application of hydrolase enzyme in fabric preparation

Enzyme name Substrate attacked Textile application

Amylase Starch Starch desizing

Cellulase Cellulose Stone wash/ bio-polishing

Bionishing for hand modications

Carbonization of wood

Pectinase Pectin Bio scour replacing caustic

Catalase Peroxides In situ peroxide decomposition without

any rinse in bleach bathProtease Protein molecules /

peptide bonds

Degumming of silk

Bioantifelting of wool

Lipases Fats and Oils Hydrophilicity of cotton and polyester




Table 2.3 Application of oxidoreductase in fabric preparation

Enzyme name Substrate attacked Textile applicationLaccase Colour

Chromophore and

pigments

Discoloration of coloured efuent

Bio-bleaching of lignin containing

bres like kenaf and jute

Bio-bleaching of indigo in denim

for various effects

Peroxidases Colour

Chromophore and

pigments

Bio-bleaching of wood pulp

Glucose oxidases Pigments In situ generation of hydrogenperoxide for bleaching of cotton

AZO reductase Colour Chromophore and

pigment

Discoloration of AZO dyes

efuent

Peroxidase ostreatus Colour Chromophore and

pigment

Discoloration of Remazol of

basic dye efuent

2.5.1 Amylase

Amylases are hydrolase class of enzymes, which hydrolyze 1–4 α glucosidic

linkage of amylase and amylopectin of starch to convert them into soluble

dextrins (Kearsley and Dziedzic 1995). Table 2.4 shows the type and

application of amylases in textile processing.

Table 2.4 Type of amylases and their applications

Type of amylases Applications

Thermostable

amylases

Amylases which catalyze starch hydrolysis in

the temperature range of 70–110°C and at pH

6.0–6.8.Conventional

amylases

Amylases which catalyze starch hydrolysis in

the temperature range of 50–70°C and at pH

6.0–6.8.

Low temperature

amylases

Majority of fungal amylases which catalyze

starch hydrolysis in the temperature range of

30–70°C and at pH 6.0–6.8.

2.5.2 CellulaseCellulases are hydrolase class of enzymes which cleavage 1–4β glucosidic

linkage of cellobiose chain or cellulose. The commercially available cellulases

are a mixture of enzymes viz., Endogluconases, Exogluconases and Cellobiases.




2H2O

2 → 2H

2O + O

2 [2.1]

Catalase is a heam-containing enzyme (Sumner and Dounce 1937;

Schroeder et al. 1969). Thus, in addition to the protein part of the molecule

the enzyme contains a non-protein part, which is a derivative of heam and

includes the metal iron (Chelikani et al. 2004). Peroxidases effectively

degrade the hydrogen peroxide at varied pH between 3 to 9 and temperature

range 30–80°C.

2.5.6 Laccase

Laccases are oxidoreductase class of enzymes, belonging to bluoxidase-

copper metalloenzymes. Laccases are generally active at pH 3–5 and in the

optimal temperature range of 30–50°C. They oxidize using molecular oxygen

as electron acceptor from the substrate. Their special property of oxidation of

indigo pigments is made use of in textile industries.

2.6 Applications of enzymes in textile industry

2.6.1 Bio-singeing

This mode of nishing has been specically developed to achieve clearer

pile on terry towel goods. A treatment with an enzyme, which is a powerful

cellulase composition, gives clearer look to the pile, improves absorbency

and softness (Koo et al. 1994). Earlier, desizing was carried out by steeping

the fabric with mineral acid, which affected the cellulose as well as the

colour. Use of enzymes here led to reaction with the starch only and thus they

assumed considerable signicance. Explaining the action of enzymes, the

food consumed by human body was digested due to secretion of the enzyme.

At the enzyme–substrate complex level, the concentration of the reactants became large and accelerated the reaction while reducing the activation

energy barrier. Thus, the reaction which took place at higher temperature and

severe conditions could be carried out at relatively lower temperatures and

milder conditions.

2.6.2 Bio-desizing

Before the fabric can be dyed, the applied sizing agent and the natural non-

cellulosic materials present in the cotton must be removed. Before the discoveryof amylase enzymes, the only alternative to remove the starch based sizing

was extended treatment with caustic soda at high temperature. The chemical

treatment was not totally effective in removing the starch (which leads to

imperfections in dyeing) and also results in a degradation of the cotton bre




resulting in destruction of the natural, soft feel, or hand, of the cotton (Etters

1999; Emre and Merih 2004). The use of amylases to remove starch-based

sizing agents has decreased the use of harsh chemicals in the textile industry,

resulting in a lower discharge of waste chemicals to the environment, improved

the safety of working conditions for textile workers and has raised the quality

of the fabric. New enzymatic processes are being developed (cellulase,

hemicellulase, pectinase and lipase), which offer the potential to totally

replace the use of other chemicals in textile preparation processes (Lange

1997; Etters et al. 2003). Complete removal of starch-containing size without

bre damage is best obtained by using enzymatic desizing agents. Formerly

amylase derived from mold, pancreas or malt where used in desizing. Todayliquid bacterial amylase preparations dominate. The enzymatic desizing

process can be divided into three stages; (a) Impregnation: enzyme solution

is absorbed by the fabric. This stage involves thorough wetting of fabric with

enzyme solution at a temperature of 70°C or higher with a liquid pick up

of 1 liter per kg fabric. Under these conditions there is sufcient enzyme

stability (temperature, pH, calcium ion level govern the stability). During

this stage gelatinization of the size (starch) is to the highest possible extent,

(b) Incubation: enzyme breaks down the size. Long incubation time allows a

low enzyme concentration, (c) After-wash: breakdown products from the sizeare removed from the fabric (Vigneswaran et al. 2012). The desizing process

is not nished until the size breakdown products have been removed from the

fabric. This is best obtained by a subsequent detergent wash (with NaOH) at

the highest possible temperature (Falholt and Olsen 1998).

2.6.3 Bio-scouring

Cotton could be treated with bioscouring enzyme although the techno-

economical parameters were not conductive (Li and Hardin 1998). But, it

had a bright future due to rigorous efuent treatment since the disposal of

both caustic soda and soda ash was causing environmental concern. The

enzymes helped removal of waxes, pectins, sizes and other impurities on the

surface of the fabric. Combination of pectinase and lipase gave best results,

but cost of the latter was a deterrent (Emilla et al. 1998; Calafell and Garriga

2004). Advantages of bioscouring were lower BOD, COD, TDS, and the

alkaline media of water, extent of cotton weight loss, which was a boon to

the knitting industry, lower alteration of cotton morphology i.e. less damagesince it was specic to pectin and waxes and not cellulose besides increased

softness (Gulrajani and Venkatraj 1986). The lone disadvantage was that

the cotton motes were not removed, which warranted peroxide bleaching.




lipases were used to bleach indigo. Earlier denim was bleached with chlorine

to get lighter denim or wash down effect. Lipase combination was used

successfully and if this could be extended to other colours, this would become

an important enzyme in future (Schrag and Cygler 1997; Varanasi et al. 2001).

The advantages were environment friendly application, and cellulose was not

affected. A bio-bleaching or lipase treatment on denim gave an authentic wash

resulting in an excellent look, which was better than a neutral wash and a grey

cast, which was used in bleaching (Nalankilli and Sundar 2002). Amylase

and lipase were used for desizing and cellulase for aberration. Laccase was

introduced for bleaching of indigo.

2.6.6 Peroxide killers

It ensured shade quality particularly with reactive dyes, reduced the complexity

of treatment after peroxide bleaching and conserved water. In case of reactive

dyeing, after bleaching it was vital that the peroxide residues must be cleared

out of the system and as such there were no fool proof ways of such clearance,

which entailed several rinsing operations or reduction treatments (Isobe et al.

2006). Empirically, it was difcult to know how much quantity of reducing

agent was required to react with the peroxide left in the bath. In the event eitherof them happened to be excess, it might affect the dyeing. Therefore, after

bleaching, the bath should be neutralized with peroxide killers like peroxidase

or catalase followed dyeing with reactive dyes (Maehly and Chance 1954;

Murthy et al. 1981). They did not affect reactive dyes and only react with the

peroxide. These catalysts were fastest acting type as one molecule of catalyst

destroyed ve million molecules of peroxide or 700 times its own weight of

peroxide.

2.6.7 Enzyme effect on color

Hydrolases and oxireductases constituted important class of enzymes which

dealt with colour in textile application. Due to effect of enzyme and physical

aberration of cellulose, the exposed areas became white as well as indigo

dyed (Andreaus et al. 2000). This kind of effect on denim was called ‘salt’

and ‘pepper’ effect. The more contrast, better was the denim wash. Some of

the denims had blue or greyer cast because they were woven with one up or

two down and one of the yarn was coloured while the other wasn’t. Thus,the effect was created with the combination of the hydrolysis of 1–4 glucose

linkage in cellulose and the abrasion e.g. turbulence of friction of metal to

metal or bre to bre led to denim appearance (Tyndall 1996). Combination

of enzyme, sand blasting and bleach evolved a fashion recently. Sand blasting




was enzyme treatments which subject the denim fabric to sand at high

pressure with consequent exposure of white area while blowing off surface

colour followed by a treatment of the fabric again with enzyme, leading to a

salt and pepper effect and bleached to reduce the colour value (Buschie-diller

et al. 1994). Furthermore, after sand blasting, treatment with enzyme followed

by over dyeing of the abraded areas produced typical effects on denim.

2.6.8 Bio-polishing

It was perceived that bio-polishing and fading or bio-polishing and wash

down were two different operations. But both of them basically employed the

same action (Hassan et al. 1996). They degraded the cellulose due to abrasion

or friction between bre to bre or bre to metal resulting in removal rst

from cellulose and then surface bleeding. Bio-polishing before dyeing could

increase depth apparently due to clarity of shade. Bio-polishing or cellulase

enzyme treatment of lyocel type of regenerated cellulose could produce peach

like effect (Kumar et al. 2008). Bio-polishing give cleaner appearance to the

garment besides wash down effect. If it was sulphur or pigment dyed goods

or ring dyed fabric, wash down effect as well as cleaning of fabric surface

could be obtained. The result surface hair was removed, reduced pilling, better print registration and colour brightness. Size of cellulase enzyme was

about 8nm as also the size of cellulose monomer, which was in similar region.

Bio-polishing, a technique rst adopted by the Danish Firm, Novo Nordisk

for the nishing treatment of cellulosic fabrics with cellulase enzymes. The

main objectives of the bio-polishing is to upgrade the quality of the fabric by

removing the protruded bres from the surface and modication of the surface

structure of the bre, thereby making it soft and smooth (Mehra et al. 1993).

In conventional process protruded bres are removed by singing process and

smoothness imparted by chemical treatment. The conventional methods are

temporary, bres return on the surface of the fabric and chemicals are removed

after few washing and fuzz is formed. The fuzz on the surface spoils the fabric

appearance and generates customer’s dissatisfaction whereas biopolishing is

permanent and it not only keeps the fabric in good condition after repeated

washing but also enhances feel, colour, drapability etc consequently products

become more attractive to the customer and fetch better prices.

Examples of some cellulases are Aspergillus Niger, Trichoderma

longibrachiatum, Fusarium solani and Trichoderma viride. The enzymes are bimolecular of about 20 amino acids with molecular weight ranging from

12,000 to 1,50,000 and therefore they are too large to penetrate the interior

of a cellulosic bre. Hence, only 1,4 β-glucosidic bonds at the surface of

cellulose bre are affected (Pedersen et al. 1992). This results in removal




of surface hairs which are responsible for improvement in the hand and

feel of the fabric due to surface etching. Biopolishing or bio nishing can

be performed continuously and in batch form but the treatment conditions

are more easily controlled in batch processes for which winches, jiggers, jet

or over ow machines are suitable. In principle, bionishing can be carried

out along with any other stage of textile nishing, with dyeing for example

provided that both processes are subject to identical conditions. However, it is

best carried out after bleaching and before dyeing.

Advantages of bio-polishing

• Improved pilling resistance

• A clearer, lint and fuzz-free surface structure • Improved drape ability and softness

• The effects are durable

• Slight improvement in absorbency

• Fashionable effects on fabric like distressed look of denim

2.6.9 Bio-carbonizing

Polyester/cellulosic blends after dyeing and/ or printing are occasionally

treated with strong solution of sulphuric acid to dissolve cellulosic component.The resultant goods are soft and have a peculiar uffy feel. This process is

risky due to highly corrosive acid that is also difcult to treat in an Efuent

Treatment Plant (ETP). The goods are treated with cellulose enzyme based

formulation to achieve dissolution of cellulosic bres. In the bio-carbonizing

process the goods are treated with a cellulose enzyme based formulation to

achieve dissolution of cellulosic component. The goods are padded in a warm

solution of this product and batched on a roll under normal conditions and are

washed off after 12–16 hours. This process offers an eco-friendly option to the

obnoxious use of strong acids.

2.6.10 Degumming of silk

Silk is made up of two types of proteins like fibroin and sericin (Ross et al.

2003). In the case of enzymatic treatment, a sericin specic protein was used

to degum the silk without causing damage, impart softness and increase dye

uptake of about 30%. If silk was degummed by alkaline treatment, there was

damage to fibroin and heavy weight loss (Puente and Lopez-Otin 2004).

2.6.11 Textile auxiliaries

Textile auxiliaries such as dyes could be produced by fermentation or from

plants in the future (before invention of synthetic dyes in the nineteenth century




many of the colours used to dye textiles came from plants e.g. wood, indigo and

madder). Many microorganisms produce pigments during their growth, which

are substantive as indicated by the permanent staining that is often associated

with mildew growth on textiles and plastics (Paul F Hamlyn 1995). It is not

unusual for some species to produce up to 30% of their dry weight as pigment.

Several for these microbial pigments have been shown to be benzoquinone,

naphthoquinone, anthraquinone, perinaphthenone and benzo uoranthene

quinone derivatives, resembling in some instances the important group of

vat dyes. Microorganisms would therefore seem to offer great potential for

the direct production of novel textile dyes of dye intermediates by controlled

fermentation techniques replacing chemical syntheses, which have inherentwaste disposal problems (e.g. toxic heavy metal compounds). The production

and evaluation of microbial pigments as textile colorants is currently being

investigated. Another biotechnological route for producing pigments for use

in the food, cosmetics of textile industries is from plant cell culture. One of the

major success stories of plant biotechnology so far has been the commercial

production since 1983 in Japan of the red pigment shikonin, which has

been incorporated into new range of cosmetics. Traditionally, shikonin was

extracted from the roots of ve year old plants of the species Lithosperum

erythrorhiz where it makes up about 1 to 2 per cent of the dry weight of theroot, tissue culture, pigment, yields of about 15 per cent of the dry weight of

the roof cells has been achieved.

2.6.12 Decolorization of dye water efuent

In textile dyeing as well as other industrial applications, large amounts of

dyestuffs are used. As a characteristic of the textile processing industry, a

wide range of structurally diverse dyes can be used in a single factory, and

therefore efuents from the industry are extremely variable in composition

(Abadulla 2000). This underlines the need for a largely unspecic process for

treating textile waste. It is known that 90% of reactive dyes entering activated

sludge sewage treatment plants will through unchanged and be discharged in

to rivers. High COD and BOD, suspended solids and intense colour due to the

extensive use of dyes characterize wastewater from textile industry, especially

process houses. This type of water must be treated before discharging it into

the environment. The water must be decolorized; harmful chemicals must be

converted into harmless chemicals. Biological treatments have been used toreduce the COD of textile efuents (Menzes and Desai 2004). Physical and

chemical treatments are effective for colour removal but use more energy and

chemicals than biological processes. They also concentrate the pollution into

solid or liquid side streams that require additional treatments or disposal, on




the contrary biological processes completely mineralize pollutants and are

cheaper. Instead of using the chemical treatments, various biological methods

can be used to treat the water from the textile industry. These methods include,

biosorption, use of enzymes, aerobic and anaerobic treatments etc. Only

biotechnological solutions can offer complete destruction of the dyestuff, with

a co-reduction in BOD and COD. In addition, the biotechnological approach

makes efcient use of the limited development space available in many

traditional dye house sites. The synthetic dyes are designed in such a way that

they become resistant to microbial degradation under the aerobic conditions.

Also the water solubility and the high molecular weight inhibit the permeation

through biological cell membranes. Anaerobic processes convert the organiccontaminants principally occupy less space; treat wastes containing up to

30,000 mg/l of COD, have lower running costs and produce less sludge.

Azo dyes are susceptible to anaerobic biodegradation but reduction of azo

compounds can result in odor problems. Biological systems, such as biolters

and bioscrubbers, are now available for the removal of odor and other volatile

compounds. The dyes can be removed by biosorption on apple pomace and

wheat straw. The experimental results showed that one gram of apple pomace

and one gram of wheat straw, with a particle size of 600 μm, where suitable

adsorbents for the removal of dyes from efuents. Apple pomace had a greatercapacity to absorb the reactive dyes, compared to wheat straw.

2.6.13 Finishing of cotton knits

Cellulase enzyme treatments increasingly nd applications in cotton hosiery

sector to enhance aesthetic feel as well as surface clarity. Ultrazyme Super is an

enzyme –based formulation, well suited for use in winches or high turbulence

soft ow machines. Adequate caution must be exercised to deactivate residual

enzyme by elevating temperatures to around 80–85°C, otherwise the reaction

would continue to take place resulting in loss of physical strength of goods.

2.6.14 Bio-denim washing

Another use of cellulase enzyme is in the fading of denims. Denims are

manufactured from indigo dyed warp yarns. The dyes are mainly absorbed on

the surface of the bre, a phenomenon technically termed as ring dyeing. The

bre surface etching with cellulase enzymes results in exposure of the undyedcore of the bres which gives a faded look to the denim. The dye removal is

further facilitated by the mechanical abrasion. Earlier the effect was obtained

by washing denim with pumice stones. Pumice stones are soft, light and

porous in nature. About 1–2 kg pumice stones per pair of jeans were used to




get the desired worn out look. Though stone washing gives the desired result

but it has got several disadvantages. The major problem with stone washing is

that lot of sludge gets deposited in the efuent tank due to worning of pumice.

The sludge has to be separated from efuent water and disposed off. The use

of stones was, therefore, replaced by cellulase enzymes. When indigo dye is

released to the wash liquor during washing, the solution turns dark blue. Indigo

dye has two amino groups which are capable of getting protonated in an acidic

media. Due to protonation, the dyestuff gains an overall positive charge on the

contrary; cellulose maintains its negative charges in an acidic media. Positive

and negative charges attract one another in solution. Therefore, in acidic pH

the afnity of indigo for cotton increases. Some of this indigo redeposit on thewhiter parts of the denim fabric which spoils the colour contrast of the stone

wash effect. This phenomenon is known as “back-staining”. Back-staining

problem is more evident with acid cellulases. The use of neutral cellulases

is recommended to control the back staining problem because of their better

control in decolouration effect and resistance to back staining. Some auxiliary

chemicals help in controlling the back staining effect, for e.g. Sandoclear IDS

Liq is claimed to be very efcient in removing back-staining. Treatment with

proteases during rinsing or at the end of the cellulase washing step results

in signicant reduction of back staining and improved contrast. The use ofenzyme for denim washing has the following advantages over pumice stone

washing.

Advantages of bio-denim washing

• Superior garment quality

• Increased load handling (30–35%)

• Environment friendly processing

• Less damage to seams, edges and badges

• Extra softener not required • Less equipment wear

• Easy handling of oors and sewers

• No handling of pumice/ceramic stones

2.7 Features of enzyme application in textile

processing

Biotechnology is a multidisciplinary eld, which has been considered in

several national development programs as one of the strategic areas and as

source of considerable amount of new products with high impact in textile

industries (Kirk 2005). Enzymes are a sustainable alternative to the use of harsh

chemicals in industry. Because enzymes work under moderate conditions,




such as warm temperatures and neutral pH, they reduce energy consumption

by eliminating the need to maintain extreme environments, as required by

many chemically catalyzed reactions. Reducing energy consumption leads to

decreased greenhouse gas emissions by power stations. Enzymes also reduce

water consumption and chemical waste production during manufacturing

processes (Lopmundra and Nayak 2004). Because enzymes react specically

and minimize the production of by-products, they offer minimal risk to

humans, wildlife, and the environment. Enzymes are both economically and

environmentally feasible because they are safely inactivated and create little

or no waste; rather than being discarded, end-product enzymatic material may

be treated and used as fertilizer for farmers’ crops. Bioprocessing done withenzymes on organic cotton fabrics will produce 100% eco-friendly garments

and apparel, which is very advantageous for the health of the consumers and

the environment (Nallankilli 1992). Bioprocessing with its pervasive eld of

application surely going to conquer the world of textiles and will make it to

rich the pinnacle of its performance. There are few to enunciate, however

many such potentials are yet to explore. Bio-processing in textiles provides to

be a boon to the ever changing conditions of the ecology as well as economy

(Vigneswaran and Keerthivasan 2009).

• Extremely specic nature of reactions involved, with practically noside effects.

• Low energy requirements, mild conditions of use, safe to handle,

non-corrosive in their applications.

• On account of lesser quantities of chemicals used in process as well

as ease of biodegradability of enzymes results in reduced loads on

ETP plants.

• Enzymes under unfavorable conditions of pH or temperatures

chemically remain in same form but their physical congurationmay get altered i.e. they get “denatured” and lose their activity. For

this reason live steam must never be injected in a bath containing

enzymes and any addition of chemicals to the enzymes bath must be

done in pre-diluted form.

• Compatibility with ionic surfactants is limited and must be checked

before use. Nonionic wetting agents with appropriate cloud points

must be selected for high working efciency as well as for uniformity

of end results.

• High sensitivity to pH, heavy metal contaminations and also toeffective temperature range.

• Intense cautions are required in use.




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Abstract: This chapter discusses the need and scope biotechnology for enzymatictreatment (bioprocessing) of natural textile bres such as cotton, jute, ax, wool

and silk to improve their physical and functional properties and also brief thechemical structural changes and improvement of surface characteristics havebeen outlined. The chapter also discusses the previous researchers and scientistsinvolved in production and characterization of various enzymes such as alphaamylase, lipase, protease, pectinase and cellulase, with various factors thatinuence the growth of microbes and yield of enzymes. Firstly, the applicationof enzymes on cotton fabric such as desizing, scouring and bleaching, theirinuence on structure and properties of bres and fabrics have also discussed.The chapter then discusses the enzyme treatment of jute and ax both bre andfabric forms have been discussed on their improvement of functional and physicalcharacteristics. Secondly, the application of wool and silk natural bres have

been discussed with previous researchers and scientists who have made attempton process like degumming and wool nishing on improvement of quality andfunctional aspects.

Keywords: Natural bre, cotton, jute, ax, wool nishing, silk degumming,pectinase, lipase, protease

3.1 Introduction

The awareness regarding environment conscious among the people has

made the scientists and technologists to look at textile processing in adifferent perspective. The textile industry was identied as a key sector

where opportunities available from adapting biotechnology are high but

current awareness of biotechnology is less. At present in textile processing

for cotton fabric the alpha amylase enzyme can be successfully used for

preparatory process like desizing. These enzymatic processes are gives

the similar results as that of conventional methods. Enzymatic desizing

processes can also identify for reduce the water consumption, power energy,

pollution, time, and increasing quality. Recently, considerable efforts have been put to use enzymes in the ax retting process to control the process

to produce linen bres of consistent quality. Previously pre-treatment of

the ax with sulphur dioxide gas brings breakdown of the woody straw

material and now to speed up enzyme retting whilst preventing excessive

3

Bioprocessing of natural bres




bacterial or fungal deterioration of the ax bre. Wool bre pretreatments,

the carbonization process in which vegetable matter in wool is degraded

by treatment with strong acid and then subjected to mechanical crushing

can, in principle, be replaced by selective enzyme degradation of the

impurities.

The international wool secretariat (IWS) together with, Novozymes,

has been developing the use of protease enzymes for a range of wool

nishing treatments aimed at increased comfort (reduced prickle, greater

softness) as well as improved surface appearance and pilling performance.

The improved enzyme treatments will allow more selective removal of

parts of the wool cuticle, there by modifying the luster, handle and feltingcharacteristics without degradation or weakening of the wool bre as a

whole and without the need for environmentally damaging pre-chlorination

treatment. Silk is made up of two types of proteins like broin and sericin.

In the case of enzymatic treatment, a sericin specic protein was used to

degum the silk without causing damage, impart softness and increase dye

uptake of about 30%. If silk was degummed by alkaline treatment, there

was damage to broin and heavy weight loss. Protease enzymes similar

to those being developed for wool processing are already being used for

the degumming of silk and for producing sand washed effects on silkgarments. Treatment of silk/cellulosic blend is claimed to produce some

unique effects. Proteases are also being used to wash down printing screens

after use in order to remove the proteinaceous gums, which are used for

thickening of printing pastes. The use of enzymes in textile processing

and after care is already the best established example of the application of

biotechnology to textiles and is likely to continue to provide some of the

most immediate and possibly dramatic illustrations of its potential in the

near- to medium-term future.

3.2 Bioprocessing of cotton and their

characteristics

3.2.1 Cotton bres physical and chemical properties

3.2.1.1 Physical properties of cotton

Cotton morphology

A cotton bre appears as a very ne, regular, look like a twisted ribbonor a collapsed and twisted tube structure under normal microscope view

(Fig. 3.1). These twists are called as ‘convolutions’ , about sixty convolutions

per centimeter in a normal cotton bre. The cross sectional view of a cotton

bre is having ‘bean’ or ‘kidney-shaped’ structure. The cotton bre is a single



Bioprocessing of natural bres 55

plant cell and composed of concentric layers. A mature cotton bre has the six

parts namely (i) cuticle, (ii) primary wall, (iii) winding layer, (iv) secondary

wall, (v) lumen wall, and (vi) lumen. The ‘cuticle’ is the outer waxy layer,

which contains pectin’s and proteinous materials. It serves as a smooth, water

resistant coating, which protects the bre. This cuticle layer is removed from

the bre by scouring during textile wet processing. The “primary wall”

is the original thin cell wall, mainly cellulose; made up of a network of

ne brils. The “winding layer” is the rst layer of secondary thickening

(Fig. 3.2). It differs in structure from both the primary wall and the remainder

of the secondary wall. It consists of brils aligned at 40–70 degree angles

to the bre axis in an open netting type of pattern. The “secondary wall”consists of concentric layers of cellulose, which constitute the main portion

of the cotton bre. After the bre has attained its maximum diameter, new

layers of cellulose are added to form the secondary wall and the brils

are deposited at 70–80 degree angles to the bre axis. The “lumen wall”

separates the secondary wall from the lumen and appears to be more

resistant to certain reagents than the secondary wall layers. The “lumen”

is the hollow canal that runs the length of the cotton bre. It is lled with

living protoplast during the growth period. After the cotton bre matures and

the boll opens, the protoplast dries up, and the lumen naturally collapses,leaving a central void, or pore space, in each bre. This internal structure

makes cotton bres accessible to liquids and vapors. The capillary action of

the brils pulls liquid in, where it is held in pores between the brils. This

structure accounts for cotton’s wickability and unique absorbing capacity.

The cotton bre is tapered on one end and brillated on the other, where

it was joined to the cotton seed and it provides the cotton bre with a soft

touch or feel to human skin.

Fig. 3.1 Microscopic view of cotton bre (longitudinal view)




Fig. 3.2 Morphological structure of cotton bre

Cotton bre length and uniformity

The cotton bre length and uniformity is basically depending on the type of

variety, cultivation environment and plantation period. Usually, cotton bre

length is described as “the average length of the longer one-half of the bres

(upper half mean length)”. Typical lengths of upland cottons might range from

0.79 to 1.36 inches (Fig. 3.3). The length of the cotton bre which determinesthe neness of the yarn and quality of fabric can be producable in the textile

process. Length uniformity or uniformity ratio is determined as “a ratio

between the mean length and the upper half mean length of the bres and is

expressed as a percentage”. Typical comparisons are illustrated in Table 3.1.

Low uniformity index shows that there might be a high content of short bres,

which lowers the quality of the textile product manufacturing.

Fig. 3.3 Cotton bre length and uniformity




ner bre result in more bres per cross section, which may turn to produce

stronger yarns. Dye absorbency and retention varies with the maturity of the

cotton bres. The greater the maturity, the better the absorbency and retention

behavior for water and other liquids.

Table 3.4 Cotton bre micronaire measurements

Cotton quality Micronaire

Fine 3.7–4.2

Average 4.3–4.9

Coarse >5.0

Color and trash

The color of cotton bres is basically determined from two parameters such as

(i) degree of reectance (Rd) and (ii) yellowness (+b). Degree of reectance

(Rd) shows the brightness of the cotton bres and yellowness depicts the

degree of cotton pigmentation. The color of the cotton bres is affected by

climatic conditions, impact of insects and fungi, type of soil, storage conditions

etc. There are ve recognized groups of color for cotton bres namely white,

gray, spotted, tinged, and yellow stained. A trash measurement describes the

amount of non-lint materials in the cotton bre. The values of trash contentshould be within the range from 0 to 1.6% for high quality cotton. Trash

content is highly correlated to leaf grade of the cotton sample.

Neps

A nep is a small tangled bre knot often caused by processing. The neps can

be measured by the AFIS (Advanced Fibre Information System) tester and

reported as the total number of neps per 0.5 grams of the bre and average size

in millimeters. Usually, the nep formation reects the mechanical processing

stage, especially from the point of view of the quality and condition of themachinery used.

Optical properties

The cotton bres show double refraction when observed in polarized light.

Even though various effects can be observed; second order yellow and

second order blue are the characteristic colors of cellulosic bres. A typical

birefringence value of cotton is 0.047.

3.2.1.2 Chemical properties of cotton

The cotton bre swells in a high humidity environment, in water and in

concentrated solutions of certain acids, salts and bases. The swelling effect

is usually attributed to the sorption of highly hydrated ions. The moisture




regain for cotton is about 7.1~8.5% and the moisture content is 7~8%. Cotton

is attacked by hot dilute or cold concentrated acid solutions. Acid hydrolysis

of cellulose produces hydro-celluloses. The cold weak acids do not affect the

cotton bre. The cotton bres show excellent resistance to alkalis and also

there are a few other solvents that will dissolve cotton completely. One of

them is a copper complex of cuprammonium hydroxide and cupriethylene

diamine (Schweitzer’s reagent). Cotton degradation is usually attributed to

oxidation, hydrolysis or both. Also, cotton bre can degrade by exposure to

visible and ultraviolet light, especially in the presence of high temperatures

around 250~397°C and humidity. Especially the cotton bres are extremely

susceptible to any biological degradation (microorganisms, fungi etc.)Chemical structure and composition of cotton bres

Cellulose chemistry

Cellulose is a polymer made up of a long chain of glucose molecules linked

by C-1 to C-4 oxygen bridges with elimination of water (glycoside bonds).

The anhydroglucose units are linked together as beta-cellobiose; therefore,

anhydro-beta-cellobiose is the repeating unit of the polymer chain (Fig. 3.4).

The number of repeat units linked together to form the cellulose polymer

is referred to as the “degree of polymerization” (DP). The cellulose chainswithin cotton bres tend to be held in place by hydrogen bonding. These

hydrogen bonds occur between the hydroxyl groups of adjacent molecules

and are most prevalent between the parallel, closely packed molecules

in the crystalline areas of the cotton bre. The three hydroxyl groups, one

primary and two secondary, in each repeating cellobiose unit of cellulose

are chemically reactive. These groups can undergo substitution reactions in

procedures designed to modify the cellulose bres or in the application of

dyes and nishes for cross linking. The hydroxyl groups are also serving as

sorption sites for water molecules, response of their strength to variations incotton bre moisture content.

Fig. 3.4 Chemical structure of cotton cellulose (one repeat unit of cellobiose unit)




In contrast, the strength of cotton generally increases with increased

moisture content. The cotton bre is not a thermoplastic bre; therefore, it

has no glass transition temperature and remains exible even at very low

temperatures. At elevated temperatures, cotton decomposes instead of

melting; long exposure to dry heat above 300°F (149°C) causes cotton bres

to decompose gradually, and temperatures above 475°F (246°C) cause rapid

deterioration. The degree of polymerization (DP) of cotton is range 5,000–

12,000. The cellulose shows approximately 66% crystallinity, which can be

determined by X-ray diffraction, infrared spectroscopy and density methods.

Each crystal unit consists of ve chains of anhydroglucose units, parallel to

the cotton bril axis.Cotton chemical composition

The chemical composition of cotton bre consists of cellulose, protein, ash,

wax, sugar, organic acids, and water (Table 3.5). The non-cellulose chemicals

of cotton are usually located in the cuticle of the cotton bre. The non-

cellulose chemicals of cotton consist of protein, ash, wax, sugar and organic

acids (Peterson 1977). In the cotton bre, wax is found on the outer surface

of the bre; primarily long chains of fatty acids and alcohols. The cotton

wax serves as a protective barrier for the cotton bre (Church and Woodhead2006). The sugar comes from two sources plant sugar and sugar from insects.

The plant sugars occur from the growth process of the cotton plant. The plant

sugars consist of monosaccharide, glucose and fructose. The insect sugars

are mainly for whiteies, the insect sugars can cause stickiness, which can

lead to problems in the textile mills. The organic acids are found in the cotton

bre as metabolic residues. The non-cellulose chemicals of cotton bre are

removed by using selective solvents. Some of these solvents include: hexane,

chloroform, sodium hydroxide solutions, non-polar solvents, hot ethanol, and

plain water (Wakelyn et al. 1975; Edward and Bharat 2004). After removingall the non-cellulose chemicals, the cotton bre is approximately ninety-nine

percent cellulose.

Table 3.5 Cotton bre composition

Composition percent

Cellulose 80–90%

Water 6–8%

Waxes and fats 0.5–1%Hemicelluloses and pectin’s 4–6%

Proteins 0–1.5%

Ash 1–1.8%




The chemical composition of cotton, when picked, is about 94 percent

cellulose; in nished fabrics is 99 percent cellulose. The cotton bre contains

carbon, hydrogen, and oxygen with reactive hydroxyl groups. Glucose is the

basic unit of the cellulose molecule. The cotton bre may have as many as

10,000 glucose monomers per molecule. The molecular chains are arranged

in long spiral linear chains within the bre. The strength of a cotton bre is

directly related to chain length. Hydrogen bonding occurs between cellulose

chains in a cotton bre. There are three hydroxyl groups that protrude from

the ring formed by one oxygen and ve carbon atoms. These groups are

polar meaning the electrons surrounding the atoms are not evenly distributed

(Allen et al. 2006). The hydrogen atoms of the hydroxyl group are attracted tomany of the oxygen atoms of the cellulose. This attraction is called ‘hydrogen

bonding’ . The bonding of hydrogen’s within the ordered regions of the brils

causes the molecules to draw closer to each other which increases the strength

of the cotton bre. Hydrogen bonding also aids in moisture absorption. Cotton

ranks among the most absorbent bres because of hydrogen bonding which

contributes to cotton’s comfort. The chemical reactivity of cellulose is related

to the hydroxyl groups of the glucose unit. The moisture, dyes, and many

chemical nishes cause these groups to readily react. Chemicals like chlorine

bleaches attack the oxygen atom between or within the two ring units breakingthe molecular chain of the cellulose substances.

3.2.2 Desizing of cotton fabric

For cotton fabrics, traditional desizing is being carried out by high temperature

washing process and high concentrations of surfactants (Lu 2005). As the

process proceeds, the viscosity of the washing liquor rises rapidly because

sizing agents dissolve. Therefore, large amounts of hot water are required.

The use of industrial enzymes for desizing represents a major improvement

because they cleave the biopolymers used a sizing agents into sub units as

oligosaccharides (Warke and Chandratre 2003). In some cases, this is a rst

step in bio-preparation of cotton fabrics (Buschie-Diller et al. 1994; Tzanko

et al. 2001). Anaerobial microbial cultures in the desizing of cotton fabrics

was studied and reported that the bioreactor from wastewater and microbial

culture for desizing of cotton was performed up to 73% desizing efciency

at 55°C and time of 60 min and it depends mainly on the temperature and

reaction time of the process and need for optimizing the process conditions forachieving better desizing and low energy (Heikinheimo et al. 2003).

The mechanical properties of synthetic size materials are better than

starch-based materials. Among different synthetic sizes, polyvinyl alcohol

(PVA) exhibits overall better performance (Bayard 1983). Sizing is a process




used for the application of a lm forming polymer to provide temporary

protection to the warp yarns from abrasive and other types of stresses

generated on the weaving machines in order to reduce the warp breakages.

Sizing helps in forming a coating which encapsulates the yarn, embeds the

protruding bres and also causes some inter-bre binding by penetration.

The spun yarns being hairy usually require size add on exceeding 8–10%;

depending on the fabric to be woven. The small addition of synthetic binder

to starch causes plasticization and increases the adhesion. Major drawback

of the starch, brittleness of the lm, can be reduced by providing internal

plasticization (Shah et al. 1976; Moghe and Khera 2005).

Enzymatic degradation of polyvinyl alcohol (PVA) was studied in thedesizing of cotton fabrics. A mixture of two different PVA-degrading enzyme

activities, including PVA oxidizing, was partially puried from the culture

ltrate of a PVA-degrading mixed culture by ionic exchange chromatography.

Optimum conditions for PVA-degradation by using this enzyme mixture were

reported at pH 8·0 and temperatures ranging from 30°C to 55°C. The cotton

fabrics sized with a PVA solution (25 g dm –3) were desized with the enzyme

mixture after a 1 h treatment at 30°C and pH 8·0. In this case, similar amounts

of residual PVA in cotton fabrics were detected in comparison with the

conventional desizing process which uses hot water at 80°C, 30 min (TatsumaMori et al. 1999). Several factors affecting the starch-size removal were

studied and are (a) the effectiveness of enzymatic desizing can be enhanced

by raising the desizing temperature up to 70°C; prolonging the desizing time

up to 60 min; increasing the material-to-liquor ratio up to 20:1; increasing

the Aquazym® 240-L dosage up to 6 g/L; treating at pH 7 (Nabil et al. 2004;

Csiszar et al. 2001). All enzymes work within a range of temperature specic

to the organism. Increases in temperature generally lead to increase in reaction

rates (Daniel et al. 2010). Most enzymes are sensitive to pH and have specicranges of activity. The pH can stop enzyme activity by denaturating (altering)

the three dimensional shape of the enzyme. Most enzymes function between

a pH of 6 and 10 (Li and Hardin 1999).

3.2.2.1 Alpha amylase enzyme

Alpha amylase enzymes used in the desizing of cotton fabrics, are obtained

from different sources with different activity levels (Bernfeld 1955). There

are two kinds of amylases available in sources namely endoamylase andexoamylases. The endoamylases cleave α, 1-4 glycosidic bonds present in the

inner parts of amylose, while exoamylases act on external glucose residues of

starch molecules (Naik and Paul 1997). Exo-enzymes include alpha amylases




(1,4 β D glucan glucanohydrolase, EC 3.2.1.2): exomaltotriohydrolases

(EC 3.2.1.95), exomaltohexohydrolases (EC 3.2.1.98) and glucoamylases

(EC 3.2.1.3). Endo enzymes include alpha amylases (1, 4 β D glucan,

glucanohydrolase, EC 3.2.1.1), pullulanases (pullulan 6 glucanohydrolase EC

3.2.1.41) and isoamylases (glycogen 6 glucanohydrolase, EC 3.2.1.68).

3.2.2.2 Alpha amylases – culture and production

Alpha amylase used in the desizing of cotton fabric for hydrolyzing the starch

component were cultured from many sources like bacterial, fungal and yeast.

Many published reports on amylase production concentrate on bacterial sources

like Bacillus subtilis (Pettier and Beckord 1945; Tomazic and Klibanov 1969;

Asgar et al. 2007), Bacillus cerens (Anto et al. 2006), Bacillus lichenijormis

(lyer 2004), Bacillus stearothermophilus (Hartman and Tetrault 1955;

Srivastava and Baruah 1986), Bacillus amyloliquifaciens (Declerck et al. 2000;

Declerck et al. 2003; Gangadharan et al. 2006), Bacillus thennooleovorans

(Malhotra et al. 2000), Bacillus macerans, Bacillus coagulans, Bacillus

circulans (Okudubo et al. 1964; Bliesmer and Hartman 1973; Mamo and

Gessesse 1999; Lee et al. 2000; Teodoro and Martins 2000; Santos and

Martins 2003; Kiran et al. 2005; Ajayl and Fagade 2008). Fungal sources like Aspergillus awaniori, Aspergillus niger (Abu et al. 2005), Thermoactinomyces

thalpophilus (Shaw et al. 1995) and Saccharomyces cerevisae, Penicillium

fellutanum and Thennomyces lanoginosus (Kunanmneni et al. 2005) due to

wide distribution and simple nutritional requirements, though amylases have

been isolated from mammalian pancreas and yeasts.

Growth of microorganisms and production of alpha amylases, in the

ferment, are highly inuenced by moisture, temperature and pH of the culture

medium (Mamo and Gessesse 1999; Teodoro and Martins 2000; Hartman and

Tetrault 1955; Kiran et al. 2005; Ajayl and Fagade 2008; Anto et al. 2006;

Gangadharan et al. 2006). At lower incubation temperatures higher yields

are observed, which reduce with increase in temperatures (Shaw et al. 1995;

Horikoshi 1999). Many alpha amylases have calcium, in their structure, for

maintaining the structural integrity and stability during hydrolytic reaction

(Hartman and Tetrault 1955; Vallee et al. 1959; Srivastava and Baruah 1986;

Malhotra et al. 2000; Hagihara et al. 2001; Lan et al. 2007). However, certain

alkali mutant Bacillus sp strains, thermophile amylases do not contain calcium

ions, whose activities are dependent on sodium ions, and they exhibit optimumactivities at 85–90°C with higher diffusion rate and lower contamination risks

(Campbell 1955; Hyun and Zeikus 1985; Mamo and Gessesse 1999; Hagihara

et al. 2001; Richardson et al. 2002; Varavinit et al. 2002; Callen et al. 2007).




3.2.2.3 Enzymatic desizing of cotton fabric

Activity of alpha amylases have been studied extensively using pure starch asa model compound (Opwis et al. 2000; Aranjo et al. 2004; Ibrahim et al. 2004;

Baks et al. 2006; Lee et al. 2006; Liu et al. 2000; Reshmi et al. 2006; Tester

2006; Shewale and Pandit 2007) and attempts have been made to analyse

the hydrolytic activities of pancreatic, malt, bacterial amylases and cellulases,

in desizing (Appleyard 1953; Fetouh et al. 1974; Khalil et al. 1974; Shah

and Sadhu 1976; Bayard 1983; Bhatawdekar 1983; Levene and Prozan 1992;

Hahn et al. 1998; Opwis et al. 2000; Feitkenhauer and Meyer 2003; Ali and

Khan 2005). Amylase assisted desizing of textile materials is carried out in

machines such as jigger, jets, pad-batch and pad-stream ranges, employing

different levels of mechanical agitations (Radhakrishnaiah et al. 1999).

Among various steps involved in enzyme reaction, hydrolysis of starch needs

longer time, depending upon the activity levels of enzyme and temperature

conditions used in desizing. Composition, properties of starch (Azevedo et

al. 2003; Aranjo et al. 2004; Moghe and Khera 2005), ingredients added in

size mix (Tomazic and Klibanov 1969; Shah and Sadhu 1976; Lange 1997;

Azevedo et al. 2003; Declerek et al. 2003) and process conditions employed

in desizing (Fetouh et al. 1974; Khalil et al. 1974; Levene and Prozan 1992;Mori et al. 1997; Ibrahim et al. 2004) have marked inuence on the efciency

of desizing. Common waxes do not inactivate amylases but prevent quick

wetting, penetration of enzymes and, other factors that affect the efciency

of size removal include viscosity of starch, amount of size applied, fabric

construction and method of washing off (Shamey and Hussein 2005).

3.2.2.4 Assessment of enzyme desizing

The assessment of alpha amylase enzyme-based desized cotton fabric has been carried out by two methods namely (a) Tegawa scale, a qualitative

spotting test using iodine solution and (b) percent weight loss (Fielf 1931;

Scott 1940; Lorentz and Oltmanns 1970; Bayard 1983; Hyun and Zeikus

1985; Shukla and Jaipura 2004). Amylose binds, ~20% of its weight of iodine

at 20° C, shows deep blue colour, violet and reddish brown, pale yellowish

brown for undegraded starch, partially degraded dextrin, degraded dextrin and

completely hydrolyzed starch, while amylopectin binds 2% iodine only.

3.2.3 Scouring of cotton fabrics

The scouring process is to make the cotton material hydrophilic, before it

undergoes other processes like bleaching, dyeing and printing in the textile

wet processing (Holme 2001; Thiagarajan and Selvakumar 2008). A desired




hydrophilicity during the scouring can be achieved by removing non-cellulosic

material from the cotton fabric, especially from the cuticle (waxes and fats)

and the primary wall (e.g. pectin, protein and organic acids). More precisely

scouring not only removes non-cellulosic material from cotton bres but also

removes substances that have adhered to the cotton bres during the production

of the yarn or fabric. Substances like, dirt, lint, pesticides, oils, and any sizing

agent applied to yarns to facilitate weaving (Eisisi et al. 1990; Ammayappan

et al. 2003). Effective scouring is essential for subsequent processing of any

cotton made substrate, regardless of its natural source. Even today, alkaline

scouring of cotton is still the most widespread commercial technique for

removing or rupturing the bre cuticle to make the bre absorbent for thecotton processing (Churi et al. 2004). Although sodium hydroxide is used

generally for the scouring, sodium carbonate and calcium hydroxide are

also mentioned as a scouring agent (Hsieh and Cram 1999). Scouring of

cotton fabric is typically done with a hot solution (90°C to 100°C) of sodium

hydroxide (± 1 mol/L) for up to one hour (Emilla Csiszar et al. 1998). The

concentration of alkali used and the time and temperature conditions needed

depend on the desired quality of the scoured fabric. Other chemicals for

instance, wetting agents, emulsifying agents and chelating agents (Nallankilli

et al. 2008; Tyndall 1996) are also included in typical preparation baths forscouring. Wetting agents act by reducing the surface tension of water enabling

improved penetration of the chemicals into the cotton fabric (Gulrajani and

Venkatraj 1986). Emulsifying agents assist in removing waxy materials.

Chelating agents remove polyvalent metal ions such as calcium, magnesium,

iron or other salts that can have a harmful effect on subsequent wet-processing

operations.

3.2.3.1 Drawbacks associated with alkaline scouring The scouring process requires large quantities of chemicals, energy and water

and is rather time consuming (Yonghua and Hardin 1997). Owing to the high

sodium hydroxide concentration and its corrosive nature, intensive rinsing is

required that leads to high water consumption. The use of high concentrations

of sodium hydroxide also requires the neutralization of wastewater, which

requires additional acid chemicals. Furthermore, the alkaline efuent requires

special handling because of very high BOD and COD values. Apart from the

above wet processing problems, the biggest drawback of alkaline scouringis a non-specic degradation of cellulose that produces fabrics of lower

tensile strength and therefore of lower quality (Wang et al. 2006). Moreover

alkaline scouring is hazardous to the workers and creates an unpleasant work

atmosphere. Although, alkaline scouring is effective and the costs of NaOH




are low, this process can be improved considerably to meet today’s energy and

environmental demands (Anon 1982).

3.2.3.2 Overview of enzymatic scouring

Enzymes are substrate specic bio-catalysts; they operate best at ambient

pressures, mild temperatures and often at a neutral pH range. Enzymes are

gaining an increasingly important role as a tool in various wet textile pre-

treatment and nishing processes (Alat 2001; Anon 2001; Carlier 2001).

Biocatalysts have been proven to be a exible and reliable tool in wet

textile processing and a promising technology to fulll the expected future

requirements. Enzymatic scouring has been investigated extensively by

various institutes and laboratories now for nearly one decade (Hartzell and

Hsieh 1998; Emilla Csiszar et al. 1998; Qiang Wang et al. 2006). Initial

investigations explored the possibility of cotton scouring with enzymes, to

see if cotton could be made hydrophilic in a reasonable time. Extracellular

enzymes involved in the degradation of the plant cell wall’s outer layer during

the invasion of the plant, excreted by phyto-pathogenic fungi and by bacteria

have been considered as candidates. Different enzymes like pectinases such

as lyases (EC 4.2.2.2); polygalacturonase endo acting type (EC 3.2.1.15)and polygalacturonase exo acting type (EC 3.2.1.67), proteases (EC 3.4.21-

25), cellulases such as endoglucanases (EC 3.3.1.4); cellobiohydrolases (EC

3.2.1.91), xylanases (EC 3.2.1.8), lipases (EC 3.1.1.3) and recently cutinases

(EC 3.1.1.74) have been examined to degrade and subsequently remove the

natural component present in the outer layer of cotton bres (Buschie-Diller

et al. 1998; Hartzell and Hsieh et al. 1998; Yonghua and Hardin 1997). These

studies incorporated staining tests, scanning electron microscopy (SEM),

weight loss analysis, cotton wax residue and nitrogen content analysis.

The scheme essentially contains the impregnation of cotton fabric with

one or more enzymes in presence or absence of surfactants and chelators,

followed by a high temperature rinsing step. The enzyme incubation time

used was up to 24 hours depending on other process conditions and the

density of the fabric. Lipases were found to be less effective in fullling this

task (Dahod 1987; El-Shafei and Rezkallah 1997). Proteases were found to

be efcient to improve whiteness rather than hydrophilicity (Ellaiah et al.

2002; Hsieh and Cram 1999). Cellulases were the only enzymes reported to

improve the wettability efciently when applied without any other treatmentor in combination with other enzymes. However, cellulase also cause a

decrease in bre strength and hence a decrease in fabric quality (Ghose 1987;

Emilla Csiszar et al. 1998; Emre Karapinar and Merih 2004). The best results

have been obtained by alkaline pectinases or pectinases in combination with




cellulase (Agarwal et al. 2007). Especially bacterial alkaline pectinase, a

pectate lyase (EC 4.2.2.2) has been proven to be effective (Ghanem et al. 2000;

Etters et al. 2003). Pectin acts as cement in the primary wall of cotton bres.

After enzymatic destabilisation of a pectin structure, the different components

present in the primary wall layer can be removed easily in subsequent rinsing

steps (Hardin and Kim 2000). A proper interpretation of the enzymatic action

on cotton bres on a molecular basis was not possible because of the lack of

structural knowledge of cotton bre (Bora and Kalita 2007).

The effect of scouring with enzymes and caustic soda on the mechanical

and surface characteristics of cotton ring-and rotor-spun yarns have been

studied (Tyagi et al. 2007). For yarn structures, the exural rigidity, hairiness,yarn-to-metal friction and dye uptake increase markedly on scouring with

enzymes and sodium hydroxide. Conventional scouring with NaOH renders

the yarns strikingly strong and less extensible. However, the tenacity of both

types of yarns is considerably reduced on enzymatic scouring. Scouring

causes a marked increase in dye uptake of ring-and rotor-spun yarns; the

increase is, however, more in NaOH scoured yarns than in the yarns spun with

identical processing conditions but scoured with enzymes (Tyagi and Sharma

2008). The dye uptake decreases marginally with increasing rotor speed. The

effect of opening roller speed on uptake of dye is also minimal. The physical properties of different varieties of cotton bres of various origins have been

studied after extraction using solvents and alkali scouring with reference to

enzyme scouring process (Nallankilli et al. 2008). Enzyme scoured cotton

samples show comparable results with that of solvent extracted and alkali

scoured samples in terms of bre neness, weight loss, moisture regain,

strength and elongation. The scouring processes improve the properties of the

bres in the order: solvent extraction < enzyme scouring < ammonium oxalate

extraction < alkali scouring. Three different scouring methods are applied to open-end and ring

spun yarns prior to hydrogen peroxide bleaching with and without metal

ions present. The scouring procedures include a penetrating treatment with

aqueous sodium hydroxide solution, a non-swelling solvent extraction, and a

bre surface affecting treatment with pectinase enzymes (Hathway and Sekins

1958). Properties of the treated yarns and bre damage are studied after each

process step. Conventional scouring with sodium hydroxide followed by

peroxide bleaching causes the highest deterioration on the molecular level, but

results in a high level of whiteness, solvent extracted yams exhibit superiortensile strength, which is preserved more or less unchanged throughout any

subsequent treatment. The bioscouring process with enzymes renders the

yams strikingly soft. The effects are generally more pronounced for open-end




spun yams, suggesting that differences in yarn structure may have an impact

on effective scouring (Buschle-Diller et al. 1998).

Cellulases and pectinases are used to treat raw cotton bres and unscoured

cotton fabrics (Kumar et al. 2008). The structural changes in the surfaces of

cotton caused by the enzymatic treatments and resulting properties are the main

focus of this study. Both staining tests and microscopy observations conrm

that the cuticle is removed by the pectinases and cellulases. All evidence

of bre weight loss and fabric water absorbency shows that signicant

changes in cotton bre and fabric properties occur with mild treatment

conditions. Physical changes in the cotton surfaces are clearly revealed by

scanning electron micrographs. All bre weight loss values stemming fromthe enzymatic treatments are smaller than or near the cotton cuticle weight.

Sufcient water absorbency for textile processing is obtained with mild

enzymatic treatments, corresponding to a small but statistically signicant

bre weight loss (Yonghua and Hardin 1998).

Yonghua and Hardin (1998) have also studied the application of cellulase

and pectinase enzymes on the cotton bres and resulting that the cuticle is

removed by the pectinases and cellulase combination. All evidence of bre

weight loss and fabric water absorbency shows that signicant changes

in cotton bre and fabric properties occur with mild treatment conditions.Sufcient water absorbency for textile processing is obtained with mild

enzymatic treatments, corresponding to a small but statistically signicant bre

weight loss (Aiteromem 2008). Commercial cellulases may contain mixtures

of different cellulase components and properties of cotton fabrics treated with

cellulases vary with the nature of these mixtures. The study reports the effect

of treatments with cellulase monocomponents such as endoglucanase I and

II and cellobiohydrolase I and II from Trichoderma reesei on the molecular

and supramolecular structures of cotton cellulose as revealed by hydrogen bonding patterns, and bre pore size distribution (Marie-Alice et al. 2003).

Relationships between the hydrolysis rate with cellulase and the swelling

degree of the cellulose bres of different samples was studied (Chahal 1985).

The water retention value (WRV) determined by the centrifugal method was

used as a measure of the swelling degree. A linear relationship was observed

between the hydrolysis rate and the WRV of the untreated pulps of different

chemical composition and crystallinity. A similar linearity was observed also

for pulps treated mechanically or chemically, but the slope of the regression

line differed with the treating methods, and it decreased in proportion with thedegree of degradation of the bre structure (Yoshitaka and Kenichiro 1968;

Reese et al. 1957). The effect of scouring at a higher temperature is to increase

the rate of enzymatic degradation without affecting the relative and important




differences in the resistance to biodegradation of the different regions of the

secondary wall (Kassenbeck 1970; Rousselle 1998).

3.2.3.3 Pectinases in bioscouring of cotton

Pectinase is an enzyme that catalyses hydrolysis/depolymerisation of the

glycosidic bonds in the pectin polymers, classied according to their preferential

substrates (high or low methylesteried pectin and polygalacturonic acid /

pectate) and their reaction mechanism (Kristensen 2001; Jayam et al. 2005).

Pectinases are either endo-acting, cutting the polymer at random sites within

the chain to give a mixture of oligomers or exo-acting attacking from one end

of the polymer and producing monomers or dimers, identied by the rate of

release of reducing sugars (Friend and Chang 1982). Pectinases have been

produced using solid state and submerged fermentation (Arguelles et al. 1995;

Pandey et al 1999; Maldonado and Saad 2000; Andersen et al. 2001; Alves

et al. 2002; Pererra et al. 1993; Martin et al. 2004; Csiszar et al. 2007), with

various bacterial (Bjo et al. 2002; Andersen et al. 2002; Vallee et al. 1959),

fungal (Arguelles et al. 1995; Sharma and Gupta 2001; Alves et al. 2002; Pyc

et al. 2003; Csiszar et al. 2007) sources and different schools of thought exist

to demonstrate their relative merits and demerits.Alkaline pectinases are produced predominantly from Bacillus and

Pseudomonas (Lawson and Hsieh 2000; Shuen-Fuh et al. 1996), while

Aspergillus appears to be the major acid pectinase producer (Csiszar et al.

2007). Comparisons have been made in the past, to assess the performance

of acid pectinases (Pyc et al. 2003; Calafell and Garriga 2004; Canal et al.

2004; Sahin and Gursoy 2005; Agrawal et al. 2007) and alkaline pectinases

(Bruhlmann 1995; Etters 1999; Etters et al. 1999; Almeida et al. 2003; Canal

et al. 2004; Agrawal et al. 2007; Wan et al. 2007; Wang et al. 2007a; Wang

et al. 2007b; Wang et al. 2007c). Effects of various carbon sources, carbon

supplements and other components in the culture medium have been well

documented (Pererra et al. 1993; Kashyap et al. 2000; Maldonado and Saad

2000; Miller et al. 2003; Presa 2007).

Pectin

Pectin is a complex carbohydrate, which is found both in the cell walls of

plants, and between the cell walls, helping to regulate the ow of water

in between cells and keeping them rigid (Solbak et al. 2005). Pectins are

considered as intracellular adhesives, combining hydrophobic molecules

like proteins, waxes, hemicelluloses with various degrees of branching and,

the three dimensional structure of pectin breaks the cellulose array in cotton

bres (Mc Call and Jurgens 1951; Anon 1954; Potikha et al. 1999; Hardin and




Kim 2000; Lawson and Hsieh 2000; Devries and Visser 2001; Brushwood

2003; Gambler 2003). Pectic acid, present in the primary wall of cotton bres,

contains negatively charged galacturonic acid residues, forms a bridge with

calcium (pectates), thereby holding the inner primary wall of cotton that

gives stability at high temperature (Whistler et al. 1940; Chandrika 1999).

Figure 3.5 shows the pectolytic activity of pectin lyase, pectin methyl esterase

and polygalacturonase enzymes.

Fig. 3.5 Pectolytic activity of pectin lyase, pectin methyl esterase and

polygalacturonase enzymes

Pectinase treatments and evaluation methods

In enzymatic scouring of cotton, nonionic surfactants are used to overcome

hydrophobicity of the substrate, which assist enzymes to penetrate through

micro-pores or cracks and help them to orient themselves in favorable

positions for catalytic actions (Sahin and Gursoy 2005), while ionic surfactants

complex with enzymes and disrupt their structure to different extents (Tzanov

et al. 2000). Concentration of pectinase, pH, time and temperature range usedin scouring, inuence efciency of the process (Etters et al. 1999; Canal et al.

2004). Microscopic observations (Li and Hardin 1998b; Sahin and Gursoy

2005), absorbency (Li and Hardin 1998b; Lenting et al. 2002; Wang et al.

2007b; Wang et al. 2007c), selective staining methods (Li and Hardin 1998a;




Canal et al. 2004; Calafell et al. 2005), weight loss (Hathway and Sekins 1958;

Li and Hardin 1999; Lenting et al. 2002; Lenting and Warmoeskerken 2004;

Ramasamy and Kandasamy 2004; Calafell et al. 2005; Sahin and Gursoy

2005; Schnitzhofer et al. 2006), residual cotton wax and nitrogen content

have been used for the assessment of bio-scoured fabrics (Boer 1980; Etters

et al. 1999; Lawson and Hsieh 2000; Degari et al. 2002; Lenting et al. 2002;

Brushwood 2003; Choe et al. 2004; Chung et al. 2004; Church and Woodhead

2005; Bargel et al. 2006).

3.2.3.4 Lipases in processing of cotton fabrics

Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3) are widely distributed

among the animals, plants and microorganisms, capable of hydrolyzing ester

bonds of oils, fats and certain waxy substances (Gupta et al. 2004; Pera et al.

2006). Lipases catalyse reactions including synthesis, transesterication of

glycerides and phosphoglycerides as well as a variety of non-glycerides, ester

bonds (Inoue et al. 2003; Sammour 2005).

Lipase – culture medium

Amount of lipases produced, in a medium, depends on several environmental

factors such as incubation temperature, pH, nitrogen and carbon sources,

concentration of inorganic salts, availability of moisture and oxygen (Lie et

al. 1991; Ghanem et al. 2000; Sharmaa et al. 2001; Kashmiri et al. 2006; Mala

et al. 2007). Plant oils like olive, soybean, sunower, sesame, cottonseed,

rapseed, corn and peanut, have been attempted for lipase production with

Ophiostolna piceae (fungal source) (Sharmaa et al. 2001; Sammour 2005).

Bacillus sp also produces equivalent activity levels as that of fungi at relatively

lower nutritional levels (Shah et al. 2007; Abada 2008). It has been reported

that lipases produced from Penicillium chrysogenum exhibit activity in the pH range of 6.0 to 8.2 and, about 80% of activity retention in the pH range of

5.0 - 6.0 (Sharmaa et al. 2001; Schafer et al. 2006), while lipases produced by

Bacillus exhibit maximum activity at 37° C with stability in the temperature

range of 30–50°C and wider pH range 5 to 12 (Shah et al. 2007; Abada 2008).

Applications of lipases in cotton processing

The term cotton wax includes all lipids found in the cotton bre surface,

including waxes, fats and oils (Lawson and Hsieh 2000; Brushwood 2003;

Bargel et al. 2006). Cuticular lipids, of cotton, are complex mixtures ofaliphatic and aromatic components, most of them resembling derivatives

of nacyl alkanes. Benzene, chloroform, carbon tetrachloride, ethanol,

isopropyl alcohol, alcohol-benzene mixture, and trichloroethylene have been

recommended for extraction of the cotton wax (Kettering et al. 1946). Lipase




activity is often determined with reagents comprising a low concentration of

buffer and a diglyceride dissolved therein (Dahod 1987; Kokusho et al. 1982;

Obendorf et al. 2001; Varanasi et al. 2001; Obendorf et al. 2003; Pera et al

2006; Imamura 2007). Lipases with, activity of pH > 6.5 at 30–60°C, ability to

withstand many surfactants (linear alkyl benzene sulphonates) and proteases,

meant for detergent formulations, can also withstand bleaching agents like

hydrogen peroxide and peracids (Fujii et al. 1986). Lipases increase lipid

removal from all morphological locations on the cotton bres including lumen

and bre surfaces (Obendorf et al. 2003).

3.2.3.5 Proteases in cotton fabric processing Proteins that occur (~ 1.3%) in the lumen and primary wall of the cotton bres,

are the residual, dead protoplasm from biosynthesis, composed of several

proteins and peptides, formed by various amino acids, rather than single

protein (Usharani and Muthuraj 2010). Proteases belong to the sub-class of

peptide hydrolase or peptidase and more conveniently proteases are classied

into serine, cysteine, aspartic and metalloproteases (Gupta et al. 2002).

Protease culture

Proteases are generally produced by submerged fermentation, though solid

state fermentation offers certain advantages in terms of reduced energy

consumption during extraction of enzymes from the culture (Battaglino et

al. 1991; Dahot 1993; Uttrup and Conrad 1999; Alves et al. 2002). Though

addition of glucose to the media increases the growth of Penicillium

aeruginosa, Penicillium expansum, there is a marked reduction in the amount

of protease production, due to catabolite repression (Wang et al. 1974). Rice

husk is used as the carbon source and corn steep liquor increases the yield

of protease signicantly (Battaglino et al. 1991). Soybean meal, corn steepliquor, tryptone and casein serve as excellent nitrogen sources in protease

production. Though low pH of the medium does not limit the growth of

Aspergillus oryzae, it limits protease yields, slightly above 6.5. Attempts have

been made to analyse the gene coding for the expression of enzymes, for

inserting into a suitable host cell or organism for the development of detergent

formulations and cleaning agents (Lassen et al. 2007).

Removal of cotton proteins and proteases

Most of the nitrogen containing compounds of cotton can be removed by amild alkali boil and a very low residue remains in scoured and bleached cotton

(Wakelyn 1975; Hartzell and Hsieh 1998; Naja et al. 2005). Denaturation

of protein substances by ageing, heating and oxidation makes them less

accessible to enzymatic degradation (Andrade et al. 2002). Pretreatments with




boiling water enhance the accessibility of proteins in cotton beneath the waxes

and improve scouring efciency. Since protein contents are high among the

non-cellulosics in cotton bre, potential of proteolytic enzymes as scouring

agents for raw cotton cannot be underestimated (Hartzell and Hsieh 1998;

Silva et al. 2006). Hydrolysates produced in the protease reactions depend on

the source of proteases with 9 - 12 major amino acids (Wang et al. 2007a).

Nitrogen content of untreated cotton ~ 0.4% reduces to ~ 0.2% after treatment

with proteases (Andrade et al. 2002). Water absorption and retention capacity

and K/S values have also been advocated for characterization of protease

treated fabrics (Sae et al. 2007). Though protease treatments in both scouring

and detergency offer a range of advantages, there are certain limitations, also,in terms of proteolysis of enzymes in the crude cultures (Pererra et al. 1993).

3.2.3.6 Fabric weight loss and strength loss

Cotton bres, free from pectic substances show, no damage in tensile strength

and uidity (Whistler et al. 1940). Pectinase of Aspergillus niger, at pH 4.2,

40° C shows weight loss ~ 4 to 5.5%, however, lower weight losses in the

range of 0.31% to 1.04% has also been reported. Strength loss of about 3.23%

(warp) and 2.24% (weft) has been reported in fabrics, while the loss of singleyarn strength has been reported at 2.6% (Calafell and Garriga 2004; Calafell

et al. 2005; Sahin and Gursoy 2005; Schnitzhofer et al. 2006).

3.2.3.7 Fabric absorbency, wettability and dyeability

Practically no difference in absorbency and wettability has been reported

between alkali and bioscoured samples, alkaline pectinases and certain acidic

pectinases (Etters 1999; Lenting et al. 2002; Pyc et al. 2003; Canal et al.

2004; Calafell et al. 2005; Sahin and Gursoy 2005; Schnitzhofer et al. 2006;Agrawal et al. 2007). Drop absorbency, similar to that of alkali scour, (~ 1

sec), has been reported in most of the literature, except in certain cases where

acidic pectinases have been used in the process (Calafell and Garriga 2004).

Degree of whiteness (CIE) of bioscoured fabrics shows lower value compared

to alkali scoured cotton fabrics (Canal et al. 2004; Calafell et al. 2005). When

long chain pectin (fully methylated pectic acid) is completely degraded to

galacturonic acid, the iodine reducing value increases from zero to 8.95

(Karmakar 1998). Dyeing of samples scoured with commercial pectinasesshows no difference in absorbance values and colour depth compared to

the alkali scoured samples however, substantively of the dyes is lower on

bioprepared fabric, due to ‘benecial wax’ that remains in the bres (Etters

1999; Calafell et al. 2005). Ruthenium red, a basic dye, selectively binds the




carboxyl oxygen of galacturonide moiety and hydroxy oxygen of an adjacent

galacturonide in the pectate chain, a characteristic reaction that helps to

measure free carboxyl groups available in cotton bres (Li and Hardin 1998a;

Li and Hardin 1999; Lenting et al. 2002; Calafell et al. 2005; Chinnadurai and

Selvakumar 2009).

3.2.3.8 Mixed enzymatic process in scouring of cotton

fabrics

Binary combinations of enzymes

Combinations of pectinases with protease, hemicellulase, cellulase and lipasehave been attempted, which are not efcient in scouring process when used

alone. Combinations of amylases with other enzymes, preparatory chemicals

have been attempted in the past to combine scouring or bleaching (Etters

1999; Tzanov et al. 2000; Lu 2005; Opwis et al. 2006; Kuilderd and Wu

2008; Lenting 2008). In the case of amylase desizing, addition of hydrogen

peroxide improves whiteness, while neutral cellulases increase weight

loss and desizing efciency. Alkaliphile amylases with sodium hydroxide

and hydrogen peroxide have been recommended for a combined desizing-

scouring-bleaching process (Etters 1999; Csiszar et al. 2007). Integrateddesizing and scouring using a-amylase and polygalacturonate lyase process

involves two steps, in which fabrics are desized rst, using a-amylases, then

by a combination treatment of amylase and pectate lyase at 45 – 55° C at

pH of 8.5 - 9.0, followed by washing in presence of chelating agent at 90 –

100° C (Lenting 2008). Simultaneous desizing and scouring using amylase

and pectinase obtained from single source, i.e. Bacillus and different sources

have been attempted in the past (Lenting and Warmoeskerken 2004; Dalvi et

al. 2007). Higher weight loss values (7.0 to 15.3%), better drop absorbency(~1 sec), dye absorption ( KIS values of 7.45 against 6.9 of acid desized

samples) have been reported with higher concentration of enzymes and longer

incubation time (Dalvi et al. 2007). Whenever a very high whiteness in the

fabrics is not required, desizing is combined with bleaching and scouring,

incorporating protease, cellulase and pectinase enzymes (Lange et al. 2001;

Miller et al. 2003).

Combinations of pectinase or cellulase with hemicellulases like

arabinases or, pectinases with hemicellulase activities have been used for

scouring. Crude mixtures of xylanase, cellulolytic and pectinolytic enzymesdestroy lignocellulosic structure of the seed coat fragments, produce soluble

forms of lignin (Kalum and Andersen 2000; Csiszar et al. 2001a; Lange et al.

2001 Opwis et al. 2006). However, such combinations does not improve the

drop absorbency (~ 290 sec), while no differences are observed in terms of




strength loss, whiteness index, compared to commercially scoured samples.

Hemicellulases, in the pectinase scouring is expected to give strong action

against seed coat fragments but their combinations with cellulase prove to

show detrimental effects in terms of strength loss (Csiszar et al. 2001 b).

Pectinase and cellulase act synergistically to improve the wettability within

shorter process time, in which addition of non-ionic surfactants, mechanical

agitations enhance their reactions (Saravannan et al. 2008). Presence of

cellulase does not increase the pectin removal but results in higher weight

losses (Lu 2005; Schnitzhofer et al. 2006). Cellulase containing pectinases

results in at ridges, concave grooves with polished surface in presence of

surfactants, while alkali scoured fabrics often show fuzzy and blurred surface(Li and Hardin 1998b; Li and Hardin 1999; Hsieh et al. 2002; Lu 2005;

Schnitzhofer et al. 2006). In cellulase assisted protease scouring, hydrolysis

of cellulosic chains in primary wall is expected to enhance scouring action of

protease synergistically (Diller et al. 1999; Guha and Shah 2001).

Scouring using acid and alkaline pectinases, combined bleaching together

with peracetic acid has been attempted in one bath, as one step or two step

processes to yield commercially acceptable whiteness (Liu et al. 2000; Patra

et al. 2004; Wan 2007). Combined scouring and bleaching process using

peroxide in a single step or two steps has also been advocated, by addinghydrogen peroxide at the end of pectinase scouring at higher pH (Tavcer

et al. 2005) and such process is capable of retaining fabric strength up to

~ 90% with Hunter whiteness index and yellowness index of 84 and 16,

respectively. An attempt has been made to utilize desize bath for bleaching

using immobilized glucose oxidase in an aerated system at pH 10–11 and

temperature of 90° C (Diller et al. 1999; Tzanko et al. 2000; Kuilderd and

Wu 2008). However, such combined desizing and bleaching using glucose

oxidase, often, results in non-uniform wetting properties, though wicking(~2.0 cm) and average drop absorbency (~1 sec) show similar values as that

of commercial processes (Kuilderd and Wu 2008). Single bath dyeing and

biopreparation either simultaneously or sequentially using pectate lyase, at

alkaline pH ( > 8), suitable for reactive dyes which result in wash fastness,

rubbing fastness grades at least 3.5 to 4.0 (Liu et al. 2000).

Ternary and quadruplet combinations of enzymes

In many situations, combinations of three or four different enzymes including

alpha amylase, pectinase, protease, cellulase, glucose oxidase and varioushemicellulases have been attempted since pectinases alone is often ineffective

in removal of impurities from cotton bres and to improve absorbency of the

scoured samples (Cziszar et al. 1998; Diller et al. 1999; Traore and Diller

2000; Degari et al. 2002). Presence of xylanase and pectinase in commercial




cellulase preparations also facilitates removal of seed coats up to 70 - 85%

(Cziszar et al. 1998). Protease and lipase are used together with pectate lyase

to remove spinning, coning and slashing lubricants from the fabrics (Li and

Hardin 1998a; Lange et al. 2001; Miller et al. 2003; Wang et al. 2007a). Lipase

or protease treatments, alone, do not improve wetting or water retention

values (Buchert et al. 2000). Few research have been carried out (Diller et

al. 1999; Traore and Diller 2000) to study the effects of combined enzymes

on the efciency of scouring, using lipase, pectinase, xylanase and cellulase,

with strong agitation levels that result in weight loss up to 13.9%. Higher

wickability is observed in the case of pectinase and xylanase combinations

than pectinase and cellulase, pectinase and lipase combinations and thehighest wicking observed in all-enzymes combination (Kim et al. 2005).

Two-step scouring of cotton has also been suggested, with lipase and protease

in the rst step and cellulase in the second step. Pectinase scouring produces 18-

fold higher amounts of reducing sugars and galacturonic acid than any of the

two step processes, while lipase / proteases / cellulase scouring produces 5-fold

higher amounts of amino acids than the pectinase scouring (Sae et al. 2007;

Karapinar and Sariisik 2004). Pectinase - cellulase or, pectinase - cellulase with

protease, or pectinase - cellulase - xylanase produces better scouring results than

those individual enzymes, in terms of dyeability, K/S and fastness properties.However, after hydrogen peroxide bleach, all the combinations (Wang et al.

2007a) shows similar CIE whiteness index (68 – 70).

3.2.4 Bleaching of cotton fabrics

Conventional bleaching methods have been reviewed in many occasions,

(Shenai 1996; Dickinson 1979; Chinta et al. 1993; Hayhurst and Smith

1995; Chakraborthy et al. 1998; Pardeshi 2000; Singh 2000; Menezes and

Chaudhari 2005; Maekawa et al. 2007), oxidative chemical pretreatments are

effective in degrading colourants and other impurities, though such methods

often lead to oxidative degradation of substrates. However, peroxide bleaches

are often referred to as the “colour safe bleaches”, due to minimal degradation

of substrates.

3.2.4.1 Enzymatic bleaching

Many attempts have been made to utilize various enzymes that belongto oxidoreductases, in bleaching of cotton fabrics (Nalankilli and Sundar

2002; Tzanko et al. 2002; Opwis et al. 2006; Diller and Traore 1998; Anis

et al. 2009; Anis et al. 2009) and post-bleaching processes (Jensen 1998).

Peroxidases are used to activate oxidizing agents like hydrogen peroxide,




however rapid deactivation of these agents in bleaching process does not

guarantee satisfactory bleaching effects (Bernards et al. 2004). The bleaching

was done with a commercial desized and alkaline scoured cotton fabric.

As described in the introduction peroxidases (POD) are used in textile

decoloration processes (Xia and Li 2009), but their activity is limited by the

hydrogen peroxide concentration, which attack the POD during the reactions

(Allen et al. 2006). To maintain the activity of the POD over a long time

period the new combined system of Glucose Oxidase GOD and glucose as

the hydrogen peroxide source and additional the POD as an oxidation catalyst

was used exemplarily for the slow decoloration of Sirius Supra Blue® FGG

200%. Figure 3.6 shows the results of different bleaching procedures of cotton.The starting material has a degree of whiteness (according to Berger) of 55.

Bleaching of cotton with POD (Baylase®RP) in the presence of hydrogen

peroxide fails, the degree of whiteness remains at 55, because the POD is

inactivated by H2O

2 after a short time. Using the combined system with GOD,

glucose and POD (Baylase®RP) the degree of whiteness increases up to 64.

Applying GOD and the new chlor-peroxidase from ASA-Spezialenzyme

simultaneously in the presence of glucose a degree of whiteness of 66 can be

reached. Compared to a conventional non-enzymatic bleaching process using

only hydrogen peroxide at high pH-value and high temperature the bleachingresult is not satisfactory but the investigations show that this environment-

friendly enzymatic bleaching procedure at low temperature and a pH-value

near to neutral basically works.

Fig. 3.6 Bleaching of cotton with glucose oxidase, glucose and different peroxidases




An enzymatic cotton bleaching process is not realized up to now because

of different problems such as the use of catalase contaminated GODs

or the inactivation of PODs by hydrogen peroxide (Gubitz and Cavaco

2001). Therefore a new concept was developed combining the use of two

oxidoreductases, GOD and POD, in textile decoloration and bleaching

processes. Under gentle conditions in terms of temperature, pH-value and

even the peroxide concentration different peroxidases (commercial POD from

Lanxess and a new chloro-peroxidase from ASA-Spezialenzyme) are stable

over a long period of time, what offers the possibility to use this enzyme

cascade also in heterogeneous systems such as the bleaching of cotton fabrics.

In future further investigations have to be carried out to open this environment-friendly concept for an even economic attractive application in large-scale

industrial manufacturing.

3.2.4.2 Effects of process parameters on enzyme

treatments

Besides the nature of substrates, efciency of hydrolysis is also inuenced

by process conditions (Hemmpel 1991; Tyndall 1996; Paulo et al. 1996;

Andreaus et al. 1999), co-reactants present in the process (Cavedon et al.1990; Hemmpel 1991; Ueda et al. 1994; Traore and Diller 1999; Heikinheimo

et al. 2003) and mechanical agitations employed in the reaction systems

(Pendersen et al. 1992; Tyndall 1996; Koo et al. 1994; Lee et al. 1996; Paulo

1998; Andreaus et al. 1999; Traore and Diller 1999; Lee et al. 2000; Cortez

et al. 2001; Tzanko et al. 2001; Heikinheimo et al. 2003). Material to liquor

ratio of process bath alters the efciency of all the components exhibited by

weight loss values and, little changes are observed in the range of 1: 10 to 1:

40 (Paulo et al. 1996). Mechanical actions, winch machines and jet systems, between fabrics and equipment or surface to surface contact of fabrics enhance

reactivity of cellulases by improving two way mass transfers and enhance

weight loss, removal of weakened bres from surfaces of yams and fabrics,

thereby facilitating a clean surface to the fabrics (Ogiwara and Arai 1968;

Tyndall 1996; Paulo and Almeida 1994; Lee et al. 1996; Gama et al. 1998;

Andreaus et al. 1999; Traore and Diller 1999; Lee et al. 2000; Heikinheimo et

al. 2003; Ramkumar and Abdalah 2001).

3.2.5 Cellulases on cotton fabrics

The cotton fabric, treated with cellulases, is aimed to remove cellulosic

impurities, individual and loose bre ends that protrude from fabric surfaces

and to provide an enhanced appearance and handle, with or without the aid




of mechanical agitations but without degrading the properties of the cotton

fabrics signicantly. Cellulase enzymes are complex mixtures of three

major constituents enzymes namely, endo l-4 α D glucanases (ED) (EC

3.2.1.4), which randomly cleave internal glucosidic bonds, 1-4, α D glucan

cellobiohydrolases (CBH) (EC 3.2.1.91), which cleave them into cellobiases.

Hydrolysis of cellobiose into the glucose end product is completed by β

glucosidases or cellobiases (EC 3.2.1.21), which split cellobiose units into

soluble glucose monomers and complete hydrolysis of native celluloses,

largely, depends on the synergistic actions of these three component enzymes.

Figure 3.7 shows the structure of cellulose and catalysis of cellulase enzyme

on cotton structure.

Fig. 3.7 Structure of cellulose and catalysis of cellulase enzyme on cotton structure




The cellulase producing bacteria include Cellulomonas, Clostridium,

Pseudomonas, Streptomyces, Thermonospora, Ruminococcus but bacterial

cellulases digest cotton bres to lower levels compared to amorphous celluloses

(Cavedon et al. 1990; Lu et al. 2005; Ray et al. 2006). Cellulases have two

functionally distinct domains, in their structure namely catalytic domain and

cellulose substrate binding domain, linked by an inter domain, glycosylated

linker peptide either at the N or C terminal of the protein; cellulose binding

domains exhibit different afnities, specicities, some binding to crystalline

cellulose, while others restrict themselves to the disordered regions. Catalytic

domain has an active site in the shape of a tunnel or cleft where hydrolytic

reactions take place (Cavedon et a1 1990; Lee et al. 1996).

3.2.5.1 Biotreatment with cellulase and xylanase

Recently published results indicate that enzymes, mainly cellulases and

several non-cellulolytic enzymes like lipases, proteases, and pectinases

may be used effectively in the cleaning processes of cotton (Hartzell and

Hsieh 1998). Raw cotton contains approx. 10% of non-cellulosics such as

waxes, pectins, proteins, non-cellulosic polysaccharides, inorganics, lignin

containing impurities, colouring materials, etc. Depending upon variety andcultivation conditions, these impurities are mainly located in the outer layers

of the bre in the cuticle and the primary wall. Traditional cleaning procedure

applies concentrated sodium hydroxide solution alkaline scouring and

additional hydrogen peroxide and or sodium hypochlorite solutions bleaching

to eliminate these impurities (Li and Hardin 1998). The cellulase enzymatic

treatment prior to the alkaline scouring process enhanced both the removal

and degradation of seed coat fragment impurities of cotton fabrics. When

consecutive cellulase treatment and conventional alkaline scouring were

combined, the increase in whiteness of the fabrics was signicantly improved

(Csiszar et al. 1998). Cellulase pretreatment also allowed the reduction of the

hydrogen-peroxide consumption in the consecutive chemical bleaching step.

The effect of seven commercial cellulase and hemicellulase enzymes

was tested on fabric weight loss, reducing sugar liberation and change in

degree of polymerisation (DP) of desized cotton fabrics. The changes in bre

surface were monitored by scanning electron microscopy (Csiszar 2001).

Greige cotton fabric, 122 GSM was used for the experiments after amylase

enzymatic desizing. Cellusoft L, Viscozyme 120 L, Celluclast 1.5 L, CellulaseEBT, Pulpzyme HC, Denimax L and Denimax Acid L enzymes were supplied

by Novo-Nordisk, Copenhagen, Denmark. The enzyme activities were

measured at 50°C from the products using internationally recognized methods

(Table 3.6). Desized cotton fabric was subjected to biotreatment with seven




commercial cellulase and hemicellulase enzymes in non-agitated and agitated

systems at 50°C for 0.5–4 h. The enzymes performed better in agitated bath

than in non-agitated ones. All enzymes at 1 grl concentration in 2 h caused

weight loss less than 6%. Those three enzymes Celluclast 1.5, Cellusoft L and

Cellulase EBT which exhibited the highest lter paper activity FPA showed

the most aggressive action on cotton in agitated system at 1 grl concentration

when time of treatment exceeded 2 h.

Table 3.6 Activity values of commercial enzymes

Enzyme Cellulase Endoglucanase Xylanase Glucosidase

Cellusoft L 100 37,000 5,000 76Viscozyme 120L 8 12,700 800 5

Celluclast 1.5L 67 28,000 2300 11

Pulpzyme HC 0.02 33 120,000 0.2

Denimax L 7 2500 16,700 87

Denimax Acid L 18 6300 1000 12

Cellulase (EBT) 108 73,200 12,900 81

Note: Enzyme activity unit IU/ml at pH 4.8

Effect of enzymatic treatment on fabric weight loss

Enzymatic degradation of cotton is generally characterized by weight loss.

Enzymes Celluclast 1.5L and Cellulase EBT cause the most signicant

weight losses nearly 2.5% in 4 hours biotreatment. There is no signicant

effect of increase in enzyme concentration from 0.5 to 1 gpl on weight loss

of cotton fabric in non-agitated system. In agitated system at 0.5 gpl enzyme

concentrations the cotton fabric weight losses are less than 1.5% for enzymes

Viscozyme 120L, Pulpzyme HC and Denimax enzymes. Except for Denimax

Acid L, the weight loss is practically independent from the treatment time.Enzymes Celluclast 1.5L and Cellulase EBT caused the highest weight losses.

Increase in reaction time resulted in increase in fabric weight loss. In agitated

system, Cellulase EBT is the most aggressive and higher weight loss on the

cellulosic materials. Depending on the time of treatment, the weight loss

is 1.8–6.3%. These data suggest that agitation has a signicant impact on

enzymatic degradation of raw cotton fabric (Ghose 1987). Weight loss values

are much higher in agitated solutions than in non agitated ones. The increase

of enzyme concentration from 0.5 to 1 gpl has smaller effect on weight loss

than the agitation of the solution.

Reducing sugars released by enzymatic treatment

There is a close correlation between the weight loss of cotton fabric and the

amount of liberated reducing sugars measured in the bath. In non-agitated




system, enzymes Cellusoft L, Pulpzyme HC, Denimax L and Denimax Acid L

do not produce reducing end groups in the solutions. Enzymes Celluclast 1.5L

and Cellulase EBT having high FPA which cause the highest fabric weight

loss and liberate the most reducing sugars. The amount of released reducing

sugar by these two enzymes increases with the time of treatment. Increase in

reaction time also results in increase in reducing sugar production (Cavaco-

Paulo 1996). The amount of reducing sugars liberated by the different enzymes

is higher in agitated system than those of in non-agitated one. Cellulase

EBT produces the highest level of reducing sugars of all enzymes tested.

The amount of reducing sugars formed by Celluclast 1.5L increases almost

linearly with the time of treatment. The maximum weight loss values are near2%, and the reducing sugar liberation is also not signicant. It is likely that

mainly the surface brils, small protruding bres, seed-coat fragments and

certain constituents of cuticle are degraded. At longer duration e.g. 4 h., these

enzymes, especially Cellulase EBT liberate signicant amount of reducing

sugars. Due to the damage of the outer layer of cotton bre, cellulase can

reach the primary cell wall and start to degrade the less crystalline cellulose

constituents, releasing reducing sugars into the solution. Agitation of the

enzyme solution increases the cellulose degrading effect for all enzymes. The

degradation of crystalline cellulose components in the cotton primary wall issignicant when the treatment time exceeds 1 h.

Figures 3.8–3.10 represent the scanning electron microscope view of

desized, cellulase enzyme treated cotton fabrics, after biotreatment of cotton

fabric, cracks and cavities, which might be the consequence of a serious damage

in the main body of cotton bre and the bre surface variations are consistent

with the results of the activity nature of the applied enzymes (Csiszar et al.

2001). The effect of seven commercial cellulase and hemicellulase enzymes

was investigated on the weight loss and reducing sugar liberation of desizedcotton fabrics. The bre surface was characterized by scanning electron

microscopy. Biotreatment in non-agitated system at 0.5–1.0 gpl enzyme

concentration for 0.5–4 h did not cause signicant degradation in cotton bre.

The weight loss was less than 3% and only a small amount of reducing sugar

was produced. These results suggest that mainly the surface brils, small

bres, seed-coat fragments, water-extractable materials and other natural

impurities have been degraded signicantly (Bailey and Nevalainen 1981;

Ioelovich and Leykin 2008).

Biopolishing of cotton fabric with commercial enzymes

The research work has been made to process the cotton fabrics with commercial

enzymes with specic treatment conditions which are available in the

commercial market (Gubitz and Cavaco2001). The physical properties of bio-




processed cotton fabrics and their fastness properties such as washing, rubbing

and perspiration characteristics were studied before and after treatments with

cellulase enzymes. The cotton fabric particulars 3/1 twill weave, 220 GSM,

and fabric width 120 cm was selected for commercial enzymatic treatments.

The functions of commercial enzyme and process conditions are given in

Table 3.7.

Table 3.7 Process conditions for bio-processing of cotton fabric

Parameter Bio-desizing Bio-

scouring

Bio-

bleaching

Bio-washing

Enzyme Palkozyme HT Lixazyme D Palkoperox Palkostone ultra

Concentration 6 g/l 0.5–1% 0.5–1 g/l 0.5%, 1.0%, 1.5%

pH 5.5–7.5 6.5–7.0 5–10 4.5–5.0

Temperature 50–75°C 55–65°C 60–75°C 45–55°C

Time 30 min 45 min 30 min 30 min

MLR 1 : 8 1 : 8 1 : 8 1 : 8

Other

chemicals(concentration)

Wetting agent

(1–3 g/l)

Stabilizer

(0.5–1%)

Stabilizer

(0.1–0.3%)

Non-ionic

detergent(0.5–1 g/l)

Fig. 3.8 Scanning electron micrograph of desized cotton fabrics

[Source: Csiszar et al. 2001]




Fig. 3.9 Scanning electron micrograph of a cotton bre treated with celluclast1.5l

enzyme at 1gpl, 2 h, agitated bath [Source: Csiszar et al. 2001]

Fig. 3.10 Scanning electron micrograph of cotton bre treated with cellulase (EBT)

enzyme at 1 gpl, 2 h, agitated bath [Source: Csiszar et al. 2001]

Process sequence for bio-processing of cotton fabrics is given below:

Desizing→ Defuzzing→ Scouring→ Bleaching→ Dyeing→ Enzyme

washing à Dryer

Procedure

Load the fabric→ add water→ heat to 50–55°C→ add cellulase (required %)

→ treat for specic time and pH→ drain à rinse twice→ dry




The bio-processing of 100% cotton fabric was processed with commercial

enzymes and their physical properties such as tensile behavior, elongation to

break, fabric thickness and fabric mass of grey stage and after Bioprocessing

are given in Table 3.8.

Table 3.8 Physical properties of grey and bio-processed cotton fabrics

Properties Grey fabric Bioprocessed cotton fabric

Tensile strength (lbs)

(a) Warp direction 133.5 132

(b) Weft direction 94 92Elongation to break (%)

(a) Warp direction 17.7 18.5

(b) Weft direction 15.3 15.8

Fabric thickness (mm) 0.454 0.425

Fabric GSM 124.5 122.5

Hypothesis 1: The tensile strength of the bio-processed cotton fabric was

noticed loss in warp and weft way direction of 1.12% and 2.23% respectivelywhen compared with grey cotton fabric. It may be due to loss of fabric weight

in bio-scouring and bio-washing process. The cellulase enzymes break the

cellubiose structure in the cotton bre during biopolishing (Hassan et al.

1996). The elongation to break behavior of the bio-processed cotton fabric

was noticed improvement in warp and weft way direction of 4.52% and

3.26% respectively when compared to grey cotton fabric. The fabric mass (in

terms of grams per square meter) of the bio-processed cotton fabric was also

decreased as 6.28% when compared to grey cotton fabric, it may be due to theconcentration of the enzymes and treatment conditions.

Hypothesis 2: Fastness properties of the bioprocesses cotton fabrics dyed

with reactive blue dye with 3% shade and then treated with various concentrations

of enzyme washing of 0.5%, 1% and 1.5% using acid enzyme. The washing

fastness ISO-method 3 reactive blue dye and various concentrations of 0.5%,

1% and 1.5% with acid enzyme washed cotton fabrics show good value of

3.5–4.0. The rubbing fastness of reactive dyed fabric & enzyme washed fabric

of both wet rubbing and dry rubbing shows 3.5–4.5 (Fig. 3.11). Bio-processed

cotton fabric tested with acid and alkali perspiration was noticed well (Fig.3.12). From the above research work, the processing of enzyme for treatment

at various stages to be taken special care for getting good quality of the bio-

processed cotton fabrics and also the processes are mainly depends on the

enzymes concentration, treatment time, temperature and pH of the system.




Fig. 3.11 Rubbing fastness of bio-processed cotton fabric

Fig. 3.12 Acid and alkali perspiration fastness of bio-processed cotton fabric




3.3 Bioprocessing of jute and their characteristics

3.3.1 Jute bre grades, properties and applications

3.3.1.1 Jute bre – grading and classication

Jute bre, the current grading system rst separates C. capsularis and C.

olitorius into white and tossa categories respectively and then further classies

each into ve grades denoted by the letters A to E. The highest prices are

paid for Grade A, although a special grade may be introduced for which a

higher price can be demanded. The principal criteria are color, luster, strength,

cleanliness, and freedom from retting defects. From a spinning point of view,color is irrelevant but certain end-users traditionally prefer bres in particular

colors for the sake of appearance.

3.3.1.2 Physical properties of jute bres

Jute is 100% bio-degradable and thus environment friendly and it has some

unique physical properties like high tenacity, bulkiness, sound & heat

insulation property, low thermal conductivity, antistatic property etc. Due

to these qualities, jute bre is more suited for the manufacture of technicaltextiles in certain specic areas. Moreover, the image of jute as a hard and

unattractive bre does not affect its usage in technical textiles. The use of jute

was primarily conned to marginal and small manufacturers and growers, but

now it is used as important raw materials for several industries. At present

time, jute is termed as a favorite fabric for packaging materials and furnishings

and also as golden bres for national and international fashion world. Jute

bres are used for making mats, gunny cloth, cordage, hangings, paper, and

decorative articles. Prevalent uses of jute in handicraft stuff, in order to give an

aesthetic appeal, have made it popular across the globe. It is the second most

important vegetable bre after cotton, in terms of usage, global consumption,

production and availability. The jute characteristics are listed below;

• 100% biodegradable, environmental friendly and recyclable.

• High tensile strength and low extensibility

• Better breathability of fabrics suitable for packaging of agricultural

commodities

• High insulating and anti static properties

• Moderate moisture regain and low thermal conductivity• High moisture absorption capacity

• Flexibility

Jute bre has some standard physical properties. These are

• Ultimate jute length: 1.5 to 4 mm




• Ultimate diameter of jute: 0.015 to 0.002 mm

• Jute bre length: 150 to 300 cm (5 to 12 feet)

• Jute Color: Jute bre can be white, yellow, brown or grey

• Strength of jute: 3.5 to 5 grams per denier

• Moisture regain of jute: 13.75% (Standard)

• Elasticity: Breaking extension 1.8% and elastic recovery very low

• Resiliency: Poor

• Dimensional stability of jute: Good on average

• Fibre density: 1.47 grams/cc

• Fibre tenacity: 4.2 grams per denier

• Average toughness index: 0.02 • Average stiffness: 330 grams per denier

3.3.1.3 Jute bre structure

In jute plant, the rings of bre cell bundles form a tubular mesh that encases

the entire stem from top to bottom. Two layers can usually be distinguished,

connected together by lateral bre bundles, so that the whole sheath is really

a lattice in three dimensions. The cell bundles form the links of the mesh,

but each link extends only for a few centimeters before it divides or joins upwith another link. After extraction from the plant, the bre sheath forms a at

ribbon in three dimensions. When a transverse section of a single jute bre is

examined under the microscope, the cell structure is seen clearly. Each cell is

roughly polygonal in shape, with a central hole, or lumen, comprising about

10% of the cell area of cross section.

• In longitudinal view, the bre appears the overlapping of the cells

along the length of the bre. The cells are rmly attached to one

another laterally, and the regions at the interface of two cells are

termed the middle lamella. Separation of cells can be effected by

chemical means, and they are then seen to be threadlike bodies

ranging from 0.75 to 5 mm in length, with an average of about 2.3

mm. The cells are some 200 times longer than they are board, and

in common terminologies are referred to as ‘ultimale cells’ . A single

bre thus comprises a bundle of ultimates.

• Transverse selections of single bres show that the number of ultimate

cells in a bundle ranges from a minimum of 8 or 9 to a maximum

of 20–25. Bundles containing up to 50 ultimate cells are sometimesreported, but in such cases whether the bre is truly single in the

botanical sense or is comprised of two bres adhering together. A

minimum number of cells in the cross section are evidently necessary

to provide a coherent and continuous overlapping structure.




3.3.1.4 Chemical structure and composition of jute bres

Chemical structure of jute breJute is a strong, stiff, natural bre. Jute bres are aggregates of single cells

consisting of α-cellulose, which are cemented by lignin and hemicellulose.

Lignin is responsible for the dark colour, branching patterns and harshness.

The spinnability of the bre is greatly reduced due to poor elongation and

high exural rigidity. Jute is the common name given to the bre extracted

from the stems of plants belonging to the genus Corchorus, family Tiliaceae.

A retted jute bre has three principal chemical constituents, namely cellulose,

hemicelluloses, and lignin. The lignin can be almost completely removed by

chlorination methods in which a soluble chloro-lignin complex is formed,

and the hemicelluloses are then dissolved out of the remaining holocellulose

by treatment with dilute alkali. The nal insoluble residue is the cellulose

constituent, which invariably contains traces of sugar residues other than

glucose. The hemicelluloses consist of polysaccarides of comparatively low

molecular weight built up from hexoses, pentoses, and uronic acid residues. In

jute, capsularis and olilorius have similar analyses, although small differences

occur among different bre samples.

For bre extracted from jute plants grown in Bangladesh, the range ofcomposition has been given as lignin, 12–14%; alpha-cellulose, 58–63%;

and hemicellulose, 21–24% (Kundu et al. 1996). In addition, analysis of the

hemicellulose isolated from alpha-cellulose and lignin gives xylan, 8–12.5%;

galactan, 2–4%; glucuronic acid, 3–4%; together with traces of araban and

rhamnosan. The chemical composition of jute at different stages of plant

growth is given in Table 3.9. The insoluble residue of cellulose has the

composition glucosan, 55–59%; xylan, 1.8–3.0%; glucuronic acid, 0.8–1.2%;

together with traces of galactan, araban, mannan, and rhamnosan. As well

as the three principal constituents, jute contains minor constituents such as

fats and waxes, 0.4–0.8%; inorganic matter, 0.6–1.2%; nitrogenous matter,

0.8–1.5%; and traces of pigments. In total, these amount to about 2%. The

detailed molecular structure of the hemicellulose component is not known

with certainty, although in the isolated material the major part is stated to

consist of a straight chain of D-xylose residues, with two side-branches of

D-xylose residues, whose position and length are uncertain (Ghosh and Dutta

1983). In addition, there are other side branches formed from single residues

of methyl glucuronic acid, to the extent of one for every seven xylose units.The third major constituent, lignin, is a long-chain substance of high molecular

weight which, like the hemicelluloses, varies in composition from one type

of vegetable material to another. The molecular chains are built up from

comparatively simple organic units which may differ from different sources




as well as in the way in which they are combined. Most of the studies in lignin

have been concerned with wood; the bast bres have been rather neglected.

It seems unlikely, however, any major differences will exist between jute

and wood lignin, but in any case many details of the molecular structure still

remain unresolved. Later, Mukherjee et al. (1993) working at the Indian Jute

Industries Research Association in Calcutta, studied the surface characteristics

of jute bre at different stages of growth (Emilia Csiszar et al. 1998). At the

early stages of growth, they found that there was an incomplete formation of

the middle lamella in the cell wall and that the parallel bundles of brils were

oriented as an angle with respect to the bre axis that gradually decreased

with growth.

Table 3.9 Chemical composition of jute at different stages of plant growth

Component Pre-bud Bud Flower Small pod Large pod

α cellulose 58.3 57.6 59.4 58.7 59.1

Holocellulose 86.8 87.8 87.3 87.1 86.8

Xylan 15.5 14.8 14.4 13.7 13.9

Lignin 12.1 12.7 12.4 12.0 12.0

Ash 0.57 0.53 0.47 0.67 0.47

Iron 0.020 0.018 0.009 0.011 0.008

Reed Length (ft) 6.6 9.1 9.3 9.6 10.7

Chemical composition of jute bre

Cellulose > 65%

Hemi-cellulose > 22.5%

Lignin > 11%

Fat and Wax > 0.3%

Water Soluble Materials > 1.2%Total = 100%

3.3.2 Changing scenario in multifunctional applications

Today jute can be dened as an eco-friendly natural bre with versatile

application prospects ranging from low value geo-textiles to high value

carpet, apparel, composites, decorative, upholstery furnishings, fancy non-

woven for new products, decorative color boards etc. Possible application

areas for technical textiles and suitable jute products are given in Table 3.10.Jute with its unique versatility rightfully deserves to be branded as the “bre

for the future”. Jute bre has deserved for many advantages such as (a) good

insulating and antistatic properties, (b) low thermal conductivity, (c) moderate

moisture regain, (d) acoustic insulating property and (e) can be blended with




productivity have indeed taken place. But it is now timely to consider what

new innovations would assist the spread of jute materials into textile uses

outside the traditional elds of packaging and carpets. Developments to

breed Corchorus or Hibiscus plants containing bres of signicantly lower

linear density would allow yarns of lower count than is now feasible, to be

spun and so enable the production of light-weight fabrics. Such jute fabrics

could have increased potential for decorative and furnishing uses, especially

if the constraint of color instability could rst be removed and a process

devised to produce additional elasticity that is more permanent basis than

that of woolenized jute. Research is being conducted in the areas of paper

and composite production in several countries, and it is expected that wholenew markets will be developed for jute as well as for many other agro-based

bres.

3.3.2.2 Chemical modication for jute property

improvement

The performance of any lingo-cellulosic bre is restricted by the properties of

the bre itself. Agro-based substances will change dimensions with changes in

moisture content, will burn, and will be degraded by organisms and ultravioletradiation. The agro-based jute bre can be used in property enhanced

applications, it is important to understand the properties of the components of

the cell wall and their contributions to bre properties. All agro-based bres

are three-dimensional, polymeric composites made up primarily of cellulose,

hemicelluloses, lignin, and small amounts of extractives and ash (Coll et al.

1993). The cell wall polymers and their matrix make up the cell wall and in

general are responsible for the physical and chemical properties of the jute

bre (Fig. 3.13). Agro-based bres change dimensions with changing moisture content

because the cell wall polymers contain hydroxyl and other oxygen-containing

groups that attract moisture through hydrogen bonding. The hemicelluloses

are mainly responsible for moisture sorption, but the accessible cellulose,

non-crystalline cellulose, lignin, and surface of crystalline cellulose also play

major roles. Moisture swells the cell wall, and the bre expands until the cell

wall is saturated with water. Beyond this saturation point, moisture exists as

free water in the void structure and does not contribute to further expansion.

This process is reversible, and the bre shrinks as it loses moisture. Agro-basedbres are degraded biologically because organisms recognize the carbohydrate

polymers (mainly by hemicelluloses) in the cell wall and have very specic

enzyme systems capable of hydrolyzing these polymers into digestible units.




Biodegradation of the high molecular weight cellulose weakens the bre cell

wall because crystalline cellulose is primarily responsible for the strength of

the cell wall. Strength is lost as the cellulose polymer undergoes degradation

through oxidation, hydrolysis, and dehydration reactions (Basu and Rekha

1962).

Fig. 3.13 Chemical structure of jute bre

Jute bres are exposed outdoors to ultraviolet light undergo photochemical

degradation. This degradation takes place primarily in the lignin component,

which is responsible for the characteristic color changes. The lignin acts as an




adhesive in the cell walls, holding the cellulose bres together. The surface

becomes richer in cellulose content as the lignin degrades. In comparison

to lignin, cellulose is much less susceptible to ultraviolet light degradation.

After the lignin has been degraded, the poorly bonded carbohydrate-rich

bres erode easily from the surface, which exposes new lignin to further

degradative reactions. In time, this “weathering” process causes the surface

of the composite to become rough and can account for a signicant loss in

surface bres. Dimensional stability can be greatly improved by bulking the

bre cell wail either with simple bonded chemicals or by impregnation with

water-soluble polymers. For example acetylation of the cell wall polymers

using acetic anhydride produces a bre composite with greatly improveddimensional stability and biological resistance. The same level of stabilization

can also be achieved by using water-soluble phenol formaldehyde polymers

followed by curing.

Biological resistance of jute fibre-based materials can be improved by

several methods (Chakraborty et al. 1995). Bonding chemicals to the cell

wall polymers increases resistance due to the lowering of the equilibrium

moisture content below the point needed for microorganism attack and by

changing the conformation and configuration requirements of the enzyme-

substrate reactions. Toxic chemicals can also be added to the compositeto stop biological attack. This is the basis for the wood preservation

industry (Chakraborty and Sinha 2001). Resistance to ultraviolet radiation

can be improved by bonding chemicals to the cell wall polymers, which

reduces lignin degradation, or by adding polymers to the cell matrix to

help hold the degraded fibre structure together so that water-leaching of

the undegraded carbohydrate polymers cannot occur. Fire retardants can

be bonded to the fibre cell wall to greatly improve the fire performance.

Soluble inorganic salts or polymers containing nitrogen and phosphoruscan also be used.

3.3.3 Chemical and bio-chemical softening treatment of

jute fabric

Samanta et al. 2006 have been studied the untreated and H2O

2 bleached

jute fabrics subjected to softening treatment with selective chemical and

bio-chemical agents like NaOH, HCl, K 2S

2O

8 , cellulase enzyme/mixed

enzyme (mixture of cellulase, xylanase and pectinase), cationic softener,amino-silicone softener and poly-oxo-ethylene emulsion softener under

specied treatment conditions (Chattopadhaya et al. 1997; Basu et al.

2007). Plain-weave raw jute fabric with 67ends/dm and 59 picks/dm having

warp count 207 Tex and weft count 241 Tex and fabric aerial density of




256 g/m2 with thickness of 0.90 mm were used for desizing and scouring

and conventional 3% H2O

2 bleaching processes (Basu and Chattopadhyay

1996).

3.3.3.1 Alkali treatment on jute fabric

Raw untreated jute fabric was treated with 1%, 5%, 10% and 18% aqueous

solution of NaOH using material to liquor ratio (MLR) 1:20 for 1 min or 30

min duration either at room temperature or at boil (100°C). NaOH treated

jute fabric samples were neutralized in each case by dipping the treated fabric

in 1.5% acetic acid solution for 15 min and then washed with plain water

followed by squeezing and nal air drying (Pedersen et al. 1992).

3.3.3.2 Enzyme treatment with cellulase and specic

mixed enzyme

Conventional scouring and H2O

2 bleaching enhances enzyme activity on

jute or jute/cotton union fabrics and hence all the enzyme treatment was

performed on bleached jute fabrics. 3% H2O

2 bleached jute fabric was treated

with cellulase enzyme and also with mixed enzyme (mixture of a xed proportion of cellulase, xylanase and pectinase enzyme) varying over all

enzyme application dosages of 2%, 4%, 6% and 8% (owf) at 55ºC for 2 h with

pH 4.8–5.0 as per method using 1/20th part of sodium-acetate and acetic acid

buffer solution) in rotating (with 40 rpm) beakers using SASMIRA launder-

o-meter. After 2 h enzyme treatment, in each case temperature was raised to

90ºC for 15 min for de-activation of the enzyme (Kyohei et al. 2002). Then the

enzyme treated fabric samples were thoroughly washed with plain water and

dried in air. In a separate set of experiment 1 ml, 2 ml, and 3 ml of cellulase

enzyme (35 unit/ml) was added in commercially available said mixed enzyme

to prepare a specic mixed enzyme enriched in cellulase enzyme content and

4%(owf) over all dose of this specic mixed enzyme was applied on bleached

jute fabric.

3.3.3.3 Effect of treatment with alkali

Action of alkali (NaOH) and consequent effect on jute is commonly

known, however, to utilize this treatment for softening of jute fabric priorto its bleaching is a relatively new approach (Fig. 3.14). With increase

in concentrations of NaOH (1%-18%w/w) or with increase of treatment

temperature for dilute NaOH (1%-5%) treatment, there is increase in

weight loss, treatment shrinkage, elongation at break, crease recovery




angle, yellowness index and K/S value along with consequent reduction

in initial modulus, tenacity, bending length, exural rigidity, bending

modulus, whiteness index and brightness index. However, 18% NaOH

treatment of jute fabric (under slack condition) at room temperature

(30°C) for 30 min renders the fabric an optimum balance parameters

with maximum reduction in bending length, exural rigidity and bending

modulus with moderate loss of tenacity and maximum increase in breaking

extension, despite substantial weight loss and maximum surface browning

(causing maximum increase in yellowness index value); while this surface

darkening can be eliminated by post bleaching treatment with H2O

2 under

specic conditions (Fig. 3.15).

Fig. 3.14 Chemical structure of jute bre after alkali treatment




Fig. 3.15 Degrading the lignin composition by chemical treatment of jute bre

3.3.3.4 Effect of treatment with cellulase enzyme and

specic mixed enzyme system

Scoured and conventional 3% H2O

2 bleached jute fabric is more responsive to

enzyme action than untreated or even alkali pretreated jute fabric (Pedersenet al. 1992). Hence, a comparative study of softening action of scoured and

bleached jute fabric has been studied using different concentrations of (a)

single cellulase enzyme solution (35 unit/ml) and (b) commercially available

mixed enzyme solution (a mixture of 35 unit/ml of cellulase, 96 unit/ml of

xylanase and 136 unit/ml of pectinase) and (c) specic mixtures of varying

amount of (a) with prexed amount of (b) ie, cellulase enriched formulations

of specic mixed enzyme.

3.3.4 Biosoftening of jute bre with white rot fungi and

specic enzymatic systems

The research investigated the effect of using white rot fungi namely,

Phanerochaete chrysosporium (Kerem et al. 1992) and Ceriporiopsis

subvermispora (Blanchette et al. 1997), cellulase enzyme and a mixture of

enzymes (cellulase, xylanases and pectinases), under specic treatment

conditions on the physical characteristics of jute bres (Akin et al. 1995).

Raw jute reed of Tossa Daisee variety (grade TD3) having average neness of2.4 Tex was used for the study. Biosoftening of jute with commercial enzymes

such as cellulase (35 unit/ml) and mixed enzymes (mixture of 35 unit /ml of

cellulase; 96 unit/ml of xylanase; 136 unit/ml of pectinase) were used.




tartrate (pH 5) supplemented with 0.1 mM H2O

2 (Wariishi 1992). One unit

of enzyme activity is dened as the amount of the enzyme required for the

transformation of 1 μmol of substrate per minute.

3.3.4.3 Evaluation of physical characteristics

The physical characteristics such as tenacity, elongation % and exural

rigidity for the raw jute and enzyme treated jute bres were evaluated using

standard textile testing instruments following standard testing procedures.

Tensile properties of bre

Tensile properties, such as tenacity and elongation % were evaluated using acomputer aided Instron tensile tester after conditioning the sample at 65 ± 2%

RH and 27 ± 2°C for 24 hours. A total length of 100 mm of bre was tested

for tenacity and elongation %.

Flexural rigidity measurement

The 5 mg weight of 7 cm long jute bres in bundle form were mounted on

a horizontal platform in such a way that it overhung in the front for a xed

length of 5 cm like a cantilever. A small vertical load of 2.5 mg in the form of

a xed pre-weighted synthetic gum tape was stuck to the free end of the bre bundle. It was observed that the bre bundle bends downward on application

of the known load. The exural rigidity in (mg/mm2) was calculated from the

angular deection of the bre bundle measured on a circular scale tted round

the horizontal platform (Samanta et al. 2005). Flexural rigidity was calculated

using the formula;

Flexural rigidity (mg.mm2) = (L2 / 2Ø)x (W2 + (W

1/3)) [3.1]

Where, L – length of overhung bre bundle (in mm), Ø – angle deection

(in radian), W1

– weight of the bundle in (mg/mm), W2

– the xed load (in mg)

at the free end of bre bundle.

Color spectroscopy analysis

The whiteness index, yellowness index, brightness index and reectance

value of the enzyme bre samples were measured using JAYPAK Color

Spectroscopy (Model 4800) with CIE 76, observer 10 degree at D65 light

source in the range between 400nm and 700nm.

The Brightness Index (ISO-2470-1977) was calculated using the

following formula,

Brightness Index =Reectance value of the substrate at 457 nm

Reectance value of the standard white tile at 457 nm

× 100 [3.2]




Scanning electron microscopy

The morphological structure of the bre samples were studied using JEOL

scanning electron Microscopy (Model JSM -5200) with magnications of

500X.

3.3.4.4 Effect of white rot fungi on Lignin degradation

Cellulose and lignin contents in raw jute were 51.54% and 11.50%,

respectively. Reduction of lignin was observed to be 44.16% and 25.91%

for P. chrysosporium and C. subvermispora respectively after a period of

30 days. P. chrysosporium exhibited a higher cellulose reduction of 32.63%

in comparison with 27.92% reduction by C subvermispora in 30 days

(Table 3.11). Degradation of lignin by white rot fungi enables them to gain

access to the holocellulose, the actual carbon and energy source of the fungi

(Ghosh and Gangopadhyay 2000). The mycelium which had grown over the

solid substrate jute bre had depleted the available nitrogen and in turn had

triggered the development of ligninolytic system.

Table 3.11 Effect of white rot fungi on lignin and cellulose content of jute

Incubation period (days)

Lignin Cellulose

Treatments 30 45 60 30 45 60

Control 9.99 ±

0.449

8.39 ±

0.215

8.05 ±

0.353

34.76 ±

0.05

29.46 ±

0.11

28.68 ±

0.330

P .

chrysosporium

7.75 ±

0.355

7.62 ±

0.808

7.39 ±

0.465

28.78 ±

0.05

27.15 ±

0.132

25.71 ±

0.005

C

subvermispora

8.52 ±

0.638

8.29 ±

1.010

7.89 ±

0.397

34.72 ±

0.10

29.00 ±

0.112

28.37 ±

0.200

Note: Mean values of 3 replicates; Values in % w/w

Optimization of incubation period for biosoftening

Biosoftening of the jute bre was carried out using P. chrysosporium and

C. subvermispora for varying incubation periods of 30, 45 and 60 days.

Maximum improvement in color and softness of the bre was achieved in 30

days with both the organisms. Change in color was not observed visually in

less than 30 days. No signicant increase in lignin degradation was observed

in the samples incubated for 45 and 60 days. As the incubation time increases,

there is a chance of deterioration of the bre strength due to the production of

cellulolytic and pectinolytic enzymes. Hence, 30 days incubation time can be

considered as the optimum period required for degrading lignin effectively.




Flexural rigidity

It is tangible that the stiffness of raw jute bre, as indicated by the exural

rigidity value of 60.69 cN/mm2 is reduced substantially to 37.81 cN/mm2 in

case of P. chrysosporium and to 34.14 cN/mm2 in case of C. subvermispora due

to biosoftening (Kundu et al. 1993; 1996). This marked reduction in exural

rigidity in terms of percentage is 37.84% and 34.14% for P. chrysosporium

and C. subvermispora respectively. It is chiey responsible for bringing in

suppleness and softness in the biosoftened jute bres. This behavior is in

agreement with the ndings of Ghosh et al. (2004), who had employed caustic

alkali and softened jute bres by oxidation at different operating conditions.

Lignin is the component, which imparts brittleness to the bre. Decrease inlignin content causes an increase in the softness of bre, which enhances the

cross linking and adhesion forces between the jute bres. From Table 3.14,

it is quite noticeable that the stiffness of raw jute bre, as indicated by the

exural rigidity value of 60.69 mg.mm2 is reduced substantially to 20.49%,

25.47% & 34.67% in case of cellulase and 13.61%, 29.87% & 36.97% in case

of mixed enzyme at 2%, 3% & 4% concentration due to biosoftening

Scanning electron microscopy

The SEM photographs of raw jute and biosoftened jute bres are depicted(Fig. 3.16) and jute bre was treated with P. chrysosporium enzyme and

Fig. 3.16(b) shows that the brils in the peripheral cells are opened up in

biosoftened bres (Vigneswaran and Jayapriya 2008). Delignication

is initiated by hyphae growing on the inner surface of the cell wall and

penetrating into the secondary wall towards the middle lamellae as a result

of which the cells tend to separate. From Fig. 3.16(c) and (d), it is jute bre

with cellulase enzymes and Fig. 3.16(e) and (f), it is jute bres treated with

mixed enzyme system, it is seen that the surface, when compared to that of

raw bres, becomes even in case of biosoftened bres. The smoothness ofthe surface is sensed while handling the bres. It is quite noticeable that the

rate of degradation of jute bres at the surface level was observed at higher

rate as the concentration of the enzyme increases. In biosoftening process,

some weight loss was observed, which however does not yet indicate any

bre damage. Only with prolonged treatment duration, degradation occurs in

the accessible bre surfaces. This process can eventually lead to signicant

bre deterioration, indicated by a high weight and strength loss or cracks in

brillar direction and can lead to extensive surface peeling.




(a)

(d)

(b)

(e)

(c)

(f)

Fig. 3.16 SEM images of (a) raw jute bre, (b) jute bre treated with fungus

phanerochaete chrysosporium, (c) jute bre treated with 2% cellulase, (d) jute bre

treated with 4% cellulase, (e) jute bre treated with 2% mixed enzyme, (f) jute bre

treated with 4% mixed enzyme [Source: Vigneswaran and Jayapriya 2008]

Statistical analysisANOVA (at 95% condence interval) was performed in order to statistically

compare the extent of variation between the treated and the control bres as

shown in Table 3.15.

Table 3.15 Anova analysis

Samples P-value Tenacity,

(cN/tex)

P-value Elongation

(cN/tex)

Control and 2% cellulase 0.157385 0.203454

Control and 3% mixed enzyme 0.037583 0.029658



Control and 3% cellulase 0.157385 0.057583

Control and 4% cellulase 3.46E-05 0.008798

Articial Neural Network (ANN)

Neural networks are used for modeling non-linear problems and to predict theoutput values for a given input parameters from their training values. Most

of the textile processes and the related quality assessments are non-linear

in nature and hence neural networks nd application in textile technology.

ANNs are typically composed of interconnected “units” which serve as




(ii) Input parameters

(i) Enzyme concentration

(ii) Process time

(iii) Process temperature

(iii) Output parameters

(i) Jute bre tenacity

(ii) Jute bre elongation to break

(iii) Jute bre exural rigidity

(b) Training of neural network

For training, the raw jute bres were treated with various concentration andtime and temperatures with cellulase and mixed enzyme system. Then the

physical characteristics such as tenacity, elongation to break and exural

rigidity of the jute bres were evaluated with standard testing procedures and

their values are trained by using feed backward propagation algorithm. For the

error back propagation net, the sigmoid function is essentially for non linear

function. Training process of the neural network developed was started with

5000 preliminary cycles to optimize the ANN prediction accuracy. The best

structure is one that gives lowest training error and it is found to be minimum

error percent (Fig. 3.18). The training of the network was further continuedin order to reduce the training error. The average training error of 1%was

obtained and terminated at this stage since beyond this reduction in training

error was not appreciable.

Fig. 3.18 Schematic diagram of ANN used in the study




(c) Testing of neural network

For testing the prediction accuracy of the neural network a known specications

and process parameters were evaluated and their error percentage was

compared with predicted sample values which are given in Table 3.16. It can

be observed that mean absolute error with respect prediction is around 1%.

Figure 3.19 represents the neural network training of cellulase and mixed

enzymatic systems and their performance levels.

Fig. 3.19 Neural network training of cellulase enzyme system

and their performance level

Table 3.16 Articial neural network (ANN) analysis

Cellulase enzyme system

Input layers Trained sample results (actual) ANN output results (predicted) Error %

Conc. Temp Time Tenacity Elongat ion Flexural Tenacity Elongation Flexural Tenacity Elongation Flexural

2% 40 60 35.17 1.878 48.25 34.819 1.899 48.099 0.998 1.134 0.312

2% 50 90 33.24 1.897 46.57 33.655 1.904 47.044 1.248 0.395 1.017

2% 60 120 32.45 1.905 45.24 33.075 1.922 45.730 1.928 0.939 1.083

3% 40 60 32.96 1.932 45.23 32.844 1.922 45.247 0.350 0.471 0.037

3% 50 90 31.45 1.978 44.85 31.174 1.951 43.687 0.877 1.339 2.592

3% 60 120 31.01 1.997 42.54 31.364 1.977 41.849 1.142 0.991 1.623

4% 40 60 31.00 1.958 39.65 30.317 1.933 40.127 2.202 1.270 1.203

4% 50 90 30.47 1.998 38.61 30.258 2.012 39.328 0.693 0.715 1.859

4% 60 120 29.45 2.004 37.56 29.396 2.039 37.387 0.181 1.781 0.459

Average 1.068 1.004 1.131

Contd...




Mixed enzyme system

Input layers Trained sample results(actual) ANN output results(predicted) Error %

Conc. Temp Time Tenacity Elongat ion Flexural Tenacity Elongation Flexural Tenacity Elongation Flexural

2% 40 60 34.04 1.924 52.43 34.711 1.881 52.610 1.972 2.203 0.343

2% 50 90 33.82 1.877 46.57 33.509 1.910 47.680 0.915 1.795 2.384

2% 60 120 31.45 1.805 43.24 32.180 1.843 44.438 2.322 2.105 2.772

3% 40 60 33.46 1.953 42.56 33.332 1.939 43.810 0.384 0.701 2.931

3% 50 90 30.50 1.998 44.85 30.949 1.972 43.438 1.474 1.261 3.148

3% 60 120 31.01 2.197 38.54 30.586 2.155 38.114 1.362 1.911 1.105

4% 40 60 29.46 1.962 38.25 29.735 2.002 38.476 0.933 2.054 0.591

4% 50 90 28.47 2.004 34.61 28.457 2.033 35.360 0.045 1.462 2.169

4% 60 120 26.45 2.004 32.56 26.332 2.060 32.618 0.453 2.824 0.178

Average 1.095 1.818 1.738

Color spectroscopy analysis

Whiteness index of the cellulase and mixed enzyme treated jute bres

showed signicant improvement when compared to raw jute (Fig. 3.20).The percentage improvement of the whiteness index of cellulase enzyme

treated jute bres were noticed at 12.35%, 16.02% & 23.47% and those of

the mixed enzyme treated jute bres were noticed at 14.66%, 15.49% &

26.37% respectively at 2%, 3% and 4% concentration levels. The reason for

the improvement of whiteness index are mainly due to the delignication of

the jute bres and uniform/smooth surface obtained by enzymatic hydrolysis

of the specic enzyme treatment conditions. The yellowness characteristics

of the raw jute bres are mainly reduced by the cellulase and mixed enzyme

hydrolysis treatment. It was noticed that reduction level of cellulase treated

jute bres were shown at 54.90%, 58.02% & 54.97% and for the mixed

enzyme system it was noticed at 52.84%, 55.58% & 58.02% respectively

at various concentration of the enzyme/s. Further research is required to

improve the whiteness and reduction level of yellowness of the jute bres for

the making them apparel worthy. The brightness index of the cellulase and

mixed enzyme treated jute bres were better when compared to raw jute and

control bleached jute bres (Reid and Paice 1998). It is also observed that

the brightness values increase as the concentration of the enzyme treatmentincreases because of the reduction of yellowness index and the improvement

of the surface smoothness of the jute bres.

Contd...




Fig. 3.21 Reectance characteristics of raw jute, cellulase andmixed enzyme treated jute bres

Practical application of the developed neural network

When the target of the physical properties of the jute bres is predetermined

then we can start the input parameters such as the enzyme concentration, time

and temperature of the process condition which will predict the desired quality

of the jute bre properties in either cellulase or mixed enzymatic systems.

3.3.4 Prospective research

The mixed enzyme treated jute bres have noticed a higher rate of surface level

degradation when compared to the cellulase enzyme treatment (Vigneswaran

and Jayapriya 2008). The improvement in elongation, as a result of the

breakdown of cellulase and mixed enzyme in the jute bres, was noticed up to

5.26% and 5.48% respectively at 4% concentration of the enzyme treatments.

The exural rigidity of the mixed enzyme treated jute bres was noticed

signicant reduction level than the raw jute and cellulase enzyme treated jute

bres (Ghosh and Dutta 1983). Due to the reduction in the exural rigidity,

soft and smooth texture has been observed more in the enzyme treated jute

bres than in raw jute (Ghosh and Dutta 1980 and 1983). The white rot fungi

Phanerochaete chrysosporium and Ceriporiopsis subvermispora are found to




be degrading lignin effectively without any loss of appreciable cellulose in

the biosoftening process spanning for a period of 30 days which is considered

as the optimum period of incubation (Tien and Kirk 1988). Biosoftening of

jute bres using these fungi decreases the tensile characteristics of jute bre

with 12.26% decrease in tenacity and 14.62% increase in elongation in case

of P. chrysosporium, while the usage of C.subvermispora as 16.86% decrease

in tenacity and a 6.45% increase in elongation with respect to raw jute. The

bending characteristics of the jute bre, when compared with that of raw jute,

are affected favorably by reduction in exural rigidity to the extent of 37.84%

in case of P. chrysosporium and 34.14% in case of C. subvermispora. These

improvements, with respect to increase in tenacity and elongation percentageand decrease in exural rigidity will essentially improve the spinnability of the

bre. This research work has proven that biosoftening of jute bre can be done

effectively in an ecofriendly manner using Phanerochaete chrysosporium and

Ceriporiopsis subvermispora.

The action of these enzymes is limited to the surface level degradation,

while the white rot fungi increases the high degree of depolymerization of lignin

and this improves the quality of the jute bres thereby making the development

of the apparel worthy and it widened the scope for using the bre effectively in

the manufacture of value added products like jute-based fabrics and composites(Macmillan et al. 1995). The neural network trained using the input and the

output parameters related to application of cellulase and mixed an enzymatic

system which gives an average error of 1% with respect to the enzymatic

process. The trained network also gives the same error% when testing of the

network was carried out with an enzyme and jute bres, which were not used

for training the network. Hence, the neural network developed can be used to

determine the tenacity, elongation to break and exural rigidity of the jute bres

for producing the expected quality with enzymatic treatment on the jute bres. Hassan et al. (1996) studied the jute-cotton blended fabric treated

with commercial cellulases, xylanases and pectinases individually and in

combination at various concentrations in order to smooth and soften the fabric.

Enzyme treatment was carried out at 50°C in the presence of 0.1 M phosphate

buffer (pH 7.0), for 3 h. Enzymatic activities were evaluated by the release of

reducing sugars and changes in surface appearance of the fabric. Addition of

commercial cellulases alone extensively removed protruding jute and cotton

bres from the fabric, whereas addition of commercial pectinases or xylanases

mainly loosened the protruding long jute bre bundle. Combined treatment

of pectinases and xylanases with reduced amounts of cellulases was equally

effective as high levels of cellulases in the removal of surface protruding bres.

The amount of reducing sugar released correlated with removal of bres from




the fabric surface. Thus, the fabric surface was smoother in enzyme-treated

samples compared to untreated control and treated with mixtures of enzymes

were more effective than cellulase alone.

With increase in the concentration of enzyme solutions for either single

(a) cellulase enzyme or (b) specic mixed enzyme or (c) mixture of (a) and

(b) with higher cellulase concentration, there is notable increase in weight loss

and treatment shrinkage, marginal increase in breaking extension, whiteness

index (with consequent small reduction in yellowness index and K/S value) and

brightness index, showing a measurable reduction in bending length, overall

exural rigidity and overall bending modulus with consequent decrease in fabric

tenacity to some extent. A close look on to the overall bending property indicatethat, treatment with the specic mixed enzyme system (c) mixture of 105 unit/

ml of cellulase, 96unit/ml of xylanase and 136 unit/ml of pectinase enzymes

give an overall balanced improvement in the physical property parameters,

while further increase in the cellulase concentrations do not show any further

notable improvement besides a marginal improvement in brightness index with

small increase in weight loss and tenacity loss (Samanta et al. 2006).

3.4 Bioprocessing of ax and their characteristics

3.4.1 Flax bre

Flax ( Linum usitatissimum L., Linaceae), which has been grown throughout the

world for millennia, is the source of products for existing, high-value markets in

the textile, composites, paper/pulp, and industrial/nutritional oil sectors (Sharma

and Van Sumere 1992). Flax is the source of industrial bres and, as currently

processed, results in long-line and short (i.e., tow) bres (Van den Oever et al.

2000). Long line bre is used in manufacturing high value linen apparel, while

short staple bre has historically been the waste from long line bre and usedfor lower value products. Flax is a bast bre and length varies from about 25

to 150 cm (18 to 55inches) and average 12–16 micrometers in strand diameter.

There are two varieties of ax available, (a) shorter tow bres used for coarser

fabrics and (b) longer line bres used for ner fabrics. Flax bres can usually be

identied by their “nodes” which add to the exibility and texture of the fabric.

The cross-section of the linen bre is made up of irregular polygonal shapes

which contribute to the coarse texture of the fabric.

3.4.1.1 Flax bre structure

The physical ax bre form being present in composite materials ranges

from bre bundles to elementary bres, or to even further opened-up shapes




(Fig. 3.22). The mechanical properties of these different bre forms differ

strongly. Flax bre bundles are being obtained after the rst isolation

processes called ‘breaking’ and ‘scutching’. These bres bundles have an

acceptable price-performance ratio and are often commercially used in

natural bre mat reinforced thermoplastic (NMT) and thermoset composites

(Heijenrath and Peijs 1996). Their lateral strength is rather poor compared

with their axial strength, mainly due to the weak pectin bonds between the

so-called ‘technical bres’. The really strong bres are the elementary bres,

which have an average tensile strength up to 1500 MPa (Van den Oever et al.

2000). Flax bres look like small lengths of bamboo under a microscope. The

cellulose molecules in ax bres are folded back and forth in a fairly regulararrangement and responsible properties of crystallinity. The ax bres are

composed of closely packed “ultimate cells” of the brillar structure that are

cemented together with holocellulose and lignin. The ultimate cells under a

microscope and abraded bres often show ultimate cells sticking away from

the surface.

Fig. 3.22 Flax bre structure

3.4.1.2 Flax properties

Finer ax bres are called as ‘linen’. Linen fabric feels cool to the touch. It

is smooth, making the nished fabric lint-free and gets softer the more it is




washed. Linen has poor elasticity and does not spring back readily, explaining

why it wrinkles so easily (Domier 1997). Linen fabrics have a high natural

luster; their natural color ranges between shades of ivory, ecru, tan, or grey.

Pure white linen is created by heavy bleaching. Linen typically has a thick

and thin character with a crisp and textured feel to it, but it can range from

stiff and rough, to soft and smooth. When properly prepared, linen fabric has

the ability to absorb and lose water rapidly. It can gain up to 20% moisture

without feeling damp. It is a very durable, strong fabric, and it is stronger

in wet than dry. The bres do not stretch and are resistant to damage from

abrasion. However, because linen bres have a very low elasticity, the fabric

will eventually break if it is folded and ironed at the same place repeatedly.Mildew, perspiration, and bleach can also damage the ax fabric, but it is

resistant to moths and carpet beetles. Linen is relatively easy to take care of,

since it resists dirt and stains, has no lint or pilling tendency, and can be dry-

cleaned, machine-washed or steamed. It can withstand high temperatures, and

has only moderate initial shrinkage.

3.4.1.3 Applications

Linen fabrics are being used for wide range from bed and bath fabrics (tablecloths, dish towels, bed sheets, etc.), home and commercial furnishing items

(wallpaper/wall coverings, upholstery, window treatments, etc.), apparel items

(suits, dresses, skirts, shirts, etc.), to industrial products (luggage, canvases,

sewing thread, etc.). A linen handkerchief, pressed and folded to display the

corners, was a standard decoration of a well-dressed man’s suit during most

of the rst part of the 20th century. Currently researchers are working on a

cotton/ax blend to create new yarns which will improve the feel of denim

during hot and humid weather. Linen fabric is one of the preferred traditional

supports for oil painting (Burger et al. 1995). Linen is also used extensively

by artisan bakers. Paper made of linen can be very strong and crisp that is

made from 25% linen and 75% cotton. The excellent mechanical properties

of ax, combined with the added functionalities they bring, make them a very

attractive potential material for bre reinforced composites. Over the past 30

years the end use for linen has changed dramatically. Approximately 70% of

linen production in the 1990s was for apparel textiles whereas in the 1970s

only about 5% was used for fashion fabrics.

3.4.2 Need of bioprocessing of ax

Natural bres are mainly formed out of cellulose which is surrounded by a

hydrophobic layer inhibiting their wetting. This hydrophobic layer constituted




from so called “natural impurities” (pectin, hemicellulose, lignin, proteins,

waxes, fats and mineral compounds) must be removed to render a valuable

hydrophilic property to natural bres. Flax bres, which together with ramie,

jute, hemp belong to the grow-up of bast bres, are special among textile

materials due to their properties. Flax bres, besides cellulose (65 - 80%)

contain non-cellulosic substances such as hemicellulose and lignin. Lignin

being a constituent of this non-cellulosic matter is a large, cross-linked

macromolecule with molecular mass in excess of 10,000 amu. It is relatively

hydrophobic and aromatic in nature. In primary ax bre, lignin occurs in the

primary wall and in the outer part of the secondary wall. Lignin is resistant

to mineral acid activity and it is sparingly soluble. It can dissolve when itis initially transformed into derivatives by chlorination and oxidation and

then by bleaching. Lignin presence in ax bre affects its rigidity due to

incrustation in amorphous areas of cellulose. Traditionally these “impurities”

are effectively removed by chemical scouring in water solutions of sodium

hydroxide at an elevated temperature of 98°C, and time of 60 min.), which is

harmful for the environment. Application, for this purpose, of equally efcient

ecological biotechnological methods eliminates these problems.

Many researchers are made attempts in the application of enzymatic pre-

treatment of fabrics made of natural bres showed a possibility of substitutingtraditional alkali scouring of cotton woven fabrics. In particular, the

employment of pectinolytic enzymes happened to be effective in removing

non-cellulosic substances from cotton and linen fabrics (Sójka-Ledakowicz

et al. 2005). Enzyme such as laccase is active during the decomposing of the

lignin-cellulose complex. Hence, the research task attempts to apply laccase

complex in the treatment of woven fabrics made of ax bres. Laccase (EC

1.10.3.2, p-diphenol oxidase) is an extracellular blue oxidase capable of

oxidizing phenols and aromatic amines by reducing molecular oxygen to water by a multicopper system (Thurston 1994). Laccase occurs in certain plants

and bacteria, but the enzyme is particularly abundant in white-rot fungi and

it is assumed to comprise a lignin biodegradable complex. From among other

microorganisms it is the best lignin degrader (Hatakka 2001). It degrades wood

by a simultaneous attack of lignin and cellulose/hemicellulose or selectively

degrades lignin far more than polysaccharides (Eriksson et al. 1990; Kuhad et

al. 1997). Laccase seems to be one of the most important enzymes in lignin

degradation (Kawai and Ohashi 1993) since it can attack polymeric lignin,

degrade the framework structure loosely, introduce additional hydrophilicgroups, and produce water-soluble material (Iimura et al. 1999). In the

presence of suitable redox mediators (e.g. 1-hydroxybenzotriazole), laccase

is even able to oxidize recalcitrant non-phenolic lignin units. This capability

has generally extended their use to a series of biotechnological applications,




all of them related to the degradation of structurally diverse aromatic

compounds. Laccase is currently being investigated by many researchers with

respect to litter mineralization (Dedeyan et al. 2000), dye detoxication and

decolorisation (Abadulla et al. 2000; Wesenberg et al. 2003), the bleaching

of paper pulp (Bourbonnais et al. 1997; Ander and Messner 1998) and

bio-scouring of ax bre (Ossola and Galante 2004). Sharma et al. (2005)

conducted tests on enzyme application for scouring dew retted ax roving.

From literature analysis it turns out that research performed up to now has

referred mainly to ax roving. Former research carried out by the Textile

Research Institute concerned the removal of impurities from fabrics made of

natural bres applying pectinolytic enzyme complex.

3.4.2.1 Flax rippling and retting

The quality of the linen fabric is mainly dependent upon growing conditions

and harvesting techniques. For getting the longest possible bres, ax is either

hand-harvested by pulling up the entire plant or stalks are cut very close to

the stem root. After harvesting, the seeds are removed through a mechanized

process called ‘rippling’ or ‘winnowing’. The ax bres must then be loosened

from the stalk. This is achieved through retting. This is a process which uses bacteria to decompose the pectin that binds the bres together. Natural retting

methods take place in tanks and pools, or directly in the elds. There are

also chemical retting methods; these are faster, but are typically more harmful

to the environment and to the bres themselves. After retting, the stalks are

ready for scutching, it removes the woody portion of the stalks by crushing

them between two metal rollers, so that the parts of the stalk can be separated.

The bres are removed and the other parts such as linseed, shive, and tow are

set aside for other uses (Friesen 1986). Next the bres are heckled: the short

bres are separated with heckling combs by ‘combing’ them away, to leave

behind only the long, soft ax bres. After the ax bres have been separated

and processed, they are typically spun into yarns and woven or knit into linen

textiles. These textiles can then be bleached, dyed, printed on, or nished with

a number of treatments or coatings.

Retting, which is the separation of bast bres from the core tissues, is

preeminent in ax bre processing, as it affects quality and yield (Pallesen

1996). Two traditional methods used commercially to ret ax for industrial

grade bres are water- and dew-retting (Sharma and Van Sumere 1992).Water-retting results in high quality bre but was discontinued in western

countries several decades ago because of the extensive stench and pollution

from fermentation products and the high cost of drying. Dew-retting is now

the accepted practice in most countries and supplies the linen used in high




quality textiles. Enzymes have been considered as a method to improve retting

(Van den Oever et al. 2000).

In water-retting, ax stems are submerged in rivers and lakes, and

anaerobic bacteria colonize the ax stems and degrade pectins and other

matrix compounds, thus freeing bres from the core tissues. Dew-retting is

an art that depends upon the removal of matrix materials from the cellulosic

bres before cellulolytic, and therefore weakening, of the bres occurs. This

process is dependent mostly upon plant cell-wall degrading enzymes produced

by indigenous, aerobic fungal consortia (Henriksson et al. 1997a; Fila et al.

2001). In dew-retting, ax plants are pulled from the soil and laid out in elds

for selective attack by the fungi over several weeks.Disadvantages of dew-retting are its dependence on particular geographical

regions that have the appropriate moisture and temperature ranges for retting,

coarser and lower quality bre than water retting, poor consistency in bre

characteristics, and occupation of agricultural elds for several weeks (Van

den Oever et al. 2000). Further, dew-retting results in a heavily contaminated

bre that is dusty and particularly problematic in textile mills. Chemical retting

(Van Sumere 1992), enzyme-retting (Akin et al. 1996), and steam explosion

techniques (Kessler and Kohler 1996) are bre extraction methods that have

previously been investigated. Because of problems with both water- and dew-retting, a long-term objective for improving the ax bre industry has been

development of enzyme-retting (Hamilton 1986; Schunke et al. 1995). In

the 1980s, extensive research was undertaken in Europe to develop enzyme-

retting as a method to replace dew-retting. The strategy of the research was

to replace the anaerobic bacteria with enzyme mixtures in controlled tanks,

thereby producing ax of water-retted quality but without the negative aspects

of stench and pollution.

Research resulted in development of the commercial enzyme mixtureFlaxzyme from Novo Nordisk (Denmark), several patents pertaining to enzyme-

retting (Akkawi 1990), and a pilot scale, tank method (Van den Oever et al.

2000). Enzyme-retting produced bres having the neness, strength, color, and

waxiness comparable to the best water-retted bre. Advantages of the enzyme

method were: (1) time savings of 4–5 days, (2) increased yield of ca 2% over

water-retting, and (3) bre consistency. The ax plant supplies both industrial

oil (i.e., linseed oil) and bast bre used to produce textiles, composites, and

paper/pulp. Linen has occupied a prominent place in textiles for centuries.

Flax can be grown in many locations and is environmentally friendly.Flax production in the South Atlantic region has the potential to enhance rural

economic growth and to supply a domestic source to the bre industries of the

United States. Flax is well known to grow in a cool and moist climate (Sharma

and Van Sumere 1992; Elhaak et al. 1999).




3.4.3 Bioscouring of ax bres

The nature of the ax bres and their characteristics were examined using ascanning electron microscope, it was evident that large bre bundles could

be observed as well as big hollow and porous structures and cracks were

clearly visible. Moreover, the surface of these ax bres dose not appears to

be smooth and totally free of debris. At maturity, the typical bast bres show

high cellulose content (60% to 70%), but not as high as cotton. Moreover,

in the bast tissue, these cells are part of a three dimensional structure rich in

pectin (Bledzki et al. 1996). By attacking this macromolecule (pectin), it eases

the decortication process and liberates bre bundles with few cells per bundle,

if retting is optimal.

3.4.3.1 Pectin, pectate lyase and bioscouring process

Pectin is found in the middle lamella of the primary cell wall of practically all

higher plant tissues. It is composed of a homo-galacturonan backbone (smooth

region) interrupted by heavily branched regions of rhamno-galacturonan

(hairy region). Homogalacturonan consists of α-1,4-linked D-galacturonic

acid (GalpA) residues, which can be methyl-esteried. Both pectin lyase (EC4.2.2.10) and pectate lyase (EC 4.2.2.2) are able to depolymerize the smooth

region of pectin by ß-elimination (Kozlowski et al. 2006). Although pectate

lyase cleaves between two α-1,4-D-GalpA residues and pectin lyase cleaves

between two methylesteried α-1,4-D-GalpA residues, this distinction on

substrate specicity is not strict. Pectate lyase is generally active on pectin

with a moderate degree of methyl esterication (DM) (0–50%), whereas

pectin lyase activity often increases with increasing DM. Developing a

biotechnology based on the utilization of an engineered enzyme (pectate

lyase) for the production of high quality bast bres from ax straw, i.e. betterneness and cleanliness, proper strength, modulus and length (Denis Rho

et al. 2008). Pectate lyases are secreted enzymes produced by a variety of

plant-pathogenic bacteria. These pectinolytic enzymes, as well as various

endo-polygalacturonase, are useful retting or bioscouring catalysts for the

processing of natural hemp and ax bres, and cotton fabric.

The bast bre bundles were then treated by soaking, using the newly

engineered pectate lyase (Fig. 3.23). Bioscouring of bast bre bundles was

performed at 37ºC and pH 8.5 in a non-agitated system. Scanning electron

microscopy (SEM) was used as a technique to monitor the bre surface

morphology and a chemical method was used to monitor the progress of the

enzymatic reaction. The tests show that the enzyme treatment produced an

effect that translates in a signicant increase of the relative percent of cellulose




from 34% to 58%, with a concomitant decrease of the relative pectin content

from 7.5% to 4.5% (Rana et al. 1998). However, the apparent efciency

of the bioscouring process was not as efcient as the chemical scouring

process (end point results: cellulose 68% and pectin 1.6%). An improvement

in bre cleanliness and separation of the bre bundles (diameter of 60 µm

to 100 µm) into ultimate bres (diameter of 17 µm) can also be observed

(Fig. 3.24). A commercial enzyme preparation (Bioscour 3000, used

traditionally in the cotton industry for degumming cotton bres) was used as a

control experiment. An improvement in bre cleanliness and separation of the

bre bundles into ultimate bres were observed but, bioprocessing conditions,

such as, enzyme-bre ratio, Ca++ concentration, temperature, and pH, as wellas the utilization of a cutinase for a better surface treatment of the bres is

among other parameters that need to be optimized.

Fig. 3.23 Mechanically decorticated ax bres, the bath in which the bres are

soaked, and the enzyme-treated bres [Source: Denis Rho et al. 2008]

Fig. 3.24 SEM micrographs show the ax bres before (left) and after (right) the

enzyme treatment performed with the engineered pectate lyase at a concentration of

1.2% (2.5 IU/ml). [Source: Denis Rho et al. 2008]




3.4.3.2 Eco-friendly approach for ax processing

John et al. (2002) have made an attempt to develop an enzyme-retting pilot plant method to replace traditional methods thus producing ax bres

with specic properties for industrial uses. Fundamental knowledge of the

structural and chemical characteristics of ax are important for designing

a strategy using enzymes to produce bres with specic properties

required for industrial applications. Flax bres, which occur in the bast

(cortex) region of the stem, lay between the protective cuticle/epidermis

barrier and the lignied core tissues (Van den Oever et al. 2000). The

ultimate bres (i.e., individual elongated bre cells) occur in bundles

that, intermixed with parenchyma tissues, form a ring around the lignied

core cells of the stem (Akin et al. 1997). Pectin serves as a glue to hold

bres together in bundles and the bundles to non-bre tissues (Van den

Oever et al. 2000). Calcium levels are especially high in the protective

barrier of the ax stem and likely help stabilize pectins and thereby plant

tissues in that location. These structural/chemical characteristics indicate

specic regions that serve well to protect the stem and must be breached

by enzymes for effective retting.

The new environment-friendly processing system requires harvestedax stems to be crimped between uted rollers thus splitting the stalk

both longitudinally and transversely. Briey, the procedure uses a

pectinase-rich, commercial enzyme mixture plus chelator, e.g., 50 mM

ethylene diamine tetra acetic acid (EDTA), applied to crimped ax stems

that are then incubated for 24 hours at 40°C. Enzyme was required for

ease of bre removal with chelator (e.g. EDTA) scavenging and binding

exposed calcium ions. During incubation, the chelators and enzymes work

concurrently to further separate the bre bundles. Following incubation,

the enzyme-retted ax stalks are rinsed with water to remove the enzyme

solution and the solubilized portion of stalk. Retted ax stalks are then

dried with circulating heated air (Foulk et al. 2004). By controlling all

processing steps, uniform ax bres of known properties are produced.

Enzyme-retted bres were then mechanically cleaned and characterized

for properties relevant to textile bres. In contrast to traditional linen

production, the current US textile industry requires short staple, rened

bres. Enzyme-retting and additional processing produces short staple

bres of more consistent quality reduces the environmental pollutionthrough excess dirt and dust of dew-retted ax, does not limit the process

to geographical regions of particular temperature and moisture, and allows

elds to be harvested and made ready for subsequent crops in a known

time-frame. This retting could be carried out in facilities near farms to




et al. 2000). Color was measured for both dew-retted and enzyme-retted

bre and seed ax (Table 3.18). Enzyme retting produced bres that were

substantially lighter than dew-retted bres for both bre and seed ax, with

enzyme retted bre ax the lightest. Color data for the samples varied, with

the seed ax having considerably greater redness and yellowness (Table

3.18). Results suggest opportunities to modify color properties of ax by

retting methods and ax type. Comparing various enzyme- and dew-retted

bre samples indicated that signicantly different properties of strength and

neness resulted with the various formulations.

Table 3.17 Properties of Flax bres retted and processed by various means

[Akin et al. 2000]

Sample Strength

(g/tex)

Fineness

(micronaire)

European long line, dew-retted 38 ± 5 8.0

South Carolina grown, dew-retted 24 ± 2 4.2

Ariane bre ax, spray-enzyme retted 27 ± 4 7.1

Shirley-cleaned 18 ± 2 4.6

Ariane bre ax, spray-enzyme retted 33 ± 5 7.8

Shirley-cleaned 27 ± 2 4.8

Seed ax. Spray-enzyme retted 21 ± 1 4.1

Upland cotton 21 to 25 3.7 to 4.2

Table 3.18 CIELAB color of dew- and enzyme-retted ax bres [Akin et al. 2000]

Retted sample CIELAB values

L* a* b*

Fibre ax

Dew 59.9 2.5 10.3

Enzyme 76.2 2.3 14.0

Seed ax

Dew 60.5 3.9 13.0

Enzyme 67.9 4.6 17.1

3.4.4 Bio-scouring of linen fabrics with laccase complex

Presently biotechnology plays an important role especially in the eld of

environmental protection. In the textile industry enzymes are often used in

many technological processes as they are ecological. Flax bres are special




among textile raw materials due to their properties (Buschie-Diller et al.

1994). Flax bres, besides cellulose, contain non-cellulosic substances such

as hemicellulose, lignin, pectins, waxes and fats. Enzyme such as laccase

is active during the decomposing of lignin-cellulose complex. Hence, the

research task attempts to apply laccase complex in the treatment of woven

fabrics made of ax bres (Kan et al. 2007). The possibility and effectiveness

of applying laccase complex produced by Cerrena unicolor strain in the

scouring processes of linen fabrics was studied. The tests performed proved

that the pre-treatment with laccase complex from Cerrena unicolor provides

a high level of water sorption capabilities in of linen fabrics and with laccase

can be an alternative to traditional chemical scouring. Jadwiga et al. 2007was made attempt using linen woven fabric (plain weave, mass per unit area

223 g/m2) with laccase enzymes. In the enzymatic treatment of ax bres,

laccase enzyme produced by Cerrena unicolor (Bull. Ex Fr.) strain 137, which

belongs to white rot fungi, was used.

3.4.4.1 Enzymatic pre-treatment of linen fabric

Linen woven fabric before enzymatic treatment was washed in water bath

at 60–65°C for 60 min in order to remove sizing agents. Linen woven fabricwas subjected to pre-treatment in Linitest laboratory dyeing apparatus,

using different amounts of laccase enzymes produced by Cerrena unicolor .

Enzymatic pre-treatment of woven fabric was performed in baths of varying

concentration of laccase enzymes from 2.4 to 7.5 U/g fabric in optimal

treatment conditions: pH 5.3 (acetate buffer), temperature of 60°C, time 30–

120 min; liquid ratio 10:1. Enzymes inactivation occurred in water bath at a

temperature of 98°C for 5 min.

3.4.4.2 Traditional alkali treatment

Traditional alkali-scouring pre-treatment was performed in Linitest laboratory

dyeing apparatus at the liquid ratio of 10:1 in a bath containing sodium

hydroxide 1.8 g/l. Process conditions: temperature of 98°C, time of 60 min;

rinsing temperature of 80°C, time of 10 min.

3.4.4.3 Bleaching treatment

Linen woven fabrics after the bio- and chemical scouring were subjected to

two-stage bleaching in baths containing hydrogen peroxide 35%, 10.0 ml/l

stabilizer, anionic agent 0.7 g/l sodium hydroxide 2.0 g/l process conditions:

temperature of 98°C, time of 60 min, liquid ratio of 10:1.




3.4.4.4 Enzyme stability, temperature and pH optima

Laccase from Cerrena unicolor was found to be relatively thermostable.Surprisingly, at 50°C, the enzyme did not lose almost any activity within 1

hour and at 70°C still 10% of its activity remained after 60 min of incubation.

A higher temperature of 80°C, however, caused the rapid inactivation

of laccase (95% activity loss within 10 min). Laccase also seems to be

relatively stable during long time storage in a refrigerator at 4°C. After 6

months, the activity of laccase was almost unchanged. At an alkaline pH of

10, the enzyme was also stable while an acidic pH of 3 caused the partial

inactivation of laccase.

3.4.4.5 Water sorption characteristics

The water sorption ability of linen fabrics was determined on the basis of

sorption coefcients dened by the method of sorption curve analysis. For

comparison purposes the tests of raw woven fabrics and fabrics after traditional

alkali boiling off were performed. It has been stated that woven fabric made

of ax bres after enzymatic pre-treatment (using laccase from Cerrena

unicolor ) are characterized with higher sorption values when compared towoven fabrics after alkali boiling-off.

3.4.4.6 Whiteness characteristics of linen fabrics

For woven fabrics made of ax bres and bleached after enzymatic pre-

treatment, comparable or even higher whiteness degree was obtained when

compared to fabrics bleached after traditional alkali treatment (Table 3.19).

This conrms the effectiveness of applied bio-treatment. Pretreatment with

laccase complex from Cerrena unicolor provides a high level of watersorption capabilities in linen fabrics. As is known, the ability of bres to

absorb liquids is an important parameter of flax textile fabrics during their

processing (bleaching, dyeing). The tests performed have conrmed the

usefulness of laccase produced by Cerrena unicolor in purifying woven

fabrics made of ax bres (Jadwiga et al. 2007). Efcient removal of lignin

from ax bre facilitates the penetration of oxidizing whitening agents

into bre structure. After bio-treatment, comparable whiteness degrees are

obtained as compared to the ones after alkali scouring. From the results

conrm that linen woven fabric treatment with laccase enzymatic complex

from Cerrena unicolor can be used as better alternative to traditional

chemical treatment.




Table 3.19 Comparison of bleaching of linen fabric with alkali and bio-treatments

[Jadwiga et al. 2007]

Process conditions Whiteness

coefcient (W)

Whiteness digital

assessment (TV)

Fabric after alkali scouring 36.5 –3.5

Bio-pre-treatment 2.4 U/g

fabric

30 min 37.2 –3.1

60 min 37.9 –3.0

90 min 38.1 –3.0

Bio-pre-treatment 5.0 U/g

fabric

30 min 37.9 –3.0

60 min 35.6 –3.2

90 min 38.3 –3.0

3.5 Bioprocessing of wool and their characteristics

3.5.1 Wool bre and their classication

The wool bre is natural hair grown on sheep and is composed of protein

substance called as ‘keratin’ . Wool is composed of carbon, hydrogen, nitrogen

and this is the only animal bre, which contains sulfur in addition. The wool

bres have crimps or curls, which create pockets and give the wool a spongyfeel and create insulation for the wearer (Feughelman 1997). The outside

surface of the wool bre consists of a series of serrated scales, which overlap

each other like the scales of a sh. Wool is the only bre with such serration’s

which make it possible for the bres to cling together and produce felt for

extreme cool climatic condition (Morton and Hearle 1993).

3.5.1.1 Wool bre – Classication

The quality of wool bres produced is based on the breeding conditions, theweather, food, general care etc. For example, excessive moisture dries out

natural grease. Similarly the cold weather produces harder and heavier bres.

The wool could be classied in two different ways.

3.5.1.2 Classication by sheep

The wool is classied according to the sheep from which it is sheared as given

below:

Merino wool: Merino sheep originated in Spain yields the best quality

wool. These bres are strong, ne and elastic bre which is relatively short,

ranging from 1 to 5 inches (25–125 mm). Among the different wool bres,

merino wool has the greatest amount of crimp and has maximum number




of scales. These two factors contribute to its superior warmth and spinning

qualities. Merino is used for the best types of wool clothing.

Class – Two wool: This class of sheep originates from England, Scotland,

Ireland and Wales. The bres are comparatively strong, ne, and elastic and

range from 2 to 8 inches (50–200mm) in length. They have a large number of

scales per inch and have good crimp.

Class – Three wool: This class of sheep originates from United Kingdom.

The bres are coarser and have fewer scales and less crimp when compared

to earlier varieties of wool bres and are about 4 to 18 inches long. They are

smoother, more lustrous, less elastic and resilient. They are of good quality,

used for clothing. Class – Four wool: This class is a group of mongrel sheep sometimes

referred to as half-breeds. The bres are about 1 to 16 inches (25–400 mm)

long, are coarse and hair like, and have relatively few scales and little crimp.

The bres are smoother and more lustrous. This wool is less desirable, with

the least elasticity and strength. It is used mainly for carpets, rugs, and

inexpensive low-grade clothing.

3.5.1.3 Classication by eece

Wool shearing is the process by which the woolen eece of a sheep is

removed. Sheep are generally shorn of their eeces in the spring, but the time

of shearing varies in different parts of the world (Heine and Hocker 1995).

Sheep are not washed before shearing. They are sometimes dipped into an

antiseptic bath as required conditions.

The classication by eece is as follows:

Lamb’s wool: The eece obtained by shearing the lamb of six to eight months

old for the rst time is known as lamb’s wool. It is also referred to as eece

wool, or rst clip. As the bre has not been cut, it has a natural, tapered end

that gives it a softer feel.

Hogget wool: Hogget wool is the one obtained from sheep about twelve

to fourteen months old that have not been previously shorn. The bre is ne,

soft, resilient, and mature, and has tapered ends. These are primarily used for

warp yarns.

Wether wool: Wether wool is the one obtained from the sheep older than

14 months. The shearing is not done for the rst time and in fact these eeces

are obtained after the rst shearing. These eeces contain much soil and dirt. Pulled wool: Pulled wool is taken from animals originally slaughtered for

meat. The wool is pulled from the pelt of the slaughtered sheep using various

chemicals. The bres of pulled wool are of low quality and produce a low-

grade cloth.




Dead wool: This is the wool obtained from the sheep that have died of age

or accidentally killed. This type of wool bre known should not be confused

for pulled wool. Dead wool bre is decidedly inferior in grade; it is used in

low-grade cloth.

Cotty wool: This type of wool is obtained from the sheep that are exposed

to severe weather. As discussed; the severe weather conditions hamper the

qualities of the eece obtained. The cotty wool is of a poor grade and is hard

and brittle.

Tag locks: The torn, ragged, or discolored parts of a eece are known as

tag locks. These are usually sold separately as an inferior grade of wool.

3.5.2 Wool bres properties and their composition

3.5.2.1 Physical and chemical properties of wool bres

Wool morphology

Wool is the thick, wavy and brous protective covering of sheep. It consists

of the insoluble protein, known as ‘keratin’ . The wool bre grows from the

follicle situated in the dermis (the middle layer of skin). Wool bre consists

mainly of three morphological components: the cuticle or skin, the cortex, andmedulla in the centre (Lowry et al. 1951). High quality wool bre (ne wool)

does not contain the medulla (central core of hard cells) and has a hollow

centre. Fleece obtained from sheep is called grease wool or raw wool. Though

wool bres are more or less cylindrical, the surface consists of overlapping

and interlocking scales of the cuticle (El-Sayed et al. 2002). The serrated wool

bres tend to interlock and cling together imparting felting qualities to the

wool (Fig. 3.25).

Fig. 3.25 Longitudinal view of wool bre

The cortex comprises spindle-shaped cortex cells that are separated from

each other by a cell membrane complex. Wool cuticle cells (overlapping cells

that surround the cortex) are subdivided into two main layers, namely the




exocuticle and endocuticle (Fig. 3.26). The outer surface of the scale of the

cuticle is covered by a very thin membrane called the epicuticle (Jones and

Leeder 1975). Below this hydrophobic epicuticle is the exocuticle, a cystine-

rich component forming about two-thirds of the scale structure. The exocuticle

just below the epicuticle is referred to as the ‘A’ layer, having a distinctly

higher cystine component than the rest of the exocuticle (known as the ‘B’

layer). Below the exocuticle, forming the remainder of the scale structure is

the endocuticle and then a thin layer of intercellular cement (Feughelman

1997). Figure 3.27 shows the cell membrane structure of wool bre.

Fig. 3.26 Cross-section diagram of a merino wool bre

Fig. 3.27 Schematic of a wool bre showing cuticle and cortical cells

Staple length: It is the total length of a bre in its natural condition. It is

obtained by measuring the natural staple without stretching the crimps out of

the bre.




Fibre length: It is the total length of a wool bre after removing the

crimps or waviness by straightening it.

Crimpiness: It refers to the waviness of the wool bre. Its number varies

from 2 to 12 per cm depending upon the quality. It is a valuable property in

spinning and increases the elasticity of the yarn and fabric. Crimps are more

pronounced in ne wool.

Elasticity: The property of wool bres to return to their original or natural

form after being stretched or compressed. Wool is quite elastic, and therefore,

resists wrinkling, bagging and tearing.

Lustre: It is the ability of wool to reect light. Wool with lustre, when

dyed, has a brighter appearance than wool without lustre. Coarse wool withfewer scales has more lustre than ne wool because of smoothness of bre.

Strength: It is the property of wool bre to undergo processing without

breaking. Wool bre and fabrics are usually strong and durable.

Conductivity: Wool is one of the best bres for retaining body heat and

also for keeping external heat out. This is because of its insulating nature i.e.

it is a poor conductor of heat.

Dyeing properties: Wool is one of the easiest bres to dye because dyes

penetrate the bre easily and remain permanently.

Softness: Softer bres consist of numerous, small scales which t overone another loosely and produce fabrics which are softer to touch.

Inammability: Wool is slower to burn, and on burning, it gives off a

pungent odour and forms a bead when burning ceases.

Action of chemicals: Alkalis weaken the wool and may even dissolve it

completely. Dilute acids do not act upon wool, and wool is generally dyed

with acid colours.

Moisture: Wool readily absorbs and gives off moisture. Under normal

conditions, the moisture content varies from 12% to 17%. Warm: You only have to look at a Highland sheep breed in its dense,

long eece standing in the show to understand the special thermal capacity of

wool. It seems warm to the touch, while cotton feel cool, for example.

Hygroscopic: Wool absorbs, retains and releases moisture without

affecting its thermal properties. This makes is perfect for use in breathable

ancient structures, and anywhere where moisture is a concern.

Acoustic absorption: Wool is very good at soaking up reverberated sound,

particularly in the range of the human voice. It makes a fabulous acoustic

cloud material and why it is so good inside quality speakers. Biodegradable: As a completely natural material made from keratin, like

human hair, wool will eventually break down completely, leaving no residue

and damaging nothing. It is therefore good for gardening and use in places

where safe, chemical-free environment are important.




Nitrogen slow release – As it biodegrades, wool releases nitrogen slowly

into its surroundings, feeding nearby plants and organisms that need nitrogen-

rich conditions.

3.5.2.2 Properties of woollen clothing

Wool, one of the oldest textile bres known, has survived the test of time because

of its unique natural properties. Today there are many other textile bres, but as

yet science has not been able to produce another bre containing all the natural

properties of wool. Wool remains unique; a masterpiece of design.

Wool insulates against heat and cold Wool absorbs moisture vapor and wool clothing provides superior comfort in

both hot and cold weather. In cold weather even a little moisture on the skin

becomes cold, quickly reducing body temperature (Rippon 2003). However,

by absorbing body moisture a dry layer of air is left next the skin and it helps

to hold in body heat. In addition the crimp in the wool bres makes them stand

apart from each other.

As a result, little pockets of still air are trapped between the bres. This

lining of air trapped inside the fabric acts as an insulator. Still air is one of

the best insulators found in nature. The absorption/evaporation process works

in hot weather to help keep the body cooler. Evaporation of perspiration is

the body’s natural cooling device. Wool helps this process along. Its thirsty

cells absorb body vapour and help reduce skin temperature. Also, much of the

outdoor heat is blocked out because of wool’s insulating barrier of air pockets.

This means that the body is kept at an even temperature.

Wool – healthy in nature

Wool has the ability to insulate against heat and cold, it protects against

sudden changes of temperature, and it lets body breathe. Wool can absorb up

to 30 percent of its own weight in moisture before it becomes really damp. As

moisture is absorbed; heat is generated so that the wool remains warm rather

than cold and clammy.

Wool – water repellent property

While wool can absorb moisture, it repels liquids. The scales on the outside of

the bre cause liquid to roll off the surface of the wool fabric.

Wool – re resistant propertyWool is naturally safe. It does not have to be specially treated to become non-

ammable. While it can catch alight, it will not are up nor support a ame.

Instead of burning freely, once the ame is removed a cold ash is left which

can be brushed away immediately. Wool for clothing (particularly children)




will protect from accidents associated with re. Firemen wear wool uniforms,

and re-ghters in rural areas should always ensure they dress themselves in

wool before rushing to ght a re. Wool is self extinguishing because of its

high Limiting Oxygen Index (LOI=25.2), which means to completely burn

wool an oxygen content of 25.2% is necessary whereas normal air only has a

21% content.

Wool – elastic property

Wool has natural elasticity, greater than that of any other bre, makes it

comfortable to wear because it ts the shape of the body. Wool can be twisted,

turned and stretched, and yet it returns to its natural shape. A wool bre

when dry can be extended by about 30 percent. When wet it will stretch by between 60 and 70 percent. This means that a wool garment gives freedom of

movement, especially important for children’s clothes and sportswear, when

ease of movement is all important.

Wool – static electricity

Wool naturally absorbs moisture from the air; the tendency to collect static

electricity is reduced. Walking across a wool carpet, less likely to receive a

shock when touch a grounded object. Wool garments are much less likely to

“spark” or cling to the body.

Wool – noise insulating property

Wool is a wonderful insulator against noise; because it absorbs sound and

reduces noise level considerably. For this reason wool wallpaper is often used

in ofces, restaurants, airport terminals, etc. Wool is also an ideal material

used in such places as concert halls to attain the best acoustics possible.

Wool resists dirt

Wool resists dirt, retains its appearance, and stays cleaner longer. Its abilityto absorb moisture prevents a build-up of static electricity and therefore wool

does not attract lint and dust from the air. Furthermore the crimp in the wool

bre and the scales on the outside of the bre assist in keeping dirt from

penetrating the surface.

3.5.2.3 Wool quality – assessment

Fineness/Grade: In general, grade refers to the average diameter or thickness

of the wool bre. Three systems of wool grading are commonly used namely(i) the American or Blood system; (ii) the English or Spinning Count system;

and (iii) the Micron system. The American Blood Grade System was developed

in the early 1800s and originally represented the amount of ne-wool Merino

genetics (Spanish origin) present in the native coarse-wool sheep. The English




system of grading wool uses a measurement called the “spinning count” and is

based on the number of “hanks” of yarn which could be spun from one pound

of clean wool on the equipment available at the time the system was developed.

Increased emphasis on an exact and highly descriptive method of describing

wool grade has produced a measuring system in which individual bres are

accurately measured. The unit of measure is the micron. Traditionally, the

standard method of measuring wool bres is by a micro-projection technique

in which short longitudinal sections of the bres are projected onto a screen at

500-fold magnication. Technological advancements like electro-optical and

image analysis machines have greatly improved the efciency and accuracy

of bre diameter measurement. All three systems are measures of averagebre diameter and can be used interchangeably as shown in Table 3.20, but

the micron system is the system used internationally and preferred by wool

buyers and manufacturers.

Table 3.20 Classication of wool bres based on quality/grade

Type of Wool American (or)

Blood Grade

English or Spinning

Count Grade

Microns

(average bre

diameter)

Fine Fine Finer than 80s Under 17.70

Fine Fine 80s 17.70–19.14

Fine Fine 70s 19.15–20.59

Fine Fine 64s 20.60–22.04

Medium 1/2 Blood 62s 22.05–23.49

Medium 1/2 Blood 60s 23.50–24.94

Medium 3/8 Blood 58s 24.95–26.39

Medium 3/8 Blood 56s 26.40–27.84

Medium 1/4 Blood 54s 27.85–29.29

Medium 1/4 Blood 50s 29.30–30.99

Coarse Low 1/4 Blood 48s 31.00–32.69

Coarse Low 1/4 Blood 46s 32.70–34.39

Coarse Common 44s 34.40–36.19

Very coarse Braid 40s 36.20–38.09

Very coarse Braid 36s 38.10–40.20

Very coarse Braid Coarser than 36s Over 40.20

3.5.3 Chemical structure of wool bre

The principal component of wool is a protein molecule called ‘keratin’. All

protein molecules consist of long chains of small molecular units, known




as ‘amino acids’. Figure 3.28 shows the general structure of an amino acid .

Each keratin molecule in wool consists of many hundreds of amino acid units,

arranged in an irregular order, although not a random one by analogy (De Boos

1988). The order in keratin determines how the molecules t together, giving

the bre strength and exibility. It has been estimated that wool contains more

than 170 different proteins. These are not uniformly distributed throughout the

bre; proteins of different structures are located in specic regions (Fig. 3.29).

This heterogeneous composition is responsible for the different physical and

chemical properties of the various regions of wool. The proteins in wool are

composed of amino acids; so called because they contain basic amino (-NH)

and acidic carboxyl (–COOH) groups.

Fig. 3.28 General structure of an amino acid

Fig. 3.29 Formation of a polypeptide by reaction of amino acids (R1, R

2 and R

3 may

be the same or different side groups)

In wool, individual polypeptide chains are joined together to form

proteins by a variety of covalent (chemical bonds), called ‘cross links’ , and

non-covalent physical interactions (Fig. 3.30). The most important cross

links are the sulphur containing disulphide bonds, which are formed duringbre growth by a process called “keratinisation”. These make keratin bres

insoluble in water and more stable to chemical and physical attack than

other types of proteins (Schafer 1994). Disulphide bonds are involved in

the chemical reactions that occur in the ‘setting’ of fabrics during nishing.




In this process, disulphide cross links are rearranged to give wool fabrics

smooth-drying properties so that ironing is not required after laundering.

Another type of crosslink is the isopeptide bond, formed between amino

acids containing acidic or basic groups. In addition to the chemical cross

links, some other types of interactions also help to stabilize the bre under

both wet and dry conditions. These arise from interactions between the side

groups of the amino acids that constitute wool proteins. Thus, hydrophobic

interactions occur between hydrocarbon side groups; and ionic interactions

occur between groups that can exchange protons. These ionic interactions or

‘salt linkages’ between acidic (carboxyl) and basic (amino) side chains are the

most important of the non-covalent interactions. The most important of thenon-covalent interactions are the ionic, or ‘salt linkages’ between acidic

(carboxyl) and basic (amino) side groups.

Fig. 3.30 Bonding structure of wool bre

3.5.4 Wool bre – enzyme reactions

The necessity to use more environmental friendly processes leads to the

replacement of conventional chemical textile bre treatments by enzymatic

ones. In the case of wool bre, there are many attempts to substitute

the conventional chlorine treatment by an enzymatic process capable of

providing the fabric with the same characteristics, like anti-shrinking andanti-pilling behaviour. This could be achieved by using proteases, which

would degrade the outermost layer of wool bre (the cuticle) responsible

for wool’s undesirable physical properties (Silva and Cavaco Paulo 2003).

The wool cuticle resistance is thought to be due to the naturally occurring




covalent isopeptide cross links, as well as to covalently attached lipid (Heine

and Hocker 1995). Alkali, chlorination and amine treatments are capable of

removing some of the bound lipid (Earle et al. 1971). These treatments alter

the surface properties of the bre by reducing its hydrophobic nature and

enhance textile properties such as dye uptake, polymer adhesion in shrink

resist treatments and electrical conductivity (Brack 1998). The diffusion of

serine proteases into wool fabrics and yarns was studied (Carla et al. 2005)

and reported that the proteases used free subtilisin and subtilisin- PEG (the

same enzyme that was covalently cross linked to polyethylene glycol) were

shown the adsorption and diffusion facilitated by the pre-treatment performed,

being the alkaline surfactant washing and bleaching the most effective inwhat concerns enzyme adsorption. Furthermore, this study suggests that

the diffusion of proteases into wool is dependent on the size of the protease

(Grebeshova et al. 1999). The free enzyme penetrates into wool bre cortex

while the modied bigger enzyme is retained only at the surface, in the cuticle

layer. Also, proteins without proteolytic activity do not adsorb considerably

on wool due to its hydrophobic nature, therefore the diffusion is facilitated by

hydrolytic action (Cortez et al. 2004). These results have important practical

implications for the establishment of enzymatic wool nishing processes,

since they allow for control of the enzyme hydrolysis, which was the majordrawback of this environmental friendly option to the conventional chlorine

treatments (Pascual and Julia 2001).

Heine and Hocker (1995) have suggested that either the enzyme has to be

controlled (for example, diffusion control by enzyme immobilization) or the

enzyme has to be specially “designed” (for example, by genetic engineering)

in such a way that only a distinct part of the substrate is altered. The research

work analyses and compares the behaviour of two proteases, native subtilisin

and polyethylene glycol (PEG)-subtilisin, which differ essentially in theirsize, in the hydrolytic attack to wool bres. To contrast with the adsorption

and diffusion of the enzymes, two water soluble proteins without catalytic

activity, namely bovine serum albumin (BSA) and carbonic anhydrase,

were used. The major objective of the study was to understand the nature of

enzyme–wool interactions which lead to wool degradation, and investigate the

possibility of using an enzymatic process for wool nishing, which would be

an environmental friendly alternative to the conventional chlorine treatments.

3.5.4.1 Enzymes, proteins and reagents

The enzymes used in the study were the proteases Subtilisin Carlsberg

(Protease type VIII), (E.C.3.4.21.62) and PEG-subtilisin, a subtilisin that

was modied by covalent coupling to polyethylene glycol (6 moles PEG/




Fig. 3.32 SEM microphotographs of the wool bres after the alkaline pre-treatments:

(a) surfactant washing, and (b) surfactant and bleaching washing [Source: Silva et al.

2005]

3.5.4.3 Effect of enzyme size

The subsequent studies were performed with 100% wool fabric subjected toan alkaline surfactant washing followed by bleaching. The enzymes used were

the native subtilisin and subtilisin-PEG. The protein concentrations used were

low, so that the surface was never saturated with the enzyme. The research study

was performed using an enzyme concentration of 40 mg/l and for this reason

a longer time had to be employed in order to better understand the differences

in the behaviour of the two enzymes (Silva et al. 2005). Therefore, a study

conducted for 168 h was performed, where protein adsorption and Tyrosine

formation were monitored. The study observed that the subtilisin-PEG is not

being adsorbed (only about 7% of protein adsorption was attained) while free

subtilisin had about 50% of adsorption into wool fabric. The differences are

also noticeable in the formation of Tyrosine equivalents. The subtilisin that

was covalently coupled to PEG showed a very low release of amino-acids

into media. Comparing to free subtilisin, the amount of amino-acids produced

in Tyrosine equivalents was much higher, indicating wool bre degradation

by the enzyme. The control test run simultaneously with free subtilisin and

the inhibitor anti pain showed no adsorption and no Tyrosine formation,

conrming that the adsorption of the protease into wool was assisted by theenzymatic action. This result was also conrmed by the determination of the

bres strength resistance using a dynamometer.

The maximum tensile strength supported by the yarns was lower for

free subtilisin, indicating higher bre degradation. To follow the diffusion of




the enzymes into fabrics, they were uorescently labelled with FITC. After

covalently coupling the enzymes to a uorescent dye (FITC), an extensive

dialysis was performed until no release of free dye into solution was veried.

Then, after enzymatic treatment, a microtome was used to cut thin layers

of the wool bre entrapped in a non-uorescent resin. The free subtilisin

penetrates completely inside the bre cortex while uorescently labeled

subtilisin-PEG only appears at the surface of some bres (in the cuticle layer).

A similar result was found by Nolte et al. 1996 when studying the effect of

Alcalase, a commercial protease, in wool tops in untreated and Hercosett-

treated wool (wool that was treated by the application of a water-soluble resin

after chlorination). They found that after a 50 h treatment, the uorescentlylabeled alcalase had fully penetrated the untreated-bre cortex, while it was

retained only at or near the surface of Hercosett-treated bres after an identical

treatment process. To compare with the adsorption of the different size

enzymes, the proteins BSA and Carbonic Anhydrase, with average molecular

weights of 66 and 29 kDa, respectively, were also tested for adsorption on

wool at several concentrations (Fig. 3.33). These two proteins showed no

adsorption on wool, thus the isotherms could not be formulated.

Fig. 3.33 Fluorescence microphotographs of bre cross-sections of wool treated with

FITC-labeled subtilisin (a) and subtilisin-PEG (b) [Source: Silva et al. 2005]

The large enzyme molecule is not able to enter in contact with substrate

and to form the intermediate enzyme-substrate complex, because of steric

constraints. It is known that proteases hydrolyze mainly the inside of thebre rather than cuticle (Sawada and Ueda 2001). This fact is due to the high

hydrophobicity of the external surface of wool on one hand, and the fatty layer

overlapping the cuticles, on the other. Thus, proteases degrade preferentially

the intercellular cement, penetrating under favorable conditions relatively




quickly into the bre cortex (Nolte et al. 1996). In this study it seems that

subtilisin-PEG hydrolyzed just the cuticle layer of wool bre, explaining the

low release of amino-acids and the higher tensile strength resistance of the

bre. To support this idea, wool bre samples treated with these two enzymes

were washed for 3 consecutive cycles in a rota-wash machine and felting was

evaluated visually. It seems that wool bre treated with subtilisin-PEG felted

less, highlighting the idea that it had its cuticle layer partially removed. This

fact could be very useful in wool nishing, where only the cuticle layer is

intended to be hydrolyzed. The dimension of the protease is a self-limiting

factor for the undesirable hydrolysis of wool bre cortex, thus overcoming

the major drawback of wool enzymatic nishing: the difculty in controllingenzyme hydrolysis process.

Hypothesis: The adsorption of a native and a modied subtilisin on

wool was studied (Suzana Jus 2007) and reported the alkaline peroxide pre-

treatment improves the enzyme diffusion on wool. This diffusion seems to

be facilitated by the hydrolytic attack, since proteins without activity could

not adsorb considerably on wool. Subtilisin-PEG, the protease, hydrolyzed

just the cuticle layer of wool, fact that was conrmed by the lower release

of amino acids into media and the higher tensile strength and lower felting

of the bre. Thus, the production of diffusion-controlled enzymes might bea solution for a future enzymatic wool treatment process, which would be an

environmental friendly alternative to the conventional chlorine treatments.

3.5.5 Wool nishing – surface modication

The use of proteases modied with the soluble polymer polyethylene glycol

(PEG) in the bio-nishing process of wool bres was studied to analyse the

enzyme action on the outer parts of wool bres. Different proteolytic enzymes

from Bacillus lentus and Bacillus subtilis in native and PEG-modied forms

were investigated and their inuence on the modication of wool bres

morphology surface, chemical structure, as well as the hydrolysis of wool

proteins, the physico-mechanical properties, and the sorption properties of

1:2 metal complex dye during dyeing were studied (Suzana jus et al. 2007).

Modied enzyme products have a benet effect on the wool bres felting

behaviours (14%) in the case when PEG-modied B. lentus is used, without

markedly bre damage expressed by tensile strength and weight loss of the

bre.Handle improvement and shrink-proong treatment are the most

important quality enhancing steps in wool nishing processes (Riva et al.

1999; Heine 2002). However one of the most important of the shrink-proong

treatments (chlorine-Hercosett) still use chlorine, which leads to the pollution




of efuents with adsorbable organic halogen (AOX) by-products and water

pollution (Haefely 1989). This process involves a chlorination treatment

to modify the surface of the wool bres, followed by the application of a

cationic polyamide, and has led to the availability of machine washable wool

garments (Lewis 1992). Looking after alternative, bre-processing enzymes

have an important role in an ecological and economic way (Simpson and

Crawshaw 2002; Gaertner and Puigserver 1992). Numerous investigations

have indicated that proteolytic enzymes mostly subtilisin or papain (serine-

proteases and sulphydryl proteases) preferential degrade the inner parts of

the wool bre (Heine and Hocker 1995; Rao et al. 1998). Nevertheless there

is currently considerable interest in the use of proteases to achieve highershrinking resistance, increasing substrate smoothness and softness, as well as

better dyeability of the enzymatically treated wool bres (Shen et al. 1999;

Heine et al. 1998).

Enzyme diffusion plays a much more decisive role in the heterogeneous

system of soluble enzyme and solid substrate than it does in a homogenous

system, where both enzyme and substrate are soluble (El-Sayed et al. 2001).

The kinetics does not only depend on the concentration of the reaction

partners, the temperature and the pH value of the liquor, but also on the

diffusion of the enzyme to, and into, the solid phase of the substrate and the

diffusion of the reaction products out of the solid phase into the liquor (Silva

et al. 2005; Tzanov et al. 2003). The problem of the enzymatic treatment on

wool with proteases, which having diffused into the interior of the bre, is

that the protease hydrolysis is not limited to the bre surface but it hydrolysis

parts of the endocuticle and proteins in the cell membrane complex (CMC)

causing unacceptable strength and weight lost of the bre (Schroeder et al.

2006). Heine et al. (1998) describes the hydrolytic attack of protease enzymes

on wool, which attacks preferential the non-keratinous parts, i.e. parts of theendocuticle, inter macro-brillar material and the CMC of the wool (Heine

et al. 1998). The uncontrolled degradation of the CMC led to a complete

disintegration of the wool structure resulting in the bre brillation. Heine

and Hocker have suggested that the enzyme diffusion could be controlled

with the possibility of enzyme immobilization or with genetic engineering of

microorganisms. Kodera et al. (1998) published the possibility of chemical

modication of proteins by conjunction with synthetic macromolecules.

Chemically modied proteases (Silva et al. 2005; Schroeder et al. 2006)showed important improvement in the wool quality after treatment. Polymers

attached onto the proteins, namely increase the molecular mass, occupy larger

volumes in an aqueous environment, and restrict the enzyme action on the

bre surface affecting the diffusion behaviour of wool bres (Bahi et al. 2007).




In this contribution, the effects of two alkaline proteases ( Bacillus lentus and

Bacillus subtilis) and their PEG conjugates with different molecular weights,

on the hydrolytic attack to the wool bre surface were studied.

The inuence of two protease enzymes from different sources ( B. lentus

and B. subtilis) and their PEG conjugates of the same specic proteolytic

activity were studied. The impact of enzyme treatment on the wool bres

morphology, physico-mechanical properties and the sorption properties using

the 1:2 metal complex dyes were evaluated (Suzana jus et al. 2007). Australian

merino wool-top was used which having the following characteristics: neness

of bre 19.5 microns, width of bre 70 mm, the pH of water extract 7.1, and

0.4% of fat content. Enzyme treatment was performed using alkalophilic andthermostable proteases from B. lentus (Genencor, Netherlands) and B. subtilis

(Novozymes, Denmark) used without further purication except during the

chemical modication procedure.

3.5.5.1 Application of protease enzyme

Enzymes are applied potentially to all stages of textile processing, starting

from bre retting to fabric nishing since 1970s, and its application has also

reached a wide range of textile products (Buchert and Pere 2000; Heine andHocker 1995). Protease enzymes constitute a major application in the detergent

industry for removing protein stains (Grebeshova et al. 1999; Moreira et al.

2002). Alkaline-stable protease enzymes are applied to woolen textiles under

alkaline conditions, i.e., pH from 8.2 to 8.8, for improving softness, handle,

drape, and pilling resistance with a slight reduction in mechanical properties

(Heine 2002; Riva et al. 1993; Sawada and Ueda 2000). It penetrates into the

amorphous region of wool bre and decreases the amount of the ordered alpha

helix region (Wojciechowska et al. 2004), as well as removes the surface

fatty acids (Kantouch et al. 2005). It decreases the resistance of wool bre

against dye diffusion and therefore decreases the apparent activation energy

for the dyestuff during dyeing when compared with the untreated fabric (Riva

et al. 2002). Neutral-stable protease enzymes are also applied under neutral

reducing conditions, i.e., pH from 6.2 to 6.8, to preferentially modify the

cuticle scales of wool and thus have been used to impart softness and antishrink

nish (El-Sayed et al. 2002; Riva et al. 2006). Even though enzymes impart

a good handle to woolen textiles, their industrial application is limited, since

they need special care and also reduce the mechanical properties of treated products.

Ammayappan et al. (2011) have studied the wool/cotton union fabric

treated with specic selected enzymes namely Savinase-16.0L-Ex (EC

3.4.21.62) and Papain-URPP (EC3.4.22.2) for analyzing the mechanical and




handle properties of wool/cotton union fabrics. The weight loss due to savinase

and papain enzyme treatments is 3.6% and 3.9%, respectively, and so there is

no signicant difference in the weight loss due to these two enzyme treatments.

The extent of modication of cuticle scales in wool bre is generally better in

papain enzyme treatment than in savinase enzyme treatment, since papain is

applied in the presence of a reducing agent. Along with enzyme modication,

the reducing agent also generates more acidic functional groups on wool bre,

which additionally inuence the adhesion, spreading and xation of nishing

chemicals on the surface of the wool bre (Cardamone et al. 2005; Walawska

et al. 2006). The selected alkaline stable and neutral-stable enzyme treatments

on this union fabric have distinct and progressive effects on its performance properties such as handle, softness, comfort, and mechanical properties. The

extent of modication of wool bre in cuticle as well as in cortical level

is better in papain enzyme treatment than in savinase enzyme treatment

(Fig. 3.34).

(a) (b)

Fig. 3.34 SEM photographs of (a) Savinase-treated wool bre and (c) Papain-treated

wool bre [Source: Silva et al. 2005]

3.5.5.2 Enzymatic treatment of wool

Wool top was incubated with the proteases or their conjugates at 50°C at

medium bath migration, for 30–180 min in a solution of 0.1M sodium borate

buffer (pH 8.5), using the same amount of proteolytic activity (25 mU/ml)

against azocasein. After the enzymatic treatment a solution of distilled water

and acetic acid (100 ml/l) was used to reduce the pH of the treatment baths

in order to denaturate the enzymes. Reference samples (REF) were run with

wool top treated in the buffer solution without the enzymes. The proteolytic

autolysis of the used enzymes (E-BL, E-MBL, E-BS and E-MBS) was

monitored in the absence of wool.




3.5.5.3 Effectiveness of wool felting

The effectiveness of PEG-modied proteases from different sources wasanalyzed and to change wool bres morphology in order to increase anti-

felting properties of wool. In order to elaborate the effect of chemical

modication of proteases on wool bres morphology and chemical changes,

the enzymatic treatment was performed with an equal amount of proteolytic

activity (25 mU/ml) using native and modied protease against azocasein

were used. Treatment of wool bres with native proteases from (BL and BS)

showed an increased release of proteins from wool bres compared to their

modied forms (MBL and MBS) (Fig. 3.35). Native proteases from B. lentus

exhibit higher activity (8.5%) on wool bre than the protease from B. subtilis.

The attachment of PEG onto the proteases reduced their activity towards high

molecular weight substrates, resulting in reduced bre destruction (Suzana

Jus et al. 2007; Jovancic et al. 2001).

Fig. 3.35 SEM images of enzyme-treated wool bres using native and PEG-modied

proteases of B. Lentus (BL) and B. Subtilis (BS) and their corresponding conjugates(MBL and MBS) after 180 min of incubation [Source: Suzana Jus et al. 2007]

The higher rate of native enzyme diffusion into the inner part of the wool

bre resulted in almost complete degradation of the bre at longer incubation




time (180 min) due to nearly complete hydrolytic degradation of the non-

keratinous parts of the wool bre deviating and creasing of the cuticle scales

(Fig. 3.35). After 180 min of incubation time the native B. subtilis enzyme (BS)

showed a high weight loss (15%), compared to the reference sample (REF),

whereas negligible increasing weight (1%) is obtained using its modicated

form (MBS). The B. lentus protease (BL) showed similar but considerable

lower impact on wool bre degradation. The negligible weight increase in

the case of modied proteases could be the consequence of the deposition of

PEG-modied enzymes or even free remaining PEG on the bre surface.

3.5.5.4 Tensile strengthThe analysis of tensile strength on the enzymatic treated wool bres have

conrmed a high degree of bre damage after long-term incubation period

and a benet impact at bre surface with smoothness by shorter incubation

period. However, the enzymatic treatment had a limited effect on the felting

behaviour of wool bres (Riva et al. 1993). It was obvious that the diameter

of the felt-ball increased after the enzymatic treatment which was more

pronounced after the application of native B. lentus protease (from 22.78mm

for untreated wool to 24.16mm after 30 min incubation period) indicatinga decrease in wool felting by 27.6%. Using the PEG conjugate of the B.

lentus protease the felting behaviour did not decrease signicantly and was

comparable to the sample treated with modied B. lentus for 180 min. In

the case of native B. subtilis protease the felting of wool decreased for 5.6%

after 30 min and 19.9% after longer incubation time (180 min). Unfortunately

the high loss of tenacity and weight of the wool bre are useless for bre

processing. Consequently, after 30 min of treatment the application of native

B. subtilis protease was observed less impact on the wool felting (23.18 mm)

compared to the native B. lentus protease (24.16 mm), but in comparison

with the untreated wool sample (22.78 mm) a felting decrease about 8% was

obtained. Based on test results of PEG onto the protease enzymes resulted in

a successful treatment of the wool bre surface without markedly changes of

the weight loss and the bre tenacity, and with improvement of felting in all

cases, compare to the untreated and native proteases treated wool.

3.5.6 Handle and dyeability of wool

Hand and dyeing behaviour are important quality aspects for wool (Chapman

1976). Common nishing processes, such as oxidation of the wool surface by

means of chlorination to increase the dyeing afnity or application of softening

agents to modify the handle, improve these properties (Denby 1974). A great




disadvantage of these processes is the environmental pollution. Enzymes can

be used in order to develop environmentally friendly alternative processes.

Since wool mainly consists of proteins and lipids especially proteases and

lipases have been investigated for wool bre modication. Wool treatment

with Mesophilic proteases leads to a reduced felting tendency and an increased

dyeing afnity (Heine et al. 1998). Both the cuticle and the cortex of the bre

are modied by proteolytic enzymes. Moreover, the handle of wool top and

yarn can be improved by the reduction of the bending modulus as a result

of a partial hydrolysis caused by proteases (Bishop et al. 1998). Enzymes

from extremophilic micro-organisms (extremozymes) such as thermo-, halo-

, psychro-, alkali-, or acidophilic micro-organisms perform best under thecorresponding extreme conditions (Sunna et al. 1996; Sunna and Antranikian

1997). Therefore, the industrial use of extremozymes is regarded as promising.

Aqueous treatments under constant temperature and pH conditions are most

suitable for application of these enzymes. In the textile industry alkaliphilic

enzymes could be used in alkaline washing processes and thermophilic and/

or acidophilic enzymes under high temperature conditions and in acid dyeing

processes respectively (Bajaj 2002). A positive side effect of the high process

temperatures suitable for thermophilic enzymes is the elevated diffusion rate.

For economical reasons it is aimed at combining the extremozymes treatmentwith the procedures already established in the textile industry.

3.5.7 Properties of wool bre after esperase enzyme

treatment

Karin Schumacher et al. (2001) have studied the handle and dyeability of

wool bres and material used in the study was merino wool top, average

bre diameter 21.3 µm. The enzyme used was Esperase 8.0 L, type A (Novo Nordisk) 8.0 U g−1 resp. 7.8 U ml−1.

3.5.7.1 Degree of whiteness

Wool top and fabric were treated under the conditions of the pre-washing

step on the laboratory dyeing machine either with or without adding protease

(Chikkodi 1995). The degree of whiteness of the wool samples increases with

the amount of protease used. The reference wool top and reference fabric

show the lowest degree of whiteness. The difference between the degree ofwhiteness of references and samples is signicant. Increasing the amount of

protease from 9.8 to 19.5 mU g−1 does not signicantly enhance the degree

of whiteness though.




3.5.7.2 Dye uptake

Dyeing kinetics using the dyestuff Lanasol Blue was monitored during wooltop and fabric dyeing processes. The protease treated fabric absorbs approx.

17% more dyestuff than the reference. The residual dye content of the control

sample bath is about 50%. Fabric treatment with 19.5 mU g−1 protease leads

to a 20% higher dyestuff uptake compared with the control sample (about

53% uptake). Even though protease treatment of wool with both 9.8 and

19.5 mU g−1 of enzyme increases the dye uptake the resulting differences in

colour values (DE D65) to the corresponding references only amount to about

1.5 for wool top as well as for fabric. This can be explained by the fact that the

way of dye uptake of reference and protease treated wool is different. Figures

3.36 and 3.37 show bre cross sections of dyed control sample and protease

treated fabric respectively. The increased dyestuff uptake of the protease

treated fabric leads to a more even and more intensive dyeing. Additionally

more bres of the protease treated fabric are completely dyed whereas most

bres of the control sample show merely ring dyeing.

Fig. 3.36 Cross-sections of pre-washed reference; 2% (owf) Lanasol Blue 8G dyeing

(1 h) [Source: Karin Schumacher et al. 2001]




Fig. 3.37 Cross-sections of protease treated (19.5 mU g –1) pre-washed sample; 2%

(owf) Lanasol Blue 8G dyeing (1 h) [Source: Karin Schumacher et al. 2001]

3.5.7.3 Tensile strength

Compared with the control sample the loss in tensile strength of the enzyme

treated (9.8 and 19.5 mU g−1) bre bundles of wool top amounts to approx.

30%. The values of the tensile strength of yarn bundles of control sample

and protease (9.8 and 19.5 mU g−1) treated wool fabric measured before

and after dyeing does not differ signicantly (De Boos and White 1978). The

prewashing step leads to a loss in tensile strength of 10% and the dyeing step

to another 10% compared to standard wool top for reference as well as for

enzyme pre-treated fabric.

3.5.7.4 Colour fastness

The wash fastness and the acid and alkaline fastness to perspiration were

determined for all wool fabrics. The values of wash fastness of control and

protease treated wool fabric do not differ. The fastness values are optimal (it is

5) for staining of wool and polyester. However the colour change is 4 for both.

The acid fastness to perspiration shows the same tendencies as the alkaline




fastness to perspiration though on the whole better values are obtained. The

values of alkaline fastness to perspiration for the references of fabric 1–3

are about 0.5–1 higher than the corresponding values of the protease pre-

treated samples for the staining of wool, polyester and cotton. The values for

staining on cotton are the worst (2–3 and 2 respectively) while the values for

colour change are 4–5 for both references and enzyme treated samples (Karin

Schumacher et al. 2001).

3.6 Bioprocessing of silk and their characteristics

3.6.1 IntroductionSilk is a natural protein bre which composed mainly of broin and produced

by certain insect larvae to form cocoons. The best known type of silk is obtained

from the cocoons of the larvae of the mulberry silkworm Bombyx mori reared

in captivity (sericulture). The shimmering appearance of silk is due to the

triangular prism-like structure of the silk bre, which allows silk cloth to

refract incoming light at different angles, thus producing different colors. Silk

proteins belong to a class of unique, high molecular weight, block copolymer

like proteins that have found widespread use in biomaterials and regenerativemedicine. The useful features of these proteins, including self-assembly,

robust mechanical properties, biocompatibility and biodegradability can be

enhanced through a variety of modications. These modications provide

attachment of growth factors, cell binding domains and other polymers to

silk, expanding the range of cell and tissue engineering applications.

3.6.2 Types of silk

There are two main types of the silk: ‘mulberry silk’ (produced by the Bombyx Mori) (Fig. 3.38), also called ‘cultivated silk’, and second namely ‘wild silk’,

also called as ‘Tussah silk’. Mulberry silk is produced by silkworm larvae

cultivated in provided habitats and fed with freshly picked mulberry leaves

(Li et al. 2005). Cultivated silk is different from Tussah silk as the Tussah

variety is purely fed on oak leaves. Cultivated silks are ne, almost white

(when degummed) with soft laments of lustre (Tan et al. 2001). Wild silks,

on the other hand, are coarser, more irregular and brownish in appearance,

and are never as white as the cultivated silk lament. Nearly 80–85% of the

world’s silk production consists of cultivated silk. Mulberry silkworms can be

divided in three groups: (a) univoltine breed (one generation per year) which

is usually found in Europe where due to the cold climate the eggs are dormant

in winter and they are hatched in spring, (b) bivoltine breed (two generations




per year) usually found in Japan China and Korea, where the climate is

suitable for developing two life cycle per year and (c) multivoltine breed

(up to eight generations per year) usually found in tropical zone. The major

groups of silkworms fall under the univoltine and bivoltine categories. Silk

producing insects have been classied on the basis of morphological clues,

such as follicular imprints on the chorine egg, arrangement of tubercular setae

on the larvae, and karyo typing data (Jolly and Sen 1969; Jolly et al. 1970).

Classication based on phenotypic attributes is sometimes misleading because

morphological features may vary with the environment (Souche and Patole

2000). Molecular marker-based analysis has been developed to distinguish

genetic diversity among silkworm species (Tan et al. 2001; Mahendran et al.2005 and 2006). Most commercially exploited silk moths belong to either the

family Bombycidae or Saturniidae, in the order lepidoptera.

Fig. 3.38 Mulberry silkworms

The nest quality raw silk and the highest bre production are obtained

from the commonly domesticated silkworm, Bombyx mori, which feeds on

the leaves of the mulberry plant, Morus spp. Other than the domesticated B.

mori, silk bre production is reported from the wild non-mulberry saturniid

variety of silkworms. Saturniid silks are of three types: tasar, muga, and eri

(Table 3.21).

Table 3.21 Commercially exploited sericigenous insects of the world

Common Name Scientic Name Origin

Mulberry Silkworm Bombyx mori China

Tropical Tasar Silkworm Antheraea Mylitta India

Oak Tasar Silkworm Antheraea proylel India

Eri Silkworm Philosamia ricini India

Muga Silkworm Antheraea assama India

Oak Tasar Silkworm Antheraea yamamal Japan

Oak Tasar Silkworm Antheraea pernyi China




The tasar silkworms are of two categories namely (i) Indian tropical

tasar, Antheraea mylitta, which feeds on the leaves of Terminalia arjuna,

Terminalia tomantosa, and Shorea robusta, and (ii) the Chinese temperate

oak tasar, Antheraea pernyi, which feeds on the leaves of Quercus spp. and

Philosamia spp. Indian tropical tasar (Tussah) is copperish colour, coarse silk

mainly used for furnishings and interiors. It is less lustrous than mulberry silk,

but has its own feel and appeal. Oak tasar is a ner variety of tasar silk (Kundu

et al. 2008). Muga silk is produced by the multivoltine silkworm, Antheraea

assamensis (also called A. assama), which feeds mainly on Machilus spp.

Muga is a golden yellow colour silk. Muga culture is specic to the state of

Assam (India) and an integral part of the tradition and culture of that state.The muga silk, a high value product is used in products like sarees, mekhalas,

chaddars, etc. Eri silk is produced by Philosamia spp (Samia spp.), whose

primary host plant is the castor ( Ricinus spp.). The luster and regularity of B.

mori silk makes it superior to the silk produced by the non mulberry saturniid

silkworms, although non-mulberry silk bres are also used commercially due

to their higher tensile strength and larger cocoon sizes. Spider also produced

silk bres that are strong and ne, but have not been utilized in the textile

industries (Kundu et al. 2008).

3.6.3 Physical properties of silk

Silk, from the bombyx mori silkworm, have a triangular cross section with

rounded corners which is having 5–10 μm wide. The at surfaces of the brils

reect light at many angles, giving silk a natural shine; has a soft texture and

smooth surface. Silk is one of the strongest natural bres but it loses up to

20% of its strength when wet. It has a good moisture regain of 11% and its

elasticity is moderate to poor. Silk material can be weakened if exposed to too

much sunlight and it may also be attacked by insects due its protein content.

The commercial silk which is available in market for moth and spider variety

is given in Table 3.22.

Table 3.22 Physical properties of silk bres derived from moth and spider variety

Comparison of silk bres Linear density

(dtex)

Diameter

(μm)

Coefcient

variation (%)

Moth: 1.17 12.9 24.8%

Spider: Argiope aurentia 0.14 3.57 14.8%

Some of the physical properties of silk are given below:

1. Color: The color of silk bre could be yellow, brow, green or grey.

2. Tensile strength: The strength of silk is greatly affected by moisture;




the wet strength is 75–85%, which is higher than dry strength.

3. Elongation at break: 20–25% at break.

4. Specic gravity: 1.25 to 1.34

5. Moisture regain: 11–13%

6. Effect of heat: Silk will withstand at higher temperatures than wool.

It will remain unaffected for prolonged periods at 140°C. Silk

decomposes at 175°C.

7. Effect of sun light: Sun light tends to encourage the decomposition of

silk by atmospheric oxygen.

8. Lustre: Silk is bright due to its triangular cross section.

3.6.4 Chemical properties of silk

The chemical properties of the silk bre are given below:

1. Effect of acids: The Fibroin of silk can be decomposed by strong

acids into its constitute amino acids. In moderate concentration, acids

cause a contraction in silk. Dilute acids do not attack silk under mild

conditions.

2. Effect of alkalis: Silk is less readily damaged by alkalis than wool.

Weak alkalis such as soap, borax and ammonia cause little appreciabledamage. Silk dissolves in solutions of concentrated caustic alkalis.

3. Effect of organic solvent: Silk is insoluble in the dry-cleaning solvents

in common use.

5. Effect insects: Insect does not affect silk.

6. Effect of mildew: Silk is affected by mildew slightly.

3.6.5 Chemical composition of silk

The chemical composition of raw silk obtained from the silk worm Bombyxmori is presented (Table 3.23). Silk is produced in several countries and

the bres from different regions contain different amounts of sericin which

exhibits diverse chemical and physical properties (Gulrajani 1992).

Table 3.23 Composition of raw silk from the silk worm Bombyx mori

Component %

Fibroin 70–80

Sericin 20–30Wax 0.4–0.8

Carbohydrate 1.2–1.6

Inorganic matter 0.7

Pigments 0.2




3.6.6 Structure of the silk bre

Silk lament extruded from silk worm consists of two main proteins, namely(i) sericin and (ii) broin. Fibroin is being the structural center of the silk,

and sericin being the sticky material surrounding it. Fibroin is made up of the

amino acids Gly-Ser-Gly-Ala-Gly-Ala and forms beta pleated sheets. High

proportion (50%) of glycine, which is an amino acid, allows tight packing;

strong and resistant to breaking. The tensile strength of silk is due to the many

interceded hydrogen bonds and when stretched the force is applied to these

numerous bonds resist and they do not break. The silk lament is resistant

to most mineral acids, except for sulfuric acid; and chlorine bleach destroys

silk fabrics. The cocoons of the mulberry silkworm B. mori are composed of

two major types of proteins: broins and sericin. Fibroin, the ‘core’ protein

constitutes over 70% of the cocoon and is a hydrophobic glycoprotein

(Sinohara et al. 1971) secreted from the posterior part of the silk gland (PSG)

(Prudhomme et al. 1985). The broin, rich in glycine (43.7%), alanine (28.8%)

and serine (11.9%), is composed of a heavy chain (~325 kDa), a light chain

(~25 kDa) and a glycoprotein, P25, with molar ration of 6:6:1. The heavy and

light chains are linked by a disulde bond. P25 associates with disulde-linked

heavy and light chains primarily by non-covalently hydrophobic interactions,and plays an important role in maintaining integrity of the complex (Inoue

et al. 2000). The light chain has a non-repetitive sequence and plays only a

marginal role in the bre. The heavy chain contains very long stretches of

Gly-X repeats (with residue X being Ala in 64%, Ser in 22%, Tyrin 10%,

Val in 3%, and Tyrin 1.3%) that consist of 12 repetitive domains (R01–R12)

separated by short linkers. It is an antiparallel, hydrogen bonded β-sheet and

yields the X-ray diffracting structure called the “crystalline” component of

silk broin (Zhou et al. 2001). Silk is a typical representative of β-sheet.

Each domain consists of sub-domain hexapeptides including: GAGAGS,

GAGAGY, GAGAGA or GAGTGA (G is glycine, A is alanine, S is serine and

Y is tyrosine) (Vepari and Kaplan 2007). In contrast, the 151 residues of the

N-terminal, 50 residues of the C-terminal, and the 42–43 residues separating

the 12 domains are non-repetitive and ‘amorphous’ (Li et al. 2012). Silk

broin can exist as three structural morphologies termed silk I, II, and III;

where silk I is a water soluble form and silk II is an insoluble form consisting

of extended β-sheets. The silk III structure is helical and is observed at the

air-water interface. In the silk II form, the 12 repetitive domains form antiparallel b-sheets stabilized by hydrogen bonding (Teramoto et al. 2006). Due

to the highly oriented and crystalline structure of Silk II, silk broin bre is

hydrophobic and has impressive mechanical properties. When controllably

spun, its mechanical property may be nearly as impressive as spider dragline




silk (Shao and Vollrath 2002).

Sericin is a protein that surrounds broin, which becomes silk. The section

indicated in blue represents sericin, which surrounds broin depicted in red

(Fig. 3.39). Sericin, the ‘glue’ proteins constitute 20–30% of the cocoon, and

are hot water-soluble glycoproteins that hold the bres (broin) together to

form the environmentally stable broin-sericin composite cocoon structure

(Jin and Kaplan 2003; Sinohara 1979; Vollrath and Knight 2001). Sericin,

secreted in the mid-region of the silk gland, comprises different polypeptides

ranging in weight from 24 to 400 kDa depending on gene coding and post-

translational modications and are characterized by unusually high serine

content (40%) along with signicant amounts of glycine (16%), (Sprague1975; Takasu et al. 2002). Three major fractions of sericin have been isolated

from the cocoon, with molecular weights 150, 250, and 400 kDa (Colomban

et al. 2008). Sericin remains in a partially unfolded state, with 35% β-sheet

and 63% random coil, and with no α-helical content (Teramoto et al. 2006).

The amino acid compositions of broin and sericin have been published,

with somewhat differences from paper to paper for some specic amino acid

contents (Freddi et al. 1999; Gulrajani 1992).

Fig. 3.39 Cross-sectional view of silk

3.6.7 Need and industrial practice of degumming

processThe silk lament spun by the silkworm (Bombyx mori) is composed

of two broin laments held together by a cementing layer of sericin.

Fibroin and sericin account for about 75 wt% and 25 wt% of the raw silk,




respectively. The degumming process consists in removing the sericin layer

prior to dyeing or printing and nishing to get bright lustrous fabrics and

garment. The degumming of silk has conventionally been carried out under

alkaline conditions at a pH of 10 to 11 near boil. In recent years two new

processes have emerged. They are the “H.T.-H.P. Degumming” and “Enzyme

Degumming”. High temperature high pressure degumming requires special

pressured equipment and is energy-intensive process. Enzymatic degumming

is emerging as an eco-friendly bre-gentle process where proteolytic enzymes

that are effective under alkaline, neutral as well as acidic conditions are being

used (Fabiani et al. 1996). With the local availability of the enzymes at a

reasonable price this process has a commercial potential in India. Being largemolecules, enzymes do not penetrate into the interstices of the fabric and

hence are suitable for yarn degumming only. A critical control of the pH and

temperature is required to realize the full potential of the enzymes requiring

use of sophisticated machinery. Since most of the enzymes are effective at a

comparatively low temperature of about 60°C, they are less energy-intensive.

The degumming waste liquor that is rich in sericin content is being used

as a raw material for the production of sericin powder (Vaithanomsat and

Kitpreechavanich 2008). The sericin powder is being used in the cosmetic

industry as moisturizer, in hair-care products and also as a sustainable naturaltextile nishes. Removal of sericin from the waste degumming liquor also

substantially reduces the efuent.

3.6.8 Degumming of silk – chemical and enzyme

methods

Degumming is at the heart of the wet processing of raw silk due to the fact

that the raw silk contains the two components broin and sericin which coversthe lament. Sericin contains some impurities, such as, waxes, fats, mineral

salts and pigments. Sericin has the same amino acid residues as broin but

the proportions contained in both components are quite different. As a result

of this, the degumming process on silk must be carefully carried out in the

appropriate conditions otherwise the broin may be damaged. The main

purposes of the degumming process are; 1) to remove the sericin from the

bre, 2) to remove impurities (eg. waxes, fats and mineral salts) affecting both

the dyeing and printing processes, 3) to make the bre highly absorbent for

dyes and chemicals, and 4) to reveal the lustre of broin while improving theappearance of the bre (Saligram 1993). The sericin has to be removed from

the bre but the broin must not be damaged in the process. Silk degumming

is a high resource consuming process as far as water and energy are concerned.

Moreover, it is ecologically questionable for the high environmental impact




of efuents. The development of an effective degumming process based on

enzymes as active agents would entail savings in terms of water, energy,

chemicals, and efuent treatment. This could be made possible by the milder

treatment conditions, the recycling of processing water, the recovery of

valuable by-products such as sericin peptides, and the lower environmental

impact of efuents (Freddi et al. 2003). However, the limitations of higher

cost of enzymes compared to chemicals and the necessary continuous use of

enzymes may limit the development of industrial processes using proteolytic

degumming methods (Long et al. 2008; Chopra et al. 1996). Silk processing

from cocoons to the nished clothing articles consists of a series of steps which

include: reeling, weaving, degumming, dyeing or printing, and nishing.Degumming is a key process during which sericin is totally removed and

silk bres gain the typical shiny aspect, soft handle, and elegant drape highly

appreciated by the consumers. In addition, the existence of sericin prevents

the penetration of dye liquor and other solutions during wet processing of

silk. Also, it is the main cause of adverse problems with biocompatibility and

hypersensitivity to silk. Furthermore, to prepare pure silk broin solution for

silk-based biomaterials, separation of silk broin bre from the sericin glue,

is a critical step, since (a) residual sericin causes inammatory responses and

(b) non-degummed bres are resistant to solubilization (Wray et al. 2011).The industrial process takes advantage of the different chemical and physical

properties of the two silk components, broin and sericin. While the former is

water-insoluble owing to its highly oriented and crystalline brous structure,

the latter is readily solubilized by boiling aqueous solutions containing soap

(Gulrajani 1992), alkali, and synthetic detergents (Syilokos and Colonna

1992). However, the higher temperature (95°C) and an alkaline pH (8–9) in the

presence of harsh chemicals in the treatment bath impose a markedly unnatural

environment on the silk, and thus cause partial degradation of broin. Fibredegradation often appears as loss of aesthetic and physical properties, such

as dull appearance, surface brillation, poor handle, drop of tensile strength,

as well as uneven dyestuff absorption during subsequent dyeing and printing

(Freddi et al. 2003). More importantly, the large consumption of water and

energy contribute to environmental pollution.

The increasing awareness of legislators and citizens for the ecological

sustainability of industrial processes has recently stimulated the interest of

scientists and technologists for the application of biotechnology to textile

processing (Gubitz and Cavaco-Paulo 2001). In recent years, various studieshave dealt with the removal of sericin by using proteolytic enzymes since

they can operate under mild conditions and low temperatures which save

energy in comparison to the traditional method (Mahmoodi et al. 2010).

Enzymes act selectively and can attack only specic parts of sericin to cause




proteolytic degradation. So the pattern of soluble sericin peptides obtained by

degumming silk changes as a function of the kinds of enzyme used, attributing

to the different target cleavage of the enzymes. Several acidic, neutral, and

alkaline proteases have been used on silk yarn as degumming agents. Alkaline

proteases performed better than acidic and neutral ones in terms of complete

and uniform sericin removal, retention of tensile properties, and improvement

of surface smoothness, handle, and lustre of silk (Gulrajani et al. 1998 and

2000). Enzyme degummed silk fabric displayed a higher degree of surface

whiteness, but higher shear and bending rigidity, lower fullness, and softness

of handle than soap and alkali degummed fabric, owing to residual sericin

remaining at the cross over points between warp and weft yarns (Chopraet al. 1996). Freddi et al. (2003) have applied acidic, neutral, and alkaline

proteases to silk degumming and found that alkaline and neutral proteases

performed better than acidic proteases in terms of complete sericin removal.

After complete sericin removal with proteolytic methods, the quality of

appearance and retention of tensile properties is expected to be superior to

those silks degummed through traditional methods due to less chemical and

physical stress applied to the silk during enzymatic processing. Nakpathom

et al. (2009) have studied the degumming of Thai Bombyx mori silk bres

with papain enzyme and alkaline/soap and reported that the former exhibitedless tensile strength drop and gave higher color depth after natural lac dyeing,

especially when degumming occurred at room temperature condition.

Alcalase, savinase, (two commercial proteolytic preparations) and their

mixtures were also proved to be feasible for degumming applications (Arami

et al. 2007). Gulrajani et al. (2000) have also studied the degumming of silk

with the combination of protease and lipase enzymes, and obtained efcient

de-waxing and degumming effects, while maintaining favorable wettability of

silk bres.Since the silk of Bombyx mori apart of the proteins broin and sericin,

also contains fats, wax etc., the combined effect of proteolytic enzymes with

a lipolytic one was investigated. Combined action of protease with lipase

(Lipolase® Ultra 50T) resulted in lower degumming efciency compared

(under the same conditions) with protease treatment only, but generated silk

fabrics with signicant improvement in whiteness after bleaching. As far as

the properties measured in the Kawabata evaluation system the combination of

proteolytic with lipolytic enzymes resulted in silk fabrics with extremely low

bending rigidity, reduced shear stiffness and with higher elasticity comparedto Marseille soap treated. This means that the fabrics were softer, less rigid

with better drape compared to conventionally treat. Addition of lipolase

caused decrease in bending and increase in shear rigidity and elasticity of

the silk fabrics compared to those treated only with protease. One of the least




explored areas for the use of proteases is the silk industry and only a few

patents have been led describing the use of proteases for the degumming of

silk (Kanehisa 2000). Sericin, which is about 25% of the total weight of raw

silk, covers the periphery of the raw silk bres, thus providing the rough texture

of the silk bres. This sericin is conventionally removed from the inner core

of broin by conducting shrink-proong and twist-setting for the silk yarns,

using starch. The process is generally expensive and therefore an alternative

method suggested is the use of enzyme preparations, such as protease, for

degumming the silk prior to dyeing. The silk-degumming efciency of an

alkaline protease from Bacillus sp. RGR-14 was studied and results were

analyzed gravimetrically (bre weight reduction) and by scanning electronmicroscopy (SEM) of treated silk bre. After 5 h of incubation of silk bre

with protease from Bacillus sp., the weight loss of silk bre was 7.5% (Puri

2001).

Increased awareness and concerns about the environment and pollution

have paved way for Eco-friendly processes in chemical processing.

Conventional soap- soda boiling method of degumming silk has many

disadvantages coupled with the convenience of time. The enzymatic

degumming processes have been attempted earlier and are gaining importance

due to less degradation of silk, ease of process control, better hand properties ofsilk besides being environmental friendly process. An attempt is made to apply

neutrol-alkaline protease enzyme Serinzyme for degumming of 4-ply lature

silk yarn and compared with the control soap-soda boil method (Yuksek et al.

2012). The degummed silk yarn is analysed for properties related to strength

and weight loss. The enzymatic degummed silk yarn showed better control

over the conventional process based on the dosage of enzyme and degumming

weight loss. Tenacity and breaking force at 1% and 2% enzyme dosage was

superior in comparison to the soap soda method, whiteness of the enzymaticdegummed samples was satisfactory.

Papain is used for boiling off cocoons and degumming of silk. Raw silk

must be degummed to remove sericin, a proteinaceous substance that covers

the bre. Degumming is typically performed in an alkaline solution containing

soap, a harsh treatment that also attacks broin structure. Several alkaline,

acidic and neutral proteases have been studied as degumming agents since

they can dissolve sericin, but are unable to affect silk bre protein. Alkaline

proteases seem to be the best for removing sericin and improving silk surface

properties like handle, shine and smoothness (Freddi et al. 2003; Arami et al.2007), although this is not in commercial use. In the past, papain was also used

to ‘shrink-proof’ wool. A successful method involved the partial hydrolysis

of the scale tips. This method also gave wool a silky lustre and added to its

value. The method was abandoned a few years ago for economic reasons.




Mulberry silk fabric has been degummed by using ve different methods

(acid, alkali, triethylamine, soap and enzyme) and the results compared in

terms of handle properties. Low stress mechanical properties tested on the

Kawabata Evaluation System (KES) system have been used to evaluate the

hand values. Soap has been taken as the standard method of degumming

and the other treatments have been compared with this method. Signicant

differences in the low stress properties have been observed for the different

treatments. Soap, alkali and triethylamine methods score over the acid and

enzymatic methods in terms of handle properties, Low shear and surface

properties characterise the acid and enzyme degummed samples indicating

non-uniform removal of gum from the interlacing areas (Chopra et al. 1996).

3.6.8.1 Silk degumming process – comparative study

A crêpe silk fabric was treated with different alkaline (3374-L, GC 897-H),

neutral (3273-C), and acid (EC 3.4 23.18) proteases with the aim to study their

effectiveness as degumming agents (Giuliano et al. 2003). Proteases were used

under optimum conditions of pH and temperature, while enzyme dosage (0.05–

2 U/g fabric) and treatment time (5–240 min) were changed in order to study

the kinetics of sericin removal. Degumming loss with soap and alkali was 27wt%. The maximum amount of sericin removed in 1 h was 17.6, 24, and 19

wt.% for 3374-L (2 U/g fabric), GC 897-H (1 U/g fabric), and 3273-C (0.1 U/g

fabric), respectively. Under the experimental conditions adopted, EC 3.4 23.18

was almost ineffective as a degumming agent. Degumming loss increased as a

function of the treatment time, reaching a value of 25 wt% with 1 U/g fabric

of 3374-L. The feasibility of degumming Persian silk with alcalase, savinase,

and mixtures of these enzymes with different alcalase/savinase weight ratios

(0/1, 0.25/0.75, 0.5/0.5, 0.75/0.25, and 1/0 g/L) was investigated (Mokhtar et

al. 2007). The enzymatic degumming process was performed at 55°C with an

operation time of approximately 30 min, whereas the soap degumming process

was carried out around the boiling point in 120 min. The evaluation of the data

was carried out through the measurement of the weight loss, strength, and

elongation of the samples. The optimum amount of sericin removed was 21.52

wt % for alcalase in 30 min, 20.08 wt % for savinase in 60 min, and 22.58 wt

% for soap in 120 min. Also, the enzymatic treatment improved properties of

the silk yarn such as the strength (33.76 cN/tex for alcalase and 32.03 cN/tex

for savinase) and elongation (20.08% for alcalase and 18.42% for savinase).The obtained values were better than the strength (29.90 cN/tex) and elongation

(18.59%) from the soap degumming method. Through the use of an enzyme

mixture (0.5/0.5 g/L), good weight loss (22.43%), strength (33.22 cN/tex), and

elongation (17.74%) were achieved in 30 min.




3.6.8.2 Silk waste water characterization

Jin-Hong et al. (2007) have developed a new effective technology for theextraction of sericin from silk wastewater. Sericin was extracted with 75%

(v/v) ethanol to obtain crude powder. Silk manufacturing is one of the textile

industrial sectors where intensive water consumption involved and therefore

a large volume of wastewater is produced. The degumming process is used to

remove external sericin prior to dyeing and is a source of waste water. This

process generally uses a synthetic soap solution at 95°C for 1 h, with 100 kg

of silk producing 22 kg of sericin. Sericin is a globular protein in the form of

a tube outside the silk broin with its molecular weight ranging between 10

and 300 kDa (Fabian et al. 1996; Zhang et al. 2004). When subjected to the

alkaline degumming process, sericin is degraded into sericin peptide. Both

the peptide and the hydrolysate of sericin have excellent moisture absorption

properties and are also involved in a lot of biological activities such as

antioxidation, tyrosinase activity inhibition and anticancer activity (Kato

et al. 1997; Chang-Kee et al. 2002). As a result, they can be used in many

elds including cosmetics, biomaterials, textiles and pharmaceuticals (Zhang

2002). High concentrations of BOD, COD and nitrogen in the degumming

waste solution make it complicated and costly to treat (Rigoni-Stern et al.1996; Fabiani et al. 1996). However, the high nitrogen and protein content in

this wastewater derived from sericin products could be recovered and used.

Vaithanomsat et al. (2008) applied Ultraltration to recover sericin from silk

degumming waste which was further hydrolyzed into sericin hydrolysate of a

similar quality to that of commercially-available sericin hydrolysate used for

cosmetics purposes. It would also be very advantageous if such a waste solution

could be used as a substrate for microbial growth or enzyme production. In

most cases, the growth media makes up approximately 40% of the production

cost of industrial enzymes. Organic nitrogen substrates such as casein, yeast

extract, soy protein or gelatin are widely used in many microbial applications

due to their favorable amino acid balance and high protein content.

3.6.8.3 Microbial protease production

As waste solution from the silk degumming process contains high nitrogen

levels, this wastewater must be treated prior to discharge. In this study, waste

solution was prepared and tested as a nutrient substrate for microbial growth

and protease production by Bacillus licheniformis TISTR 1010 and Aspergillus

avus TISTR 3130, TISTR 3366, TISTR 3135 and TISTR 3041 (Pilanee et

al. 2008). All strains were preliminarily screened for their protease activity by

growing on casein-agar plates with B. licheniformis TISTR 1010 being chosen




as the best producer of protease. Cultivation in a silk degumming solution

as the nutrient source demonstrated that the highest protease activity was

achieved at an optimum pH of 10 for 36 h. Among the culture media used, the

specic activity of released protease was best with a medium containing 6%

protein from the silk degumming waste, 1% malt extract, 1% polypeptone and

1% Na2CO

3. This study was the rst to report the use of silk degumming waste

as a nitrogen source for microorganism growth and protease production. As

such it could suggest an alternative way to convert wastes into more valuable

and marketable products.

3.6.8.4 Applications of alkaline proteasesAlkaline proteases account for a major share of the enzyme market all over

the world (Godfrey and West 1996; Kalisz 1988). Alkaline proteases from

bacteria nd numerous applications in various industrial sectors and different

companies worldwide have successfully launched several products based on

alkaline proteases (Aramwit et al. 2012). The success of detergent enzymes

has led to the discovery of a series of detergent proteases with specic uses.

Alkazym (Novodan, Copenhagen, Denmark) is an important enzyme for the

cleaning of membrane systems. Other enzymes used for membrane cleaningare Tergazyme (Alconox, New York, USA), Ultrasil (Henkel, Dusseldorf,

Germany) and P3-pardigm (Henkel-Ecolab, Dusseldorf, Germany). Pronod

153L, a protease enzyme-based cleaner is used to clean surgical instruments

fouled by blood proteins. Subtilopeptidase A is an enzyme-based optical

cleaner, presently marketed in India (Kumar et al. 1998). Sakiyama et al.

(1998) have reported the use of a protease solution for cleaning the packed

columns of stainless steel particles fouled with gelatin and lactoglobulin. In

addition to these major applications, alkaline proteases are also used to a lesser

extent for other applications, such as contact lens cleaning (Nakagawa 1994),

molecular biology for the isolation of nucleic acid (Kyon et al. 1994), pest

control (Kim et al. 1999), and degumming of silk (Kanehisa 2000; Puri 2002).

3.6.8.5 Application of silk broin

Silk broin from the silkworm, Bombyx mori, has excellent properties such

as biocompatibility, biodegradation, non-toxicity, and adsorption properties.

As a kind of ideal biomaterial, silk broin has been widely used since it wasrst utilized for sutures a long time ago. The degradation behavior of silk

biomaterials is obviously important for medical applications (Cao and Wang

2009). It can be used as a biomaterial in various forms (Chitrangada et al.

2008), such as lms (Minoura et al. 1995; Acharva et al. 2008; Kundu et al.




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Abstract: This chapter discusses the application of enzymes in synthetic bresfor improving the functional properties such as moisture and surface aspects.

Synthetic bres are being playing in major role in textile applications such asclothing, hygienic, sports, industrial uses like protection and ballistic etc. Thesesynthetic bres made from polymer source like polyester, nylon, polyethylene, andpolypropylene or regenerated from natural source such as viscose rayon, modal,lyocell and tencel have been widely accepted for their care properties, versatilityand long life. In spite of such acceptance, certain drawbacks concerning theirhand properties, thermal properties, and moisture absorbency can be enhancedby introducing hydrophobic block copolymers. However, this modication canlead to problems of longer drying time, excessive wrinkling, and wet cling. In thischapter the properties of the synthetic bres and their structural modicationsmade by the many researchers both chemical and biochemical treatments have

been discussed. The latest developments and research going on the enzymebiotechnology on various bres such as polyester, polyamide, regeneratedcellulosic bres, and biodegradable plastics have been discussed.

Keywords: Biodegradation polyester, nylon, viscose rayon, renewable polymers,regenerated cellulose, polyurethane

4.1 Introduction

Synthetic bres are man-made bres that derived from chemical resources

(Achwal 1984). Synthetic bres are continuous lament form during bre

extrusion process at the stage of manufacturing either dry or wet or melt

spinning methods, which means the bres come in long lengths. Synthetic

bres are manufactured using plant materials and minerals: viscose comes

from pine trees or petrochemicals, while acrylic, nylon and polyester come

from oil and coal. Viscose bre is obtained from the cellulose; versatility

allows imitating materials such as cotton or silk. Polyester is a synthetic

material, strong and easy to maintain. Its aspect is smooth and glossy. Nylon

bre has tough and resilient need not be pressed, and to be synthetic, wash

with warm water (So Hee Lee and Wha Soon Song 2010).

The potential of microbial enzymes for surface modication of syntheticbres has recently been assessed (Khoddami et al. 2001). The major advantages

of enzymes in polymer modication compared to chemical methods are milder

reactions leading to less damage to bres, easier control, and environment

friendly on polymer surfaces. Enzymatic hydrolysis of synthetic bres

4

Bioprocessing of synthetic bres




improves some undesired properties such as hydrophilicity, improved wearing

comfort, tendency to pilling, low dyeability, and electrostatic forces.

Poly (ethylene terephthalate) (PET) is the most widely used synthetic brefor clothes, because it has many benecial properties. However, it also has severaldisadvantages, most of which are attributable to low hydrophilicity (moistureregain of 0.4%) (Zeronian and Collins 1990). Because of such low hydrophilicity,the surfaces of PET fabrics cannot wet easily and this may causes some difcultiesin nishing, washing, and dyeing. In addition, due to the buildup of electrostaticcharge and pilling on the surface of PET fabrics, the wearing comfort of clothingis diminished. In order to solve these problems, many attempts have been madeto modify the low hydrophilicity of the surface of PET fabrics. Recent studieshave suggested new alternatives for chemical treatment; one of these involvesthe use of enzymes for eco-friendly processing (Kim and Song 2012). Enzymatichydrolysis is more advantageous than conventional chemical hydrolysis by alkalitreatment as it consumes lesser amounts of energy; further, no harsh chemicalsare required. Moreover, enzymatic hydrolysis is restricted to the bre surface

because the enzymes cannot penetrate the bre; thus, there is no decrease inbre strength (Cavalco-Paulo and Gübitz 2003). Some of the enzymes that can

potentially be applied to PET fabric hydrolysis include lipases, and cutinases,esterases. These enzymes hydrolysis on ester linkage cause producing hydroxyland carboxyl groups on the surface of the fabrics, so surface hydrophilicity ofPET fabrics could be improved. Among these, lipases have the greatest numberof industrial applications and are already regarded as effective enzymes for the

hydrolysis of PET fabrics.

The bioprocessing of synthetic bres such as polyester, nylon,

polypropylene, polyethylene, polystyrene; and semi-synthetic (regenerated)

bres such as viscose rayon, lyocell and modal have been reported (Heumann et

al. 2006). Moreover the reports of previous research work made by researcher

and scientist in the area of synthetic bre have shown very little and studiesare being focussed on improving the functional characteristics of synthetic

bres. The new innovative work on the surface characteristics of polyester

bre by using the lipase and other enzymes to improve the hydrophilic nature

of polyester is creative and will pave the new path for functional textiles.

4.2 Bioprocessing of polyester and their

characteristics

4.2.1 Polyester bres

The world leader among synthetic man-made bres is polyester bre. Polyester

was one of the great man-made bre discoveries of the forties and has been

manufactured on an industrial scale since 1947. In 1996, 24.1 million metric



Bioprocessing of synthetic bres 191

tons of man-made bres were produced worldwide. The main volume gain

took place in production of PET bres (PET lament 9%, PET staple 4%)

(Froehlich 1997). The primary drive for this growth is demand for bre and

container resin. Seventy ve percent of the entire PET production is directed

toward bre manufacturing. Hoechst, Dupont and Eastman are the three world

largest polyester producers. Additional current US Polyester Fibre Producers

are: Acordis Industrial Fibres, Inc.; AlliedSignal Inc; Cookson Fibres, Inc.;

KoSa; Intercontinental Polymers, Inc., Martin Color-Fi. Nan Ya Plastics Corp.,

Wellman, Inc. Dramatic growth in PET bre production is foreseen in Asia in

the near future (Harris 1996). The cost of polyester, with the combination of

its superior strength and resilience, is lower than that of 100% cellulosic andregenerated rayon. Polyester bres are hydrophobic, which is desirable for

light weight facing fabrics used in the disposable industry.

4.2.2 Polyester bres – chemical structure

Polyester bre is a “manufactured bre in which the bre forming substance is

any long chain synthetic polymer composed at least 85% by weight of an ester of

a dihydric alcohol (HOROH) and terephthalic acid (p–HOOC–C6H

4COOH)”.

Figure 4.1 shows the chemical structure of polyethylene terephthalate (PET).The most widely used polyester bre is made from the linear polymer poly

(ethylene terephtalate), and this polyester class is generally referred to simply

as PET. High strength, high modulus, low shrinkage, heat set stability, light

fastness and chemical resistance account for the great versatility of PET.

Fig. 4.1 Chemical structure of polyethylene terephthalate (PET)

4.2.3 Polyester bre characteristics

The polyester bre has unique characteristics when compared to other

synthetic bres because it has the following characteristics such as

• Strong

• Resistant to stretching and shrinking

• Resistant to most chemicals• Quick drying

• Crisp and resilient

• Wrinkle resistant

• Mildew resistant




• Abrasion resistant

• Retains heat-set pleats and crease

• Easily washed

4.2.4 Polyester polymer formation

Polyethylene teraphthalate (PET) is a condensation polymer and is industrially

produced by either terephthalic acid (TPA) or dimethyl terephthalate (DMT)

with ethylene glycol (EG) (Hearl 1969). The polymerization processes

includes: (a) Terephthalic acid (TPA), produced directly from p-xylene with

bromide-controlled oxidation, (b) Dimethyl terephthalate (DMT), made in the

early stages by esterication of terephthalic acid. However, a different processinvolving two oxidation and esterication stages now accounts for most DMT,

and (c) Ethylene glycol (EG) initially generated as an intermediate product

by oxidation of ethylene. Further ethylene glycol is obtained by reaction of

ethylene oxide with water.

4. 2.5 Synthesis of polymer

4.2.5.1 Linear polyesters

Linear polyester, PET is polymerized by one of the following two ways: Ester interchange: Monomers are diethyl terephtalate (DET) and ethylene

glycol (EG). Direct etherication: Monomers are terephthalic acid (TPA) and

ethylene glycol (EG). Both ester interchange and direct esterication processes

are combined with polycondensation steps either batch-wise or continuously.

Batch-wise systems need two-reaction vessels – one for esterication or ester

interchange, the other for polymerization. Continuous systems need at least

three vessels – one for esterication or shear interchange, another for reducing

excess glycols, the other for polymerization.

4.2.5.2 Branched and cross linked polyesters

In the polymer formation process, if glycerol is allowed to react with a di-acid

or its anhydride each glycerol will generate one branch point. Such molecules

can grow to very high molecular weight. If internal coupling occurs (reaction

of a hydroxyl group and an acid function from branches of the same or

different molecule), the polymer will become cross linked.

4.2.6 Fibre formation

The sequences for production of PET bres and yarns depend on the different

ways of polymerization (continuous, batch-wise, and solid-phase) and

spinning (low or high wind-up speed) processes.




4.2.6.1 Spinning process

The degree of polymerization of PET is controlled, depending on its end-uses.PET for industrial bres has a higher degree of polymerization, higher molecular

weight and higher viscosity. The normal molecular weight range lies between

15,000 and 20,000. With the normal extrusion temperature (280–290°C), it has

a low shear viscosity is 1000–3000 poise. Low molecular weight PET is spun

at 265°C, whereas ultrahigh molecular weight PET is spun at 300°C or above.

The degree of orientation is generally proportional to the wind-up speeds in the

spinning process. Theoretically, the maximum orientation along with increase

in productivity is obtained at a wind-up speed of 8000–10,000 m/min.

4.2.6.2 Drawing process

To produce uniform PET, the drawing process is carried out at temperature

above the glass transition temperature (80–90°C). Since the drawing process

gives additional orientation to products, the draw ratios (3:1–6:1) vary

according to the nal end-uses. For higher tenacities, the higher draw ratios

are required. In addition to orientation, crystallinity may be developed during

the drawing at the temperature range of 140–220°C.

4.2.7 Structural composition of PET

The one of the distinguishing characteristics of PET is attributed to the

benzene rings in the polymer chain. The aromatic character leads to chain

stiffness, preventing the deformation of disordered regions, which results in

weak van der Waals interaction forces between chains. Due to this, PET is

difcult to be crystallized. Polyester bres may be considered to be composed

of crystalline, oriented semi crystalline and non-crystalline (amorphous)

regions. The aromatic, carboxyl and aliphatic molecular groups are nearly

planar in conguration and exist in a side-by-side arrangement. The cohesion

of PET chains is a result of hydrogen bonds and van der Waals interactions,

caused by dipole interaction, induction and dispersion forces among the

chains (Lewin and Pearce 1985). The capacity to form useful bres and the

tendency to crystallize depend on these forces of attraction. The interactive

forces create inexible tight packing among macromolecules, showing high

modulus, strength, and resistance to moisture, dyestuffs and solvents. The

limited exibility in the macromolecule is mainly due to the ethylene group.

4.2.7.1 Relationship between structure, properties and

processing parameters of PET bres

Properties of polyester bres are strongly affected by bre structure. The

bre structure, which has a strong inuence on the applicability of the




bre, depends heavily on the process parameters of bre formation such as

spinning speed, hot drawing (stretching), stress relaxation and heat setting

(stabilization) speed. As the stress in the spinning is increased by higher

wind-up speed, the PET molecules are extended, resulting in better as-spun

uniformity, lower elongation and higher strength, greater orientation and high

crystallinity (Morton et al. 1975). Hot drawing accomplishes the same effect

and allows even higher degrees of orientation and crystallinity. Relaxation is

the releasing of strains and stresses of the extended molecules, which results

in reduced shrinkage in drawn bres. Heat stabilization is the treatment to

“set” the molecular structure, enabling the bres to resist further dimensional

changes. Final bre structure depends considerably on the temperature, rateof stretching; draw ratio (degree of stretch), relaxation ratio and heat setting

condition. The crystalline and non-crystalline orientation and the percentage

of crystallinity can be adjusted signicantly in response to these process

parameters.

4.2.8 Mechanical properties of PET bres

As the degree of bre stretch is increased (yielding higher crystallinity and

molecular orientation), so are properties such as tensile strength and initialYoung’s modulus. At the same time, ultimate extensibility, i.e., elongation

is usually reduced. An increase of molecular weight further increases the

tensile properties, modulus, and elongation. Typical physical and mechanical

properties of PET bres are given (Table 4.1).

Table 4.1 Physical properties of polyester bres

Type of material /

Property

Filament yarn Staple and tow

Regular

tenacity

High

tenacity

Regular

tenacity

High

tenacity

Breaking tenacity (N/tex) 0.35–0.5 0.62–0.85 0.35–0.47 0.48–0.61

Breaking elongation 24–50 10–20 35–60 17–40

Elastic recovery at

5% elongation (%)

88–93 90 75–85 75–85

Initial modulus (N/tex) 6.6–8.8 10.2–10.6 2.2–3.5 4.0–4.9

Specic gravity 1.38 1.39 1.38 1.38

Moisture regain (%) 0.4 0.4 0.4 0.4

Melting temperature (oC) 258–263 258–263 258–263 258–263




chain scission at ester linkages (Fig. 4.2), although a radical mechanism has

also been proposed. The degradation products can undergo further changes,

but at ordinary processing temperatures a certain proportion of carboxyl

groups are introduced into the polymer structure. Color formation upon

degradation has been attributed to the formation of polyenaldehydes from

acetaldehyde and from a further breakdown of poly(vinyl ester)s. Polyester

bres display good resistance to sunlight but long-term degradation appears

to be initiated by ultraviolet radiation. However, if protected from daylight

by glass, PET bre gives excellent performance, when enhanced by an UV

stabilizer, in curtains and automobile interiors. Although PET is ammable,

the fabric usually melts and drops away instead of spreading the ame. PETbre will burn, however, in blends with cotton, which supports combustion.

Polyester has good oxidative and thermal resistance. Color forming species

are produced and carboxyl end groups are increased.

Fig. 4.2 Thermal degradation of polyester – chain molecular mechanism

4.2.12 Dyeing properties of PET bres

Because of its rigid structure, well-developed crystallinity and lack of reactive

dye sites, PET absorbs very little dye in conventional dye systems. This is

particularly true for the highly crystalline (highly drawn), high tenacity–high

modulus bres. Polyester bres are therefore dyed almost exclusively with

disperse dyes. A considerable amount of research work has been done to

improve the dye ability of PET bres. Polymerizing a third monomer, such

as dimethyl ester, has successfully produced a cationic dye able polyester

bre into the macro-molecular chain. This third monomer has introduced

functional groups as the sites to which the cationic dyes can be attached (Pal

et al. 1993). The third monomer also contributes to disturbing the regularity ofPET polymer chains, so as to make the structure of cationic dye able polyester

less compact than that of normal PET bres. The disturbed structure is good

for the penetration of dyes into the bre. The disadvantage of adding a third

monomer is the decrease of the tensile strength.




A new dyeing process for polyester bre at low temperature (40°C

and below) has been reported (Fite et al. 1995). This method employs a

disperse dye in a micro-emulsion of a small proportion of alkyl halogen and

phosphoglyceride. The main advantage of this method is low temperature

processing, but there remains the environmental problem that is produced

by using toxic carriers. Another approach has been introduced by Saus et al.

(1993a). The textile industry uses large amounts of water in dyeing processes

emitting organic compounds into the environment. Due to this problem

dyeing process for polyester bre was developed, in which supercritical CO2

is used as a transfer medium (Saus 1992b). This gives an option avoiding

water discharge. It is low in cost, non-toxic, non-ammable and recyclable.

4.2.13 Applications of PET bres

DuPont Company produced the rst US commercial polyester bre in 1953.

Since polyester bre has a lot of special characteristics, most of them are used

in the following three major areas:

• Apparel: Every form of clothing

• Home furnishings: Carpets, curtains, draperies, sheets and

pillowcases, wall coverings, and upholstery • Other uses: Hoses, power belting, ropes and nets, thread, tire cord,

auto upholstery, sails, oppy disk liners, and brell for various

products including pillows and furniture

• Composites made of 100% polyester bres are widely used as

ltration media. Its layered structure gives excellent tear strength, a

smooth, bre-free surface and edge stability.

4.2.14 Chemical method of PET hydrolysisOne of the surface modications is the controlled alkaline hydrolysis of the

polyester fabric. The action of a strong base leads to the cleavage of ester

linkages on the bre surface. The result is the formation of terminal hydroxyl

and carboxylase groups on the bre surface. Hydrolysis is believed to

increase the number of polar functional groups on the bre surface. In a study

by Zeronian and Collins (1990), on a 22-hour treatment with 10% aqueous

NaOH at 60°C, a weight loss of 25.3% was observed for semi-dull polyester

bres and, under the same treatment conditions, a weight loss of 81.4% wasobserved for bright polyester bres. In a comparative study by Namboori and

Haith (1968), treating polyester bres with alkalies and various alkoxides

showed the weight loss in the following order: sodium hydroxide (NaOH)

< tertiary butoxide < secondary propoxide < methoxide and ethoxide. In a




study by Chidambaram, Venkatraj, and Manisankar (2004), alkali treatment

of a polyester fabric was carried out in a solution of 0.3% NaOH in ethanol

(organic solvent medium). The fabric was squeezed to a 50% pick up and then

stored after covering it with a nylon lm for 24 hours at room temperature.

There was a 21% weight loss in the treated fabric, and the fabric attained

uniform silk-like handle. In a study by Zeronian and Collins (1990), a

polyester fabric was treated with a solution containing 60 g/l of alkali and

about 5% ethanol. The fabric was squeezed to a 50% pick up and then was

stored at room temperature after subjecting it to a few rotations to minimize

the uneven deposition or drying of the alkali solution on the fabric. In this

case, the fabric suffered a weight loss of 21%. Bendak and El-Marsa (1991)studied the topochemical degradation of polyester bres by pretreatment

with methanolic NaOH solutions. The methanolic medium and the higher

temperature of the pretreatment resulted in a steeper loss in the weight of the

fabrics. The pretreated samples, which had lost about 5–8% of their original

weight, showed a signicant decrease in the wicking time and also a relatively

slight improvement in reducing the sinking time. The pretreatment of bres

did not signicantly alter the moisture regain characteristics – only a small

part of the bres seemed to be affected by the pretreatment.

In a study by Samanta, Chattopadhyay, Konar, and Sharma (2003), pretreatment and post-treatment of micro-denier polyester fabrics with

selective chemicals were studied for improving the surface depth of the color.

It was observed that to achieve a 15% weight loss, the optimum treatment

conditions were 5% NaOH plus 0.1% hexamethylene diamine, 60 min, and

90°C.In a study by Achwal (1984) on treatment of polyester fabrics with 4%

NaOH in aqueous medium, weight losses of 10.7%, 15%, and 17% were

observed at 30, 45, and 60 min, respectively, at a material-to liquor (M:L)

ratio of 1:60 and at 90°C temperature with stearyl trimethyl ammoniumchloride (10 g/l) as catalyst. Whereas weight losses of 6%, 8.5%, and 10.5%

were observed at 30, 45, and 60 min, respectively, under the same treatment

conditions without the catalyst. The contribution of the surface wetting ability

and the pore structure to the liquid retention ability of brous materials were

analyzed by Hsieh, Miller, and Thompson (1996) using regular and micro-

denier poly(ethylene terephthalate) (PET) fabrics by treatment with an

aqueous NaOH solution. The hydrolysis time and the alkali concentration

of PET fabrics showed improved water wettability and liquid retention

characteristics. A theoretical model based on the surface reaction concept todescribe the kinetics of polyester bre dissolution in alkaline solutions has

shown that the weight loss is not a simple linear function of time. In their

study, Kallay et al. (1990) treated PET fabrics with an aqueous NaOH solution

at 100°C and reported that the reaction was a rst-order process with respect




to the surface area of the bres and the concentration of hydroxyl ions. In the

studies by Bruce (1984) and Gorrafa (1980), the treatment of a polyester fabric

with an aqueous NaOH solution caused a decrease in the weight and breaking

strength of the fabric and the handle quality improved with the increase in

the NaOH concentration, treatment time, and temperature. In view of these

studies, it is clear that many researchers have attempted the modication of

the polyester bres/ring spun yarn/fabric either by copolymerization or by

alkali/acid/organic solvents treatments (El-Gendy 2004; Kish and Nouri

1999; Shohola and Tumuli 1993; Yang 2003) to overcome their disadvantages.

With the above background information in mind, the alkaline hydrolysis of

polyester rotor spun yarns was carried out with the objective to study theinuence of various treatment conditions and yarn twist parameters on the

physical characteristics of polyester rotor spun yarns. Hence, characterization

of the alkali treated polyester spun yarns was also carried out to understand

the optimum or balanced process parameters and yarn parameters to get the

most desirable modications.

The alkaline hydrolysis of polyester spun yarns by treating with aqueous

sodium hydroxide (NaOH) solution (with and without catalyst) has been

studied at various concentrations, times, and temperatures (Vigneswaran and

Anbumani 2011). The changes in the physical characteristics of the parentand the alkali-treated polyester spun yarns, such as weight loss, strength

loss, abrasion resistance, wicking behavior, coefcient of friction, and

exural rigidity were analyzed. The rates of strength loss and weight loss

of the polyester spun yarns showed an increasing trend as the concentration

of NaOH increased from 5% to 15% and the temperature rose from 60°C to

100°C. The absorbency properties in terms of the wicking behavior of the

alkali-treated yarns increased compared with those of the parent yarn. Due to

the loss of mass from the bre surface, a decreasing trend was observed in theabrasion resistance and exural rigidity of the alkali-treated polyester rotor

spun yarns when compared with the parent yarn. The polyester rotor spun

yarns treated with 10% NaOH at 60°C gave better results for the physical

properties compared with the yarns subjected to the same treatment at 100°C

under comparable treatment conditions. This research work will enhance

further study on improving comfort characteristics and developing new

products in functional apparel using polyester spun yarns.

4.2.15 Enzymatic hydrolysis of polyester using lipase

The application of lipase enzyme on the biodegradation of polyester was

investigated by many researchers in the past 10 years. Lipolytic hydrolysis

of polyester as well as copolymers and blends containing polyester for the




purpose of biodegradation has been studied (Khoddami et al. 2001). In a study

reported in 1998 (Hiseh and Cram 1998), it was reported that the lipase enzymes

had the ability of improving the wettability and absorbency of polyester fabrics.

Estrases (carboxylic ester hydrolyses) are a group of lipases that are able to

play the role of a catalyst in the hydrolysis of many uncharged carboxylic

esters. Aryl estrases and Ali estrases constitute two major types of estrases.

Ali-estrases, which are also called Bestrases (EC 3.1.1.1) can break the ester

linkages of aliphatic compounds such as butyrate and tributyrine. Aryl estrases,

or A-estrases (EC 3.1.1.2) can hydrolyze the linkages in aromatic compounds

(Krisch 1971; Reed and Underkoer 1966) such as polyethylene terephthalate

(PET) (Hiseh and Cram 1998). Identication of the process parameters affectingthe enzymatic hydrolysis of polyester bres is important, because ideally, it is

required to improve the quality of polyester fabrics without considerable loss of

strength and other desired characteristics by means of controlling the conditions

of the selective enzyme reaction carefully.

In order to make the bio-treatment process more applicable to polyester

bres, more research is needed in this eld. As the results of previous works

showed that the enzymatic hydrolysis was limited to the surface of polyester

bres, and this could be related to the compact structure of drawn polyester

(Khoddami et al. 2001; Khalili et al. 2001), therefore, the choice of undrawnyarn could explain whether enzymes would penetrate more in the less ordered

structure of polyester bres. For this purpose, two polyester multilament

yarns were drawn with four levels of draw ratio; each draw ratio at four levels

of temperature, and then hydrolyzed with the same conditions. Khoddami et

al. (2001) have been studied the hydrolysis of polyester lament yarn using

lipase enzyme at various enzymatic process conditions. The material used in

this study consisted of two polyester multilament yarns, namely 180 denier

with 20 laments and 280 denier with 34 laments. The lipase enzyme is produced by submerged fermentation of a genetically modied Aspergillus

microorganism (Novozym 1999). The lipase enzyme hydrolysis process

conditions are given (Table 4.2).

Table 4.2 Lipase enzyme hydrolysis conditions for polyester

Hydrolysis Process condition

Polyester sample weight (kg) 5 × 10 –3

Enzyme conc. (kg/m3) 0.5

Temperature (°C) 40

Time (seconds) 15 × 60

pH 8–8.5

MLR 1:10




In order to hydrolyze the polyester, the phosphate buffer solution was

prepared rst and then the required amount of enzyme was added to the buffer

solution. After adding the samples to the hydrolysis bath, the temperature was

increased to 40°C in 5 × 60s and hydrolysis continued for 15 × 60s. At the end,

the polyester lament yarn hanks were treated in an acid buffer in order to

denature and deactivate the lipase enzyme (Khoddami et al. 2001). This was

followed by cold and warm rinsing. Finally the polyester yarn samples were

dried in air.

4.2.15.1 Testing procedure

The weight loss was measured by weighing the samples before and after

hydrolysis treatment with an accuracy of ±0.0001 g. Before weighing, the

samples were dried at 60°C for 12 × 3600s. Measurement of tenacity and

elongation-at break were carried out according to the standard test method

of ASTM D2256. For the determination of moisture regain and other

experiments, the samples were conditioned at 20 ± 2°C and 65 ± 2% relative

humidity (RH) for 72 × 3600s.

4.2.15.2 Weight loss

The weight loss of the polyester samples drawn with different conditions

as well as that of POY yarns are analyzed after lipase enzyme treatments.

The weight loss for both yams is less than 0.5%. No trend can be established

for either draw ratio or temperature (Nagata 1996). Considering an average

weight loss of 0.41% for 180 denier and 0.27% for 280 denier drawn yarns. It

can be observed that on the whole drawing at different temperatures increases

the weight loss by about 0.1% for both yams when compared with hydrolyzed

POY samples. Considering a weight loss of 0.3 and 0.14 for 180 and 250

denier POY yams, respectively, it is concluded that drawing at different

temperatures does not lead to considerable weight loss. The relative increase

in weight loss due to drawing can be related to the decrease of lament

diameter, which leads to an increase of specic surface area. The effects of

specic surface area on the rate of enzymatic hydrolysis are well known. It

is also pointed out that due to the big size of the enzymes molecules and

the compact structure of polyester bres, the enzymatic reaction is limited

to the surface of bres (Khalili et al. 2001). Therefore, it can be said that the

effect of increase in accessible surface area is more important than the internal

structure of polyester bres.




4.2.15.3 Tensile strength

The percentage strength loss due to hydrolysis for both polyester yarns drawnwith draw ratios of 1.4 and 1.85 at 90°C and 140°C lies between 3.5% and

10%. No trend can be established for the results. It should be noted that in

spite of the drawing effect which lead to higher strength (Walter et al. 1995).

These strength losses show the negative effect of enzymatic hydrolysis on

the strength of polyester laments. The percentage decrease of elongation-at-

break due to hydrolysis was between 7% and 25%.

4.2.15.4 Moisture regainTable 4.3 shows the result of the moisture absorption measurements. As it can

be seen the moisture absorption increases due to hydrolysis. This agrees with

the results already obtained for the hydrolysis of polyester fabric (Khoddami et

al. 2001). It shows that higher draw ratios lead to higher moisture absorption,

which again is related to higher accessible surface area.

Table 4.3 Increased moisture regain % of polyester after enzymatic hydrolysis

Type of PET yarn Temperature (oC)

Draw ratio

90oC 140oC

180/20 1.46 4.8 4.28

1.85 9.18 26.67

280/34 1.46 21.05 10.91

1.85 49.24 33.70

4.2.15.5 SEM micrograghs

Figure 4.3 shows the SEM micrographs of some of the hydrolyzed and

unhydrolyzed POY and drawn yams. It is evident that the drawn yarns

have been affected by the enzymatic hydrolysis more than the POY yarns.

Results of this research showed that the weight loss of polyester yarn hanks

is generally less than 0.5%. The loss of strength, due to the hydrolysis

had a maximum value of 10%, the moisture absorption of the hydrolyzed

samples increased by up to 50%. The decrease of elongation-at-break due to

hydrolysis was between 7% and 25% for drawn yarns. The observation of

surface of hydrolyzed and unhydrolyzed yarns show that due to the big sizeof the enzymes molecules and compact structure of polyester, the hydrolysis

effect is limited to the surface of the substrate. Therefore, the intensity of

hydrolysis is more affected by the specic surface area of the substrate

rather than the effects of drawing.




to detect collagenase activity. The technique is very sensitive to the optical

properties of the dielectric adjacent to the metal layer and can be used to

detect changes in the refractive index or the thickness of thin organic lms

(Homola et al. 1999).

4.2.16.1 Biodegradable polymers

Three types of biodegradable materials, which can be dissolved under

the catalytic action of a single enzyme, were chosen for the study (Sumner

and Krause 2001). Figure 4.4 shows the rst biodegradable polymer

under study as the poly (ester amide) based on bis (L-phenylalanine)alkylene di-esters. This system was chosen because the esters of N-acyl-L

amino acids are readily cleaved by chymotrypsin, i.e. they are hydrolyzed

105 times faster than the corresponding amides. The enzymatic hydrolysis

of powdered material and of lms of the polymer at pH 8 and 37°C has

previously been investigated (Arabuli et al. 1994). This study showed that

>50% of ester groups were cleaved within 4 h by the action of 540 units

of chymotrypsin. The starting rate of hydrolysis of the lm was similar

to that of a powdered substrate. Chymotrypsin is a suitable enzyme labelfor immunosensing since it is virtually never present in peripheral blood

samples. The second polymer is dextran hydrogels, the degradation of dextran

hydrogels in the presence of different concentrations of dextranase has been

investigated by Brøndsted et al. (1995, 1996). Hydrogels were obtained by

cross-linking dextran with diisocyanates (Fig. 4.5). Disks of the material,

2 mm thick, were reported to dissolve completely within 4 h or 20 min at

concentrations of dextranase of 31 or 1000 units/ml, respectively. The rate

of degradation can be adjusted by the degree of cross-linking. The higher the

cross-linking density the longer is the dissolution time. Since hydrogels are

strongly hydrated.

Fig. 4.4 Chemical structure of biodegradable PET bre




Fig. 4.5 Dextran hydrogel cross linked with hexamethylene diisocyanates

The third polymer under investigation was poly (trimethylene) succinate

(Fig. 4.6). This polyester is degraded by the enzyme lipase, which cleaves

naturally occurring ester groups (Walter et al. 1995). The dissolution of poly

(trimethylene) succinate powder and lms was investigated and comparing

ester bond cleavage and weight loss measurements. Oligomers with an

average length of ve to six monomers were released from the polymer bulk.The enzyme activity for the interaction of lipase with an insoluble substrate

was found to be highly reproducible (Walter et al. 1995).

Fig. 4.6 Structure of poly (trimethylene) succinate

4.2.16.2 Preparation of polymer lms

Films of poly (ester amide) ranging from 5 to 10 nm thick were spin coated

at a speed of 3000 rpm onto the gold surface from solutions of the polymer in

chloroform. Films of different thickness were produced by varying the amount

of the poly (ester amide) in the solutions. Dextran (2.5 g, 3.57 × 10−5 mol) was

dissolved in 14 ml DMSO. Some 0.03 ml hexamethylene diisocyanate wereadded under continuous stirring (Sumner et al. 2001). The resulting solution

was immediately applied to an SPR substrate and spun at a speed of 3500

rpm for 40s. The substrates were then left on a hotplate at 70°C overnight for

the cross-linking reaction to proceed. Films of poly (trimethylene) succinate




were produced by spin coating at 3000 rpm from a 0.13 wt. % solution of the

polymer in acetone. All poly (ester amide) and poly (trimethylene) succinate

lms were left to dry at room temperature for at least 24 hours before analysis.

4.2.16.3 Effect of enzyme concentration

Degradation of the polymer lms was shown to be dependent on the enzyme

concentration for all three polymer enzyme systems. The change in the SPR

angle with time is presented for the degradation of dextran hydrogel lms at

different enzyme concentrations. The lower the enzyme concentrations used,

the smaller the rate of degradation. After an initial period, the degradation was

roughly linear with time.

4.2.16.4 Effect of lm thickness

Poly (ester amide) lms ranging from 5 to 10 nm thickness were degraded

by chymotrypsin solutions with a concentration of 4 × 10−8 mol dm−3. For

the range of thickness used in this study, a linear relationship between the

lm thickness and the total change of the SPR angle was observed. Films

of different thickness were found to degrade at the same rate. These results

agree with the accepted mechanism of chymotrypsinolysis (Arabuli et al.

1994). Once the chymotrypsin had adsorbed onto the surface of the polymer

lm, degradation occurred at a constant rate. A linear relationship between

enzyme concentrations and the rates of the enzyme catalysed dissolution of

three biodegradable materials has been established. The poly (ester amide)

chymotrypsin and the dextran hydrogel–dextranase systems have been

proven to be the most sensitive and have therefore been selected for further

development. Since the dextran hydrogel degraded faster at low enzyme

concentrations, it has potential for further improving the lower limit ofdetection (Sumner et al. 2000).

4.2.17 In vitro enzyme catalysis on polyester

Enzyme technology has signicantly expanded in scope and impact over

the past 10 years to include organic transformations in non-traditional

environments. This review focuses on a relatively new but rapidly expanding

research activity where in vitro enzyme catalysis is used for the synthesis of

polyesters (Gross et al. 2001). Aspects include enzyme-catalyzed step-growthcondensation reactions, chain-growth ring-opening polymerizations, and

corresponding transesterication of macromolecular substrates are discussed.

Increasingly, research has been carried out to explore the ability of enzymes

to function in non-traditional media such as in organic solvents. Researchers




have found that many enzymes can function with specicity on a surprisingly

broad range of synthetic polymers. Prior to 1995, almost all of these activities

were directed towards developing knowledge on organic transformations of

small molecules. More recently, the use of enzymes for polymer synthesis is

gaining attention and is showing great promise. Enzyme-catalyzed polyester

synthesis based on lipase-catalyzed condensation polymerizations, a

pioneering study by Margolin et al. (1987) described the synthesis of optically

active oligoesters by exploiting lipase enantioselectivity. These researchers

prepared oligoesters by porcine pancreatic lipase (PPL) catalyzed reactions

between both a racemic di-ester and an achiral diol, or, a racemic diol and

an achiral diester. In both cases, trimers and tetramers of type AA-BB-AAand AA-BB-AA-BB-AA and very low quantities of higher oligomers were

formed. They observed formation of hydroxyl-capped oligomers since an

excess of the diol was used. Wallace and Morrow (1989) were also early

contributors to this new eld. They recognized the importance of stoichiometry

and studied polycondensation using equimolar quantities of trihaloalkyl

diesters and primary diols. Halogenated alcohols such as 2,2,2-trichloroethyl

activated the acyl donor and thereby improved the polymerization kinetics.

They also removed by products periodically during the reactions to facilitate

the growth of chains. Wallace and Morrow 1989; Morrow and Wallace 1992investigated PPL-catalyzed copolymerization of bis (2,2,2-trichloroethyl)

trans-3,4-epoxyadipate and 1,4-butanediol. They reported the synthesis

of product with Mw = 7.9–103 g/mol after 120 hours. In addition, the

polyesters formed by Wallace and Morrow (1989) had high optical purity

(>95%). Knani et al. (1993) studied the inuence of enzyme type, solvent,

concentration, reaction time, and other parameters on the self-condensation

of methyl hydroxyhexanoate. They observed no chain growth with aromatic

monomers. Reactions conducted in bulk gave oligoesters with longer chainlength than similar reactions conducted in solvents. Chaudhary et al. (1997)

summarized work carried out using lipase-catalysis for condensation-type

polymerizations. They emphasized that moderate molecular weight polyesters

required efcient methods to shift the thermodynamic equilibrium towards

product formation. For example, Novozyme-435 was used to catalyze the

solvent less copolymerization of divinyl adipate and 1,4-butanediol to form

a polyester with Mw = 23.2–103 g/mol. From this work and that by others, it

was concluded that the product molecular weight and end-group structure are

a function of: (1) Enzyme water content

(2) The enzyme/ substrate ratio

(3) Monomer-substrate stoichiometry, and

(4) Reaction temperature




Later, Rodney et al. (1999) described Novozyme-435-catalyzed AA-BB

type condensation polymerizations to form aromatic polyesters.

4.2.17.1 Enzyme-catalyzed ring-opening polymerizations

Ring-opening polymerizations of lactones and cyclic carbonates circumvents

the generation of leaving groups that can limit propagation kinetics and product

molecular weight (Gross et al. 2001). Publications have appeared on lipase-

catalyzed ring-opening polymerizations of caprolactone (CL) (Uyama and

Kobayashi 1993; Macdonald et al. 1995: Kumar and Gross 2000a), valerolactone

(VL) (Uyama and Kobayashi 1993), methyl-valerolactone (Kullmer et al.

1998), methyl--caprolactone (Kullmer et al. 1998), propiolactone (Nobes et

al. 1996), methyl--propiolactone, methyl--propiolactone (Svikrin et al. 1996),

butyrolactone (Dong et al. 1998; Nobes et al. 1996), 8-octanolide (Kobayashi

et al. 1998), and others. In all of these reports, the polymerizations proceeded

with slow propagation kinetics and gave low molecular weight products.

However, as will be seen in certain examples below, in the few years since

these reports, signicant improvements in polymerization efciencies and

product molecular weights have been made. Lipase catalyzed polymerizations

of macro lactones have in some cases proved advantageous relative to chemical preparative routes. Kobayashi and coworkers were the rst to investigate

lipase-catalyzed polymerization of undecanolide (UDL) (Uyama et al. 1996),

dodecanolide (DDL) (Uyama et al. 1995a), and pentadecanolide (PDL) (Uyama

et al. 1996) (12-, 13-, and 16-membered lactones). Screening of enzymes for

the polymerization of UDL, DDL, and PDL using lipases including those from

Aspergillus niger, Candida cylindracea (lipase B), Candida rugosa, Rhizopus

delmar, Rhizopus javanicus, Pseudomonas uorescens (lipase P, Cosmo Bio.)

Pseudomonas sp. (lipase PS, Amano) as well as phospholipase and PPL were

carried out (Uyama et al. 1995b). Quantitative conversions of UDL to poly(UDL)

were achieved within 120 h using lipase P and PS. The highest number average

molecular weight reported by these workers was for poly (DDL) synthesis

(Mn = 25.0–103 g/mol, 75°C, 120 h) using the immobilized lipase PS from

a Pseudomonas sp. (lipase PS, Toyobe Co.) (Uyama et al. 1995b). Bisht et al.

(1997a) reported that, by using lipase PS-30 immobilized on celite, the solvent

less polymerization of PDL gave poly(PDL) having Mn 62.0–103 g/mol and

PDI 1.9. Kumar et al. (2000b) reported poly (PDL) having Mn of 86.4–103 g/

mol using Novozyme-435 as catalyst in low levels of toluene. Copolymerization of two or more monomers is an important strategy

for the “tailoring” of polymeric materials. Since in vitro enzyme-catalyzed

polymerization is a relatively new area of study, copolymerizations have

thus far received little attention. Namekawa et al. (1996) reported the lipase-




catalyzed copolymerization of propiolactone and CL. Uyama et al. (1996)

studied Pseudomonas uorescens lipase-catalyzed copolymerizations of PDL

with DDL, UDL, VL, and CL. The rates of these reactions were slow and

yielded low molecular weight copolymers (Mn<6000 g/mol). Later, Kobayashi

et al. (1998) reported the formation of copolymers from VL and CL using the

lipase from P. uorescens. In addition, the lipase from Candida antarctica

was used for the copolymerization of 8-OL with CL and DDL in isooctane.

The latter copolymerization gave %-monomer conversions of about 80% in

48 hours at 60°C (lipase 0.10 g/1.0 mmol monomer) and products with Mn <

9.0, 103 g/mol (Kobayashi et al. 1998). Matsumura et al. (1999) reported the

preparation of polyester copolymers also. Thus far, the kinetics and mechanism of lipase-catalyzed ring-opening

polymerizations have been investigated with only a limited set of monomers

and enzyme-catalyst systems. However, for these systems, work has been

conducted to begin developing knowledge of how reaction parameters such

as solvent, temperature, enzyme concentration, and water and monomer

concentration inuence the rate of propagation, molecular weight and

polydispersity. Henderson and Gross (2000) and Bisht et al. (1998) believe

that lipase-catalyzed ring-opening polymerization proceeds by the nucleophilic

attack of lipase serine residues at lactone carbonyl groups. This results in theformation of an enzyme-activated monomer (EAM) complex. Polymer chain

growth or propagation takes place when the hydroxyl terminal group of a chain

acts as the nucleophile that reacts with the EAM complex to give a product that

is elongated by one repeat unit (Fig. 4.7). Lipase-catalyzed polymerization of

-CL was found to share many features with that of an immortal polymerization.

Fig. 4.7 Mechanism of lipase catalysed polymerization




Considering the requirement that two macromolecules must be in close

proximity at the lipase active site for these reactions, it is surprising that they

occur so rapidly (Bankova et al. 2000). Apparently, lipase catalysis results in

the cleavage of internal ester linkages along chains. The resulting enzyme-

activated-polymer complex (EAP) then reacts with terminal hydroxyl groups

at chain ends to form ester linkages. These reactions can be used to reshufe

the repeat unit sequence distribution between different aliphatic polyester

chains (Kumar and Gross 2000c). Integration of these powerful new tools

in enzyme engineering with the current challenges in enzyme-catalyzed

polymerizations will surely become a prominent activity in future years that

will empower current activities.

4.2.18 Biodegradation of polyester-based polyurethane

polymer

Polyurethane (PUR), which is a kind of plastic used as base material in various

industries (Yukie Akutsu et al. 1998). PUR is synthesized by condensation of

polyol and polyisocyanate (Fig. 4.8). There are two types of PUR, named

according to the polyol used in the synthesis: PUR which uses polyester as

polyol in synthesis is called polyester PUR, and the one which uses polyetheris called polyether PUR. PUR is a material which is resistant to microbial

attack, but generally, the ester-type PUR is more easily degraded than the

ether-type PUR (Morton and Surman 1994). Several kinds of fungi and

bacteria that can degrade ester-type PUR have been reported (Crabbe et al.

1994; Darby and Kaplan 1968; Kay et al. 1991; Pathirana and Seal 1984). In

all cases, the degradation was considered to be initiated by hydrolysis of the

ester bond by some hydrolytic enzyme(s), such as esterase.

Fig. 4.8 Structure of poluurethane (pur)

In solid-polyester biodegradation, degradation of poly (3-hydroxyalkanoate)

(PHA) by PHA depolymerase is well-known (Kanesawa et al. 1994; Kasuya




and Doi 1994; Kita et al. 1995). This enzyme, which is one of the esterases, has a

hydrophobic surface-binding domain and a catalytic domain and accomplishes

the degradation of PHA lm via two steps (Kasuya et al. 1996; Nakajima-

Kambe et al. 1997). The rst step is adsorption of the enzyme on the surface of

the lm via the surface-binding domain, and the second step is hydrolysis of the

ester bond. Pathirana and Seal (23) reported the activities of several enzymes,

namely, esterase, protease, and urease, in culture broth during ester-type PUR

degradation by fungi. Crabbe et al. (1994) also obtained fractions containing

esterase activity from the broth of a fungus culture which could degrade colloidal

PUR. In the case of bacteria, Kay et al. (1993) have detected extracellular

esterase activity in the medium during PUR degradation by a Corynebacterium

sp. Previously (Nakajima-Kambe et al. 1995) a bacterium, strain TB-35, which

could utilize solid polyester PUR as the sole carbon and nitrogen source, was

isolated and identied as Comamonas acidovorans. Esterase plays a major role

in PUR degradation by this strain (Nakajima-Kambe et al. 1997). This strain

constitutively secreted two kinds of extracellular esterase: one is secreted into

the culture broth, and the other is bound to the cell surface. Only the cell-bound

esterase could degrade the PUR.

4.2.18.1 MaterialsThe polyester PUR was synthesized by reacting poly(diethylene glycol adipate)

with 2,4-tolylene diisocyanate under anhydrous conditions (Nakajima-Kambe

et al. 1995).

4.2.18.2 Bacterial strains and cultivation

Comamonas acidovorans TB-35, which had been isolated as a degrader of

solid polyester PUR, was used (Akutsuet et al. 1998). One loopful of cells

was used to inoculate three 500-ml Erlenmeyer asks, each containing 100ml of basal medium and 1.0 g of PUR cubes (5 by 5 by 5 mm) as the sole

carbon source. Cultivation was performed under the following conditions:

aeration, 1.5 liters/min; temperature, 30°C; and agitation, 500 rpm. Cells were

harvested after 7 days’ cultivation and were stored at 280°C until use.

4.2.18.3 Degradation of polyurethane polymer

The ester bonds of the PUR were hydrolyzed by the PUR esterase of strain

TB-35 and that adipic acid and diethylene glycol was released as degradation products. This observation suggests that this enzyme is a novel plastic-

degrading esterase with properties different from those of lipase or PHA

depolymerase. Figure 4.9 shows the scanning electron micrograph of PUR in

which (A) Undegraded control; (B and C) PUR after degradation by 0.02 and




0.2 U of PUR esterase, respectively, for 24 h. The puried PUR esterase had

a high hydrophobicity, surface-binding domain and a catalytic domain. The

degradation of the PUR was done by two steps. The rst step is hydrophobic

adsorption of the enzyme to the surface of the PUR via the surface-binding

domain, and the second step is hydrolysis of the ester bond. From this

observation, it was thought that the surface binding site and catalytic site of

the PUR esterase exist in three-dimensionally close positions, unlike those

of PHA depolymerase. In this model, the catalytic domain can gain access to

the PUR surface even if the PUR surface has been saturated by the enzyme

molecules. However, since the number of adsorbable enzyme molecules per

surface area of the PUR is xed (Fig. 4.10), the PUR degradation activityremained constant. Furthermore, in contrast to PHA depolymerase, which is

strictly extracellular, the PUR esterase is bound to the cell surface; therefore,

the PUR esterase may have a cell surface-binding domain in addition to

catalytic and PUR surface-binding domains.

Fig. 4.9 SEM view of polyurethane (PUR); (a) undegraded control; (b and c) pur after

degradation by 0.02 and 0.2 u of PUR esterase, respectively, for 24 h.




Fig. 4.10 Kinetic models of the surface binding and hydrolysis of PHA by PHA

depolymerase (a) and PUR by PUR esterase (b) [Source: Mukai et al. 1993]

Recently the potential of enzymes for surface hydrophilisation and/

or functionalisation of polyethyleneterephthalate (PET) and polyamide(PA) have been discovered (Sonja Heumann et al. 2006). However, there

was no correlation between enzyme class/activity (e.g. esterase, lipase,

cutinase) and surface hydrolysis of these polymers. Enzymes active on

the model substrates bis (benzoyloxyethyl) terephthalate and adipic acid

bishexyl-amide were also capable of increasing the hydrophilicity of PET

and PA. When dosed at the identical activity on 4-nitrophenyl butyrate,

only enzymes from Thermobida fusca, Aspergillus sp., Beauveria sp. and

commercial enzymes (TEXAZYME PES sp5 and Lipase PS) increased thehydrophilicity of PET bres. Hydrophilicity of bres was greatly improved

based on increases in rising height of up to 4.3 cm and the relative decrease

of water absorption time between control and sample of the water was up to

76%.




4.3 Bioprocessing of polyamide and their

characteristics4.3.1 Nylon bres

Nylon (Polyamide), invented in 1928 by Wallace Carothers (DuPont) is

considered to be the rst engineering thermoplastic (Pezzin and Gechele

1964). Nylon is created when a condensation reaction occurs between amino

acids, dibasic acids and diamines. Commercially nylon is commonly used in

the production of tire cords, rope, belts, lter cloths, sports equipment and

bristles. Nylon was the rst truly synthetic bre and commercialized in 1939.

Nylon was developed in the 1930s by scientists at Du Pont, headed by anAmerican chemist Wallace Hume Caruthers (1896–1937). It is a polyamide

bre, derived from a diamine and a dicarboxylic acid. The two most

common versions are nylon 66 (polyhexamethylene adiamide) and nylon 6

(Polycaprolactam, a cyclic nylon intermediate). Raw materials for these are

variable and sources used commercially are benzene (from coke production

or oil rening), furfural (from oat hulls or corn cobs) or 1,4-butadiene (from

oil rening). Figure 4.11 shows the chemical reaction of Nylon 6 and Nylon

66 polymers (Galanty and Bujtas 1992).

Fig. 4.11 Chemical reaction of nylon 6 and nylon 66 polymers

Nylon is produced by melt spinning and is available in staple, tow,

monolament, and multi-lament form. The nylon bre has outstanding

durability and excellent physical properties. Nylons are semi-crystalline

polymers. The amide group -(-CO-NH-)- provides hydrogen bonding between

polyamide chains, giving nylon high strength at elevated temperatures,

toughness at low temperatures, combined with its other properties, such as

stiffness, wear and abrasion resistance, low friction coefcient and good

chemical resistance. These properties have made nylons the strongest of allman-made bres in common use. Because nylons offer good mechanical and

thermal properties, they are also a very important engineering thermoplastic.

For example, 35% of total nylon produced is used in the automobile industry

(Meplestor 1997). There are several commercial nylon products, such as




nylon 6, 11, 12, 6/6, 6/10, 6/12, and so on. Of these, the most widely used

nylon products in the textile industry are formed of nylon 6 and nylon 6/6. The

others are mainly used in tubing extrusion, injection molding, and coatings of

metal objects. Nylon’s outstanding characteristic in the textile industry is its

versatility. It can be made strong enough to stand up under the punishment

tire cords must endure, ne enough for sheer, high fashion hosiery, and light

enough for parachute cloth and backpacker’s tents. Nylon is used both alone

and in blends with other bres, where its chief contributions are strength and

abrasion resistance. Nylon washes easily, dries quickly, needs little pressing,

and holds its shape well since it neither shrinks nor stretches.

4.3.2 Properties of nylon bres

Tenacity-elongation at break ranges from 8.8g/d-18% to 4.3 g/d-45% (Han et

al. 1987). Its tensile strength is higher than that of wool, silk, rayon, or cotton.

The other properties of nylon bres are

• 100% elastic under 8% of extension

• Specic gravity of 1.14

• Melting point of 263oC

• Extremely chemically stable • No mildew or bacterial effects

• 4–4.5% of moisture regain

• Degraded by light

• Permanent set by heat and steam

• Abrasion resistant

• Easy to wash

• Resilient

• Filament yarn provides smooth, soft, long lasting fabrics

• Spun yarn lend fabrics light weight and warmth

4.3.3 Crystalline structure

Both nylon 6 and nylon 66 are semi-crystalline polymers. These linear aliphatic

polyamides are able to crystallize mostly because of strong intermolecular

hydrogen bonds through the amide groups, and because of Vander walls forces

between the methylene chains. Since these unique structural and thermo-

mechanical properties of nylons are dominated by the hydrogen bonds inthese polyamides, quantum chemistry can be used to determine the hydrogen

bond potential. For the α-form of nylon 6, adjacent chains are parallel and

the hydrogen bonding is between adjacent chains within the same sheet

(bisecting the CH2 angles). For formation of nylon 6, the chains are parallel




and the hydrogen bonding is between chains in adjacent sheets (Nagata and

Kiyotsukuri 1992). In nylon 66, the chains have no directionality. Research

results have shown that the stable crystalline structure is the form comprised

of stacks of planar sheets of hydrogen bonded extended chains. It also appears

that young’s modulus of the α-form is higher than the γ-form. Mechanical,

thermal and optical properties of bres are strongly affected by orientation and

crystallinity. Basically, higher bre orientation and crystallinity will produce

better properties. Crystallinity of nylons can be controlled by nucleation, i.e.,

seeding the molten polymer to produce uniform sized smaller spherulites. This

results in increased tensile yield strength, exural modulus, creep resistance,

and hardness, but some loss in elongation and impact resistance. Anotherimportant benet obtained from nucleation is decrease of setup time during

processing (Galanty and Bujtas 1992).

4.3.4 Environmental degradation properties

The -COOH and -NH2 end-groups in nylons are sensitive to light, heat, oxygen,

acids and alkali. When exposed to elevated temperatures, unmodied nylons

undergo molecular weight degradation, which results in loss of mechanical

properties. The degradation is highly time/temperature dependent (Shibusawa1996). By adding heat stabilizer, nylon can be used at elevated temperature

for long-term performance. Exposure to UV light results in degradation

nylon over an extended period of time; it appears that adding carbon black

can reduce the radiation degradation. Nylons are chemical resistance to

hydrocarbons, aromatic and strong acids, bases, and phenols attack aliphatic

solvents, but them. They also are gradually attacked hydrolytically by hot

water. Newly developed sulfonation of nylon 6 bre (El Garf and El kemry

1997) by 2,5 dichlorobenzene sulfonyl chloride (DSBC) has a great effect on

the heat and chemical stability of the bres. It reported that the modied bre

is non-melting up to 1000°C, and does not burn when put it in direct ame

(but chars without losing bre form). It does not dissolve in formic acid and

concentrated mineral acid. Its glass transition temperature is about 500°C.

4.3.5 Biodegradation of polyamides

4.3.5.1 Nylon 4

It has been reported that nylon 4 was degraded in the soil (Hashimoto et al.

1994) and in the activated sludge (Kawasaki et al. 2005). The results conrmed

that Nylon 4 is readily degradable in the environment. Furthermore, the

biodegradability of nylon 4 and nylon 6 blends was investigated in compost




and activated sludge. The nylon 4 in the blend was completely degraded

in 4 months while nylon 6 was not degraded (Hashimoto et al. 2002).

Recently, Yamano et al. (2008) was able to isolate polyamide 4 degrading

microorganisms (ND-10 and ND-11) from activated sludge. The strains were

identied as Pseudomonas sp. The supernatant from the culture broth of strain

ND-11 degraded completely the emulsied nylon 4 in 24 h and produced γ-

amino butyric acid (GABA) as degradation product.

4.3.5.2 Nylon 6

Polyamide (nylon) has excellent mechanical and thermal properties, good

chemical resistance and low permeability to gases, but it is known to be

resistant to degradation in the natural environment. The poor biodegradability

of nylon in comparison with aliphatic polyesters is probably due to its

strong inter-chain interactions caused by the hydrogen bonds between

molecular chains of nylon. Some microorganisms such as Flavobacterium

sp. (Kinoshita et al. 1975) and Pseudomonas sp. (NK87) (Kanagawa et

al. 1989) have been reported to degrade oligomers of nylon 6. Moreover,

some white rot fungal strains were reported to degrade nylon 66 through

oxidation processes (Deguchi et al. 1998). The biodegradation of nylon-6utilized in particular intrauterine devices (IUD) in vivo enzyme system has

been reported (Hudson and Crugnola 1987). For periods of two years or

longer, 67% of these devices examined had breaks in the coating of the tail

string. From results using synthesized specimens of 14C-labeled nylon-6,6

exposed, in vitro, to a number of enzyme solutions, nylon-6,6 was found to be

unaffected by esterase, but degraded by papain, trypsin, and chymotrypsin,

though the extent of degradation was small (Smith et al. 1987). Recently,

biochemical studies on the biodegradation of nylon-6,6 by a lignin-degrading

fungus were reported (Deguchi et al. 1998; Nomura et al. 2001). A nylon-

degrading enzyme was found in the culture medium of a white rot fungus

strain. The characteristics of the puried protein such as molecular weight,

absorption spectrum, and requirements for 2,6-dimethoxyphenol oxidation

were found to be identical to those of manganese peroxidase. The nylon-

degrading activity did not depend on exogenous H2O

2, but was inhibited

by catalase. These features are identical to those of the reaction catalyzed

by horseradish peroxidase. From the nuclear magnetic resonance (NMR)

analysis of the degradation products, it was proposed that the methylenegroup adjacent to the nitrogen atom in the polymer chain was attacked by

the enzyme, and subsequently the reaction proceeded auto-oxidatively (Fig.

4.12) (Nomura et al. 2001).




Fig. 4.12 Proposed mechanisms of nylon degradation by fungus peroxidase

[Source: Nomura et al. 2001].

A nylon-degrading enzyme found in the extracellular medium of a

ligninolytic culture of the white rot fungus strain IZU-154 was puried by ion-

exchange chromatography, gel ltration chromatography, and hydrophobic

chromatography (Tetsuya Deguchi et al. 1998). The characteristics of

the puried protein (i.e., molecular weight, absorption spectrum, and

requirements for 2,6-dimethoxyphenol oxidation) were identical to those of

manganese peroxidase, which was previously characterized as a key enzyme

in the ligninolytic systems of many white rot fungi. Nylon degradation is

catalyzed by manganese peroxidase (MnP). However, the reaction mechanism

for nylon degradation differed signicantly from the reaction mechanism

reported for manganese peroxidase. The nylon-degrading activity did notdepend on exogenous H

2O

2 but nevertheless was inhibited by catalase, and

superoxide dismutase inhibited the nylon-degrading activity strongly. These

features are identical to those of the peroxidase-oxidase reaction catalyzed

by horseradish peroxidase. In addition, α-hydroxy acids which are known to

accelerate the manganese peroxidase reaction inhibited the nylon-degrading

activity strongly. Degradation of nylon-6 bre was also investigated. Drastic

and regular erosion in the nylon surface was observed, suggesting that nylon

is degraded to soluble oligomers and that nylon is degraded selectively. Figure

4.13 shows the variations in surface properties after degradation of nylonbres. After 1 day of incubation, the smooth surface of the bre became rough,

but there was no change in diameter (Fig. 4.13b), indicating that the surface of

the bre was stripped. Subsequently, many horizontal grooves were observed




4.3.6 Enzymes in dye reduction on polyamide fabrics

Classical processes of indigo dyeing use sodium dithionite as reducing agent,causing enormous environmental problems. New process of indigo reduction

using NADH dependent reductases from Bacillus subtilis in the presence of

redox mediators is studied (Mojca Božič 2009). The efciency of mediated

enzymatic indigo reduction on the dyeing of decitex polyamides 6 and 6,6

was studied at 60°C, pH 7 and 11, and different indigo concentrations. The

color values and color fastness properties (to wash, light and perspiration)

were evaluated and compared to chemically indigo-dyed polyamides. The

results indicated that the dyeing properties were pH, time and polyamide

type dependent, resulting in the greater color depth of morphologically more

amorphous polyamide 6,6 dyed at pH 11 for 90 min. Both the alkaline and

acid perspiration fastness properties of the dyeing were very good, whereas

the dyeing displayed poorer fastness of 3—4 to light and wash.

4.4 Bioprocessing of regenerated cellulosic and

their characteristics

4.4.1 Viscose rayon

Viscose rayon is the regenerated cellulosic man-made bre and has a long

tradition. The primary raw material is cellulose derived from wood. The wood

of the Southern Pine grown in Florida is particularly suitable as it contains

60–70% cellulose (Lenz et al. 1993). Various processes turn the cellulose into

a thick spinning solution which is pressed through ne spinnerets. Extremely

thin continuous threads are produced, viscose lament yarns, mainly used

for making silk-like textiles. Viscose is made up for linings and mostly lightsummer wear, such as dresses, skirts, blouses, shirts, jackets and trousers.

There are also viscose staple bres, made by cutting the laments into specic

lengths. Viscose staple bres are mostly processed into cotton, wool or linen

like yarns and fabrics. Viscose has many characteristics, the properties varying

according to the method of processing. Textile fabrics made of viscose can be

dyed and printed extremely well and exhibit exceptionally brilliant colors.

Viscose breathes actively, regulates temperature well, making it especially

pleasant to the skin and have a silk-like brightness, soft, graceful ow and

high moisture absorption. The physical and chemical properties of viscose

rayon are given in Tables 4.4 and 4.5, respectively. Viscose rayon is a bre

made from regenerated wood cellulose. Viscose rayon is structurally similar

to cotton, which is almost pure cellulose (Bochek et al. 1997). Cellulose




is a linear polymer of β-D-glucose units with the empirical formula of

(C6H

10O

5)n. To prepare viscose, the cellulose is treated with sodium hydroxide

to form “alkali cellulose,” which has the chemical formula [C6H

9O

4-ONa]

n.

The alkali cellulose is then treated with carbon disulde to form a solution of

sodium cellulose xanthate, which is called viscose (Liu et al. 2001).

Table 4.4 Physical properties of viscose rayon

Tensile strength Tensile strength of the viscose rayon bre is less when

the bre is wet than dry conditions. It is 1.5–2.4 gpd in the

dry state and 0.7–1.2 gpd in the wet state.

Elasticity The elasticity of viscose rayon is less than 2–3%. This is

very important in handling viscose yarns during weaving,

wet processing when sudden tensions are applied.

Moisture absorption Viscose rayon absorbs more moisture than cotton bre.

Moisture content of cotton is 6–8% at 70°F and 65%

relative humidity, and for viscose rayon it is 11–13%

under the same conditions.

Elongation at break Viscose rayon has 15–30% elongation at break, while

high tenacity rayon has only 9–17% elongation at break.

Density The density of viscose rayon is 1.53 g/cc.

Action of heat and light At 300°F or more, viscose rayon loses its strength andbegins to decompose at 350–400°F. Prolonged exposure

to sunlight also weakens the bre due to moisture and

ultraviolet light of the sunlight.

Table 4.5 Chemical properties of viscose rayon

Action of acids Acids like H2SO

4, HCl breaks the cellulose to

hydrocellulose. Oxidizing agents like Na(OCl)2, Bleaching

powder, K2Cr

2O

7, KMnO

4 form oxycellulose. Organic acids

can be safely used in 1 to 2 percent concentration withoutinjury to the bre. Inorganic acids such as hydrochloric

and nitric can be used in strong concentrations. Oxalic

acid for removal of iron stains is not recommended except

at temperatures lower than 150°F.

Action of detergent Ordinary soaps in usual textile concentration have no

direct effect on regenerated cellulose materials. Improper

use of soap or use of poorly made soap results in rancidity

and odor in rayon fabrics or yarns.

Action of dry heat Most regenerated celluloses, under the inuence of heat

as well as light, show rapid loss in strength, this changebeing accompanied by an increase in copper number and

alkali solubility.

Action of solvents Textile solvents can be used on viscose rayon without

any deteriorating effect. Viscose rayon dissolves in

cuprammonium hydroxide solution.Contd...




Effect of iron Contact with iron in the form of ferrous hydroxide weakensviscose rayon yarns. Therefore staining, marking or

touching of rayon to iron or iron surface should be avoided.

Action of

microorganisms

Microorganisms (moulds, mildew, fungus, bacteria) affect

the colour, strength, dyeing properties and luster of rayon.

Clean and dry viscose rayon is rarely attacked by moulds

and mildew.

4.4.2 Application of viscose rayon bre

The viscose rayon is mainly used in four sectors namely apparels, home

furnishings, industrial uses and medical applications; (i) Apparel: Accessories,

blouses, dresses, jackets, lingerie, linings, millinery, slacks, sport shirts,

sportswear, suits, ties, work clothes, (ii) Home furnishings: Bedspreads,

blankets, curtains, draperies, sheets, slipcovers, tablecloths, upholstery,

(iii) Industrial uses: Industrial products, medical surgical products, nonwoven

products, tire cord and (iv) Medical applications: Feminine hygiene products.

4.4.3 Enzymatic treatment of viscose fabrics

Danuta Ciechańska et al. (2002) have made an attempt an enzyme-based

process applied in nishing viscose fabrics, which are very susceptible

to pilling. A normal viscose fabric has individual loose bres ends which

protrude from surface, and impurities and fuzzes. A commercial enzyme

of cellulase type Econase CE (Rohm Enzyme Finland) and experimental

cellulases such as endoglucanase II (EGII), cellobiohydrolase I (CBHI) and

cellulase enriched with EGII (Cell. F) from the Trichoderma reesei strains

prepared and were used for modication of viscose-woven fabrics. Viscose

woven fabric was exposed to the action of the enzyme solution in an acetic

buffer at pH 4.8 at temperature of 50°C by the dynamic method in a Linites

machine under the following process conditions.

• The modulus of Econase CE activity (of endo-1,4-glucanase) to the

sample weight (E/S) equaled 100 U/g. The dosage of Econase CE

was 8 mg of total protein per gram of fabric.

• The dosages of experimental enzymes (EGII, CBHI and Cell.F) were

5 mg of total protein per gram of fabric. The time of reaction was 60min. After the treatment, the enzyme solution was ltered, and the

fabrics were washed, rst with hot water and then several times with

cold water.

• The fabrics were dried at a temperature of 20 ± 2°C.

Contd...




4.4.3.1 Analytical test

The content of reducing sugars in cellulase enzyme solutions was assessed by a colorimetric method with dinitrosalicylic acid (DNS) (Miller 1959). The

endo-1,4 glucanase activity of the cellulases was estimated by the colorimetric

method with caroboxy methyl cellulose (CMC) used as a substrate. The

fabric weight loss was assessed by the gravimetric method. The average

polymerization degree (DPv) was estimated by the viscometric method with an

alkali sodium-ferric-tartrate solution (EWNN) (Edelman and Horn 1953). The

cellulose content was determined in a sodium hydroxide solution according to

the Polish Standard. The water retention value (WRV) was estimated by the

gravimetric method. The molecular weight distribution was determined by

gel permeation chromatography. The mechanical properties of fabrics such

as breaking force, breaking force per cm and strain extension were measured

using an Instron 5544 machine in the weft direction. The appearance of the

fabric’s surface before and after enzymatic treatment was examined using a

Jeol JSM-35C (Japan) scanning electron microscope at 100 × magnication.

The results of previous studies on the biomodication of cellulosic

bres such as viscose, Lyocel, and Celsol which were conducted in the

Institute of Chemical Fibres formed the basis for the experiments concerningthe application of cellulases for nishing viscose woven fabrics (Danuta

Ciechańska et al. 2002). Investigations related to the modication of viscose

fabrics were carried out both in the presence of commercial cellualse (Econase

CE) and experimental enzymes (EGII, CBHI and CELL.F) from Trichoderma

reesei strains at 50°C for 60 min with continuous shaking. The course of the

bio-modication process was evaluated on the basis of molecular and morpho

logical characteristics as well as mechanical properties (Cavaco-Paulo et

al. 1996). Some test results of the studies concerned with the estimation of

the effect of cellulases used on the changes of fabric properties (i.e. average

polymerization degree (DPv), water retention value (WRV), weight loss,

breaking force and strain extension) are presented in Tables 4.6 and 4.7

(Ciechańska et al. 2002). Table 4.6 represents properties of viscose woven

fabric after commercial cellulase enzyme treatments and Table 4.7 represents

properties of viscose woven fabric after experimental enzymes treatments

(EGII – endoglucanase, CBHI – cellobiohydrolase, CELL.F – cellulase-

enriched endoglucanase).

On the basis of the test results obtained (Table 4.6), an insignicantdegradation process during the treatment of viscose fabric with Econase CE

was observed, which leads to a decrease in the average polymerisation degree

from DPv=324 for initial fabric to DPv=314 for the biomodied, and also to




an increase in weight loss to 1.4% (Evans et al. 1998; Pyæ et al. 1999). The

puried enzymes EGII and CBHI as well as CELL F which were used for the

treatment of viscose fabric (B) (Table 4.7) caused slight weight loss ranging

from 0.9 to1.3%, and also to an increase in the average polymerization degree,

from DPv = 502 for the initial to DPv = 547 for the modied fabric by CELL.F,

which resulted mainly from the solubilisation of low molecular fractions.

Neither the commercial nor the experimental cellulases used affected the

water retention value of the modied fabrics in comparison to the untreated

ones (Guziñska et al. 2002).

Table 4.6 Properties of viscose-woven fabric after commercial cellulase enzyme

treatments

Type of

fabric

Enzyme Weight

loss

( %)

DP WRV

(%)

Mechanical properties

Breaking

force

(daN)

Breaking

force per

cm (daN/

cm)

Strain

extension

(%)

Viscose

fabric – A

(untreated)

– – 324 80.3 19.5 7.8 36.5

Viscose

fabric – A

(treated)

Econase

CE1.35 314 79.4 16.3 6.5 41.8

Table 4.7 Properties of viscose-woven fabric after experimental enzymes treatments.

Type of

fabric

Enzyme Weight

loss

(%)

DP WRV

(%)

Mechanical properties

Breaking

force

(daN)

Breaking

force per

cm (daN/

cm)

Strain

extension

(%)

Viscose

fabric – B

(untreated)

– – 502 50.3 31.8 12.7 13.6

Viscose

fabric – B

(treated)

EG II 0.87 509 50.9 23.7 9.5 11.6

CBHI 0.90 519 50.4 30.4 12.2 13.7

CELL.F 1.32 547 49.6 27.9 11.2 13.8

The practical application of cellulase enzymes for viscose bre/fabric

modication is strongly connected with estimating the changes in mechanical properties occurring during treatment. On the basis of the results presented

(Table 4.8). It was observed that the breaking force of modied viscose fabric

(A) dropped by about 17%, whereas the breaking force of viscose fabric

(B) decreased by 25% with EGII, 4% with CBHI and 12% with CELL F in




comparison to the initial. The strain extension of viscose fabric (A) increased

by about 15% after Econase CE treatment, and that of viscose fabric (B)

decreased by about 15% with EGII, and was permanent with CBHI and

CELL. F.

Table 4.8 Molecular characteristic of modied viscose fabric after experimental

enzymes treatments

Type of

fabric

Type of

enzyme

Molecular characteristics

Mn ×

103

Mw ×

103

Pd DPw Percentage of DP fraction

200> 200–

550

>550

Viscose

fabric – B

(untreated)

– 22.3 71.3 3.2 440 41 35 24

Viscose

fabric – B

(treated)

EG II 26.5 73.7 2.8 455 36 37 27

CBHI 22.1 66.2 2.9 409 42 35 23

CELL.F 28.2 71.2 2.5 440 37 38 25

To determine the structural changes of fabrics which occurred as a result of

cellulase action, the molecular characteristic of initial and biomodied viscose

fabrics was estimated by gel permeation chromatography (GPC). Within the

studies of the biomodication process, the surface changes of viscose-woven

fabrics treated with both commercial and experimental cellulases was evaluated

using scanning electron microscopy (SEM). On the basis of the results, it can be

noted that the surface of initial viscose fabric (A) is characterized by the presence

of protruding individual bre ends and impurities, which must be removed in

order to improve the fabric quality (Cavaco-Paulo et al. 1998). Application of

cellulase type Econase CE to treat the viscose fabric cleanses its surface by

removing the individual bres as well as impurities and fuzzes. When the puried cellulases (EGII, CBHI and CELL.F) are applied, no signicant changes

in the microtopography of modied viscose fabric (B) are observed (Fig. 4.14).

To summarise, it can be concluded that for the experimental cellulases used,

it is necessary to optimize the parameters of the enzymatic process in order to

improve the effect of cleaning and smoothing of woven fabrics (Morgado and

Cavaco-Paulo 2000; Hardin et al. 1998).

4.4.4 Cellulase enzyme kinetics on viscose, lyocell andmodal

Carrillo et al. (2003) have made attempt to analyze the cellulase enzyme kinetics

on lyocell, viscose and modal bres of 1.7 dtex. The bres were washed in a

bath containing 1 g/litre of a nonionic surfactant with a 12:1 liquor ratio for




60 min. The fabrics (yarns of 56.8 tex in both warp and weft directions, plain

weave construction and weight of 283 g/m2) made with the staple bres of 1.7

dtex (average length of 38 mm), were subjected to the industrial process of

brillation by mechanical action in a jet machine. Fibrillation treatments were

carried out for 90 min at a temperature of 100°C and liquor to fabric ratio

of 10:1. The pH of the liquor was adjusted to 10 with Na2CO

3. Samples of

brillated lyocell were removed from the fabric for the enzymic treatment.

(a)

(c)

(b)

(d)

Fig. 4.14 SEM view of (a) normal viscose fabric; and enzyme modied by (b)

Endoglucanase EG II, (c) Celobiohydrolase CBH I, (d) Cellulase Cell. F. [Source: Danuta

Ciechańska et al. 2002]

Microorganism: Enzyme used was a commercial product of liquid acidic

cellulase produced by submerged fermentation of a nonpathogenic fungus. The

product was standardized to a declared activity of 400 EGU/g. determined by

a Novo Nordisk test method, AF 275. The experimental complex activity was

428.8 FPU/g of crude enzyme measured by the lter paper method (FP) as

described (Mandel et al. 1976). Enzymic hydrolysis involved 2.5 g of bre at

a temperature of 50°C and a liquor ratio of 80:1 (v/w). The enzyme hydrolysis

was carried out in a thermo stated reactor, with mechanical stirring (300 rpm) by using 0.05 M acetate buffer (pH 6). Initial enzyme concentrations of 6,

12.5 and 25 g/l were tested for different hydrolysis times (15, 30, 60 and 155

min). Finally the enzymes were deactivated by increasing the temperature to

80°C for 15 min.




Testing: Sample solutions were removed at timed intervals and

the concentration of reducing sugars as glucose was determined by a

dinitrosalycilic acid method (Miller 1959) using glucose for calibration. From

this determination the rate of bre hydrolysis were calculated. The adsorption

of cellulase onto the bres during treatment was determined by means of the

loss of protein solution, using the Coomassie Brilliant Blue G-250 adsorption

method (Bradford et al. 1976). The approach relating the initial hydrolysis

rate (V0, g/l h) and the initial enzyme concentration ([E

0], g/l) was used to

calculate the rate of substrate turnover at saturation with enzyme (Vem) and

the half-saturation constant (Ke) by linearization of the experimental results

in equation [4.1].

Vem

[E0] =

Vem

[E0]

K e + [E

0]

[4.1]

Where V0 (g/l h) is the initial reaction rate, [E

0] (g/l) is the initial enzyme

concentration, Vem (g/l h) is the maximum rate of reaction at saturation with

enzyme and K e (g/l) is the half saturation constant relative to the substrate

sites. Adsorption of cellulase enzyme protein by regenerated cellulose bres

from a 12.5 g/l solution of a cellulase complex at pH 6.0, 50°C, liquor-to-

bre ratio 80:1 onto the modal, lyocell and viscose type bres is illustrated(Fig. 4.15). The initially rapid adsorption over the rst 30–60 min was

followed by a continuous slow decrease, which is believed to be due to a new

cellulose surface created as hydrolysis of the bres proceeded.

Fig. 4.15 Adsorption of cellulase enzyme protein by regenerated

cellulose bres [Source: Carrillo et al. 2003]




The maximum adsorption value was observed for a viscose rayon which

had the lower degree of crystallinity and amorphous orientation (Colom

and Carrillo 2002) and adsorption decreased in the following order: modal,

brillated lyocell and lyocell. Figure 4.16 shows the brillation treatment of

lyocel bres by cellulase which improved cellulase adsorption probably due

to an increase in accessibility produced by the formation of brils along the

surface of the bres.

Fig. 4.16 Typical SEM micrographs of lyocell bres: untreated (left) and brillated

(right) [Source: Carrillo et al. 2003]

The kinetics of various regenerated bres hydrolysis by a cellulase

enzyme complex ([E0] treated with/12.5 g/l, at pH 6, 50°C, liquor-to-bre

ratio 80:1 was studied and the corresponding rate of production of soluble

reducing sugars with an initial enzyme concentration of 12.5 g/l (Fig. 4.17)for the different substrates. Comparing regenerated celluloses, the viscose

bres showed maximal hydrolysis with great differences mainly at extended

hydrolysis times, where the adsorption step was less important. Modal bres

had a similar behavior to lyocell bres, probably due to their high molecular

orientation (Fink et al. 2001), although showing a higher degree of hydrolysis

than the more crystalline lyocell bres. Treated lyocell bres showed

higher enzyme hydrolysis than the original (Walker and Wilson 1991). This

difference can be explained by the damaged bres on the surface and a more

open structure of brillated lyocell bres (Fig. 4.16).

Table 4.9 shows the effect of initial enzyme concentration on the degree

of saccharication of viscose, modal and treated and untreated lyocell bres

treated at 500°C, pH 6 and liquor to bre ratio80:1 and 155 min.




Fig. 4.17 Kinetics of various regenerated bres hydrolysis by a cellulase enzyme

complex [Source: Carrillo et al. 2003]

Table 4.9 Degree of hydrolysis % of regenerated cellulosic bres by cellulase enzyme

Cellulase

concentration [Eo]

Viscose Modal Lyocell

fbrillated

Lyocell

6 g/l 6.6 3.3 1.7 1.6

12.5 g/l 10.7 4.6 3.1 2.7

25 g/l 14.3 6.0 6.2 5.0

Figure 4.18 shows the relationship between maximum hydrolysis rateand enzyme concentration on the regenerated cellulosic bres. The rate of

hydrolysis increased with increasing initial enzyme concentration. It was not

proportional to the enzyme concentration due to the saturation of the catalytic

reaction surface (Eginer et al. 1985). From linearization of these curves, by

application of Eq. (1), kinetics parameters Vem and Ke have been determined

and the catalytic specicity of the enzyme has been calculated. The values

obtained are shown in Table 4.10. The half saturation constant Ke can be

interpreted as an apparent dissociation constant of the entire enzyme bound

species. This dissociation constant is higher for the viscose bre, comparing

untreated bres, because they have a more accessible cellulose substrate

(Colom and Carrillo 2002). This indicates that the concentration of the

enzyme/substrate complex (ES) at the equilibrium state is not high so enzyme




concentration is greater in the non-associated form (low enzyme afnity for

substrate) and is ready for the next catalysis reaction. Modal samples show

kinetic parameters more closely similar to those of untreated lyocell than to

viscose bres, probably due to the high amorphous orientation structure of

modal and lyocell bres (Lenz et al. 1992).

Fig. 4.18 Relationship between maximum hydrolysis rate and enzyme concentration

for regenerated cellulose bres [Source: Carrillo et al. 2003]

Table 4.10 Cellulase enzyme kinetics on various bres

Enzyme kinetics Viscose Modal Lyocell

fbrillated

Lyocell

Vem

(g/l h) 1.20 0.56 0.55 0.41

Ke (g/l) 12.7 7.63 13.29 8.79

Vem

/Kc (per h) 0.094 0.074 0.042 0.046

Lyocell bres present a low value of half saturation constant (K e),

although after the brillation treatment a signicant increase of K e was

observed because they have a more accessible cellulose structure. This changemeans that brillation treatment of lyocell bres produces a more synergistic

cellulase action by increasing the dissociation of the enzyme bound species,

leaving free enzyme for catalysis (Valldeperas et al. 2000). Moreover, the

brillation effect produces little increase of the maximum rate of hydrolysis




for lyocell, although the rate continues being lower than for viscose and modal

bres. The untreated and brillated lyocell bres have the lowest catalytic

specicity (Table 4.10) by the Vem/Ke relationship. This relationship is

directly correlated to the maximum rate of hydrolysis respect of the enzyme

afnity for substrate (related to 1/ke). The higher values for viscose suggest

that enzyme attack at the ends of viscose cellulose chain is a more specic

catalysed reaction than random cellulose scission on lyocell bres.

From the adsorption experiments results show low cellulase adsorption

on lyocell bres due to the higher orientation and crystallinity of these

regenerated cellulosic bres and ahead of viscose or modal bres (Baley

1989). Moreover, brillated lyocell bres have more binding sites on the bresurface, increasing cellulase adsorption. The kinetics of the enzymic hydrolysis

of lyocell and viscose type bres can be accurately described by steady-state

Eq. [4.1], which includes two parameters providing important mechanistic

information about cellulase hydrolysis. The morphology and structure

(crystallinity and orientation) of the different regenerated bres studied lead

to different rates of cellulosic degradation suitable for an industrial process of

cellulose conversion.

Cotton, linen, ramie, and viscose rayon fabrics along with a cotton/linen

blend were hydrolyzed with cellulase from Trichoderma viride (Buschle-Diller et al. 1994). Surface brils were eliminated by a 6-hour treatment in all

cases. The loss of brillar matter appeared to be the primary cause of weight

loss at this stage. On prolonged treatment, cotton, linen, and viscose rayon

lost weight at a faster rate than ramie and the cotton / linen blend. From the

test analysis, the fall in yam strength was progressive with increasing weight

loss for cotton and viscose, while for linen and ramie it was slight initially and

then increased sharply. Retention of strength after 48 hours’ incubation time

increased in the order viscose rayon << cotton < ramie < linen, whereas weightloss increased in the order ramie < linen < cotton < viscose rayon. X-ray

crystallinity and moisture sorption of the samples did not change after the

treatment, indicating that the mechanism of endwise attack of the cellulase at

accessible cellulose chains on crystallite surfaces appeared to apply to all four

bres. Shen et al. (2004) also studied the explicit form of the kinetic equation

for the enzymatic hydrolysis of cellulose bres with SF enzyme is derived.

This equation is effective for estimating and controlling hydrolysis reactions

of cellulose with cellulase. With this equation, it is possible to calculate the

maximum digestibility and the half maximum digestion constant. Under thesame experimental conditions, the maximum digestibility of bres treated

with SF cellulase decreases in the order viscose rayon > cotton > ax, whereas

the concentration of cellulase needed to digest the same fraction of hydrolyzed




cellulose decreases in the order ax > cotton > viscose rayon. The enzymatic

hydrolysis rate of bres is greatly affected by cellulase concentration. The

results show that the rate is proportional to cellulase concentration at a lower

level, while at too high a concentration, the rate increases very little with

increased concentration. Thus, it is not rational to select too high a cellulase

concentration for the process.

4.4.5 Wash down effect by cellulase enzyme

The study was made attempt to analyze the effects of cellulase enzymes on the

physical and aesthetic properties of viscose rayon and Tencel materials both

dyed and unnished fabrics forms which were subjected to enzymatic treatment

in a Unimac garment washing machine. The trials were conducted with three

different acid-stable cellulase enzymes at medium and high activities for 60

and 120 min. The fabrics were then analyzed for improvements in aesthetics

and deterioration in physical properties. Aesthetics of viscose rayon fabrics

were assessed using subjective hand evaluations color loss data and surface

appearance. Physical properties, after enzyme treatment, were evaluated

using weight loss, tensile strength, and tearing strength data. The viscose

rayon fabric appearance was affected by enzymatic treatment, but little tensilestrength deterioration occurred after cellulase enzyme treatment at various

concentration levels. The viscose and Tencel fabric acquired a “washed down”

look typical of denim fabric. This look might be desirable for casual garments.

The softness of these fabrics, however, was not drastically improved after

bioprocessing with cellulase. The appearance and physical properties of the

Tencel fabric were greatly changed by enzymatic treatment when compared to

viscose rayon. Substantial tensile strength losses of up to 50% were obtained;

however, the treatment dramatically improved the brillation tendency of

the Tencel fabric. The most improved appearance was obtained on fabric

that lost half its strength. Enzymatic treatment on Tencel could prove very

valuable if the proper compromise can be made between strength properties

and aesthetics with various concentrations of cellulase enzyme treatments.

4.5 Biodegradability of plastics

Plastic is a broad name given to different polymers with high molecular

weight, which can be degraded by various processes. However, consideringtheir abundance in the environment and their specicity in attacking plastics,

biodegradation of plastics by microorganisms and enzymes seems to be the

most effective process (Tokiwa 2009). With the advances in technology

and the increase in the global population, plastic materials have found wide




applications in every aspect of life and industries. However, most conventional

plastics such as polyethylene, polypropylene, polystyrene, poly(vinyl

chloride) and poly(ethylene terephthalate), are non biodegradable, and their

increasing accumulation in the environment has been a threat to the planet

(Chandra and Rustgi 1998). The rst strategy involved production of plastics

with high degree of degradability. Bio-plastics consist of either biodegradable

plastics (i.e., plastics produced from fossil materials) or bio-based plastics

(i.e., plastics synthesized from biomass or renewable resources). Figure 4.19

shows the inter-relationship between biodegradable plastics and bio-based

plastics.

Fig. 4.19 Bio-plastics comprised of biodegradable plastics and bio-based plastics

[Source: Yutaka Tokiwa et al. 2009]

Polycaprolactone (PCL) and polybutylene succinate (PBS) are petroleum

based, but they can be degraded by microorganisms. On the other hand,

poly(hydroxybutyrate) (PHB), poly(lactide) (PLA) and starch blends are

produced from biomass or renewable resources, and are thus biodegradable.Despite the fact that polyethylene (PE) and Nylon 11 (NY11) can be produced

from biomass or renewable resources, they are non-biodegradable. Acetyl

cellulose (AcC) is either biodegradable or non-biodegradable, depending

on the degree of acetylation. Biodegradable plastics are seen by many as a

promising solution to this problem because they are environment-friendly.

They can be derived from renewable feed stocks, thereby reducing greenhouse

gas emissions. For instance, polyhydroxyalkanoates (PHA) and lactic acid

(raw materials for PLA) can be produced by fermentative biotechnological

processes using agricultural products and microorganisms (Wang and Lee

1997; Tokiwa and Ugwu 2007; Tokiwa and Calabia 2008). Furthermore,

biodegradable plastics can be recycled to useful metabolites (monomers and

oligomers) by microorganisms and enzymes. A typical example can be seen




in the case of some aliphatic polyester such as PCL and PBS that can be

degraded with enzymes and microorganisms (Tokiwa et al. 1976; Tokiwa

1977a; Tokiwa and Suzuki 1977b).

4.5.1 Polymer degrading microorganisms

The enzymatic degradation of plastics by hydrolysis is a two-step process:

rst, the enzyme binds to the polymer substrate then subsequently catalyzes

a hydrolytic cleavage. Polymers are degraded into low molecular weight

oligomers, dimers and monomers and nally mineralized to CO2 and H

2O.The

clear zone method with agar plates is a widely used technique for screening

polymer degraders and for assessment of the degradation potential of different

microorganisms towards a polymer. Agar plates containing emulsied

polymers are inoculated with microorganisms and the presence of polymer

degrading microorganisms can be conrmed by the formation of clear halo

zones around the colonies.

4.5.2 Factors affecting the biodegradability of polymers

materials

The properties of polymers are associated with their biodegradability. Both

the chemical and physical properties of polymers inuence the mechanism

of biodegradation. The bre polymer surface conditions (surface area,

hydrophilic, and hydrophobic properties), the rst order structures (chemical

structure, molecular weight and molecular weight distribution) and the high

order structures (glass transition temperature, melting temperature, modulus

of elasticity, crystallinity and crystal structure) of polymers play important

roles in the biodegradation processes.

In general, polyesters with side chains are less assimilated than those

without side chains (Tokiwa et al. 1976). The molecular weight is also

important for the biodegradability because it determines many physical

properties of the polymer. Increasing the molecular weight of the polymer

decreased its degradability. PCL with higher molecular weight (Mn > 4,000)

was degraded slowly by Rhizopus delemar lipase (endo-cleavage type) than

that with low Mn (Tokiwa and Suzuki 1978; Iwata and Doi 1998). Moreover,

the morphology of polymers greatly affects their rates of biodegradation.

The degree of crystallinity is a crucial factor affecting biodegradability,since enzymes mainly attack the amorphous domains of a polymer. The

molecules in the amorphous region are loosely packed, and thus make

it more susceptible to degradation. The crystalline part of the polymers is

more resistant than the amorphous region. The rate of degradation of PLA




decreases with an increase in crystallinity of the polymer (Tsuji and Miyauchi

2001). The melting temperature (Tm) of polyesters has a strong effect on the

enzymatic degradation of polymers (Fig. 4.20). The higher the Tm, the lower

the biodegradation of the polymer (Tokiwa et al. 1979; Tokiwa and Suzuki

1981). In general, Tm is represented by the following Eq. [4.2]:

Tm = ΔH/ΔS [4.2]

Fig. 4.20 Relationship between Tm and biodegradability of polyesters by r. arrhizus

lipase [Source: Yutaka Tokiwa et al. 2009]

where ΔH was the change of enthalpy in melting and ΔS is the change

of entropy in melting. It is well known that the interactions among polymerchains mainly affect the ΔH value and that the internal rotation energies

corresponding to the rigidity (the exibility) of the polymer molecule

remarkably affect the ΔS value. The chemical structures of aliphatic polyester,

polycarbonate, polyurethane and polyamides, together with their (Tm)s are

listed in Table 4.11. The aliphatic polyesters [ester bond (-CO-O-)] and

polycarbonates [carbonate bond (-O-CO-O-)] are two typical plastic polymers

that show high potential for use as biodegradable plastics, owing to their

susceptibilities to lipolytic enzymes and microbial degradation. Compared

with aliphatic polyesters and polycarbonates, aliphatic polyurethane and

polyamides (nylon) have higher Tm values. The high (Tm)s of polyurethane

and polyamide (nylon) are caused by the large ΔH value due to the presence

of hydrogen bonds among polymer chains based on the urethane bond (-NH-




mesophilic temperatures and very few of them were capable of degrading

PHB at higher temperature (Tokiwa et al. 1988; Liu et al. 2000). Tokiwa et

al. (1992) have emphasized that composting at high temperature is one of

the most promising technologies for recycling biodegradable plastics and

thermophilic microorganisms that could degrade polymers play an important

role in the composting process. Thus, microorganisms that are capable of

degrading various kinds of polyesters at high temperatures are of interest. A

thermophilic Streptomyces sp. Isolated from soil can degrade not only PHB

but also PES, PBS. This actinomycete has higher PHB-degrading activity

than thermotolerant and thermophilic Streptomyces strains from culture

collections (Calabia and Tokiwa 2004). A thermotolerant Aspergillus sp. wasable to degrade 90% of PHB lm after ve days cultivation at 50°C (Sanchez

and Tsuchii 2000). Furthermore, several thermophilic polyester degrading

actinomycetes were isolated from different ecosystems. Out of 341 strains, 31

isolates were PHB, PCL and PES degraders and these isolates were identied

as members of the genus Actinomadura, Microbispora, Streptomyces,

Thermoactinomyces and Saccharomonospora (Tseng et al. 2007).

4.5.3.2 Poly (Lactic Acid) (PLA)

PLA ([-O(CH3)CHCO-]n) is a biodegradable and biocompatible thermoplastic

which can be produced by fermentation from renewable resources (Jang et al.

2007). It can also be synthesized either by condensation polymerization of

lactic acid or by ring opening polymerization of lactide in the presence of

a catalyst. This polymer exists in the form of three stereoisomers: poly(L-

lactide) (L-PLA), poly(D-lactide) (D-PLA) and poly(DL-lactide) (DL-PLA).

The manufacture of PLA from lactic acid was pioneered by Carothers in 1932

(Carothers and Hill 1932). Ecological studies on the abundance of PLA-

degrading microorganisms in different environments have conrmed that

PLA-degraders are not widely distributed, and thus it is less susceptible to

microbial attack compared to other microbial and synthetic aliphatic polymers

(Tansengco and Tokiwa 1998). The degradation of PLA in soil is slow and that

takes a long time for degradation to start (Uruyama et al. 2002; Ohkita and

Lee 2006). Since then, a number of research studies dealing with microbial

and enzymatic degradation of PLA have been published (Tokiwa and Calabia

2006). Many strains of genus Amycolatopsis and Saccharotrix were able

to degrade both PLA and silk broin. The main amino acid constituents ofsilk broin are L-alanine and glycine and there is a similarity between the

stereochemical position of the chiral carbon of L-lactic acid unit of PLA and

L-alanine unit in the silk broin. Silk broin is one of the natural analogues

of poly(L-lactide), thus, the PLA degrading microorganisms may probably




identify the L-lactate unit as an analogue of L-alanine unit in silk broin.

Several proteinous materials such as silk broin, elastin, gelatin and some

peptides and amino acids were found to stimulate the production of enzymes

from PLA-degrading microorganisms (Pranamuda et al. 2001; Jarerat and

Tokiwa 2001, 2003a, 2003b, 2004). Williams (1981) has investigated the

enzymatic degradation of PLA using proteinase K, bromelain and pronase.

Among these enzymes, proteinase K from Tritirachium album was the most

effective for PLA degradation. Proteinase K and other serine proteases are

capable of degrading L-PLA and DL-PLA but not D-PLA. Furthermore,

proteinase K preferentially hydrolyzes the amorphous part of L-PLA and the

rate of degradation decreases with an increase in the crystalline part (Reeveand McCarthy 1994; McDonald et al. 1996). Fukuzaki et al. (1989) reported

that the degradation of PLA oligomers was accelerated by several esterase-type

enzymes, especially Rhizopus delemar lipase. The puried PLA depolymerase

from Amycolatopsis sp. was also capable of degrading casein, silk broin,

Suc-(Ala)3-pNA but not PCL, PHB and Suc-(Gly)3-pNA (Pranamuda et al.

2001). Their studies showed that PLA depolymerase was a kind of protease

and not a lipase. It was reported that α-chymotrypsin can degrade PLA and

PEA with lower activity on poly (butylenes succinate-co-adipate) (PBS/A).

Moreover, several serine proteases such as trypsin, elastase, and subtilisinwere able to hydrolyze L-PLA (Lim 1995).

4.5.3.3 Polyethylene (PE)

PE is a stable polymer, and consists of long chains of ethylene monomers. PE

cannot be easily degraded with microorganisms. However, it was reported that

lower molecular weight PE oligomers ( Mw = 600–800) was partially degraded

by Acinetobacter sp. 351 upon dispersion, while high molecular weight PE

could not be degraded (Tsuchii et al. 1980). Furthermore, the biodegradability

of low density PE/starch blends was enhanced with compatibilizer (Biliaris

and Panayiotou 1998). Biodegradability of PE can also be improved by

blending it with biodegradable additives, photo-initiators or copolymerization

(Grifn 2007; Hakkarainen and Albertsson 2004). The initial concept of

blending PE with starch was established in UK to produce paper-like PE bag.

A few years later, the idea to blend PE with starch and photoinitiators was

conceived as a way of saving petroleum, though its biodegradability was also

taken into account. Environmental degradation of PE proceeds by synergisticaction of photo-and thermo-oxidative degradation and biological activity (i.e.,

microorganisms). When PE is subjected to thermo- and photo-oxidization,

various products such as alkanes, alkenes, ketones, aldehydes, alcohols,

carboxylic acid, keto-acids, dicarboxylic acids, lactones and esters are




released. Blending of PE with additives generally enhances auto-oxidation,

reduces the molecular weight of the polymer and then makes it easier for

microorganisms to degrade the low molecular weight materials. It is worthy

to note that despite all these attempts to enhance the biodegradation of PE

blends, the biodegradability with microorganisms on the PE part of the blends

is still very low.

4.5.3.4 Polypropylene (PP)

PP is a thermoplastic which is commonly used for plastic moldings, stationary

folders, packaging materials, plastic tubs, non-absorbable sutures, diapers

etc (Iwamoto and Tokiwa 1994). PP can be degraded when it is exposed to

ultraviolet radiation from sunlight. Furthermore, at high temperatures, PP

is oxidized. The possibility of degrading PP with microorganisms has been

investigated (Cacciari et al. 1993).

4.5.3.5 Polystyrene (PS)

PS is a synthetic hydrophobic polymer with high molecular weight. PS is

recyclable but not biodegradable. Although it was reported that PS lm was biodegraded with an Actinomycete strain, the degree of biodegradation was

very low (Mor and Silvan 2008). At room temperature, PS exists in solid

state. When it is heated above its glass transition temperature, it ows and

then turns back to solid upon cooling. PS being a transparent hard plastics

is commonly used as disposable cutleries, cups, plastic models, packing and

insulation materials.

4.5.4 Future prospects

Biodegradable polymer is an innovative means of solving the plastic disposal

problem from the standpoint of development of new materials. In general,

plastics are water-insoluble, thermo-elastic polymeric materials (Witt et al.

1995; Abou-Zeid et al. 2001). Biodegradability of plastics is affected by

both their chemical and physical properties. Beside the covalent forces of

polymer molecules, various kinds of weak forces (i.e., hydrogen bond forces,

van der Waals forces, coulombic forces, etc.) among macromolecular chains

affect not only the formation of polymer aggregates, but also the structure

and physical properties and function (reactivity) of the polymer aggregates.

The biodegradation mechanisms of plastics as shown in this review can be

applied to biomasses that are composed of polymeric materials (i.e., cellulose,

hemicellulose, lignin, chitin, silk broin, etc.). Lipolytic enzymes such as




lipase and esterase can hydrolyze not only fatty acid esters and triglycerides,

but also aliphatic polyesters. Lipolytic enzyme has an important role in the

degradation of natural aliphatic polyesters such as cutin, suberin and esteroid

in the natural environment and animal digestive tract. However, it is not

certain whether human body produces any aliphatic polyester or not.

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Abstract: This chapter discusses cotton textile processing and methods of treatingwaste water efuent in the textile industries both present and future scenario.

Industrial textile wet processing comprises various operations includes desizing,scouring, bleaching, dyeing, printing, and nishing operations. Effective efuenttreatment is an important step towards conserving water resources. The use ofenzymes is an alternative method for treatment of such recalcitrant pollutants.It evaluates different methods in which enzymes can be delivered to the targetefuent, including nanoparticles as delivery systems. It also emphasizes theneed for current and future research to focus on developing economically feasibleand environmentally sustainable wastewater treatment practices. Consumersin developed countries are demanding biodegradable and ecologically friendlytextiles. The chapter then discusses the application of enzymes in decolourationof dye house efuent water treatment using white rot fungi and laccase enzymes

studied by many researchers and scientists in the efuent treatments. Theconuence of nanoscience and enzyme technology has resulted in an upcominginterdisciplinary approach to wastewater treatment. Such innovative applicationsof enzymes can enable the utilization of these biocatalysts to their maximumpotential.

Keywords: Dye waste water, efuent treatment, decolouration, whote rot fungi,laccase, enzyme technology, biotransformation

5.1 Introduction

Cotton provides an ecologically friendly textile, but more than 50% of its

production volume is dyed with reactive dyes (Chavan 2001). Today the

main challenge for the textile industry is to modify production methods,

and move towards more eco friendly at a competitive market price, by using

safer dyes and chemicals and by reducing cost of efuent treatment/disposal.

Dyes are unfavorable from an ecological point of view, because the efuents

generated are heavily colored, which contain high concentrations of salts,

and high biological oxygen demand/chemical oxygen demand (BOD/COD)

values. In dyeing textiles, ecological standards are strictly applied throughout

processing from raw material selection to the nal apparel product. This has

become more critical since the German environmental standards regarding

dye efuents became effective (Robinson et al. 1997). Clean technology,

5

Enzymes in textile efuents




eco-mark and green chemistry are some of the most highlighted practices

in preventing and or reducing the adverse effect on environment both land

and air. Due to the nature of various chemical processing of textiles, large

volumes of wastewater with numerous pollutants are discharged. Since

the stream of water affect the aquatic eco-system in number of ways such

as depleting the dissolved oxygen content or settlement of suspended

substances in anaerobic condition, a special attention needs to be demanded.

Thus, a study on different measures, which can be adopted to treat the

wastewater discharged from textile chemical processing industries to protect

environment from possible pollution problem, has been the focus point of

many recent investigations (Seshadri et al. 1994). These wet processingoperations not only consume large amounts of energy and water, but they

also produce substantial waste products. The waste production from textile

processes, such as desizing, mercerizing, bleaching, dyeing, nishing, and

printing, with a discussion of advanced methods of efuent treatment, such

as electro-oxidation, bio-treatment, photochemical, and membrane processes

have been discussed. These dyeing industries consume large quantities of

water and produces large volumes of waste water from different steps in the

dyeing and nishing processes. Wastewater from printing and dyeing unitsis often rich in color, containing residues of reactive dyes and chemicals,

and requires proper treatment before being released into the environment

(Sivaramakrishnan 2004). The toxic effects of dyestuffs and other organic

compounds, as well as acidic and alkaline contaminants, from industrial

establishments on the general public are widely accepted. Increasing public

concern about environmental issues has led to closure of several small-

scale industries. Interest in ecologically friendly, wet-processing textile

techniques has increased in recent years because of increased awareness ofenvironmental issues throughout the world.

5.2 Textile processing operations

The process of converting raw bers into nished apparel and non-apparel

textile products is complex, so most textile mills specialize their own based

on their product manufacturing (Hashem et al. 2005). Textiles generally go

through three or four stages of production that may include yarn formation,

fabric formation, wet processing, and textile fabrication. Figure 5.1 shows

some of the steps in processing of textile bers into fabrics both cotton and

synthetics.




from the cotton and chemical residuals from previous processing. Natural

impurities include waxes, oils, proteins, mineral matter and residuals seeds.

The cotton contains a signicant amount of contaminants resulting from the

widespread use of fertilizers, insecticides and fungicides. Previous knitting

or weaving processes leave residuals of knitting oils and sizing chemicals

on the surface of the cotton bers. All these impurities must be removed

before dyeing, because they can interfere with the dyeing process. Insufcient

preparation can result in an uneven dyeing, can cause spotting or can even

damage the fabric permanently. The auxiliary chemicals used in textile wet

processing are given in Table 5.1.

Fig. 5.2 Processing steps for the conventional and enzymatic pre-treatment of cotton

Table 5.1 Auxiliary chemicals used in textile wet processing (Correia, 1994)

Description Composition Function Processing step

Salts Sodium chloride

Sodium sulphate

Neutralize zeta

potential of the ber,

retarder

Dyeing

Acids Acetic acid

Sulfuric acid

pH control Preparation,

dyeing, nishing

Bases Sodium hydroxide

Sodium carbonate

pH control Preparation,

dyeing, nishing

Buffers Phosphate pH control Dyeing

Sequestering

agents (Chelators)

EDTA Complex hardness Preparation

Dyeing

Surface active

agents

Anionic, cationic

and

non-ionic

Softeners

Emulsiers

Dyeing

Finishing

Contd...



Enzymes in textile efuents 255

Description Composition Function Processing step

Oxidising agents Hydrogen peroxide Insoluble dyes Dyeing

Reducing agents Sodium

hydrosulphite

Sodium sulphide

Soluble dyes

Remove unreacted

dyes

Dyeing

Carriers Phenyl phenols Enhance absorption Dyeing

5.2.1.1 Sizing

During sizing, chemicals are applied to the yam before the production of awoven fabric. Substances such as starch, polyvinyl alcohol (PVA), polyvinyl

acetate, carboxymethyl cellulose (CMC) and gums were used to enhance the

tensile strength and smoothness of the warp yarn.

5.2.1.2 Singeing

Singeing is a process that removes surface bers from textile fabric. These

surface bers form small ber balls on the cloth after being washed several

times. Many different systems are available but usually the goods pass through

gas-red burners at high speed.

5.2.1.3 Desizing

After the weaving process, the sizes have to be removed from the fabric because

they interfere with subsequent processing steps. Sizes have, in general, a high

biological oxygen demand (BOD) and will contribute signicantly to the

waste load of the mill’s efuent. The waste stream of the desizing operationcan contribute up to 50% of the total pollution load of a mill’s wastewater.

Three methods frequently used in textile processing are acid desizing, enzyme

desizing, and oxidative desizing. The goal of these different methods is to

hydrolyze the starch.

5.2.1.4 Scouring

Scouring is typically performed in an alkaline solution and high temperature

environment. The removal of natural impurities is based upon saponicationat high pH. Soaps and detergents added during scouring may precipitate

with calcium, magnesium and iron (3+) if present. The removal of natural

impurities can be done in a single process or can be combined with desizing

and/or bleaching.

Contd...




Dyes most commonly applied to cotton are reactive and direct dyes. Cotton/

polyester goods are dyed using reactive or direct dyes for the cotton portion

of the fabric and disperse dyes for the polyester. Several auxiliary chemicals

are added to the bath during the dyeing processes. These chemicals can be

divided into two groups: commodity chemicals and specialty chemicals.

Specialty chemicals are mixtures which have an unknown composition due to

proprietary information. The mixtures are often developed to solve problems

specic to the process.

5.2.1.8 Finishing

Finishing operations may change the properties of the textile fabric or yarn.

They can increase the softness, luster, and durability of textiles. Finishing can

also improve the water repelling and ame resistant properties of the fabric.

The characteristics of textiles can be altered by physical techniques (dry

nishing processes) or by application of chemicals (wet nishing processes).

Luster can be added by both physical and chemical methods. Characteristics

like ame or water repellency can only be obtained by wet nishing.

Table 5.2 Waste materials generated during cotton textile wet processing

Process Waste water Residual wastes

Fiber preparation Little or no wastewater

generated

Fiber waste; packaging

waste; hard waste

Yarn spinning Little or no wastewater

generated

Packaging waste; sized yarn;

ber waste; cleaning and

processing waste

Slashing/Sizing BOD; COD; metals; cleaning

waste and size

Fiber lint; yarn waste;

packaging waste

Weaving Little or no air emissionsgenerated

Packaging waste; yarn andfabric scraps; used oil

Knitting Little or no air emissions

generated

Packaging waste; yarn and

fabric scraps

Desizing BOD from water-soluble size;

synthetic size; lubricants;

biocides; anti-static

compounds

Packaging waste; ber lint;

yarn waste; cleaning materials

Scouring Disinfectants and insecticide

residue; NaOH; detergents;

fats; oils; pectin; wax; knitting

lubricants; spin nishes; spent

solvents

Little or no residual waste

generated

Bleaching Hydrogen peroxide; Sodium

silicate; organic stabilizer


generated

Contd...




Process Waste water Residual wastesSingeing Little or no wastewater

generated


generated

Mercerizing High pH; NaOH Little or no residual waste

generated

Heat setting Little or no wastewater

generated


generated

Dyeing Metals; Salt; surfactants;

toxic; organic processing

assistance; cationic materials;

color; BOD; Sulde; acidity;

alkalinity; spent solvents


generated

Printing Suspended solids; urea;

solvents; color; metals; heat;

BOD; foam

Little or printing gum and

thickener

Finishing BOD; COD; Suspended

solids; toxics; spent solvents

Fabric scraps and trimmings;

packaging waste

5.3 Textile efuent characteristicsIn the textile wet processing for natural and synthetic material operations,

lists of some wastes that may be generated at each level of textile processing

are given (Table 5.2). Wet processing of textiles involves unit operations

such as desizing, scouring, bleaching, dyeing and nishing. Different

auxiliaries are used either in solid or in liquid form to the textile product

to obtain the desired fabric effect. Cotton textiles cannot be dyed evenly

without removing its natural and added impurities, which inhibit the proper

penetration of dyes and chemicals. Thus, treatment with various chemicalagents such as enzyme, alkalis, acids, salts, surfactants, solvents, oxidizing

and reducing bleaching agents etc. are necessary prior to dyeing. In the

process of dyeing, auxiliary chemicals such as glauber salt, sodium chloride

and other bio-salts are added to exhaust the dye from the liquor to the

substrate at appropriate temperature and pH. Even after dyeing, unxed

dyestuffs and complex organic products arising out of the reaction with

textile substrate and dyes are to be adequately removed from the surface

of the yarns/fabrics to achieve the proper fastness requirements (Babu et

al. 2007). So, waste stream from the dyeing industry which is to be fed into

the efuent treatment plant essentially comprised of the above ingredients

which are used in the preparatory processes and in actual dyeing process.

Waste stream generated in this industry is essentially based on water-based

Contd...




efuent generated in the various activities of wet processing of textiles. The

main cause of generation of waste water efuent is the use of huge volume

of water either in the actual chemical processing or during re-processing in

preparatory, dyeing, printing and nishing. In fact, in a practical estimate, it

has been found that 45% material in preparatory processing, 33% in dyeing

and 22% are re-processed in nishing. The fact is that the efuent generated

in different steps is well beyond the standard and thus it is highly polluted

and dangerous. Properties of waste water from textile chemical processing

are given (Tables 5.3 and 5.4). The regulatory standrds to which efuent

needs to be treated is given (Table 5.5).

Table 5.3 Properties of waste water from textile chemical processing

Property Standard Cotton Synthetic Wool

pH 5.5–9.0 8–12 7–9 3–10

BOD, mg/l, 5 days 30–350 150–750 150–200 5000–8000

COD, mg/l, day 250 200–2400 400–650 10,000–20,000

TDS, mg/l 2100 2100–7700 1060–1080 10,000–13,000

Table 5.4 Characteristics of textile waste efuent from cotton textile mill

Characteristics Values

pH 9.8–11.8

Total alkalinity (mg/l) 17–22

BOD (mg/l) 760–900

COD (mg/l) 1,400–1,700

Total solids (mg/l) 6,000–7,000

Total chromium (mg/l) 10–13

Table 5.5 Regulatory standrds for efuent water

Quality parameters Tolerance limits

pH 5.5–9

BOD (mg/l) 30

COD (mg/l) 250

TDS (mg/l) 2100Suspended solids (mg/l) 100

Chlorides (mg/l) 1000

Sulphides Nil




5.4. Mercerization

Cotton fabric is mercerized in the gray state after bleaching to impart luster,increase strength and improve dye uptake. Essentially, mercerization is carried

out by treating cotton material with a strong solution of sodium hydroxide

(about 18–24%) and washing-off the caustic after 1 to 3 min, while holding the

material under tension. Cotton is known to undergo a longitudinal shrinkage

upon impregnation during mercerization and it can be prevented by stretching

it or holding it under tension. The material acquires the desired properties of

luster, increased strength, dye uptake, and increased absorbency. The large

concentrations of NaOH in the wash water can be recovered by membrane

techniques. Use of ZnCl2 may be as an alternative method which leads to

an increase in the weight of cotton fabric and in dye uptake. Moreover, the

process is ecologically friendly and does not require neutralization by acetic

or formic acid (Karim et al. 2006).

5.4.3 Bleaching

Natural color matter in the yarn imparts a creamy appearance to the textile

fabric. In order to obtain white yarn that facilitates producing color in pale and bright shades, it is necessary to decolorize the yarn by bleaching. Hypochlorite

is one of the oldest industrial bleaching agents. The formation of highly toxic

chlorinated organic by products during the bleaching process is reduced by

adsorbable organically bound halogen (AOX) which may result minimize the

pollution load in bleaching operation. Over the last few years, hypochlorite is

being replaced by other bleaching agents (Rott and Minke 1999).

An environmentally safe alternative to hypochlorite is peracetic acid. It

decomposes to oxygen and acetic acid, which is completely biodegradable.

One of the advantages of peracetic acid is higher brightness values with lessber damage (Rott and Minke 1999). Recently, a one-step preparatory process

for desizing, scouring, and bleaching has helped to reduce the volume of

water. The feasibility of a one-step process for desizing, scouring, bleaching,

and mercerizing of cotton fabric followed by dyeing with direct dyes has

been discussed by Slokar and Majcen (1997). Cooper (1995) has suggested

an economical and pollution free process for electrochemical mercerization

(scouring) and bleaching of textiles. The process does not require conventional

caustic soda, acids, and bleaching agents. The treatment is carried out in a low

voltage electrochemical cell. The base required for mercerization (scouring)

is produced in the cathode chamber, while an equivalent amount of acid is

produced in the anode chamber, which is used for neutralizing the fabric.

Gas diffusion electrodes simultaneously generate hydrogen peroxide for




bleaching. With a bipolar stack of electrodes, diffusion electrodes can be used as

anode or cathode or both. The process does not produce hydrogen bubbles at the

cathode, thereby avoiding hazards involving the gas (Lin and Peng 1994). An

electrochemical treatment was developed for the treatment of cotton in aqueous

solution containing sodium sulphate. In this technique, the current density was

controlled between two electrodes. At the cathode, water is reduced to hydrogen

and base, while at the anode it is oxidized to oxygen and acid. Favorable results

on mercerization (scouring) and electrochemical sanitation of unmercerized

(grey) cotton have been reported (Naumczyk et al. 1996).

5.4.4 NeutralizationAccording to Bradbury et al. (2000), has stated that the replacement of acetic

acid by formic acid for neutralization of textile cotton fabric after scouring,

mercerizing, bleaching, and reduction processes is effective, economical,

and environment-friendly. The procedure also allows a sufcient level of

neutralization in a short period of time, needs low volumes of water, and

results in low levels of BOD in the waste water efuent.

5.4.5 Dyeing

Dyeing is a process of treatment of ber or fabric with chemical pigments

to impart color. The color arises from chromophore and auxochrome groups

in the dyes, which also cause pollution (Azymezyk et al. 2007). In the wet

dyeing process, water is used to transfer dyes and in the form of steam to heat

the treatment baths. Cotton, which is the world’s most widely used ber, is a

substrate that requires a large amount of water for processing. For example,

to dye one kg of cotton fabric with reactive dyes, 0.6–0.8 kg of NaCl, 30–60

g of dyestuff, and 70–150 L of water are required (Chakraborty et al. 2005).Once the dyeing operation is over, the various treatment baths are drained,

including the highly colored dye bath, which has high concentrations of salt

and organic substances. The wastewater must be treated before reuse (Ciardelli

and Ranieri 2001). Coagulation and membrane processes (nanoltration or

reverse osmosis) are among processes suggested for treatment of the dye

waste water; however, these treatments are effective only with very dilute dye

liquors. The dye baths are generally heavily polluted. For example, wastewater

produced by reactive dyeing contains hydrolyzed reactive dyes not xed on thesubstrate (representing 20–30% of the reactive dyes applied on an average of

2 g/L). This residual amount is responsible for the coloration of the efuents,

and cannot be recycled. Dyeing auxiliaries or organic substances are non-

recyclable and contribute to the high BOD/COD of the dye water efuents.




Membrane technologies are increasingly being used in the treatment of

textile wastewater for the recovery of valuable components from the waste

stream, as well as for reusing the aqueous stream. A number of studies deal

with application of various pressure driven membrane ltration processes in

the treatment wastewater from the dyeing and nishing process (Chen et al.

2005). Measures adopted for the abatement of pollution by different dyes are

(i) use of low material-to-liquor ratios, (ii) use of trisodiumcitrate (Fiebig et

al. 1992), (iii) replacement of reducing agent (sodium hydrosulphite) with a

reducing sugar or electrochemical reduction (Maier et al. 2004), and (iv) use

of suitable dye-xing agents to reduce water pollution loads. Padma et al.

(2006) rst reported the concept of totally ecologically friendly mordents ornatural mordents during dyeing with natural dyes. Deo and Desai (1999) were

the rst to point out that natural dye shades could be built-up by a multiple

dip method that renders natural dyeing more economical. Dyeing of natural

and synthetic bers with natural dyes has been the subject of several studies.

Development of eco-friendly non-formaldehyde dye xative agents for

reactive dyes was recently reported (Bechtold et al. 2005; Sekar 1995).

5.4.6 Printing

Printing is a branch of dyeing. It is generally dened as ‘localized dyeing’.

In dyeing, color is applied in the form of a solution, whereas in printing color

is applied in the form of a thick paste of the dye. The xation of the color in

printing is brought about by a suitable after treatment of the printed textile

material (El-Molla and Schneider 2006). In the textile fabric printing operation

produces hydrocarbon efuents that must be removed before they reach the

atmosphere. Limits on emissions will become more restrictive in the future,

which makes cleaning exhausts an environmental necessity. A majority of

textile printing units prefer to use kerosene in printing because of the brilliant

prints and ease of application. Kerosene is released into the atmosphere during

printing, drying, and curing which resulting pollution of the atmosphere and

wastage of hydrocarbon products. Air laden kerosene is harmful to human

beings, as well as to the ora and fauna, in the neighborhood. Therefore, it is

imperative that as much kerosene as possible is recovered from the exhaust

pipes of the printing industry. Zachariah (1995) was developed a process for

the recovery of thin kerosene vapor. In this process, the percentage of recovery

of kerosene from the printing drier was 78.5%, and the total percentage ofrecovery of kerosene consumed for the preparation of print paste was 58.8%.

The most common chemical in reactive dye printing is urea, which leads

to a high pollution load. A number of attempts have been made to limit or

eliminate the use of urea in the print paste to reduce efuent load. Geeta et




al. (2004) developed a urea-free process in which caprolactam, PEG-400, and

PEG-600 partially or completely replaced urea in the dyeing and printing of

reactive dyes on cotton fabrics. Caprolactam in many reactive dyes can fully

replace urea, while PEG-400 and PEG-600 replaced approximately 50% of

the dyes required for xation. Other substitutes for urea include glycerin,

cellosolve, sorbitol, polycarboxylic acid, PEG-200, and PEG-4000. Printing

is mainly done by a at or rotary screen, and after every lot of printing some

residual paste is left in the wastewater. This can be reused for printing of

similar shades by adding new stock. Recently, screen free printing methods,

such as ink-jet printing and electrostatic printing, have been developed that

make use of an electronic control of color distribution on fabric. Screen-free printing methods are attractive for pollution mitigation (Lukanova and

Ganchev 2005).

5.4.7 Finishing

Both natural and synthetic textiles are subjected to a variety of nishing

processes to improve specic functional properties of the materials. These

nishing processes involve the use of a large number of nishing agents for

softening, cross-linking, and waterproong. The most of the nishing operations,formaldehyde based cross-linking agents are used for getting desired properties,

such as softness and stiffness that impart bulk and drape properties, smoothness,

and handle, to cellulosic textiles. These operations contribute to high level water

pollution. The use of formaldehyde and liberation of chemical products, and

which leads to toxicity and stream pollution. Generation of formaldehyde during

vacuum extraction has been used in the storage of resin-nished fabrics and

garments. The formaldehyde resin used as a cross-linking agent is a pollutant

and a known carcinogen. Much effort has been expended in the search for a

substitute for formaldehyde (Hashem et al. 2005). Since the late 1980s, there

has been an increase in the demand for easy-care, wrinkle-resistant (durable

press), 100% cotton apparel. The formaldehyde based chemical nishes, such

as dimethylol dihydroxyethylene urea (DMDHEU) and its etheried derivative

with lower formaldehyde concentrations, are used to impart ease of care

characteristics and durable-press properties to cotton apparels. They are cost-

effective and efcient. The free formaldehyde on the nished textile fabric is a

major drawback given the adverse effects of formaldehyde, which ranges from

a strong irritant to carcinogenic. A variety of cellulose cross-linking agents, suchas polycarboxylic acids, has been investigated to provide non-formaldehyde

easy-care nishing. Natural polymeric substances, such as natural oil and wax,

have been used for water-proong; however, textiles made from natural bers are

generally more susceptible to bio-deterioration compared with those made from




synthetic bers. Bio-polishing using cellulose enzymes is an environmentally

friendly method to improve soft handling of cellulose bers with reduced piling,

less fuzz and improved drape.

5.5 Treatment of textile efuents by various

methods

5.5.1 Present scenario

The efuent treatment method is broadly classied into three main categories:

physical, chemical and biological treatments. There are four stages: preliminary,

primary, secondary and tertiary treatments to treat the efuents. The preliminary

treatment processes are equalization and neutralization. The primary stages

involve screening, sedimentation, oatation, and occulation. Secondary

stages are used to reduce the organic load, facilitate physical/chemical

separation and biological oxidation. Tertiary stages are important because they

serve as polishing of efuent treatment. The wastewater from the dye house

is generally multi-colored. The dye efuent disposed into the land and river

water reduces the depth of penetration of sunlight into the water environment,

which in turn decreases photosynthetic activity and dissolved oxygen (DO).The adverse effects can spell disaster for aquatic life and the soil. Figure 5.4

shows the classication of waste water treatments for cotton processing. Many

dyes contain organic compounds with functional groups, such as carboxylic (–

COOH), amine (–NH2), and azo (–N=N–) groups, so treatment methods must

be tailored to the chemistry of the dyes. Particularly in the dye houses, the waste

water efuents resulting from dyed cotton fabrics with reactive dyes are highly

polluted and have high BOD/COD, coloration, and salt load.

Fig. 5.3 Activities involving water in textile processing [Source: Ramesh Babu et al. 2007]




Fig. 5.4 Classication of waste water treatment process

5.5.1.1 Primary treatment After the removal of gross solids, gritty materials and excessive quantities

of oil and grease, the next step is to remove the remaining suspended solids

as much as possible. This step is aimed at reducing the strength of the waste

water and also to facilitate secondary treatment.

Screening: In the rst screening operation, coarse suspended matters such

as rags, pieces of fabric, bres, yarns and lint are removed. Bar screens and

mechanically cleaned ne screens are mainly removing most of the bres

present in the waste efuents. The suspended bres have to be removed priorto secondary biological treatment; otherwise they may affect the secondary

treatment system. They are reported to clog trickling lters, seals or carbon

beads.

Sedimentation: The suspended matter in textile waste efuent can be

removed efciently and economically by sedimentation. This process is

particularly useful for treatment of wastes containing high percentage of

settable solids or when the waste is subjected to combined treatment with

sewage. The sedimentation tanks are designed to enable smaller and lighter

particles to settle under gravity. The most common equipment used includes

horizontal ow sedimentation tanks and centre feed circular clariers. The

settled sludge is removed from the sedimentation tanks by mechanical

scrapping into hoppers and pumping it out subsequently.




Equalization: The waste efuent streams are collected into ‘sump pit’.

Sometimes mixed efuents are stirred by rotating agitators or by blowing

compressed air from below. The pit has a conical bottom for enhancing the

settling of solid particles.

Neutralization: Normally, pH values of cotton nishing efuents are on

the alkaline side. Hence, pH value of equalized efuent should be adjusted.

Use of dilute sulphuric acid and boiler ue gas rich in carbon dioxide are not

uncommon. Since most of the secondary biological treatments are effective in

the pH 5 to 9, neutralisation step is an important process to facilitate.

Chemical coagulation and mechanical occulation: Finely divided

suspended solids and colloidal particles cannot be efciently removed bysimple sedimentation by gravity. In such cases, mechanical occulation or

chemical coagulation is employed. In mechanical occulation, the textile

waste water is passed through a tank under gentle stirring; the nely divided

suspended solids coalesce into larger particles and settle out. Specialized

equipment such as clarriocculator is also available, wherein occulation

chamber is a part of a sedimentation tank. The degree of clarication obtained

also depends on the quantity of chemicals used. In this method, 80–90% of

the total suspended matter, 40–70% of BOD, 5days, 30–60% of the COD and

80–90% of the bacteria can be removed. However, in plain sedimentation,only 50–70% of the total suspended matter and 30–40% of the organic matter

settles out. Most commonly used chemicals for chemical coagulation are

alum, ferric chloride, ferric sulphate, ferrous sulphate and lime.

5.5.1.2 Secondary treatment

The main purpose of secondary treatment is to provide BOD removal beyond

the sedimentation level of treatments. It also removes appreciable amounts of

oil and phenol. In secondary treatment, the dissolved and colloidal organic

compounds and colour present in waste water is removed or reduced and to

stabilize the organic matter. This is achieved biologically using bacteria and

other microorganisms. Textile processing efuents are amenable for biological

treatments (Vanndevivera and binanchi 1998). These processes may be

aerobic or anaerobic. In aerobic processes, bacteria and other microorganisms

consume organic matter as food (Basibuyuk and Forster 1997). They bring

about the following sequential changes: (i) Coagulation and occulation of

colloidal matter (ii) Oxidation of dissolved organic matter to carbon dioxide(iii) Degradation of nitrogenous organic matter to ammonia, which is then

converted into nitrite and eventually to nitrate. Anaerobic treatment is mainly

employed for the digestion of sludge (Bortone et al. 1995). The efciency

of this process depends upon pH, temperature, waste loading, absence of




oxygen and toxic materials. Some of the commonly used biological treatment

processes are described below:

Aerated lagoons: These are large holding tanks or ponds having a depth

of 3–5 m and are lined with cement, polythene or rubber. The efuents from

primary treatment processes are collected in these tanks and are aerated with

mechanical devices, such as oating aerators, for about 2 to 6 days. During

this time, a healthy occulent sludge is formed which brings about oxidation

of the dissolved organic matter. BOD removal to the extent of 99% could be

achieved with efcient operation. The major disadvantages are the large space

requirements and the bacterial contamination of the lagoon efuent, which

necessitates further biological purication. Trickling flters: The trickling lters usually consists of circular or

rectangular beds, 1 m to 3 m deep, made of well graded media (such as

broken stone, PVC, coal, synthetic resins, gravel or clinkers) of size 40–150

mm, over which wastewater is sprinkled uniformly on the entire bed with the

help of a slowly rotating distributor (such as rotary sprinkler) equipped with

orices or nozzles. Thus, the waste water trickles through the media. The lter

is arranged in such a fashion that air can enter at the bottom; counter current to

the efuent ow and a natural draft is produced. A gelatinous lm, comprising

of bacteria and aerobic microorganisms known as “Zooglea”, is formed onthe surface of the lter medium. The organic impurities in the waste water are

adsorbed on the gelatinous lm during its passage and then are oxidized by

the bacteria and the other micro-organisms present therein.

Activated sludge process: This is the most versatile biological oxidation

method employed for the treatment of waste water containing dissolved solids,

colloids and coarse solid organic matter. In this process, the waste water is

aerated in a reaction tank in which some microbial oc is suspended. The

aerobic bacterial ora bring about biological degradation of the waste intocarbon dioxide and water molecule, while consuming some organic matter

for synthesizing bacteria. The bacteria ora grows and remains suspended in

the form of a oc, which is called “Activated Sludge”. The efuent from the

reaction tank is separated from the sludge by settling and discharged. A part

of the sludge is recycled to the same tank to provide an effective microbial

population for a fresh treatment cycle (McMullan et al. 2001). The surplus

sludge is digested in a sludge digester, along with the primary sludge obtained

from primary sedimentation. An efcient aeration for 5–24 hours is required

for industrial wastes. BOD removal to the extent of 90–95% can be achievedin this process.

Oxidation ditch: This can be considered as a modication of the

conventional activated sludge process. The waste water, after screening in

allowed into the oxidation ditch. The mixed liquor containing the sludge




solids is rst aerated in the channel with the help of a mechanical rotor. The

usual hydraulic retention time is 12–24 hours and for solids, it is 20–30 days.

Most of the sludge formed is recycled for the subsequent treatment cycle.

Oxidation pond: An oxidation pond is a large shallow pond wherein

stabilization of organic matter in the waste is brought about mostly by bacteria

and to some extent by protozoa. The oxygen requirement for their metabolism

is provided by algae present in the pond. The algae, in turn, utilize the CO2

released by the bacteria for their photosynthesis. Oxidation ponds are also

called waste stabilization ponds.

Anaerobic digestion: Sludge is the watery residue from the primary

sedimentation tank and humus tank (from secondary treatment). Theconstituents of the sludge undergo slow fermentation or digestion by anaerobic

bacteria in a sludge digester, wherein the sludge is maintained at a temperature

of 35°C at pH 7–8 for about 30 days. CH4, CO

2 and some NH

3 are liberated as

the end products.

5.5.1.3 Tertiary treatment processes

The textile waste contains signicant quantities of non-biodegradable

chemical polymers, since the conventional treatment methods are inadequateand need for efcient tertiary treatment process.

Oxidation techniques: A variety of oxidizing agents can be used to

decolorize wastes. Sodium hypochlorite decolorizes dye bath efciently.

Though it is a low cost technique, but it forms absorbable toxic organic halides

(AOX). Ozone on decomposition generates oxygen and free radicals and the

later combines with colouring agents of efuent resulting in the destruction

of dye colours. Arslan et al. (2000) investigated the treatment of synthetic dye

house efuent by ozonisation, and hydrogen peroxide in combination with

ultraviolet light. The main disadvantage of these techniques is it requires an

effective sludge producing pretreatment.

Electrolytic precipitation and foam fractionation:Electrolytic precipitation

of concentrated dye wastes by reduction in the cathode space of an electrolytic

bath been reported although extremely long contact times were required.

Foam fractionation is experimental method based on the phenomenon that

surface-active solutes collect at gas–liquid interfaces. However, the chemical

costs make this treatment method too expensive.

Membrane technologies: Reverse osmosis and electro dialysis are theimportant examples of membrane process. The TDS from waste water can

be removed by reverse osmosis. Reverse osmosis is suitable for removing

ions and larger species from dye bath efuents with high efciency (up to

>90%), clogging of the membrane by dyes after long usage and high capital




cost are the main drawbacks of this process. Dyeing process requires use of

electrolytes along with the dyes. Neutral electrolyte like NaCl is required

to have high exhaustion of the dye. For instance, in cotton dyeing, NaCl

concentration in the dyeing bath is in the range of 25–30 g/l for deep tone and

about 15 g/l for light tone, but can be as high as 50 g/l in exceptional cases.

The exhaustion stage in reactive dyeing on cotton also requires sufcient

quantity of salt. Reverse osmosis membrane process is suitable for removing

high salt concentrations so that the treated efuent can be re-used again in

the processing. The presence of electrolytes in the washing water causes an

increase in the hydrolyzed dye afnity (for reactive dyeing on cotton) making

it difcult to extract. In electro dialysis, the dissolved salts (ionic in nature)can also be removed by impressing an electrical potential across the water,

resulting in the migration of cations and anions to respective electrodes via

anionic and cationic permeable membranes. To avoid membrane fouling it

is essential that turbidity, suspended solids, colloids and trace organics be

removed prior to electro dialysis.

Electro chemical processes: They have lower temperature requirement

than those of other equivalent non electro chemical treatment and there is no

need for additional chemical. It also can prevent the production of unwanted

side products. But, if suspended or colloidal solids were high concentration inthe waste water, they impede the electrochemical reaction. Therefore, those

materials need to be sufciently removed before electrochemical oxidation.

Ion exchange method: This is used for the removal of undesirable anions

and cations from waste water. It involves the passage of waste water through

the beds of ion exchange resins where some undesirable cations or anions of

waste water get exchanged for sodium or hydrogen ions of the resin. Most ion

exchange resins now in use are synthetic polymeric materials containing ion

groups such as sulphonyl, quarternary ammonium group etc. Photocatalytic degradation: An advanced method to decolorize a wide

range of dyes depending upon their molecular structure. In this process,

photoactive catalyst illuminates with UV light, generates highly reactive

radical, which can decompose organic compounds.

Adsorption: It is the exchange of material at the interface between two

immiscible phases in contact with one another. Adsorption appears to have

considerable potential for the removal of colour from industrial efuents.

Owen (1978) after surveying 13 textile industries has reported that adsorption

using granular activated carbon has emerged as a practical and economical process for the removal of colour from textile efuents.

Thermal evaporation: The use of sodium per sulphate has better oxidizing

potential than NaOCl in the thermal evaporator. The process is ecofriendly

since there is no sludge formation and no emission of the toxic chlorine fumes




(Pala and Tokat 2002), is often resorted to after the biological treatment

(Ledakowic et al. 2001). These methods, which only release efuents into the

environment per legal requirements, are expensive.

5.7.2 Biological aerated lters (BAF)

The growth of an organism on media that are held stationary during normal

operation and exposed to aeration. In recent years, several BAF-based

technologies have been developed to treat wastewater. Efuents from textile

industry are among wastewaters that are hard to treat satisfactorily, because

their compositions are highly variable. The strong color is most striking

characteristic of textile wastewater. If unchecked, colored wastewater can

cause a signicantly negative impact on the aquatic environment primarily

arising from increased turbidity and pollutant concentrations.

5.7.3 Coagulation occulation methods

Coagulation occulation methods are generally used to eliminate organic

substances, but the chemicals normally used in this process have no effect

on the elimination of soluble dyestuffs. Although this process effectivelyeliminates insoluble dyes (Gaehr et al. 1994).

5.7.4 Adsorption on powdered activated carbon

The adsorption on activated carbon without pretreatment is impossible

because the suspended solids rapidly clog the lter (Matsui et al. 2005). This

method is feasible only in combination with occulation decantation treatment

or a biological treatment. The combination permits a reduction of suspended

solids and organic substances, as well as a slight reduction in the color (Rozziet al. 1999).

5.7.5 Electrochemical processes

Electrochemical techniques for the treatment of dye waste are more efcient

than other treatments (Naumczyk et al. 1996). Electrochemical technology

has been applied to effectively remove acids, as well as dispersed and metal

complex dyes. Marrot and Roche (2002) have studied and reported more than

100 references in a bibliographical review of textile waste water treatment.Figure 5.5 shows the treatment operation and decision structure of waste

water treatment. The physical methods include precipitation (coagulation,

occulation, sedimentation) (Lin and Peng 1996; Solozhenko et al. 1995; Lin

and Liu 1994; McKay et al. 1987), adsorption (on activated carbon, biological




sludges) (Pala and Tokat 2002), ltration, or reverse osmosis membrane

processes (Ghayeni et al. 1998; Treffry-Goatley et al. 1983; Tinghui et al.

1983).

Fig. 5.5 Electrochemical treatment and recovery of chemicals from the textile efuent

The removal of dyes from aqueous solutions results from adsorption and

degradation of the dye-stuff following interaction with iron electrodes. If metal

complex dyes are present, dye solubility and charge are important factors thatdetermine the successful removal of heavy metals. In order to maximize dye

insolubility, pH control is crucial (Chakarborty et al. 2003; Vedavyasam 2000;

Nowak et al. 1996; Calabro et al. 1990). Electro-coagulation is an efcient

process, even at high pH, for the removal of color and total organic carbon.

The efciency of the process is strongly inuenced by the current density

and duration of the reaction. Under optimal conditions, decolorization yields

between 90 and 95%, and COD removal between 30 and 36% can be achieved.

5.7.6 Ozone treatment

This ozone treatment is widely used in water treatment; ozone (O3-UV or

O3-H

2O

2) is now used in the treatment of industrial efuents (Langlais et al.

2001; Malik and Sanyal 2004). Ozone especially attacks the double bonds that




bestow color. For this reason, decolorization of wastewater by ozone alone

does not lead to a signicant reduction in COD (Coste et al. 1996; Adams

et al. 1995). Moreover, installation of ozonation plants can entail additional

costs (Scott and Ollis 1995).

5.7.7 Membrane processes

Increasing cost of water and its proigate consumption necessitate a

treatment process that is integrated with in-plant water circuits rather than as

a subsequent treatment (Machenbach 1998). From this standpoint, membrane

ltration offers potential applications. Processes using membranes provide

very interesting possibilities for the separation of hydrolyzed dye-stuffs anddyeing auxiliaries that simultaneously reduce coloration and BOD/COD of

the wastewater. The choice of the membrane process, whether it is reverse

osmosis, nanoltration, ultra ltration or microltration, must be guided by

the quality of the nal product (Al-Malack and Anderson 1997).

5.7.8 Reverse osmosis

Reverse osmosis membranes have a retention rate of 90% or more for most

types of ionic compounds and produce a high quality of permeate (Ghayeni etal. 1998; Treffry-Goatley et al. 1983; Tinghui et al. 1983). Decoloration and

elimination of chemical auxiliaries in dye house wastewater can be carried out

in a single step by reverse osmosis. Reverse osmosis permits the removal of

all mineral salts, hydrolyzed reactive dyes, and chemical auxiliaries (Abadulla

et al. 2000).

5.7.9 Nanoltration

Nanoltration has been applied for the treatment of colored efuents fromthe textile industry (Akbari et al. 2002). A combination of adsorption and

nanoltration can be adopted for the treatment of textile dye efuents

(Chakraborty et al. 2003). Nanoltration membranes retain low molecular

weight organic compounds, divalent ions, large monovalent ions, hydrolyzed

reactive dyes, and dyeing auxiliaries (Jiraratananon et al. 2000; Xu et al. 1999;

Erswell et al. 1988). Harmful effects of high concentrations of dye and salts

in dye house efuents have frequently been reported (Tang and Chen 2002;

Koyuncu 2002; Bruggen et al. 2001).

5.7.10 Ultraltration

Ultraltration enables elimination of macromolecules and particles, but the

elimination of polluting substances, such as dyes, is never complete (it is




only between 31% and 76%) (Watters et al. 1991). Even in the best of cases,

the quality of the treated wastewater does not permit its reuse for sensitive

processes, such as dyeing of textile. Rott and Minke (1999) emphasize that

40% of the water treated by ultra ltration can be recycled to feed processes

termed “minor” in the textile industry (rinsing, washing). Ultraltration can

only be used as a pretreatment for reverse osmosis (Ciardelli and Ranieri

2001) or in combination with a biological reactor (Mignani et al. 1999).

5.7.11 Microltration

Microltration is suitable for treating dye baths containing pigment dyes (Al-

Malack and Anderson 1997), as well as for subsequent rinsing baths. Thechemicals used in dye bath, which are not ltered by microltration, will

remain in the bath. Microltration can also be used as a pretreatment for

nanoltration or reverse osmosis (Ghayeni et al. 1998). The various textile

wet processing stages in the textile industry and the methodologies adopted

for treating textile wastewater (Table 5.6) are discussed from an environmental

point of view (Ghosh and Gangopadhyay 2000).

Table 5.6 Possible treatments for cotton textile wastes and their associated

advantages and disadvantages

Process Advantages Disadvantages References

Biodegradation Rates of elimination

by oxidizable

substances about

90%

Low

biodegradability of

dyes

Pala and Tokat

2002; Ledakowicz

et al. 2001

Coagulation/

Flocculation

Elimination of

insoluble dyes

Production of

sludge blocking

lter

Gaehr et al. 1994

Adsorption onactivated carbon

Suspended solidsand organic

substances well

reduced

Cost of activatedcarbon

Arslan et al. 2000

Ozone treatment Good decolorization No reduction of the

COD

Adams et al.

1995; Scott and

Ollis 1995

Electrochemical

processes

Capacity of

adaptation to

different volumes

and pollution loads

Iron hydroxide

sludge

Lin and Peng

1994; Lin and

Chen 1997

Reverse osmosis Removal of all

mineral salts,

hydrolyzes reactive

dyes and chemical

auxiliaries

High pressure Ghayeni et al.

1998

Contd...




Process Advantages Disadvantages References

Nanoltration Separation of

organic compounds

of low molecular

weight and

divalent ions from

monovalent salts

High installation

cost

Erswell et al.

1998;

Xu et al. 1999;

Akbari et al. 2002;

Tang and Chen

2002

Ultraltration/

microltration

Low pressure Insufcient quality

of the treated

wastewater

Watters et al.

1991; Rott and

Mike 1999;

Ghayeni et al.1998

5.8 Role of enzymes in decolouration

In textile processing as well as other industrial applications, large amounts of

dyestuffs are used. The high COD and BOD, suspended solids (SS) and intense

colour due to the extensive use of synthetic dyes characterize wastewater from

textile industry, especially dye houses. The water must be decolorized; harmful

chemicals must be converted into harmless chemicals. Biological treatmentshave been used to reduce the COD of textile efuents. Physical and chemical

treatments are effective for colour removal but use more energy and chemicals

than biological processes. Instead of using the chemical treatments, various

biological methods can be used to treat the water from the textile industry.

These methods include, (i) biosorption, (ii) use of enzymes, (iii) aerobic and (iv)

anaerobic treatments. Biotechnological solutions can offer complete destruction

of the dyestuff, with a co-reduction in BOD and COD. The synthetic dyes are being

designed in such a way that they become more resistant to microbial degradation

under the aerobic conditions. The water solubility and high molecular weightinhibit the permeation through biological cell membranes. Anaerobic processes

convert the organic contaminants with less space; treat wastes containing up

to 30,000 mg/l of COD, have lower running costs and produce less sludge.

Biological systems, such as biolters and bioscrubbers, are now available for

the removal of odor and other volatile compounds. The dyes can be removed

by biosorption on apple pomace and wheat straw. Apple pomace had a greater

capacity to absorb the reactive dyes compared to wheat straw.

5.8.1 Decolorization of the dye house efuent using

enzymes

The use of lignin degrading white rot fungi has attracted increasing scientic

attention as these organisms are able to degrade a wide range of recalcitrant

Contd...




organic compounds such as polycyclic aromatic hydrocarbons, chlorophenol,

and various azo, heterocyclic and polymeric dyes (Alemzadeh et al. 2009).

The enzymes associated with the lignin degradation are identied such as (i)

laccase, (ii) lignin peroxidase, and (iii) manganese peroxidase. The lacasses are

the multicopper enzymes which catalyzes the oxidation of phenolic and non-

phenolic compounds (Alexandre and Zhulin 2000). However, the substrate

of the laccases can be extended by using mediators such as 2,2-azoinobis-(3-

ethylthiazoline-6-sulfonate) and 1-hydroxy benzotriazole. Many researchers

also identied the fungi groups which have been used for laccase production

and for the decolorization of synthetic dyes such as Trametes modesta,

trametes versicolor, trametes Hirsuta, and Sclerotium Rolfsii (Ardon et al.1996).

Trametes modesta laccase enzyme has been noticed the highest potential

to transform the textile dyes into colorless products (Heining et al. 1997).

The rate of the laccase catalyzed decolorization of the dyes increase with the

increase in temperature up to certain degree above which the dye decolorization

decreases or does not take place at all (Camarero et al. 2005). The optimum

pH for laccase catalyzed decolorization depends on the type of the dye used.

Dyes with different structures were decolorized at different rates. The structure

of the dye as well as the enzymes play major role in the decolorization ofdyes and it is evident that the laccase of trametes modesta may be used for

decolorization of textile dyestuffs, efuent treatments, and bioremediation or

as a bleaching agent. Activated sludge systems can also be used to treat the

dye house efuents (Ambatkar and Mukundan 2012). Immobilized microbe

bioreactors (IMBRs) address the need of increased microbial/waste contact,

without concomitant production of excessive biosolids, through the use of

solid but porous matrix to which a tailored microbial consortium of organisms

has been attached (Campos et al. 2001). This method may be allowed greaternumber of organisms to be available for waste degradation without the need of

a suspended population and greater increased contact between the organisms

and the waste.

5.8.1.1 Causes of recalcitrance of pollutants

The removal of coloring matter from dye efuent is a major problem faced

by industries. In general, the chemical structure of dyes contains conjugated

double bonds and aromatic rings (Zille et al. 2004). Many synthetic dyeshaving characteristics which tend to persist in the environment due to the

inherent stability of their molecular structure. Azo dyes for example, have

a characteristic azo (–N=N–) linkage which is electron withdrawing in

nature. The presence of this linkage decreases the susceptibility of azo dyes




peroxidase (E.C. 1.11.1.13) and lignin peroxidase (E.C. 1.11.1.14) are

ferric ion containing heme proteins and require peroxides like H2O

2 for

their functioning. Lignin peroxidase (Kersten et al. 1990) and manganese

peroxidase (Moturi and Singara charya 2009) are obtained from fungi.

There are various plant sources of peroxidases – like horse radish (Da Silva

et al. 2010; Maddhinni et al. 2006; Ulson de Souza et al. 2007), soyabean

(Johnson and Pokora 1994), radish (Naghibi et al. 2003), beetroot (Rudrappa

et al. 2005) and peanut (Bagirova et al. 2001). Most of these peroxidases

have been tested to determine their potential to treat synthetic and actual

wastewaters. Laccase (E.C.1.10.3.2) is a blue copper oxidase that catalyzes

the four electron reduction of molecular oxygen (O2) to water (H2O)(McGuirl and Dooley 1999). These enzymes are mainly obtained from lignin

degrading fungi such as Trametes versicolor (Adam et al. 1999) and T. Villosa

(Zille et al. 2004) as well as fungi like Fusarium solani (Abedin 2008) and

Cladospora cladosporioides (Vijayakumar et al. 2006). Azo dyes undergo

reductive splitting relatively easily under anaerobic conditions (Kalyuzhnyi

et al. 2006). The anaerobic reduction of certain azo dyes, however, yields

aromatic amines that are potentially carcinogenic (Kandelbauer et al. 2004).

The degradation of different dyes by select oxidoreductases from different

biological sources has been summarized (Table 5.7).

Table 5.7 Enzyme mediated decoloration of some dyes

Substrate(s) Enzyme Reference

3-(4 dimethyl amino-1 phenylazo)

Benzene sulfonic acid

Laccase from Trametes

villosa

Zille et al. 2004

Acid Orange 6, Acid Orange 7

Methyl Orange and Methyl Red

Mixture of bacterial

Oxidoreductases from

sludge methanogens

Kalyuzhnyi et al.

2006

Direct Yellow Horseradish peroxidase

from

Armoracia rusticana

Maddhinni et al.

2006

Acid Blue Laccase from

Cladosporium

cladosporioides

Vijaykumar et al.

2006

Tartrazine and Ponceau Azoreductase from Green

Algae

Omar 2008

Reactive Yellow, Reactive BlackReactive Red and Direct Blue

Azoreductase fromStaphylococcus arlettae

Franciscon et al.2009

While peroxidases are specic to the electron acceptor, i.e. hydrogen

peroxide or alkyl peroxides, they are not very specic towards the electron




Fig. 5.6 Response surface plots for decolouration of RY15 as a function of (a) pH and

temperature at 96 U/L; (b) enzyme concentration and temperature at pH 5; (c) pH and

enzyme concentration at 35°C [Source: Ana et al. 2009]




Fig. 5.7 Response surface plots for decolourization of RB114 as function of: (a) pH

and temperature at 96 U/L; (b) pH and enzyme concentration at 35°C [Source: Ana et

al. 2009]

5.8.2.1 Applications of laccases – wastewater treatment

Textile dye efuents are complex, containing a wide variety of dyes, natural

impurities extracted from the bers and other products such as dispersants,

leveling agents, acids, alkalis, salts and sometimes heavy metals (Laing 1991).

In general, the efuent is highly coloured with high biological oxygen demand

(BOD) and chemical oxygen demand (COD), it has a high conductivity and

is alkaline in nature. The dyeing processes have, in general, a low yield and




the percentage of the lost dye in the efuents can reach up to 50% (Pierce

1994; Pearce et al. 2003). From the available literature it can be estimated that

approximately 75% of the dyes, discharged by textile processing industries,

belong to the following classes: reactive (~36%), acid (~25%) and direct

(~15%) (Øllgaard et al. 1998). In these classes, the azo dyes are the most

important chemical class of synthetic dyes. Azo dyes are characterised by the

presence of at least one azo bond (-N=N-) bearing aromatic rings and have

high photolytic stability and resistance towards major oxidising agents (Reife

et al. 1993).

Fig. 5.8 Response surface plots for decolourization of RR239 as function of: (a) pH

and temperature at 96 U/L enzyme concentration; (b) pH and enzyme concentration at

35°C [Source: Ana et al. 2009]




at 25°C (Fig. 5.9). The manganese pyrochlorophyride methylester (MnPChlide

ME) dissolved in a Triton X-100 micellar solution also exhibited the catalytic

activity, indicating the micellar environment plays an important role in the

catalytic reaction (Dellamatrice and Monteiro 2006). The reaction rate was

accelerated by addition of imidazole. The catalytic reactions were analyzed

by Michaelis–Menten kinetics, revealing that the higher reactivity of catalyst

substrate complex is responsible for the present catalytic reaction system.

Fig. 5.9 Catalyst-substrate complex of MnP and PEG in decoloration of azo dye

Fig. 5.10 Various types of PEG and MnP and their catalytic groups

The commercial azo, triarylmethane, antraquinonic, and indigoid textile dyes

are efciently decolorized with enzyme preparations from Pleurotus ostreatus,

Schizophyllum commune, Neurospora crassa, Polyporus sp., Sclerotium rolfsii,

Trametes villosa, and Mycelioph tora thermophila, (Fig. 5.10). The nature of

substituents on the dyes benzene rings inuences enzyme activity, and hydroxyl

and amino groups enhance decolorization (Abadulla et al. 2000). The presence




Fig. 5.11 Absorption spectra of the azo dyes – Reactive Black 5 (A), Reactive Yellow27 (B), and Reactive Red 158 (C) – during decoloration by Geotrichum sp. For the black

dye the overlays correspond to Day 0, 3, 4, 5, 6, 7 and 10. For the yellow and red dyes

the overlays correspond to Day 0, 3, 7, 11, 14, 18 and 24 [Source: Cristina Máximo et

al. 2003]




One of the limiting steps for bacterial degradation of azo dyes is the

necessity for their uptake into the cell. In contrast, fungal transformations

are mediated by exo-enzymes and membrane permeation of the substrate can

be by-passed (Mustafa et al. 2005; Pereira et al. 2005). It is likely that the

phenoloxidases studied i.e. Mn peroxidase; Mn-independent peroxidase and/or

laccase were responsible for the biotransformation of the black dye. In the case

of the yellow and red dyes it is suggested that additional enzymes or factors are

involved (Tzanov et al. 2003). When the yellow and red dyes were introduced

at the start of the cultivation, the dye transformation required about 20 days.

Future work will be directed at identifying additional oxidoreductases that may

have auxiliary roles in transformation of dyes that are more recalcitrant. Thiscould lead to the development of a more effective enzymatic cocktail for dye

decoloration. The use of enzymes (Setti et al. 1999; Thurston 1994), rather than

whole fungi might be expected to be a more viable option from the point of view

of the decolorization process control at the industrial scale.

5.9 Prospects and future research

The number of studies on the biodecoloration of dyestuffs has been steadily

increasing in recent years. Several countries, including India, have introducedstrict ecological standards for textile industries. With more stringent controls

expected in the future, it is essential that control measures be implemented

to minimize efuent problems. Waste minimization is of great importance

in decreasing pollution load and production costs. This review has shown

that various methods can be applied to treat cotton textile efuents and to

minimize pollution load. Traditional technologies to treat textile wastewater

include various combinations of biological, physical, and chemical methods,

but these methods require high capital and operating costs. Technologies

based on membrane systems are among the best alternative methods that

can be adopted for large-scale ecologically friendly treatment processes.

A combination methods involving adsorption followed by nanoltration

has also been advocated. Initially, refractory organic compounds and dyes

may be electrochemically oxidized to biodegradable constituents before the

wastewater is subjected to biological treatment under aerobic conditions.

Color and odor removal may be accomplished by a second electro-oxidation

process. Microbial life, if any, may be destroyed by a photochemical treatment.

The treated water at this stage may be used for rinsing and washing purposes;however, an ion-exchange step may be introduced if the water is desired to be

used for industrial processing.

Enzymes have been employed in numerous elds primarily for their

immense catalytic potential. In wastewater treatment, enzymes can be utilized




to develop remediation processes that are environmentally less aggressive than

conventional techniques. Their versatility and efciency even in mild reaction

conditions gives them an advantage over the conventional physico-chemical

treatment methods. The biological origin of enzymes reduces their adverse

impact on the environment thereby making enzymatic wastewater treatment an

ecologically sustainable technique. Currently, efuent treatment using enzymes

on a large scale is not economically viable. However, if maximum reusability of

enzymes is achieved through the use of standardized immobilization procedures,

the running cost can be lowered considerably. The conuence of nanoscience

and enzyme technology has resulted in an upcoming interdisciplinary approach

to wastewater treatment. Such innovative applications of enzymes can enablethe utilization of these biocatalysts to their maximum potential. Future research

in this eld should emphasize on the optimization of the activity of crude enzyme

preparations and on the improvement of enzyme reusability to counteract the

high start-up and running costs.

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Abstract: This chapter provides information on safety of enzyme-handlingpractices for plant managers, industrial hygienists, occupational and safety

professionals, medical personnel and employees in the enzyme-handling industry.It includes a medical surveillance program describing measures the employercan use to help ensure employee health and safety in the workplace. Enzymeshave been used for over 35 years in the textile industry for the desizing of clothand fabric nishing. Implementing an enzyme safety program is important forlimiting exposure to enzymes and maintaining employee health and safety inthe workplace. The chapter then discusses the modern biotechnology techniquesutilized to improve microbial production strains to increase the enzyme yieldsand to make minor amino acid changes that improve the functionality of theenzyme. These changes are not known to increase the ability of enzymes tocause allergies. Work-practice controls include appropriate management systems

and operational controls, education, safe work practices and good housekeepingpractices have been discussed.

Keywords: Enzyme safety, enzyme handling, enzyme allergy, safety program,work practice, medical monitoring

6.1 Introduction

Enzymes are regulated in different legislation depending on their end uses. The

scientic literatures investigated indicate that enzymes have the potential for

sensitization of the respiratory tract. At present, no validated test method exists

to determine and to predict sensitization via the inhalative route (Basketter

1995; Cullinan and Harris 2000). The studies investigated so far revealed

that enzymes seem unlikely to be dangerous to the aquatic environment due

to their ready biodegradability and the low effects on aquatic organisms

observed. However, enzymes derived from new technologies might have

increased stability (e.g. with higher stability to temperature or pH), therefore,

the ready biodegradability of such enzymes should be proved. It is suggested,

only to perform a biodegradation test in the case that the enzyme has .unusualstability (Schweigert 2000).

The long-term safety of enzyme supplements has been explored in detail

for a hundred years. The result is that enzymes are generally found to be

highly safe with no toxic limit no matter how much you take or for how long

6

Safety and precaution in handling enzymes




you take them. There is research showing that it is the lack of enzymes that

causes problems, not taking too many (Zachariae et al. 1981). Enzymes have

very specic mechanisms and many are well known and characterized based

on their specicity. Each enzyme has a very particular function according to

their active sites. Enzymes work according to ordinary biochemical properties

and are not living organisms complete with free will and instincts (AISE

1995). Because of this, enzymes have far fewer side-effects and unknown

possible reactions than other compounds, supplements, or medications. This

feature makes them extremely safe. Also, healthy tissue and cells have natural

mechanisms protecting them from enzyme action. The body is full of checks

and balances, including lots of enzyme inhibitors, which allow enzymes tofunction properly without digesting you away. Every time you eat fresh fruits,

vegetables, or other raw food you are consuming digestive enzymes. Another

report assessed in-home use by over 4,000 consumers of detergents that

contained a protease enzyme (Bolam et al. 1971). The researchers concluded

that:

“... detergents containing proteolytic enzymes had no greater effect on

the skin than conventional detergents, even when the hand skin condition was

initially poor. The same was true in a further test on 130 housewives with

‘dishpan’ hands. No adverse reactions attributable specifcally to the enzyme products were seen. No eruptions from contact with clothes washed in enzyme

products were reported from any of the families involved in these tests.”

Enzymes and enzyme notication in the European Union and in non-EEA

countries are regulated in different legal provisions depending on their use,

e.g. as technical enzymes, food enzymes, feed enzymes, cosmetic enzymes or

medicinal products.

6.2 Enzyme safety: GeneralThe enzyme industry has developed safe host organism systems for the

production of many enzymes that could not otherwise be produced (Neidleman

1991). These safe host systems have been used since the early 1980s for the

production of different enzymes in contained manufacturing facilities. The

host organisms and their enzyme products have been tested to demonstrate

that they are safe for their intended use; this includes, but isn’t restricted to,

testing of the organism to demonstrate that it is not pathogenic and does not

produce toxins and testing of the product to demonstrate that it is safe for theintended use and to determine whether it is an irritant and how likely it is to

cause allergies when inhaled (IFBC 1990). Guidelines for safety assessments

of food and food ingredients developed through biotechnology have been

prepared by several internationally recognized scientists and expert groups



Safety and precaution in handling enzymes 301

(Organization for Economic Cooperation and Development, Health Canada,

Food and Agriculture Organization / World) (Health Canada 1994; Jonas et

al. 1996).

Using tools of modern biotechnology, the enzyme industry is continuing to

dramatically improve a wide range of production processes with both primary

and secondary benets (Jank and Haslberger 2003). The reduced consumption

of energy, water and raw material with generating less waste and fewer

environmental pollutants are among the target benets of this technology.

Additionally, many unavoidable waste materials can be effectively modied,

either for recycling as a useful raw material or as a valuable secondary product

(Kessler et al. 1992). Since the 1980s, new enzymes have been developedthrough modern biotechnology for widespread use in many products for

example in laundry detergents and paper production (Fuchs and Astwood

1996; Bajpai 1999). The use of these new enzymes in detergents enables

consumers to remove difcult stains at lower wash temperatures without

the use of harsh chemical additives, thus reducing the burden on wastewater

systems. Enzymes are simple proteins and thus are fully biodegradable and

environmentally sound (Pariza and Foster 1983). The use of new, more robust

enzymes in paper processing can signicantly reduce the need for chlorine

and chlorine dioxide bleach, which ultimately feeds into wastewater streamsas chlorinated organic compounds. Without the use of the modern tools of

biotechnology, not enough of these enzymes, as well as enzymes in other

industrial applications, can be produced to meet industry demand. Further,

these modern tools give us the opportunity to produce enzymes that can

replace other processes and/or chemicals that are less environmentally sound

and thereby continue to move towards sustainability (Pariza and Johnson

2001; Masgrau et al. 2006).

6.3 Enzyme safety program

The goal of an enzyme safety program is to maintain employee exposures

below a level that could cause an adverse health effect. The elements of a

safety program include employee training, product design, engineering

controls, work-practice controls, personal protective equipment and medical

surveillance (SDIA 1991).

6.3.1 Employee training

The following are suggestions for educating employees and contractors

about the importance of safe enzyme-handling practices. Proper training is

essential for the safe use of materials in an industrial setting (Vanhanen and




Tuomi 2000). It is important that people understand not only what is required

of them but also why. It is the responsibility of every company to ensure

that employees and contractors handling enzymes and enzyme products are

adequately trained. They must have adequate knowledge of the potential

health effects of enzymes and safe work practices associated with their use.

Information provided on enzymes should include the nature of the effect, risks

to health arising from exposure, routes of entry into the body, the anticipated

degree of exposure, and any factors that may increase risk. Employees should

know proper practices and procedures for working with enzymes and enzyme-

containing products.

Employees and contractors should understand:• Reasons for an education program

• Reasons for medical surveillance

• Health effects of enzymes

• Proper use of control measures

• Personal protection

• Emergency procedures

Instruction manuals, safe practices, and/or standard operating procedures

for the handling of enzyme preparations and enzyme products should be

written for the individual workplace at a level suitable for the work force(Grifth et al. 1969). These should take into account the types of enzymes and

equipment used and the types of products that are manufactured. All employees

and contractors involved in the use of enzyme materials must be briefed

on their potential effects and adequately trained in the relevant operations

specied in the standard operating procedures before working with these

materials. These operations should include all checks on the environmental

control systems (e.g., ventilation, static pressure, etc.) to ensure their correct

and safe operation. Only responsible, trained personnel should handle enzyme preparations, as mistakes, spills or plant malfunctions could potentially result

in the release of enzyme aerosol. All such employees should be trained in

cleaning and spill recovery procedures. In addition, all emergency response

personnel should be informed of the potential health effects of enzyme

preparations. Education and training are also required by the Occupational

Safety and Health Administration (OSHA) under its Hazard Communication

(HAZCOM) Standard (OSHA 29 CFR 1910.1200). This regulation requires

that the employer prepare a written hazard communication program and

provide employees with information and training on hazardous chemicals.To satisfy these OSHA requirements, employers may wish to document and

retain training program records, including employee attendance records,

program contents and instructor qualications. Where personnel are required

to use personal protective equipment, education and training are also required.




This training includes the proper equipment selection, as well as training in its

use and maintenance.

6.3.2 Product design

The physical form of the enzyme can greatly inuence the potential for

aerosol formation. Therefore, the product form often dictates the selection

of engineering controls, handling procedures, and the type of protective

equipment needed to provide adequate protection to the employee. Enzyme

aerosols can be in the form of liquid droplets, mists, solid particles, or dusts

(Plinke et al. 1992). Powdered enzyme formulations present the greatest

potential for exposure because they are easily aerosolized. Granular enzyme

formulations encapsulate the enzyme to prevent its release into the air. They

have low dusting capabilities, but care must be taken not to crush them. Liquid

enzyme formulations have a potential for aerosolization when any type of

mechanical agitation is taking place, such as container lling or cleaning of

spills.

6.3.3 Engineering controls

Engineering controls should be designed for the specic product form and

production process. A qualied ventilation expert should evaluate and design

the control measures. Key components of an effective program include overall

plant and equipment design, performance verication, system maintenance,

process design, and management of process changes. Engineering controls

in the form of enclosures and local exhaust ventilation are the most effective

methods to control enzyme exposures. Using enclosures and local exhaust

ventilation together will assist in isolating the enzyme preparation from the

employee. Local exhaust ventilation and enclosures should be used in thefollowing areas: locations where enzyme preparations are added into the

process, material transfer points, and where the enzyme-containing product is

packaged into containers.

6.3.4 Work practice controls

Proper work practice procedures are also important in controlling enzyme

aerosols and are often used in conjunction with engineering controls and

personal protective equipment. Operational goals in the facility should include

making sure that there are no visible dust or recurring spills; minimizing

skin contact as much as possible; avoiding prolonged temporary repairs on

equipment; and preventing, as well as containing, aerosol generation (Bannan




et al. 1983). Each site that works with enzyme preparations should have

programs which address good housekeeping and work practices. It is important

to implement work practices that do not generate enzyme aerosols or result

in direct skin contact. Safe work practices include proper enzyme transfer

procedures, cleaning procedures, spill cleanup, and good personal hygiene.

High-pressure water, steam, and vacuums without HEPA (high-efciency

particulate arresting) lters should be avoided. Spills should not be swept

or brushed, and washing facilities should be accessible and well maintained

(Newhouse et al. 1970). Good personal hygiene should be encouraged and

practiced. Initial and continuing education of employees and contractors on

the health effects of enzymes will allow better understanding and compliancewith safe work practices.

6.3.5 Personal protective equipment

Personal protective equipment should only be used as a supplement to

engineering controls and work practice controls. The exclusive use of personal

protective equipment instead of these other controls is strongly discouraged.

When needed, respiratory, eye, and skin protective equipment are used.

Respiratory protection equipment prevents exposure by ltering airborneenzyme aerosols or supplying clean air to the worker (Musk et al. 1989). The

type of respirator that should be used depends on the airborne concentration of

the enzyme, time spent in the production area, and worker activity. As in any

industrial environment where chemicals are used, eye and skin contact should

be avoided. Commonly used protective eye wear includes safety glasses with

side shields, splash goggles, or, for more protection, a face shield. The type of

eye protection should be determined by the potential for contact with enzymes

and the type of process or operation the employee is conducting. Protective

clothing is important when the potential for splashing or immersion is possible

(Rodriguez et al. 1994). Common pieces of equipment used to protect the

skin are rubber gloves, splash aprons, and full protective suits. Again, the

type of protective equipment to use depends on the potential for contact with

enzymes and type of process or operation the employee is conducting. A

qualied safety and health professional should be consulted for the selection

of personal protective equipment. Education and training is vital to ensure the

proper use of protective equipment.

6.3.6 Medical surveillance

A medical surveillance program monitors employees’ health and can provide

early detection of potential health problems. Such a program includes a medical




history, a medical examination of the employee, pulmonary function tests,

and the determination of enzyme sensitization. All these elements become

a baseline for the health and well being of the worker. There are always

many questions asked about the medical evaluation of allergies or a person’s

sensitization to specic allergens. There are two commonly used tests. The

rst is called the skin prick test which is commonly used by allergists (Nelson

et al. 1993). In this test a small drop of the specic enzyme allergen is placed

on the forearm and a sterile needle is used to gently lift the skin. A sensitized

individual will have a wheal and are appear on the arm. This is an indication of

the presence of allergic antibody (Pepys 1992). The second method is a blood

test called a Radio Allegro Sorbent Test (RAST) which evaluates a specicenzyme allergen present in a person’s blood stream. While sensitization is not

an illness, it is an indication that an employee has been exposed to sufcient

enzyme concentrations to cause the development of allergic antibody. If a

worker is found to be sensitized to an enzyme, it could mean that current work

practices, engineering controls and personal protective devices are not meeting

their goals and would need to be reevaluated by plant personnel (Zetterstrom

and Wide 1974). Sensitization does not prevent an employee from working

in an adequately controlled plant environment. Sensitized employees are at

a grater risk of developing allergy symptoms. Because of this, all employeesshould be educated in safe work practices that minimize exposure. If allergy

symptoms start to develop, the employee should be instructed to notify their

supervisor immediately (Pepys et al. 1969).

6.4 Safe handling of enzymes

Enzyme Technical Association (ETA 2000) provides information on safe

enzyme-handling practices for the textile industry. The ETA is a trade

association of the US enzyme producers and distributors, and has supported

the safety production and use of enzyme products since 1970. Exposure to

enzymes may cause irritation and/or respiratory allergies (Stenius and Wide

1969). The primary routes of exposure to enzymes are by inhalation and skin

and eye contact. Preventing exposures by these routes is the goal of an enzyme

safety program. Enzymes can be safely handled by using safe product design,

engineering controls, safe work practices, and appropriate personal protective

equipment. When handling concentrated enzyme preparations – as with most

substances used in industrial processes – care should be taken to avoid skincontact and inhalation of aerosols. Enzymes can be used safely without any

adverse health effects through the use of good work practices, engineering

controls, and appropriate personal protective equipment.




6.4.1 Skin and eye exposure

Skin and eye contract with proteolytic enzymes, those with the ability to break down complex proteins into simpler products, may cause irritation.

Other classes of enzymes are less irritating or pose no risk of irritation

(Goenin 1987). However, formulation ingredients may be irritants so it is

important to consult each product’s material safety data sheet (MSDS) for

safety precautions. Exposed areas should be protected by using hand and eye

protection and other protective clothing. When the exposure is discontinued,

the irritation should disappear. There is no evidence to indicate that enzyme

allergies are developed through skin contact (Hamann 1993).

6.4.2 Inhalation

As with any protein that is foreign to the respiratory system, repeated

inhalation of enzyme containing aerosol (dust or mist) can cause a respiratory

allergy in some individuals. There are two main stages to the development of

a respiratory allergy, which is also called Type 1 immediate hypersensitivity

(Buehler 1965; Flood et al. 1985). The rst stage is called sensitization and

this occurs when the individual is rst exposed by inhalation to the allergensuch as an enzyme, house dust, or pollen. As stated in the Soap Detergent

Association (SDA) document, “if enough enzyme is inhaled, the body will

begin to recognize the enzyme as a foreign material and will produce allergic

antibodies (Juniper et al. 1977). Once allergic antibodies are produced, the

individual is said to be sensitized”. However, sensitization is not a disease

as there are no allergic symptoms at this stage. In the second stage, clinical

allergic symptoms can occur when a sensitized individual is re-exposed to an

allergen such as an enzyme (Janssens et al. 1995; Flindt 1969). Symptoms of

enzyme allergies are no different than allergies to other materials such as housedust, animal dander, or pollen and can include sneezing, congestion, coughing,

watery eyes, or a runny nose. Symptoms will only occur if enzyme dust or

aerosols are inhaled and should disappear when the exposure is discontinued.

It is important to note that not all sensitized individuals will develop allergic

symptoms (Bruce et al. 1978). The development of allergies depends on the

susceptibility of the individual and the exposure concentration.

6.5 Symptoms of enzyme exposure6.5.1 Skin irritation

Enzymes are not skin sensitizers (Grifth et al. 1969). Prolonged skin contact

with proteolytic enzymes can cause skin irritation. The eyes can also be




irritated by contact with proteolytic enzymes. As would be expected, the more

concentrated the enzyme preparation, the greater the potential for producing

irritation upon contact. Skin irritation is most likely to appear in body areas

where perspiration occurs, i.e., hands, armpits, groin, and feet, and around

tight tting clothing areas, such as cuffs, waist, collar, and facial areas in

contact with face masks. This irritation is caused by the chemical properties

of the proteases and is not an allergic response (Gothe et al. 1972). Other types

of enzymes, i.e., non proteolytic enzymes, have not been shown to cause skin

and eye irritation. However, skin and eye contact with all enzymes should

be minimized as part of personal hygiene practices. As with any chemical,

avoid contact with enzymes if the skin is broken or irritated. Consult themanufacturer’s MSDS for information on the hazards associated with other

ingredients of the enzyme preparation (Zachariae et al. 1981). Also, inhaling

high levels of enzyme-containing aerosols may result in coughing and/

or congestion due to irritation of the mucous membranes of the respiratory

tract. Respiratory irritation is a very rare occurrence and should never occur

when adequate manufacturing controls are in place. Exposed areas should

be protected by the use of gloves and other protective clothing whenever the

potential for gross skin contact exists. The irritant response is characterized by

a weeping, red glistening appearance of the skin surfaces, usually involvinghands and ngertips, which can be painful. This reaction is due to the primary

irritant characteristic of proteases and is not an allergic response. When

exposure is discontinued, imitation should disappear.

6.5.2 Allergy

The purpose of the immune system is to protect the organism from foreign

substances such as infectious agents and tumors. An allergy is a hypersensitivity

reaction of the immune system (Norman 1992). There are several types of

allergies. Taken together, allergies occur among 20–30% of all humans.

Proteins, and therefore enzymes as well, can in some cases cause a so-called

Type I allergy which requires the development of allergic antibodies. Type

I allergy symptoms can include watery eyes, runny noses, sneezing (hay

fever), wheezing (asthma), hives (urticaria) and several reactions to food,

including vomiting and diarrhea (Marzulli and Maibach 1977). As with any

protein that is foreign to the respiratory tract, repeated inhalation of enzyme

contained in aerosols can cause an allergic response. Predicting who willdevelop an allergic response or the level and duration of exposure needed

to elicit a response is not known at this time (Zachariae et al. 1973). As with

any protein allergen, such as pollen, mild to severe symptoms may occur

and may include any, or a combination of, the following: Asthma, sneezing,




nasal or sinus congestion, coughing, watery eyes, runny nose, tightness of

the chest, hoarseness or shortness of breath. These symptoms may develop

during work hours or can be delayed, occurring even 2 or more hours after

work exposure (Pepys et al. 1985). Symptoms will occur only in an allergic

individual if enzyme aerosols are inhaled, and usually disappear within hours

or a few days after exposure is eliminated. Currently, there is no evidence to

indicate that skin contact with enzymes will cause allergic contact dermatitis

(Cronin 1987). Aside from allergies, no long-lasting effects from working

with enzymes have been found. Ordinary cold or u symptoms may resemble

enzyme allergy (McMurrain 1970).

6.5.3 Routes of exposure to allergens

Allergens can trigger the immune system through several routes of exposure,

via food, via skin contact or via inhalation (Pepys 1973). Enzymes can cause

allergies through repeated exposure via inhalation in sufcient high doses

and possibly by contact with mucosal surfaces (eyes, nose). There is limited

evidence for enzyme allergy development from contact with eyes. In addition,

only susceptible individuals will develop allergies. Exposure via food and

skin contact has not been documented to be associated with enzyme allergy(AMFEP – Association of Manufacturers of Fermentation Enzyme Products).

6.5.4 Allergy tests

There are two types of simple medical tests that can be made to determine if

an individual is sensitized to a particular enzyme (Bernstein 1972). When a

person becomes sensitized to a substance, allergic antibodies will be produced

against that substance. Sensitization by itself is not a disease, but rather an

indication of exposure to the enzyme that may lead to allergic symptoms(Sarlo et al. 1990). However, not all sensitized individuals develop allergic

symptoms. By detecting sensitization early, enzyme exposure can be controlled

to prevent allergy symptoms onset. Allergic antibodies can be detected either

through a laboratory blood test (such as Radio Allegro Sorbent Test – RAST or

Enzyme Linked Immuno Sorbent Assay – ELISA) or by a simple skin prick test

commonly used by allergists. The laboratory blood test measures the amount of

antibody in the blood, with a certain level indicating sensitization to a specic

enzyme preparation. The skin prick test consists of pricking the skin with asolution of the enzyme (antigen preparation). In a sensitized individual, a raised,

reddened area (wheal and are) will appear on the skin. If the laboratory blood

test or skin test is positive, it is an indication that sensitization has developed and

allergic symptoms may result unless precautions are taken to reduce exposure.




Pulmonary function testing is also a means to screen whether an individual has

allergic symptoms. Consult a physician for advice. Additional information on

allergy test procedures and materials is available from the enzyme manufacturer

or the Enzyme Technical Association.

6.6 Practical aspects – handling and safety

Safe handling of enzyme preparations can be accomplished through proper

work practices and use of protective equipment. When working with these

preparations, it is important to use work practices that do not generate

aerosols or that result in direct skin contact. For each work operation, careful

consideration must be given to minimizing aerosol formation and skin or eye

contact. Aerosols are formed through high-energy operations such as mixing,

grinding, washing with high water pressure or steam, and using compressed

air for cleanup operations. Sweeping, blowing, splashing, steam cleaning, and

high-pressure water ushing must be avoided. Mixing and grinding operations

should be contained as much as possible, and the areas in which they take

place should be provided with adequate local exhaust ventilation (Newhouse

et al. 1970). When handling enzyme preparations or enzyme-contaminated

equipment, avoid direct skin contact. Wear appropriate gloves when there is a potential for skin contact with enzymes. Wash enzyme-contaminated surfaces

thoroughly before handling.

6.6.1 Air monitoring

There are airs monitoring techniques available to measure the level of

enzyme dust or mist in the air. The American Conference of Governmental

Industrial Hygienists (ACGIH) has established a threshold limit value (TLV)

for only one class of enzymes, subtilisins, of 60 mg/m3 as a ceiling limit.Both low-ow and high-ow air sampling methods are available for some

enzymes. Contact the enzyme manufacturer for additional information. An

air monitoring program can evaluate the potential for employee exposure to

airborne enzymes and determine if current engineering control measures are

working properly or if additional control measures are needed. Air monitoring

is also valuable for the proper selection of respiratory protection. Enzyme

supplier may provide additional information on air monitoring for enzymes.

6.6.2 Respiratory protection

Under most operating conditions involving enzymes, respiratory protection

is not normally necessary. There are some operations, such as spill cleanup,




equipment cleaning, and equipment repairing, that may generate aerosols.

In these instances, respiratory protection may be the necessary guideline by

American Industrial Hygiene Association (AIHA 1992). The use of respiratory

protection is usually necessary when working with powdered enzymes.

Respiratory protection should also be used when indicated by your supervisor,

safety professional or medical personnel. The Occupational Safety and Health

Administration (OSHA) respiratory protection standard must be followed in

the selection, training and use of respirators. Use only National Institute of

Occupational Safety and Health (NIOSH) approved respiratory protection.

6.6.3 Protective clothing and glovesProtective clothing should be worn when there is a potential for skin or eye

contact. This clothing may include gloves, aprons, safety glasses, and outer

garments, such as coveralls or lab coats. Protective clothing is particularly

important when working with proteolytic enzymes, which are known to

cause skin irritation (Witmeur et al. 1973). Operations that may require the

use of protective clothing include spill cleanup, equipment maintenance, and

equipment cleaning. Gloves should be worn when there is a potential for skin

contact with any enzyme material. Cotton liners or cotton-lined gloves are

recommended to absorb perspiration. Protective clothing should be removed

prior to leaving the work area and should not be worn into other areas of

the facility (i.e., lunchroom, ofces) or to the home. The OSHA personal

protective equipment standard (1910.132-138) must be followed in selection,

training and use of personal protective equipment. Consult the enzyme

manufacturer and/or their MSDS for additional information on the selection

of personal protective equipment.

6.6.4 MaintenanceWhenever maintenance is to be performed on equipment that has been in

contact with enzymes, the equipment should be cleaned before the work is

begun. Use wet washing (ooding, wiping) or a vacuum system equipped

with a high-efciency particulate air lter (HEPA) to clean equipment or

spills. High-pressure cleaning (steam, air, or water) must be avoided, since

these operations are known to cause aerosol formation. Personal protective

equipment (gloves, respirators, eye protection) may be required during some

maintenance operations.

6.6.5 Spills

Care should be taken when handling enzyme preparation spills to prevent the

generation of aerosols. Workers should use appropriate personal protective




equipment as recommended by a qualied safety. Measures should be taken to

contain all spills. Vacuums using high efciency lters or a dedicated central

vacuum system are preferred. At no time should spills be swept, brushed

or dispersed by air blowing or high pressure water jets. After the spill is

collected, the area should be gently washed using cold, low pressure water or

water-spray portable vacuum cleaners. Consult a qualied health and safety

professional on spill cleanup for procedures tailored to particular industrial

settings.

6.6.6 Spill cleanup

Spilled enzymes must be removed immediately by central vacuum system,vacuums equipped with a HEPA lter, mopping, or washing. To prevent dust

or aerosol formation during cleanup, do not sweep or use high water pressure,

steam, or compressed air on spills. Use plenty of water in wet washing to

ush all enzyme material away to prevent enzyme dust generation from

dried material. Dependent upon the place and extent of the spill, respiratory

protection and protective clothing may be required during cleanup. Disposal

of spilled material should be in compliance with federal, state and local

regulations.

6.6.7 Personal cleanliness

Personal cleanliness is essential to prevent irritation from proteolytic enzymes

to skin and mucous membranes. The irritation response on skin is increased

in the presence of moisture and when the natural oils of the skin are removed.

The following procedures are recommended to prevent irritation:

1. Hands should be washed with water and mild soap before leaving the

work area and immediately after coming into contact with enzyme

materials.2. Change work clothes daily and whenever they are soiled with enzyme

material. Do not wear work clothing home.

3. Avoid touching your face and eyes with enzyme contaminated

clothing or gloves.

4. Wear cotton-lined gloves to absorb perspiration.

6.7 First-aid treatment

Skin contact: Most enzyme materials are water soluble; therefore, the exposedskin should rst be thoroughly ushed with water and then washed with a

mild soap and water. If clothes are contaminated, remove them, shower and

change into clean clothes. Immerse the contaminated clothes in water and

wash them as usual.




Inhalation: Remove the individual from exposure and monitor for

irritation or allergic symptoms. If symptoms occur, consult a physician.

Symptoms may occur as late as 2 or more hours after exposure.

Eye contact: Rinse the eyes thoroughly with water for at least 15 minutes

and then consult a physician.

6.8 Safety in enzyme therapy

Enzymes are used for wound healing because they selectively degrade

infection and dead tissue away while leaving healing tissue growing. Enzymes

are used to remove tumors because they attack the cancerous tissue and

remove it, while faciliating the growth of healthy tissue. This built-in natural

selective property of enzymes can be seen on surface wounds. It has also be

seen and measured in cell cultures and by monitoring internal wounds and

tumors (Enzyme therapy, Wolf). Enzyme therapy is currently approved in the

treatment of certain health conditions (Lopez 1994).

These include:

• Cardiovascular disorders

• Gastrointestinal conditions, particularly pancreatic insufciency and

related disorders • Replacement therapy for specic genetic disorders and deciencies

• Cancer treatment

• Debridement of wounds (degradation or cleaning out of dying or

dead/necrotic tissue)

• Removal of toxic substances from the blood

6.9 Routes of exposure and possible controls

6.9.1 Work practice control

Work-practice controls include appropriate management systems and

operational controls, education, safe work practices and good housekeeping

practices. Work-practice controls are used in combination with engineering

controls to limit employee exposure. Finally, personal protective equipment

is used to supplement engineering and work-practice controls, when both are

not feasible, or in the event of emergencies, such as spills, or during high

exposure tasks such as clean outs.

6.9.2 Management systems

Accountability for maintaining the necessary hygiene standards should

be clearly dened and assigned to the appropriate level of management.




Responsibilities for daily management of the program should be clearly

delegated to operating personnel. Companies should ensure that personnel

trained in medicine, industrial hygiene, and/or engineering verify that

these company standards are being adhered to. Access to potentially high

exposure areas such as enzyme addition areas should be restricted to qualied

employees.

6.9.3 Operational controls

Enzyme operations should seek to achieve:

• No visible dust

• No recurring spills

• No prolonged ‘temporary’ repairs

• No gross skin contact

• Prevention/containment of aerosol generation

6.10 Medical monitoring program for enzyme

workers

The medical monitoring of employees potentially exposed to enzymes attheir workplace is important. It is intended for use by companies producing

enzyme-containing cleaning product formulations. However, it may also be

applicable to other industries using enzyme technology. The intent is to assess

and monitor the health of employees working with enzymes. A program should

be designed to survey employee health to ensure that any adverse health

effects are uncovered and dealt with early. Occupational health and safety

stewardship of enzyme technology is important to prevent health allegations

that could erode public trust in the cleaning products industry. Enzymes can

cause sensitization and, in some cases, symptoms in susceptible, exposed

individuals. If enough enzymes are inhaled, the body will begin to recognize

the enzyme as a foreign material and produce allergic antibodies. Once allergic

antibodies are produced, the individual is said to be sensitized. Sensitization

by itself is not a disease. In individuals who have become sensitized, further

breathing of high levels of enzyme dust or aerosol can trigger symptoms

ranging from allergy symptoms such as watery eyes, runny nose and scratchy

throat to occupational asthma.

For a new employee or an employee transferring to enzyme work, thefollowing may be considered:

1. Medical history. Obtain a medical history of the employee. Specic

reference should be made to any history of allergies, asthma, eczema,

smoking, previous chest disease and medication use.




2. Respiratory questionnaire. Have the employee complete a respiratory

questionnaire.

3. Medical examination. On the basis of the medical history or responses

to the respiratory questionnaire, the medical examiner may decide to

conduct a physical examination.

4. Baseline pulmonary function test (PFT). A PFT should be done on

all employees before they begin working with enzymes so that a

pulmonary function baseline can be established against which future

PFT results can be compared.

5. Baseline determination of enzyme sensitivity. A baseline determination

of enzyme sensitivity may be made using either a skin prick test ofenzyme(s) that the employee will be exposed to or a blood test (e.g.,

RAST) to determine the level of antibody against the specic enzyme

allergen that is present in the blood. If a skin test is performed, a

negative (saline) and positive (histamine) control should be used as a

reference for the enzyme antigen.

6. Skin prick test for common allergens. A skin prick test for common

allergens may help determine the atopy of the individual.

6.11 Safety measures

Enzymes can be handled safely through the use of appropriate control

methods. Establishing safety programs and educating employees are the rst

steps to a safe and productive plant operation. Prior to introducing an enzyme

preparation into a consumer product, the potential for consumer exposure to

the enzyme and possible health effects should be assessed. Since enzymes

are respiratory allergens and some enzymes are irritants, both the potential

for inhaling the enzyme preparation and for skin contact should be evaluated.

Important factors that need to be considered include the following: Product

use, potential misuses, enzyme concentration, and product form (liquid,

powder, granule, and foam), duration and frequency of exposure, potential

exposure level and the no-effect level of enzyme exposure. Through the use

of proper work practices and control measures, enzymes can be handled in the

work place without any adverse health effects. All work with enzymes must

be done with care and proper precautions. Avoid generation of aerosols and

direct skin or eye contact when handling enzyme materials. Even though there

may be no visible signs of dust or aerosols, safety measures must be followedat all times. By following these relatively simple work practices and control

measures, enzymes can be handled safely.




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measuring biologically active enzyme dust in the environmental air of detergent factories’,

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Abstract: This chapter discusses the organic cotton textile standard, scope andapplication of biotechnology (enzymes) in textile wet processing for especially

organic cotton textiles. Very few research works were carried out in the pastdecade in the area of organic cotton wet processing concern, more over the globalorganic cotton textiles have been guided to process the organic cotton eithermild chemicals or enzyme technology for ecofriendly processing. The previousresearchers involved in production and characterization of various enzymes suchas alpha amylase, lipase, protease, pectinase and cellulase enzymes for cellulosicmaterials have been discussed. Application of these enzymes on organic cottonfabric for desizing, scouring and bleaching, their inuence on structure andproperties of organic cotton bres and fabrics have discussed elaborately withindividual, binary and mixed enzymatic system. The improved enzyme reactionsby the sonication and aerodynamic principle have also highlighted and reported

in single-stage enzymatic desizing, scouring and bleaching processes for organiccotton textiles. This study will provide the industrial bioscouring technologies aninsight into the properties of mixed enzymatic systems and the predictability oftheir scouring performance while deciding the recipe and process parameters.

Keywords: Organic cotton, alkaline pectinase, lipase, protease, mixed enzymes,ultrasonic, aerodynamic system

7.1 Introduction

Organic cotton usage in textile and apparel industry for development of biodiversied products in medical textiles such as wound dressing, surgical

gowns and baby care skin clothing are grown in the past years. The processing

of organic cotton in textile wet processing is recommended by the Global

Organic Textile Standard (GOTS) which framed the guidelines for textile

processing through mild chemicals and/or go with enzyme technology. In the

present industrial practices, the pectinase enzymes are used for bioscouring of

cotton materials, but drawback of the pectinase enzyme is it removes or break

downs only pectin groups in the cotton bre structure, which is not sufcient

for removal of wax, oil and fatty substances in the bre. However, recent

biotechnology and genetic engineering advances have opened opportunities for

successful applications of other enzyme systems, such as lipases, xylanases,

laccases, proteases and alkaline pectinases. The aim of this research work is to

7

Bioprocessing of organic cotton textiles




study the performance of biopreparation of organic cotton fabrics in biodesizing

and bioscouring processes through specic mixed enzymatic system.

Advances in biotechnology and enzymology have brought new lines

of research on organic cotton textiles and have accelerated the development

of enzymatic applications in textile wet processing for sustainable process.

Amongst the various stages of cotton preparation, textile wet processing is a

highly energy, water and chemicals consuming processes (Naik and Paul 1997;

Warke and Chandratre 2003). Enzymes are known for their specicity, high

efciency and ability to work under mild conditions and provide a promising

solution to ecofriendly processing challenges (Carlier 2001). But the awareness

and guideline in the textile wet processing is not still well known and describedonly to go with mild chemicals and/or process with enzymes. The sustainable

processing is the need of the textile wet processing for organic cotton fabrics to

obtain ecofriendly and environmental friendly agents and sustainable methods of

processing in order to have environmentally safe processing with less chemicals

inputs and problems in efuent disposals (Aiteromem 2008). Presently, the

pectinase enzymes used in the bioscouring of cotton textiles, but it’s having

drawback such as longer reaction time and removes only pectin groups in the

cellulosic structure of cotton bres. It is clear that advanced enzyme technology

can be used to develop a usable; more environment friendly, economicalcompetitive textile wet processing for organic cotton process to develop

hygienic and value added products in the forth coming days in the textile era.

7.2 Organic cotton

Organic cotton is grown in subtropical countries such as United States, China,

India, Pakistan, Republic of Uzbekistan, Brazil, Australia, Egypt, Argentina,

Turkey, Greece and Syria, from non-genetically modied plants (Thakur et

al. 1997). Organic cotton originates from organic agriculture and is grown

without the use of any synthetic agricultural chemicals such as fertilizers

or pesticides. Its production also promotes and enhances biodiversity and

biological cycles (Grant 2000; Vughn and Turley 1998). Organic cotton is

currently being grown successfully in many countries; the largest producers

(as of 2011) are the United States, Turkey, India and China. The strategy of

organic cotton production in global status is forecasted that the need of organic

cotton may be increased to 20% higher in the 2012–13.

7.2.1 Organic Textile Standard

The GOTS was developed by the International Working Group on the Global

Textile Standard as part of the International Conference on Organic Textiles



Bioprocessing of organic cotton textiles 321

(INTERCOT). The GOTS is emerged as a result of a technical harmonization

procedure for organic cotton processing. During last few years GOTS has

become the leading organic textile processing standard in spinning, weaving

and textile wet processing (Goyal 2009). The sizing of organic cotton fabric

recommended by the GOTS is natural sizing agents such as maize, potato

starch and thin boil starch, and in case of synthetic binders such as PVA –

polyvinyl alcohol and CMC – carboxyl methyl cellulose can be used (Behara

and Gupta 2009). In case of scouring of organic cotton fabric may be with mild

alkali or prefer to go with biochemical method or go with enzyme technology.

In case of bleaching of organic cotton fabric may use hydrogen peroxide or

biochemical method of handling with safe and ecofriendly chemicals.

7.2.2 Applications of organic cotton

Organic cotton is not only better for our bodies but also better for environment.

It makes a world of difference in the health and comfort of people, especially

those with allergies, asthma, or multiple chemical sensitivities (Nallankilli

1992). Few of the applications of organic cotton in textile and apparels are

t-shirts, shirts, trousers, underwear, vests, socks, baby wear, towels, bathrobes,

denim, bed-sheet, napkins and diapers. Applications of organic cotton inmedical textiles are wound dressing, surgical clothing’s, stockings, hygienic

and healthcare dressings etc.

7.2.3 Biotechnology – scope and importance

Enzymes are gaining an increasingly important role as a tool in various wet

textile pre-treatment and nishing processes (Tzanko et al. 2001). Biocatalysts

have proven to be a exible and reliable tool in wet textile processing and

found to be a promising technology to full the expected future requirements.Enzymatic scouring has been investigated extensively by various researchers

for nearly one decade (Buschie-Diller 1994; Traore and Buschle 2000; Nabil

Ibrahim et al. 2004; Tatsuma Mori et al. 1999; Wen-Chi et al. 1999). The organic

cotton fabric for its textile wet processing needs the use of minimum safe

chemicals to health (Gubitz and Cavaco-Paulo 2001; Warke and Chandratre

2003) and for this the alternative way is to go into enzyme technology, because

enzymes are substrate specic bio-catalysts and they operate best at an

ambient pressure, mild temperature and often at a neutral pH range (Poloncaand Petra 2009; Daniel et al. 2010). Different enzymes like pectinases such

as lyases (EC 4.2.2.2), polygalacturonase endo-acting type (EC 3.2.1.15) and

polygalacturonase exo-acting type (EC 3.2.1.67), proteases (EC 3.4.21-25),

cellulases such as endo-glucanases (EC 3.3.1.4); cellobiohydrolases (EC




3.2.1.91), xylanases (EC 3.2.1.8), lipases (EC 3.1.1.3); and recently cutinases

(EC 3.1.1.74) have been examined to degrade and subsequently remove the

natural component present in the outer layer of cotton bres (Presa and Forte

2007; Jayapriya and Vigneswaran 2010; Lenting et al. 2002).

7.2.4 Bioscouring with various enzymes on cellulosic

materials

Scouring is related to hydrophilicity and can be achieved by uncovering the

pores present in the bres, by removing waxes and other non-cellulosic materials

in the primary wall (Ramachandran and Karthik 2004). The technical feasibilityof enzymatic scouring has been recognized by many researchers over the last

decade. However, continuous enzymatic scouring process has not yet been

widely implemented by textile industries, due to inability to remove waxes from

cotton bre during enzymatic scouring. Much of the work in the area of cotton

bioscouring has been focused on investigating the utility of various enzymes.

Although several types of enzymes including pectinases (Vigneswaran and

Keerthivasan 2008; Hardin and Yanghuna 1997; Li and Hardin 1998; Etters et

al. 1999), cellulases (Vigneswaran and Jayapriya 2010; Buschie-Diller 1994),

proteases (Buchert and Pere 2000; Lin and Hsieh 2001), cutinases (Vigneswaranand Jayapriya 2010), xylanases (Traore and Buschle 2000), and lipases (Lenting

et al. 2002; Traore and Buschle 2000) have been studied. Pectinases have

proved to be the most effective and suitable enzymes for cotton bioscouring.

The mechanism of pectinase scouring reportedly assumes that the degradation

and elimination of pectins makes the loosened waxes more easily removable

with the help of mechanical agitation. This allows the cotton to achieve superior

hydrophilicity without bre deterioration (Traore and Buschle 2000).

A rationale approach is adopted to design a new efcient enzymatic scouring process (Buschle-Diller et al. 1998). Several aspects were considered such as

the specicity of enzymes, the complexity of the cotton bre substrate and

mass transfer. Different commercial as well as specially produced pectinases

were tested for bioscouring performance. Alkaline pectinases (PL and

Bioprep 3000L) work better than acidic pectinases (PGs). The pectin removal

efciency of specially produced PL was comparable to commercial Bioprep

3000L. The most important parameters, such as enzyme concentration, pH,

temperature, ionic strength and chelators, for the bioscouring process have

been evaluated (Tzanko et al. 2001; Perez et al. 2000). Hardin and Yanghuna(1997) postulated that pectin acts as a cementing material in the primary wall

of cotton bres. After enzymatic destabilization of the pectin structure, the

different components present in the primary wall layer can be removed easily

in subsequent rinsing steps.




7.3 Biodesizing of organic cotton fabrics with

alphas amylaseThe biodesizing of organic cotton fabric with indigenously produced alpha-

amylase and their enzyme kinetics, using alpha-amylase enzyme was studied

with various process parameters such as enzyme concentration, temperature

and reaction time (Vigneswaran et al. 2010). In that study, grey organic cotton

yarns of 2/40s Ne for warp and 40s Ne for weft yarns were procured from

M/s. Armstrong Mills (P) Limited, Tirupur, India. These yarns were used for

weaving of plain organic cotton fabric using a power loom. The organic cotton

fabric having 64 ends per inch, 60 picks per inch, fabric cover factor of 18.96

and average fabric mass of 120.44 g/m2 was produced. The laboratory scale

production of alpha-amylase enzyme and process variable for biodesizing are

briey discussed below.

7.3.1 Laboratory scale production of alpha-amylase

Seed culture – microbial source Bacillus cereus used in the present study was

obtained from Department of Microbiology, PSG College of Arts & Science,

Coimbatore, India. The culture was maintained on Nutrient Agar (NA) slants

and subcultured periodically.

7.3.1.1 Development of the inoculum

For the development of inoculum, the bacterial culture was transferred from

the stock to 100 mL nutrient broth and the inoculated asks were incubated

overnight at (35 ± 2)°C and 150 rpm. Cells were harvested from the broth

and their absorbance (A) was checked at 660 nm. Accordingly, cells with

inoculums size of A660 = 0.5 (10% inoculum [volume per mass]) per 5 g ofsubstrate were harvested, washed and resuspended in sterile distilled water.

7.3.1.2 Enzyme production system

The substrate used in the present study was wheat bran. Production media

containing 5 g of solid substrate and 10 mL of Bushnell–Haas (BH) mineral

salt medium in 250 mL Erlenmeyer asks were inoculated with the above

inoculum. Inoculum production media were incubated under static conditions

at (35 ± 2)°C and enzyme production was checked (Anto et al. 2006) for theenhanced production of the alpha-amylase. Glucose as an additional carbon

source (0.4 mg/g) was added into the production medium and it has been

reported that maximum enzyme production was observed after 72 hours. The

same incubation time was also maintained in our experiment.




7.3.1.3 Extraction of crude enzyme

After the incubation period, crude enzyme was extracted in 50 mL of 0.1 M phosphate buffer (pH 7) on a rotary shaker at 250 rpm for 30 min. The content

was ltered through muslin cloth. Filtrate was centrifuged at 8000 rpm for 10

min and clear brown supernatant was used as the enzyme source.

7.3.1.4 Alpha-amylase enzyme assay

Alpha-amylase was determined by incorporating a mixture of 0.5 mL of aliquot

of each enzyme source and 1% soluble starch dissolved in 0.1 M phosphate

buffer, pH 7, at 55°C for 15 min (Berneld 1955). The reaction was stopped by adding 1 mL of 3,5-dinitrosalicylic acid, followed by boiling for 10 min.

The nal volume was made up to 12 mL with distilled water and the reducing

sugar released was measured at 540 nm (Miller 1959). One unit (U) of alpha-

amylase activity was dened as the amount of enzyme that releases 1 mmol of

reducing sugar as glucose per minute, under assay conditions and expressed

as U/ml of dry substrate. All the experiments were performed in triplicates.

7.3.2 Warp yarn sizing and fabricationThe sizing of warp yarns of organic cotton was carried out using laboratory

model yarn sizing machine and the average size add-on on the warp yarn was

12.21% and then the sized warp yarns were taken into warp beam preparation

for weaving. The average size add-on the organic cotton fabric was measured

with respect to warp and weft yarn mass and was found to be 8.27%. The

aerial density of the PVA-sized organic cotton grey fabric after weaving was

found to be 130.24 grams per square metre. The size recipe of PVA starch

used for the organic cotton yarn was as follows:

Size recipe for warp yarn

• Polyvinyl alcohol (PVA) – 4 parts

• Vegetable tallow – 2 parts

• Anti-static agent – 0.3 parts

• Gum tragacanth – 2 parts

• Soap oil – 1 part

• Thin boiling starch – 40 parts

• Water – required level of pick up

7.3.3 Enzymatic desizing

The organic cotton sized fabrics were treated with alpha-amylase with various

process variables such as enzyme concentration, temperature and reaction




time. The process variables were chosen according to the Box–Behnken

method of statistical tool for process optimisation. In this present work, a

systematic statistical approach has been adopted to obtain optimum weight

loss of the sized fabric with different process conditions. The response surface

methodology was used to develop a mathematical correlation between the

enzyme concentration, temperature and time. The enzymatic desizing process

was carried out at pH 6–7 and then the fabrics were thoroughly rinsed with hot

water and cold water and dried at 80°C using hot air oven.

7.3.3.1 Design of experiment

The biodesizing experiments were conducted based on the Box–Behnken

second order design for three variables. In this experimental design, enzyme

concentration (X1), temperature (X

2) and time (X

3) were taken as independent

variables. The variables were selected at three levels: –1, 0, +1. The response

(Y) is given by a second order polynomial as shown in equation [7.1]:

k k k k 2

o i i ii i ij i j

i 1 i 1 i j i 1

Y b b X b X b X X= = ≥ =

= + + +∑ ∑ ∑∑ [7.1]

where Y – predicted response, bo – offset term, bi – linear effect, bii –squared effect and b

ij –interactive effect. The above equation was solved

using the Design-Expert (State-Ease Statistics Made-Easy, version 8.0.2,

2010) to estimate the response of the independent variables. The actual design

experiment and the corresponding actual values for each variable are listed

(Table 7.1).

Table 7.1 Design of experiments – alpha-amylase process variables

Coded values Biodesizing process variables

Enzyme concentration (%) Temperature (°C) Time (min)

–1 2 50 30

0 3 55 45

+1 4 60 60

7.3.3.2 Testing – Iodine solution

The residual presence of starch in the desized organic cotton fabric was

assessed by the iodine test as per the standard test procedure of the iodinesolution preparation. (Iodine solution was prepared as using reagent of

Potassium iodine (10 g of KI [100%] in 100 ml of water), add 0.6358 g of

iodine (100%) stir and shake, iodine is completely dissolved). Fill up to 800

ml with water then complete to 1000 ml of ethanol (Mostafa 2003).




7.3.4 Effect of biodesizing process variables – Box–

Behnken designThe Box–Behnken design of experiment was used to study the optimization

of biodesizing process conditions to get an accurate output and the predicted

% weight loss of desized organic cotton fabrics. Several factors inuence the

desizing of PVA starch such as alpha-amylase concentration, temperature

and time play a vital role. The limits for the design in terms of enzyme

concentration, temperature and time were xed after careful consideration

which plays a signicant role in the degradation of PVA starch leading to

effective desizing. The effects of enzyme concentration and temperature on

weight loss of PVA-desized organic fabric at various time intervals of (a) 30

min, (b) 45 min and (c) 60 min are shown in the 3D surface plot (Fig. 7.1).

Fig. 7.1 Effect of enzyme concentration and temperature on weight loss of desizedorganic cotton fabric at various time intervals of (a) 30 min, (b) 45 min and (c) 60 min

The empirical model was tted to the response and the lack of t test

was carried out and the polynomial equation derived from the design of




experimental is given below, taking into account the signicant interaction

effects (Table 7.2).

Table 7.2 Analysis of variance for the response surface

Source F P (Prob > F)

X1 – Enzyme concentration 93.94 0.0002

X2 – Temperature 6.63 0.0498

X3 – Time 79.84 0.0003

X1X

21.000 0.0064

X1X

30.255 0.075

X2X3 0.838 0.0037

The model F-value was 21.07 which implied that the model was

signicant and there was only 0.18% chance that a “Model F-value” of this

large a value could occur due to noise. The predicted R 2 value was 0.9743 and

is in reasonable agreement with the adjusted R 2 of 0.9281. Adequate precision

which measures the signal to noise ratio was 16.132, which is greater than

4 indicating that the model can be used to navigate the design space. From

Table 7.2, it can be observed that the enzyme concentration in the degradation

of PVA size starch in the desizing process had notably signicant differencesat F

actual > F

critical (F

2,14 values of 93.94 > 21.07), at 95% condence level. With

respect to the design of experiments, no signicant differences are found

between temperature levels of 50–60°C at Factual

< Fcritical

(F2,14

values of 6.63 <

21.07) at 95% condence level; this may be due to better stability and kinetics

of the enzyme at selected temperature ranges in the desizing process. With

respect to the reaction time in the desizing process, signicant differences were

observed at Factual

> Fcritical

(F2,14

values of 79.84 > 21.07) at 95% condence

level; this may be due to the enzyme reaction time of the degradation of PVAstarch mainly depends on the selected range of times to achieve the required

weight loss of the PVA starch in the sized fabric.

7.3.4.1 Effect of temperature and time

Figure 7.2 shows the weight loss of PVA-desized organic cotton fabric at

various enzyme concentrations. It can be seen that at any given time the

weight loss of the fabric increases with increasing temperature, and it can

also be observed that at a given reaction temperature, with increasing timethe desizing efciency increases for higher enzyme concentrations. At higher

concentration, the degradation of the PVA starch increases, and it can be

attributed to the signicant weight loss of the sized organic cotton fabric

during desizing as a result of hydrolysis. Moreover, it should be mentioned




that at a higher enzyme concentration, the increase in temperature and time

aids in faster hydrolysis resulting in quick conversion of polymer chains into

water-soluble products. Similar kind of results has also been observed by other

researchers (Mori et al. 1999; Hsieh and Cram 1999) and has been attributed

to the autocatalytic effect after certain time duration, thereby resulting in

higher weight loss.

Fig. 7.2 Effect of reaction time and temperature on weight loss of PVA-desized

organic fabric at various enzyme concentrations of (a) 2%, (b) 3% and (c) 4%

7.3.4.2 Effect of enzyme concentration and time

Figure 7.3 shows the contour plot of weight loss of the PVA starch-sized

organic cotton fabric at different temperatures. It can be seen that the weight

loss of the fabric increases with the increase in concentration of the enzymes

used in both lower and higher temperatures. Moreover, it can be seen that the




amount of hydrolysis of the size starch by the enzymatic reaction is higher at

higher temperature of 60°C indicating the hydrolysis of starch chain groups

into water-soluble groups at the elevated temperature, thereby increasing the

weight loss. However, it was noted that the effect of time is more pronounced

at elevated temperature and can be seen by the skew in the contour lines.

Fig. 7.3 Effect of enzyme concentration and reaction time on weight loss of PVA starch

desized organic fabric at various temperatures of (a) 50°C, (b) 55°C and (c) 60°C

7.3.4.3 Effect of enzyme concentration and temperature

Figure 7.4 presents the effect of enzyme concentration and temperature on the

weight loss of the PVA-sized organic cotton fabrics at various reaction times.With increase in enzyme concentration and temperature there is an increase

in desizing efciency at both lower and higher reaction time intervals, but

at higher time duration there is a higher rate of PVA starch hydrolysis with

the increase in enzyme concentration. Another interesting observation made




during the trials was the higher desizing rate of the specimen at higher enzyme

concentration, time and temperature resulting in a maximum weight loss of

8% and above.

Fig. 7.4 Contour graphs represents the effect of enzyme concentration and

temperature at various reaction times of (a) 30 min, (b) 45 min, (c) 60 min

7.3.5 Process optimization

The desizing process variables such as alpha-amylase enzyme concentration,

temperature and reaction time were optimized using Box–Behnken

experimental design and their output values are executed using Design-

Expert software 8.0. The Design-Expert software was executed to get variousoptions/predicted process parameters to achieve the required weight loss of

the fabric of 8.0% during the desizing of PVA-starched organic cotton fabric.

The software was processed and the resulting desirability and FDS-Fraction

of Design Space of design model of process (Fig. 7.5). The output result of




the Design-Expert software in achieving the desired weight loss of 8.0% in

the PVA starch desizing process is studied and out of which the software opted

the best process conditions of alpha-amylase enzyme concentration of 3.37%

at temperature of 55°C and reaction time 48 min with 1.0% desirability. From

the best opted test results, the actual weight loss of the desizing process of the

organic cotton fabric was achieved, 7.90% with an error of 1.25%.

Fig. 7.5 Fraction of Design Space (FDS) of design model of PVA desizing process

7.3.6 Characteristics of the biodesizing samples

7.3.6.1 Iodine test

The desizing efciency of the PVA starch-sized fabric was assessed by the

presence of starch on the fabric in the iodine test which indicates the depth of

color of the starch. The various enzyme concentration treated organic cotton

fabrics at 2%, 3% and 4% levels compared with grey organic sized cotton

fabric are studied and the absence or pale white color of the desized fabric is

the indication of removal of starch during the enzymatic desizing.

7.3.6.2 FTIR spectroscopic analysis

The organic cotton fabric with and without PVA starch size of fabrics were

analyzed using FTIR Spectrometer – (Schimadzu). The presence and integrity




of the PVA and other sizing compounds in the sized organic cotton fabric can

be clearly understood and the hydrolysis of the PVA starch during enzymatic

desizing at 8% level of weight loss of the organic cotton fabric sample shows

the removal of PVA groups in the specimen after desizing at 60°C reaction

temperature and 60 min time at 4% enzyme concentration at 3315 cm –1 which

is responsible for the –OH group stretching. In the test results, the transparency

(%) of the sized organic cotton fabrics are at a lower level when compared to

the grey fabric which is due to the starch and other ingredients present in the

size paste. The residual size components are analysed after desizing using

FTIR reports by differentiating the transparency (%) wave length of the grey

and desized organic cotton fabrics are studied and wave length at 1058 cm –1

,1112 cm –1 and 2362 cm –1 groups are responsible for C–C stretch from phenyl

ring, –CH2 symmetric stretching and C–H stretching in the desized organic

cotton fabrics.

7.3.7 Summary

The process optimization of desizing of PVA starch sized organic cotton

fabric has been studied and the process variables such as alpha-amylase

enzyme concentration temperature and reaction time were optimized toachieve the required desizing efciency in terms of weight loss % of fabric

and the degradation of PVA starch during the desizing process on the fabrics

was assessed by iodine test and FTIR test results. The activity of the alpha-

amylase enzymes are better and catalyze the degradation of PVA starch at

the temperature range of 50–55°C and time of 30–40 min to achieve the

required level of 8% size removal efciency. The pH of the desizing bath

has a major inuence on the improved reaction of the enzyme to catalyze the

hydrolysis of starch groups. At the higher enzyme concentration of 4% level

and higher temperature of 60°C lesser time to achieve the required desizing

efciency was observed. Process variables are optimized using the Design-

Expert software 8.0, and it will pave the way to predicting the enzyme kinetics

at various concentrations, temperatures and reaction times to achieve the

required desizing efciency with minimum error %. This study will be helpful

to the organic cotton processors for the ecofriendly and sustainable textile wet

processing using alpha-amylase enzyme in the desizing of PVA starch-based

desizing operations. This study will provide the industrial sizing technologies

an insight into the properties of PVA-based modied starch materials and the predictability of their desizing performance while deciding the size recipe and

desizing parameters.




7.4 Bioscouring of organic cotton with alkaline

pectinase7.4.1 Grey organic cotton fabric and desizing

In this study the 100% organic cotton fabric was produced which having the

specications 64 ends/inch, 60 picks/inch, 19.0 fabric cover factor and 120.4

g/m2 average fabric mass. The average size add-on on the organic cotton

fabric was measured with respect to warp and weft yarn mass; it was found to

be 8.27%. The aerial density of the organic cotton grey fabric after weaving

was found to be 130.2 g/m2. The PVA sized 100% organic cotton sized fabrics

were treated with alpha-amylase using various process variables such as3% enzyme concentration, 55°C temperature and 60 min reaction time. The

enzymatic desizing process was carried out at pH 6–7 and then the fabrics

were thoroughly rinsed with hot water followed by cold water and dried at

80°C.

7.4.2 Enzymatic scouring with alkaline pectinase

Alkaline pectinase and puried pectate lyase were selected for the degradation

of cotton pectin. Scouring experiments were performed in 1 L beaker in which

three fabric samples of 10 × 10 cm were treated using enzyme solution of

different concentrations of 2–6%, non-ionic wetting agent of 1–2%, treatment

time of 30, 45 and 60 min, and pH of 8.5–9.0. The fabric samples thereafter

were rinsed in 500 mL of water at 90°C for 15 min to inactivate the enzymes

followed by drying at 80°C to constant weight.

7.4.2.1 Design of experiment

Experiments were conducted based on the Box-Behnken second order design

for three variables (Table 7.3). The response (Y) is given by a second order

polynomial, as shown in equation [7.1].

Table 7.3 Box-Behnken experimental design

Coded

values

Bioscouring process variables

Enzyme concentration (%) Temperature (°C) Time (Min)

–1 2 50 30

0 4 55 45

+1 6 60 60




7.4.3 Testing

7.4.3.1 FTIR analysisThe organic cotton fabric with and without alkaline pectinase treatment were

analyzed using FTIR Spectrometer (Model: 8400S, Make: Schimadzu).

7.4.3.2 Pectin determination

The pectin removal of organic cotton fabrics was carried out as per the

procedure reported earlier (Wen-Chi et al. 1999). K/S values were calculated

using the following equation:

K/S = (1 – R)2 / 2R [7.2]

The lower the K/S values the lesser is pectic and proteinic substance

present in cotton bre (Ame and Toby 2005).

7.4.3.3 Fabric water absorbency

Water absorbency of organic cotton fabric treated with alkaline pectinase was

evaluated according to AATCC test method 79-2000.

7.4.3.4 Wax content

The wax content of the grey organic cotton fabric and alkaline pectinase

treated fabrics were measured as per AATCC test method 97-2009 (revised)

by solvent extraction using Soxhlet apparatus.

7.4.3.5 Weight loss

After the enzymatic treatments, the weight loss (WL) of the treated fabrics

was calculated using the following formula: %WL = (W

1 – W

2) *100 / W

1 [7.3]

where W1 and W

2 are the weights of fabric before and after enzymatic

treatment.

7.4.4 Effect of enzymatic process variables

Enzymatic treatments were carried out based on the experimental design

(Table 7.3). The effect of enzyme concentration and temperature on weight

loss of organic cotton fabric at various time intervals of (a) 45 min and (b) 60min are shown as 3D surface plot (Fig. 7.6). The empirical model was tted

to the response and lack of t test was carried out. The polynomial equation

derived from the experimental design is given below taking into account the

signicant interaction effects (Table 7.4).




Fig. 7.6 Effect of enzyme concentration and temperature on weight loss of organic

cotton fabric at various time intervals of (a) 45 min, and (b) 60 min

Table 7.4 Analysis of variance for response surface

Source F P (Prob > F)

Enzyme conc. (X1), % 267.48 0.0001

Temp. (X2), °C 26.87 0.0035Time (X

3), min 2770.93 0.0003

X1*X

21.29 0.3069

X1*X

36.39 0.0527

X2* X

30.14 0.7201

Final equation in terms of coded factors

Fabric weight loss = + 2.42 + 0.23 × X1 + 0.072 × X

2 + 0.74 × C-0.023 × X

1 ×

B-0.050 × X1 × X

3 – 7.5E-003 × X

2 × X

3 + 0.075 × X

12 +

0.088 × X22 – 0.22 × X3

2 [7.4]

Final equation in terms of actual factors

Fabric weight loss = + 6.82 + 0.163 × X1 – 0.35700 × X

2 + 0.149 × X

3 –

2.25E-003 × X1 × X

2 – 1.66E-003 × X

1 × X

3 – 1.0E-004

× X2 × + 0.018 × X

12 + 3.50E-003 × X

22 – 9.77E-004

× X3

2 [7.5]

where X1 is the enzyme concentration (%); X

2 , the temperature (°C); and

X3, the time (min).

The ‘Model F-value’ was 66.15 which imply that the model is signicantand there is only 0.01% chance that a ‘Model F-value’ of this large value

could occur due to noise. The predicted R 2 value is found to be 0.94 and is in

reasonable agreement with the adjusted R 2 value of 1.0. Adequate precision

which measures the signal to noise ratio is found to be 16.821, which is




greater than 4, indicating that the model can be used to navigate the design

space. From Table 7.4, it is observed that the enzyme concentration affects the

degradation of pectin in the scouring process, showing signicant differences

at Factual

>Fcritical

(F2,14

values of 267.48 > 66.15) at 95% condence level. With

respect to the design of experiments, there is no signicant difference found

between the temperature levels of 50–60°C at Factual

< Fcritical

(F2,14

values of

26.87 < 66.15) at 95% condence level. This may be due to better stability

and kinetics of enzyme at selected temperature ranges in the scouring

process. With respect to reaction time in the scouring process, it is found that

signicant differences occur at Factual

> Fcritical

(F2,14

values of 2770.93 > 66.15)

at 95% condence level. This may be due to the enzyme reaction time on thedegradation of pectin to achieve required weight loss of the cotton fabric.

Table 7.5 shows the predicted and actual fabric weight losses of the scoured

100% organic cotton fabric with alkaline pectinase. From the test results, it is

observed that the error % of the predicted and actual value of the experimental

design is below 3% and not found to be statistically signicant at 5%.

Table 7.5 Design model and error of experiment

Run

Enzyme

(X1) conc.%

Temp.

(X2)°C

Time

(X3)min

Weight loss , %

Error %Predicted Actual

1 4.0 60 30 1.64 1.61 1.83

2 4.0 55 45 2.42 2.48 –2.48

3 2.0 55 60 2.80 2.74 2.14

4 2.0 50 45 2.30 2.25 2.17

5 2.0 60 45 2.45 2.38 2.86

6 6.0 55 60 3.2 3.15 1.56

7 6.0 50 45 2.76 2.85 –3.268 4.0 50 30 1.44 1.42 1.39

9 4.0 50 60 2.95 3.04 –3.05

10 4.0 55 45 2.42 2.43 –0.41

11 2.0 55 30 1.25 1.22 2.40

12 6.0 55 30 1.85 1.87 –1.08

13 6.0 60 45 2.82 2.81 0.35

14 4.0 60 60 3.12 3.18 –1.92

15 4.0 55 45 2.42 2.43 –0.41

7.4.5 Effect of enzyme concentration and temperature

Figures 7.7 and 7.8 clearly indicate, as expected, a progressive weight

loss with increasing enzyme concentration due to the hydrolysis of pectin.




At higher time duration, there is higher rate of pectin and wax hydrolysis

observed with the increase in enzyme concentration. The organic cotton fabric

shows higher water absorbency rate at higher enzyme concentration of 6%,

treatment time of 60 min and temperature of 60°C with maximum weight loss

of 3.2% and above. Figure 7.8 shows the interaction of alkaline pectinase

enzyme concentration and temperature for 60 min reaction time which shows

the higher rate of pectin removal by the reaction of alkaline pectinase enzyme

at 60°C on the organic cotton bre to break the pectin components in the bre

structure. The water absorbency of fabric is in the range of 12–14 sec when

treated with 2–3% alkaline pectinase at 55°C for 30 min. The better water

absorbency is observed for 5–6% alkaline pectinase concentration at 55–60°Cfor 60 min treatment time. The lower the water absorbency time the better

scouring can be done.

Fig. 7.7 Effect of enzyme concentration and temperature at various reaction time (a)

30 min, (b) 45 min, (c) 60 min




Fig. 7.8 Interaction of enzyme concentration and temperature

7.4.6 FTIR spectroscopic analysis

The FTIR spectra of the desized organic cotton fabric and 2%, 4% and 6% pectinase enzyme treated cotton fabrics are analysed and it mainly highlights

the changes in the non-cellulosic impurities by characterizing the carboxyl

acids and esters that are present in pectin and waxes. The hydrolysis of

the pectin at 6% enzyme concentration for 45 min reaction time indicates

the maximum removal of pectin and waxes, as indicated by the peak at

3315 cm –1, responsible for the –OH group stretching, the CH stretching at

2917 cm –1, the asymmetrical COO– stretching at 1617 cm –1, and the CH

wagging at 1316 cm –1. The absorbance intensity of the characteristics peaks at

around 1736 cm –1 varies in the following order: desized fabric >2% pectinase>4% pectinase >6% pectinase fabrics.

7.4.7 Wax and pectin removal

Figure 7.9 shows the wax content of alkaline pectinase treated cotton fabric

and also it is observed that the wax present in the grey organic cotton fabric is

0.81% and the subsequent alkaline pectinase treated fabric with 2%, 4% and 6%

enzyme concentrations at 60°C temperature and 45 min reaction time shows

35.80%, 48.15% and 54.32% loss in wax content respectively. It may be dueto the fact that the alkaline pectinase enzyme degrades the pectin component

in the organic cotton bre which hydrolysis the wax component in the bre.

Figure 7.10 shows the pectin degradation level of organic cotton fabric treated

with various process conditions of time and temperature with 2–6% alkaline




enzyme concentration. It is observed that the rate of pectin removal increases

with increase in enzyme concentration and at higher time and temperature.

Fig. 7.9 Wax content of alkaline pectinase treated organic cotton fabric at 60°C for

45 min reaction time

The enzyme kinetics of alkaline pectinase at various concentrations with time

interval of 30, 45 and 60 min show that the pectin removal rate is 1.30 and 1.32times higher in case of 2–4% and 4–6% pectinase concentration respectively

at 60°C. The higher pectin removal (82.41%) is observed at 60°C and 60 min

treatment time and in addition the efcient wax removal step improves the

performance of pectinase in terms of pectin removal and hydrophilicity.

Fig. 7.10 Pectin degradation of alkaline pectinase-treated organic cotton fabric

enzyme kinetics – interaction of pectin degradation rate at 60°C





The process variables such as alkaline pectinase enzyme concentration,temperature and reaction time have been optimized using Box-Behnken

experimental design and their output values are executed using Design-Expert

software 8.0. This software was executed to get various options/predicted

process parameters to achieve required pectin degradation range of 75–82%

and weight loss of 2.80%. The output result of the Design-Expert software to

achieve the desired weight loss in the alkaline pectinase process for predicted

process variables of 9 solutions is shown (Table 7.6). The software shows the

best process conditions, such as alpha-amylase enzyme concentration 5.65%,

temperature 60°C, and reaction time 45 min with 1.0% desirability. The best

opted test results are the loss of 78.40% pectin with 2.80% weight loss at an

error of 1.21%.

It is possible to employ alkaline pectinase to scour the organic cotton fabric

to achieve better water absorbency rate of <5 sec and 52.5% wax removal by

higher pectin degradation of 75–80% using 6% enzyme concentration, 60°C

temperature, and 60 min reaction time with 3.2% fabric weight loss. The

higher concentration of alkaline pectinase enzymes shows better activity and

catalyzes the degradation of pectin groups at 55–60°C and 8.5–9.0 pH, takinglesser time to achieve required pectin hydrolysis. This study will be helpful to

the organic cotton processors for the ecofriendly and sustainable textile wet

processing using alkaline pectinase enzyme in bioscouring processes and also

will pave the way to nd better scouring with mixed enzymatic systems, to

predict required alkaline pectinase and to bring better scouring performance

of cotton fabrics.

Table 7.6 Output results of Design-Expert software

Solution

no.

Enzyme

concentration

(%)

Temperature

(°C)

Time

(min)

Fabric

weight loss

(%)

Pectin

degradation

(%)

Water

absorbency

(sec)

1 5.65 60.00 45.00 2.80079 80.21 5.999

2 5.67 59.94 45.00 2.80114 80.42 5.999

3 5.70 59.84 45.00 2.80173 79.34 6.000

4 5.72 59.78 45.00 2.80215 78.40 6.000

5 5.68 60.00 44.82 2.79741 81.07 5.999

6 5.53 60.00 45.79 2.81662 79.38 5.999

7 5.41 60.00 45.00 2.76276 80.32 6.270

8 6.00 58.20 45.00 2.79149 78.92 6.140

9 5.42 59.76 47.04 2.84409 79.43 5.999




7.5 Bioscouring of organic cotton fabric using

protease enzymeProtease enzyme from puried Pseudomonas aeruginosa was selected for the

degradation of cotton low volatile wax, oil and fatty substances. Scouring

experiments were performed in 1 L beaker in which three fabric samples of 20

× 20 cm were treated in protease enzyme solution of different concentrations

of 1–3%, non-ionic wetting agent of 1–2%, treatment time of 30 min, 45 min,

60 min and pH from 8.0 to 8.5. The fabric samples thereafter were rinsed in

500 mL of water at 90°C for 15 min, to inactivate the enzymes followed by

drying at 80°C, then to weigh the fabric using electronic balance.

7.5.2 Effect of enzymatic process variables – Box-

Behnken design

Enzymatic treatments are carried out based on the experimental design as

given in Table 7.7. The effect of enzyme concentration and temperature on

weight loss of organic cotton fabric at various time intervals of (a) 30 min, (b)

45 min, and (c) 60 min are shown as 3D surface plot (Fig. 7.11). The empirical

model was tted to the response surface and lack of t test was analysed.

The polynomial equation derived from the design of experimental is given below taking into account the signicant interaction effects of input variable

for ANOVA as given in Table 7.8.

Table 7.7 Box-Behnken experimental design – protease enzyme

Coded

values

Input variables

Enzyme

concentration, %Temperature, °C Time, min

–1 1 50 300 2 55 45

+1 3 60 60


Source Factual

P (Prob > F)

X1 – Enzyme concentration 62.78 0.0035


X3 – Time 66.76 0.0763

X1*X

20.3933 0.6602

X1*X

30.2554 0.0530

X2* X

30.6602 0.7109




The model F-value of 16.04 implies the model was signicant and there

was only 0.35% chance that could occur due to noise. The predicted R 2 value

was 0.9665 and is in reasonable agreement with the Adjusted R 2 of 1.0. From

Table 7.8, it is noticed that the enzyme concentration in the degradation of

wax component in the scouring process was noticed signicant differences at

Factual

>Fcritical

(F2,14

values of 62.78 > 16.04) at 95% condence level.

With respect to the design of experiments, there is no signicant difference

found between temperature levels of 50–60°C at Factual

< Fcritical

(F2,14

values of

4.97 < 16.04) at 95% condence level, it may be due to better stability and

kinetics of enzyme at selected temperature ranges in the scouring process.

With respect to reaction time in the scouring process, it was noticed thatsignicant differences at F

actual > F

critical (F

2,14 values of 66.76 >16.04) at 95%

condence level, it may be due to the enzyme reaction time on the degradation

of wax depends mainly on the selected range of times to achieve required

weight loss of the organic cotton fabric.

Figure 7.12 shows the predicted and actual fabric weight losses of the

bioscoured 100% organic cotton fabric treated with protease enzyme. From

the test results, it was observed that the error % of the predicted and actual

value of the experimental design was not found to be statistically signicant

at 95% condence level.

Fig. 7.12 Relationship between the actual and predicted weight loss of protease

enzyme-treated on organic cotton fabric




7.5.2 Effect of protease enzyme concentration and

temperatureFigure 7.13 clearly indicates, as expected, a progressive weight loss with

increasing protease enzyme concentration due to the hydrolysis of wax

components. At higher time duration, there is higher rate of wax degradation

observed with increase in enzyme concentration. An interesting observation

noticed during the trials that the organic cotton fabric was noticed higher water

absorbency rate at higher enzyme concentration of 3%, 60 min time and 60°C

temperature of maximum weight loss of 1.92% and above. The interaction

of protease enzyme concentration and temperature at 60°C which shows the

higher rate of wax removal by the reaction of enzyme on the organic cotton bre

to break the low volatile fatty components. The water absorbency of fabric is

in the range of 8–14 sec when treated with 2–3% protease enzyme at 55°C for

60 min. The lower the water absorbency time the better scouring can be done.

Fig. 7.13 Contour graphs represent the effect of enzyme concentration and

temperature at various reaction times of (a) 30 min (b) 45 min (c) 60 min





The FTIR spectra of the desized organic cotton fabric and 1–3% proteaseenzyme treated cotton fabrics are analyzed and it mainly highlights changes in

the non-cellulosic impurities by characterizing the carboxyl acids and esters

that are present in waxes.

The hydrolysis of the wax components at 3% enzyme concentration for

60 min reaction time indicates the maximum removal of waxes, as indicated

by the peak at 3315 cm –1, responsible for –OH group stretching, the CH

stretching at 2917 cm –1, the asymmetrical COO- stretching at 1617 cm –1, and

CH wagging at 1316 cm –1. The absorbance intensity of the characteristics

peaks at around 1736 cm –1 varied in the following order: desized fabric > 1%

protease > 2% protease > 3% protease fabrics.

7.5.4 Wax removal by protease enzyme treatment

Figure 7.14 shows the wax content of protease enzyme treated cotton fabric,

it was observed that the wax present in the grey organic cotton fabric was

0.81% and subsequent protease enzyme treated fabric at 1%, 2% and 3%

enzyme concentrations at 60°C temperature and 60 min reaction time noticed14.82%, 18.15% and 24.63%, respectively. It may be due to protease enzyme

degrading the low volatile wax, oil and fatty components in the organic cotton

bre.

Fig. 7.14 Wax removal (%) of protease treated organic cotton fabric at 60°C and 60

min reaction time




7.5.5 Process optimization – protease enzyme

The process variables such as protease enzyme concentration, temperature andreaction time was optimized using Box-Behnken experimental design and their

output values are executed using Design-Expert software 8.0. This software

was executed to get various options / predicted process parameters to achieve

required wax degradation range of 23–25% and weight loss of the fabric 1.80%.

The output result of the Design-Expert software to achieve the desired

weight loss in the protease enzyme process is shown the predicted process

variables of seven solutions which is shown (Table 7.9), out of which the

software opted best process conditions of protease enzyme concentration

of 2.95% at temperature of 60°C and reaction time of 58 min with 1.0%

desirability. The best opted test results are loss of 23.54% wax with 1.80%

weight loss at an error of 1.08%. It is possible to employ protease enzyme

to scour the organic cotton fabric to achieve better water absorbency rate

between 5 and 8 sec and 22–25% wax removal by higher wax removal of

75–80% pectin degradation at 6% enzyme concentration, 60°C, and 60 min

reaction time with 1.80% fabric weight loss. The higher concentration of

protease enzyme have noticed better active and catalyze the degradation of

wax groups at temperature range of 55–60°C and pH at 8.5–9.0 took lessertime to achieve required proteinolytic hydrolysis.


Solution

no.

Enzyme

conc.

(%)

Temp

( °C)

Time

(min)

Fabric

Weight loss

(%)

Wax

removal

(%)

Water

absorbency

(sec)

1 2.95 60.00 58.24 1.8079 23.54 8.1258

2 2.87 59.92 60.00 1.8114 22.85 6.2944

3 2.90 59.84 59.86 1.8173 23.61 8.12464 2.92 59.72 60.14 1.8215 24.52 6.2576

5 2.91 60.00 58.00 1.7627 23.64 6.5270

6 3.00 59.28 56.42 1.7949 25.04 5.6814

7 2.92 58.76 58.04 1.8449 23.85 7.6992

7.6 Bioscouring of organic cotton fabric using

lipase enzyme

Lipase enzyme from puried Pseudomonas pseudoalcaligenes was selectedfor the degradation of low volatile wax, oil and fatty substances. Scouring

experiments were performed in 1 L beaker in which three fabric samples of

20 × 20 cm were treated in an enzyme solution of different concentrations of

lipase enzyme 0.4–0.8%, non-ionic wetting agent of 1–2%, treatment time of




30 min, 45 min, 60 min and pH from 8.0 to 8.5. The fabric samples thereafter

were rinsed in 500 mL of water at 90°C for 15 min, to inactivate the enzymes

followed by drying at 80°C, then to weigh the fabric using electronic balance.

7.6.1 Effect of enzymatic process variables – Box-

Behnken design

Lipase enzymatic treatments are carried out based on the experimental design

as given in Table 7.10. The effect of enzyme concentration and temperature on

weight loss of organic cotton fabric at various time intervals of (a) 30 min, (b)

45 min, and (c) 60 min are shown 3D surface plot (Fig. 7.15). The empiricalmodel was tted to the response surface and lack of t test was analysed.

The polynomial equation derived from the design of experimental and their

signicant interaction effects between variables are given in Table 7.11.

Fig. 7.15 Effect of lipase enzyme concentration and temperature on weight loss of

bioscoured organic cotton fabric at various time intervals of (a) 30 min, (b) 45 min and

(c) 60 min




Table 7.10 Box-Behnken experimental design – Lipase enzyme

Codedvalues

Input values of the variablesEnzyme concentration

(%)

Temperature

(°C)

Time (min)

–1 0.4 50 30

0 0.6 55 45

+1 0.8 60 60


Source F P (Prob > F)X

1 – Enzyme concentration 110.73 0.0001


X3 – Time 149.64 0.0478

X1*X

20.6004 0.7624

X1*X

30.0384 0.8630

X2* X

30.2496 0.4094

Final equation in terms of coded factors

Fabric weight loss = +0.52 + 0.20 × X1 + 0.035 × X2 + 0.23 × X3 + 0.015 × X1 × X

2 + 0.075 × X

1 × X

3 + 0.035 × X

2 × X

3 – 0.055 × X

12

+ 0.025 × X22 – 0.030 × X

32 [7.8]

Final equation in terms of actual factors

Fabric weight loss = +3.42250 + 0.70 × X1 – 0.133 × X

2 – 0.013167 ×

X3 + 0.015 × X

1 × X

2 + 0.025 × X

1 × X

3 + 4.667E-004 ×

X2 × X

3 – 1.375 × X

12 + 1.00E-003 × X

22 – 1.33E-004 ×

X32 [7.9]

X1 – Enzyme concentration (%) X

2 – Temperature in °C

X3 – Time in minutes

The model Fcritical

value of 31.06 implies the model was signicant and

there was only 0.07% chance that could occur due to noise. The predicted R 2

value was 0.9824 and is in reasonable agreement with the adjusted R 2 of 1.0.

From Table 7.11, it is noticed that the enzyme concentration in the degradation

of wax component in the scouring process has signicant differences at Factual

> Fcritical (F2,14 values of 110.73 > 31.06) at 95% condence level. With respectto the design of experiments, there is no signicant difference found between

temperature levels of 50–60°C at Factual

< Fcritical

(F2,14

values of 3.39 < 31.06)

at 95% condence level; it may be due to better stability and kinetics of

enzyme at selected temperature ranges in the scouring process. With respect




to reaction time in the scouring process, signicant differences were noticed

at Factual

>Fcritical

(F2,14

values of 149.64 > 31.06) at 95% condence level; it

may be due to the enzyme reaction time on the degradation of wax depends

mainly on the selected range of times to achieve required weight loss of the

organic cotton fabric. The predicted and actual fabric weight losses of the

scoured 100% organic cotton fabric treated with protease enzyme is shown in

Fig. 7.16. From the test results, it was observed that the error % of the predicted

and actual value of the experimental design was noticed within range and not

found to be statistically signicant at 95% condence level.

Fig. 7.16 Relationship between the actual and predicted weight loss of protease

enzyme-treated organic cotton fabric

7.6.2 Effect of lipase enzyme concentration and

temperature

Figure 7.17 clearly indicates, as expected, a progressive weight loss with

increasing lipase enzyme concentration due to the hydrolysis of wax and oil

components. At higher time duration, there is higher rate of wax degradation

observed with increase in enzyme concentration. An interesting observation




noticed during the trials that the organic cotton fabric was noticed higher water

absorbency rate at higher enzyme concentration of 0.8%, 60 min reaction

time and 60°C temperature of maximum weight loss of 0.92% and above,

which shows the higher rate of wax removal by the reaction of enzyme on

the organic cotton bre to break the low volatile fatty components. The water

absorbency of fabric is in the range of 7–26 sec when treated with 0.4–0.8%

lipase enzyme at 60°C for 50–60 min reaction time.

Fig. 7.17 Contour graphs represent the effect of lipase enzyme concentration and

temperature at various reaction times of (a) 30 min, (b) 45 min (c) 60 min


The FTIR spectra of the desized organic cotton fabric and 0.4–0.8% protease

enzyme treated cotton fabrics are analyzed and it mainly highlights changes in


that are present in waxes. The hydrolysis of the wax components at 0.8%




lipase enzyme concentration for 60 min reaction time indicates the maximum

removal of waxes, as indicated by the peak at 3315 cm –1, responsible for –OH

group stretching, the CH stretching at 2917 cm –1, the asymmetrical COO–

stretching at 1617 cm –1, and CH wagging at 1316 cm –1. The absorbance

intensity of the characteristics peaks at around 1736 cm –1 varied in the

following order: desized fabric > 0.4% lipase > 0.6% lipase > 0.8% lipase

fabrics. The residual non-cellulosic components were analyzed after enzyme

treatment using FTIR reports.

7.6.4 Wax removal by lipase enzyme

The wax content of lipase enzyme treated cotton fabric was observed that

the wax present in the grey organic cotton fabric was 0.81% and subsequent

protease enzyme treated fabric at 0.4%, 0.6% and 0.8% enzyme concentrations

at 60°C temperature and 60 min reaction time noticed 8.76%, 12.42% and

18.94%, respectively (Fig. 7.18). It may be due to lipase enzyme degrading

the low volatile wax, oil and fatty components in the organic cotton bre.

Fig. 7.18 Wax content of lipase enzyme-treated organic cotton fabric at 60°C and 60

min reaction time


The process variables such as lipase enzyme concentration, temperature

and reaction time was optimized using Box-Behnken experimental design

and their output values are executed using Design-Expert software 8.0. This

software was executed to get various options / predicted process parameters




to achieve required wax degradation range of 16–19% and weight loss of the

fabric 0.80%. The output result of the Design-Expert software to achieve the

desired weight loss in the alkaline pectinase process is shown the predicted

process variables of 9 solutions which is shown in Table 7.12, out of which

the software opted best process conditions of lipase enzyme concentration

of 0.85% at temperature of 60°C and reaction time of 60 min with 1.0%

desirability. The best opted test results are loss of 16.24% pectin with 0.82%

weight loss at an error of 1.15%. It is possible to employ lipase enzyme to scour

the organic cotton fabric to achieve better water absorbency rate in the range

of 10–15 sec and 16.2% wax removal at 0.8% lipase enzyme concentration,

60°C, and 60 min reaction time with 0.82% fabric weight loss. The higherconcentration of lipase enzymes are better active and catalyze the degradation

of low volatile wax and oil groups at temperature range of 55–60°C and pH at

8.5–9.0 took lesser time to achieve required proteinolytic hydrolysis.


S. no. Enzyme

conc.

(%)

Temperature

(OC)

Time

(min)

Fabric

weight

loss (%)

Wax

removal

(%)

Water

absorbency

(sec)

1 0.85 60.00 58.24 0.82079 16.24 13.8

2 0.87 59.92 60.00 0.80412 17.52 12.2

3 0.80 59.84 59.86 0.81173 16.85 14.1

4 0.82 59.72 60.14 0.82215 16.76 13.2

5 0.88 59.64 58.62 0.79741 17.52 12.5

6 0.83 60.00 58.79 0.81662 18.94 13.8

7 0.81 60.00 58.00 0.79760 16.96 14.5

8 0.80 59.28 56.42 0.79491 16.94 13.6

9 0.82 58.76 58.04 0.83409 16.82 14.6

7.6 Binary enzyme treatment on bioscouring of

organic cotton fabric

7.6.1 Effect of binary enzyme concentration on weightloss of organic cotton fabric

The effect of binary enzyme concentration on the weight loss of the organic

cotton fabrics treated at 60°C and 60 min reaction time was studied. With




increase in enzyme concentrations, there is an increase in fabric weight loss.

Another interesting observation that occurred during the trials was that the

organic cotton fabric was noticed higher water absorbency rate at binary

enzyme concentration of 8% of pectinase and 3% of protease and the fabric

weight loss of 6.76% and above. From the test results, pectinase plays important

role in removal of pectin groups and protease and lipase plays esterication of

oil, wax and fatty substances in the organic cotton during bioscouring process.

The cellulase enzyme enhances the pectinolytic reaction of pectinase enzyme

to fasten the enzyme reaction. Another interesting observation noticed that the

pectinase and protease combination of enzyme treatment was noticed higher

pectinolytic and proteinolytic reaction at 60°C and 60 min reaction time.The protease and lipase combination of enzyme treatments was noticed the

removal of low volatile wax and fatty substances in the organic cotton.

7.6.2 Fabric wax removal – binary enzyme combinations

Figure 7.19(a) and (b) represent the wax removal analysis of various binary

mixed enzyme combinations of pectinase and protease enzymes and pectinase

and lipase enzymes treated at 60°C, 60 min reaction time at pH 8–8.5.

From the test results, it was noticed that higher wax removal was achievedat 6% pectinase and 3% protease. Protease enzyme plays important role in

degradation or breakdown of wax and oil substances present in the organic

cotton fabrics.

The regression equation of this analysis shows R 2 = 0.975; it correlated

the linear relationship between wax removal and concentration of binary

enzyme combinations at 95% condence level. The combination of protease

and lipase enzyme was carried out to optimize the better removal of wax and

oil substances at various combinations treated at 60°C, 60 min reaction time

is shown (Fig. 7.19c). From the test results, it was observed the interesting

results that wax removal rate was achieved 52.8% at 6% protease and 0.8%

lipase binary mixed enzyme treatment on the organic cotton fabric. The

pectinase and protease binary enzyme combination was observed higher

rate of pectinolytic and proteinolytic activity on organic cotton fabric when

compared with pectinase and lipase enzyme combinations. Because protease

enzyme can be removal of high and medium volatile fatty substances and

lipase enzyme responsible for low volatile fatty substance hydrolysis. The

wax and oil substances using protease and lipase responsible for proteinolyticactivity and pectinase enzyme responsible for pectinolytic activity on the

organic cotton fabric was analyzed with individual and binary mixed enzyme

combination.




Fig. 7.19 Wax removal % of organic cotton fabric treated with binary enzymes:

(a) pectinase and protease, (b) pectinase and lipase, and (c) protease and lipase




7.7 Bioscouring of organic cotton fabrics through

specifc mixed enzymatic systemThe objective is to develop an improved enzymatic cotton scouring process

on the basis of a fast enzyme reaction to efciently remove the pectin and

wax compounds from the organic cotton fabrics by specic mixed enzymatic

system.

An attempt has been made to study the pectinolytic activity of degrading

rate of pectin on the organic cotton fabric using four selective enzymes such as

alkaline pectinase, protease, lipase and cellulase enzyme with various process

parameters such as enzyme concentration, temperature and reaction time.

These process variables are selected based on the articial neural network

(ANN) using MATLAB 7.0 software design of experiment and output of

experiment was resulted with fabric physical properties such as weight loss,

water absorbency, wetting area, whiteness index, yellowness index, brightness

index. The bioscoured organic cotton fabric was tested for wax content and

pectin degradation rate on the fabric and their results were optimized with

minimum error. The test results were analyzed to predict the optimum process

parameters to achieve the required bioscouring fabric properties and removal

of pectin degrading rate and compared their results with actual trials. The performance of specic mixed enzymatic system and alkaline pectinase

enzyme during bioscouring process was assessed by ruthenium red dye test

and FTIR results to conrm the degradation of pectin on the bioscouring of

organic cotton fabrics.

7.7.1 Bioscouring with mixed enzymatic system

In this study, the bioscouring of organic cotton fabric was carried out by

selecting specic mixed enzymes namely (a) alkaline pectinase, (b) protease,(c) lipase, and (d) cellulase. These enzymes was specially screened, isolated

and puried, the selective alkaline pectinase puried from Pectate Lyase was

selected for the degradation of cotton pectin. Various experimental setups

and techniques were applied in the enzymatic scouring experiments. All

experiments were performed with demineralised water.

Scouring experiments where performed in 1 L beaker in which three

fabric samples of 10 × 10 cm were treated in an enzyme solution of different

concentrations of 2–6%, non-ionic wetting agent of 1–2%, treatment time of30 min, 45 min, 60 min and adjusted to pH of 8.5–9.0. The beaker was placed

in a temperature controlled water bath at 50°C, 55°C and 60°C. After the

treatment, the fabric samples were rinsed in 500 mL of water at 90°C for

15 min, to inactivate the enzymes. Thereafter the samples were rinsed twice




for 5 min in water at room temperature. Finally, the samples were dried at

80°C using hot air oven and weigh the fabrics using electronic balance with

accuracy ±0.01grams.

7.7.1.1 Articial Neural Network (ANN)

Neural networks are used for modeling non-linear problems and to predict the

output values for a given input parameters from their training values. Most

of the textile processes and the related quality assessments are non-linear

in nature and hence neural networks nd application in textile technology.

The software used in this study was backward feed propagation network

using MATLab 7.0. In order to carry out prediction, the network was trained

with training patterns namely input and output parameters. Input and output

parameters used for training the ANN and their selection criteria are given

below.

Input parameters

(i) Enzyme concentration

(ii) Process time

(iii) Process temperature

Output parameters

(i) Fabric weight loss

(ii) Fabric water absorbency

(iii) Fabric wetting area

(iv) Fabric whiteness index

(v) Fabric yellowness index

(vi) Fabric brightness index

7.7.1.2 Training of neural network

For training, the organic cotton fabrics were treated with various enzyme

concentration, time and temperatures with specic mixed enzyme system.

Then the physical characteristics such as fabric weight loss, water absorbency,

wetting area, whiteness index, yellowness index and brightness index of the

organic cotton fabrics were evaluated with standard testing procedures and

their values are trained by using feed backward propagation algorithm. For the

error back propagation net, the sigmoid function is essentially for non linearfunction. Training process of the neural network developed was started with

5000 preliminary cycles to optimize the ANN prediction accuracy. The best

structure is one that gives lowest training error and it is found to be minimum

error percent. The training of the network was further continued in order to




reduce the training error. The average training error of 1% was obtained and

terminated at this stage since beyond this reduction in training error was not

appreciable. Figure 7.20 shows selective articial neural network training of

specic mixed enzymatic systems and their input and output levels.

Fig. 7.20 Schematic diagram of ANN used in bioscouring of organic cotton fabric

7.7.1.3 Testing of neural network

For testing the prediction accuracy of the neural network a known specications

and process parameters were evaluated and their error percentage was

compared with predicted sample values. It can be observed that mean absolute

error with respect prediction is around 1%.

7.7.2 Fabric weight loss – effect of enzymatic process

variables

The response surface methodology is an empirical modeling technique, which

is used to evaluate the relationship between a set of controllable experimental

factors and observed results. Factors inuence the bioscouring process oforganic cotton such as enzyme concentration; temperature and time play a

vital role. The effect of enzyme concentration and temperature on weight loss

of organic cotton fabric at various time intervals of (a) 30 min, (b) 45 min and

(c) 60 min are shown 3D surface plot (Fig. 7.21).




Fig. 7.21 Fabric weight loss of bioscoured organic cotton fabric treated with various

process conditions of (a) pectinase, (b) protease, (c) lipase, and (d) cellulase

7.7.3 Effect of enzyme concentration and temperature

The effect of enzyme concentration and temperature on the weight loss of the

alkaline pectinase enzyme treated organic cotton fabrics at various reaction

times was analysed. With increase in enzyme concentration and temperature

there is an increase in fabric weight loss at both lower and higher reaction

time intervals but at higher time duration there is higher rate of pectin and wax

hydrolysis with increase in enzyme concentration. An interesting observation

noticed during the trials that the organic cotton fabric was noticed higherwater absorbency rate at higher enzyme concentration of 6%, 60 min time and

60°C temperature with maximum weight loss of 3.2% and above.





The FTIR spectra of the desized organic cotton fabric and 2%, 4%, 6% pectinase enzyme treated cotton fabrics are analyzed and found changes in


that are present in pectin and waxes. It can be clearly understood that the

presence of cellulose group peaks around 1000–1200 cm –1 and integrity of

the pectin and wax compounds in the organic cotton fabric at 1736 cm –1 and

1617 cm –1, respectively. The hydrolysis of the pectin during alkaline pectinase

enzymatic treatment (D) at 6% concentration and 45 min reaction time of

the organic cotton fabric showed the removal of pectin and wax groups in

the specimen at 3315 cm –1 which was responsible for –OH group stretching,

the –CH stretching at 2917 cm –1, the asymmetrical COO- stretching at

1617 cm –1, and CH wagging at 1316 cm –1. The absorbance intensity of the

characteristics peaks at around 1736 cm –1 varied in the following order:

desized fabric > 2% pectinase > 4% pectinase > 6% pectinase fabrics. In the

test results, the transmittance (%) of the pectinase enzyme treated organic

cotton fabrics are noticed lower level when compared to desized fabric which

was due to the degradation of pectin, waxes and non-cellulosic compounds

while pectinolytic degradation. The residual non-cellulosic components wereanalyzed after enzyme treatment using FTIR reports by differentiating the

transmittance (%) wave length. From the test results, the peaks at 1058 cm –1,

1112 cm –1 and 2362 cm –1 groups are responsible for C–C stretch from phenyl

ring, –CH2 symmetric stretching and C–H stretching in the alkaline pectinase

treated organic cotton fabrics.

7.7.5 Process optimization – specic mixed enzymatic

systemThe various specic enzymes such as alkaline pectinase, protease, lipase and

cellulase enzymes with process variables such as enzyme concentrations,

temperature and reaction time was optimized using MATLAB 7.0 software

with neural network experimental design and their output values are executed.

Tables 7.13 and 7.14 represent the process variables for training sample of

bioscoured organic cotton fabrics of their input values and output results of

actual and predicted respectively. The software was executed to get various

options / predicted process parameters to achieve required pectin degradationrange of 75–82% and weight loss of the fabric 4.80%. The software was

processed for analyzing the performance and desirability of FDS-Fraction of

Design Space of design model of process for optimized test results (Fig. 7.22a

and b). The output result of the software to achieve the desired bioscouring




of organic cotton fabric on their physical properties such as fabric weight

loss, water absorbency, wetting area, whiteness index, yellowness index, and

brightness index in the specic enzymatic system. The software opted best

process conditions of specic mixed enzymes was sample 19, which was

treated with 8% alkaline pectinase, 3%protease, 0.8%lipase and 0.8%cellulase

process condition at temperature of 55°C and reaction time 60 min, pH 8.5

with 1.0% desirability. From the best opted test results, the actual pectin and

weight loss of the bioscoured organic cotton fabric was achieved 78.40% and

4.80% respectively with error of 1.218%.

Fig. 7.22 Neural network training of mixed enzymatic system for analyzing (a) their

performance level, and (b) FDS –Fraction of Design Space level




7.7.6 Fabric Whiteness Index (FWI)

From Table 7.13, the highest whiteness index of bioscoured organic cottonfabric with specic mixed enzymatic system is achieved 52.57% at 8%

pectinase, 4%protease, 0.8% lipase and 0.4% cellulase enzyme at 60 min

reaction time, 60°C and pH 9.0 (Sample no. 29). It may be due to better

integration and higher concentration of pectinase for removal of pectin up

to 83.2% and wax/oil component removal up to 92.4% on the organic cotton

bioscoured fabrics which has higher water absorbency and lower yellowness

in nature when compared to sample no. 18. The mixed enzymes such as

pectinase, protease and lipase plays a important role for removal of pectin

and wax/oil components and also cellulase enzyme supports the exo and endo

partial surface reaction of the organic cotton fabrics.

7.7.7 Fabric Yellowness Index (FYI)

From Table 7.13, the lowest yellowness index of the bioscoured organic cotton

fabric with specic enzymatic system is achieved 13.14% at 8%pectinase,

3%protease, 0.8%lipase and 0.8%cellulase of sample no. 19 treated at 60 min

reaction time, 55°C and pH 9.5. It may be due to higher removal of pectin and

wax component in the organic cotton fabric in the enzymatic system whichhas whiteness index of 52.413. It is noticed that highest whiteness index of

organic cotton fabric show lower yellowness index in all the treated fabrics.

For sample no. 18 which has highest yellowness index of 24.371% due to

absence of pectinase and cellulase enzymes, 2%protease and 0.8%lipase.

From the test results, the pectinase plays important role in removal of pectin

for lowering the yellowness index of fabric and cellulase plays the better

mixed enzyme reaction on the organic cotton fabric during bioscouring.

7.7.8 Fabric Brightness Index (FBI)

From Table 7.13, the highest fabric brightness in bioscoured organic cotton

fabric was found in the sample no. 19 which was treated with 8% pectinase,

3% protease, 0.8% lipase and 0.8% cellulase at 60 min time, 55°C, and pH

8.5. It may be due to higher whiteness of 52.413 and lower yellowness index

of 13.14 and fabric treated higher pectinase and cellulase concentrations.

It was also noticed that higher concentration of cellulase enzyme treated

fabric observed higher brightness index due to surface smoothness of the

organic cotton fabric. The lowest brightness index of organic cotton fabric

was noticed in sample no. 18, it was treated with absence of pectinase and

cellulase, 2%protease and 0.8% lipase enzyme conditions. It was noticed that

pectinase and cellulase enzymes plays important role in brightness index of

the bioscoured organic cotton fabrics.




T a b l e 7 . 1

3 A r t i

f c i a l n e u r a l n e t w o r k – t r a i n i n

g s a m p l e s

S a m p l e n o .

I n p u t d a t a ,

S p e c i f c m i x e d e n z y m e s

O u t p u t d a t a ,

T r a i n e d s

a m p l e s f a b r i c p r o p e r t i e s ( A c t u a l )

E n z y m e c o n c e n t r a t i o n s ( % )

P r o c e s s p a r a m e t e r s

F a b r i c

W e i g h t l o s s

( % )

F a b r i c w a t e r

a b s o r b e n c y

( s e c )

F

a b r i c

w

e t t i n g

a r e a

( m m

2 )

F a b r i c

W h i t e n e s s

I n d e x

( W I )

F a b r i c

Y e l l o w

I n d e x ( Y I )

F a b r i c

B r i g h t

I n d e x

( B I )

P e c t i n

a s e

P r o t e a s e

L i p a s e

C e l l u

l a s e

T i m e

T e m p

p H

S 1

4

2

0 . 4

0 . 4

3 0

5 5

8 . 5

2 . 8

3 . 2

7 0

2 6 . 1

5 2

2 2 . 1

4 2

5

4 . 1

4 7

S 2

4

2

0 . 6

0 . 6

3 0

5 5

9 . 5

3 . 1

2 . 4

1 1 4

2 6 . 4

5 2

2 1 . 8

3 1

5

6 . 3

8 7

S 3

4

2

0 . 8

0 . 8

3 0

6 0

8 . 5

4 . 4

1 . 8

2 4 0

2 7 . 3

2 0

2 0 . 3

9 2

5

8 . 9

4 7

S 4

6

3

0 . 4

0 . 4

3 0

6 0

9

4 . 1

1 . 4

2 3 8

3 2 . 5

2 0

2 0 . 3

1 4

5

7 . 3

2 4

S 5

6

3

0 . 6

0 . 6

4 5

5 5

8 . 5

4 . 3

1 . 2

2 4 7

3 4 . 3

7 8

2 0 . 1

3 2

5

8 . 1

4 7

S 6

6

3

0 . 8

0 . 8

4 5

6 0

9

4 . 4

1 . 2

2 5 1

3 8 . 4

1 5

1 7 . 7

4 2

6

3 . 4

3 6

S 7

6

2

0 . 6

0 . 8

4 5

6 0

8 . 5

4 . 7

0 . 8

3 3 0

3 6 . 1

3 7

1 9 . 2

4 1

6

1 . 5

2 4

S 8

2

2

0 . 4

0 . 4

4 5

6 0

9

2 . 7

6 . 2

6 5

2 7 . 8

6 0

2 0 . 6

9 0

5

1 . 5

8 0

S 9

2

1

0 . 6

0 . 6

3 0

5 5

8

2 . 4

5 . 8

6 4

2 8 . 6

0 0

2 3 . 5

4 7

5

2 . 4

0 7

S 1 0

2

3

0 . 8

0 . 8

6 0

6 0

9

3 . 8

4 . 8

6 7

2 7 . 6

9 0

2 2 . 6

4 1

5

3 . 4

5 2

S 1 1

2

1

0 . 4

0 . 4

6 0

6 0

8

2 . 4

5 . 7

7 2

2 7 . 8

3 1

2 3 . 6

4 0

4

9 . 8

6 0

S 1 2

0

3

0 . 6

0 . 6

6 0

5 5

8 . 5

2 . 7

7 . 2

4 5

2 4 . 5

8 0

2 3 . 6

5 4

4

9 . 6

3 1

S 1 3

0

1

0 . 4

0 . 4

6 0

5 5

9 . 5

2 . 9

8 . 9

5 1

2 3 . 9

8 0

2 3 . 9

7 2

5

1 . 2

5 0

S 1 4

6

0

0 . 8

0 . 4

4 5

6 0

9

3 . 8

3 . 1

1 1 4

3 7 . 8

6 0

1 7 . 8

5 0

5

2 . 3

6 8

S 1 5

4

1

0 . 6

0 . 6

4 5

6 0

9

4 . 5

3 . 4

1 2 0

3 9 . 6

4 7

1 9 . 6

4 0

5

3 . 2

4 0

S 1 6

6

1

0 . 4

0

3 0

6 0

9 . 5

3 . 1

4 . 2

7 4

3 2 . 0

7 8

2 0 . 0

6 8

5

9 . 3

7 8

S 1 7

4

2

0 . 1

0

4 5

6 0

8 . 5

2 . 7

4 . 4

7 0

2 8 . 5

2 0

2 0 . 1

2 7

5

8 . 4

1 8

S 1 8

0

2

0 . 8

0

6 0

5 5

9

2 . 2

8 . 8

6 1

2 2 . 4

1 3

2 4 . 3

7 1

4

3 . 4

3 6

S 1 9

8

3

0 . 8

0 . 8

6 0

5 5

9 . 5

4 . 8

1 . 2

2 5 0

5 2 . 4

1 3

1 3 . 1

4 0

6

8 . 7

1 5

S 2 0

8

1

0 . 2

0

3 0

6 0

8 . 5

2 . 8

2 . 5

8 5

5 1 . 4

5 2

1 4 . 8

5 7

5

1 . 4

5 0

S 2 1

8

3

0

0 . 4

4 5

6 0

9 . 5

4 . 1

1 . 8

2 6 4

5 2 . 4

7 0

1 4 . 2

5 0

5

8 . 6

7 0

S 2 2

6

2

0

0 . 8

6 0

5 5

8 . 5

4 . 2

1 . 4

2 2 8

4 7 . 6

8 0

1 5 . 8

5 7

5

3 . 4

8 0

S 2 3

2

2

0 . 6

0 . 4

6 0

6 0

9

3 . 1

4 . 8

2 6 0

2 7 . 6

5 0

1 9 . 5

6 1

5

2 . 6

8 7

S 2 4

2

3

0 . 6

0 . 4

4 5

5 5

9

3 . 4

4 . 6

2 6 8

2 6 . 7

8 0

2 0 . 4

5 0

5

2 . 6

8 1

S 2 5

2

2

0 . 4

0 . 8

4 5

6 0

8

3 . 3

4 . 2

2 5 0

2 7 . 5

4 0

2 1 . 5

8 0

5

1 . 2

4 0

S 2 6

4

3

0 . 6

0 . 4

3 0

5 5

8 . 5

3 . 8

2

3 0 0

3 0 . 6

8 1

1 8 . 6

4 0

5

4 . 8

9 0

S 2 7

4

3

0 . 4

0

4 5

5 5

8 . 5

3 . 6

1 . 8

3 1 0

2 9 . 7

4 0

1 9 . 5

6 4

5

9 . 5

7 0

S 2 8

4

3

0 . 8

0 . 4

4 5

5 5

8 . 5

3 . 5

2

2 8 0

3 0 . 4

5 0

1 7 . 6

9 8

6

4 . 5

8 0

S 2 9

8

4

0 . 8

0 . 4

3 0

6 0

9 . 5

4 . 9

1 . 6

2 9 2

5 2 . 5

7 0

1 3 . 8

0 0

6

7 . 4

8 0

S 3 0

0

3

0

0 . 4

4 5

6 0

8

2 . 8

8 . 7

4 8

2 3 . 1

4 7

2 2 . 4

1 3

5

4 . 1

3 8




T a b l e 7 . 1

4 A r t

i f c i a l n e u r a l n e t w o r k – o u t p u

t d a t a a n a l y s i s ( p r e d i c t e d )

S a m p l e n o .

A N N o u t p u t d a t a , F a

b r i c p r o p e r t i e s

A

N N e r r o r %

F a b r i c

w e

i g h t

l o

s s

( % )

F a b r i c

w a t e r

a b s o r b

( s e c )

F a b r i c

w e t t i n g

a r e a

( m m 2 )

F a b r i c

W h i t e n e s s I n d e x

( W I )

F a b r i c

Y e l l o w

I n d e x ( Y I )

F a b r i c

B r i g h t I n d e x

( B I )

F a b r i c

W e i g h t

l o s s

( % )

F a b r i c

w a t e r

a b s o r b

( s e c )

F a b r i c

w e t t i n g

a r e a

( m m 2 )

F a b r i c

W h i t e n e s s I n d e x

( W I )

F a b r i c

Y e l l o w

I n d e x ( Y I )

F

a b r i c

B

r i g h t

I n d e x ( B I )

S 1

2 . 8 7 4

3 . 0

8 7

7 2

2 8 . 8

7 6

2 1 . 9

2 7

5 7 . 9

6 7

– 2 . 6

5 7

3 . 5

0 6

– 2 . 8

5 7

– 1 0 . 4

1

0 . 9

6 7

–

7 . 0

5 5

S 2

2 . 9 8 9

2 . 4

5 3

1 1 9

2 8 . 8

7 5

2 1 . 3

2 1

5 8 . 0

3 5

3 . 5

7 4

– 2 . 2

4 6

– 4 . 3

8 5

– 9 . 1

6 0

2 . 3

3 2

–

2 . 9

2 3

S 3

4 . 3 2 5

1 . 8

3 7

2 2 0

2 8 . 8

7 6

2 1 . 9

6 9

5 7 . 9

9 5

1 . 6

9 5

– 2 . 0

8 3

8 . 3

3 3

– 5 . 6

9 6

– 7 . 7

3 3

1 . 6

1 5

S 4

4 . 0 9 0

1 . 4

7 2

2 1 8

3 3 . 0

8 8

1 9 . 3

5 8

5 7 . 9

8 6

0 . 2

3 9

– 5 . 1

5 0

8 . 4

0 3

– 1 . 7

4 7

4 . 7

0 5

–

1 . 1

5 6

S 5

4 . 2 4 2

1 . 2

7 7

2 6 1

3 4 . 5

2 5

1 7 . 1

7 3

5 7 . 9

9 2

1 . 3

4 9

– 6 . 4

2 5

– 5 . 6

6 8

– 0 . 4

2 8

1 4 . 6

9 7

0 . 2

6 5

S 6

4 . 3 9 4

1 . 2

4 9

2 4 8

3 8 . 3

0 9

1 7 . 6

1 4

5 8 . 0

1 4

0 . 1

2 7

– 4 . 1

0 8

1 . 1

9 5

0 . 2

7 4

0 . 7

1 9

8 . 5

4 5

S 7

4 . 6 3 6

0 . 9

0 7

3 2 1

3 5 . 9

4 8

1 8 . 7

5 2

5 7 . 4

4 9

1 . 3

4 7

– 1 3 . 3

7 5

2 . 7

2 7

0 . 5

2 0

2 . 5

4 0

6 . 6

2 3

S 8

2 . 8 5 6

6 . 3

3 4

5 7

2 8 . 7

6 7

2 2 . 0

1 8

5 7 . 0

3 5

– 5 . 8

0 0

– 2 . 1

6 1

1 2 . 3

0 7

– 3 . 2

5 6

– 6 . 4

2 1

–

1 0 . 5

7

S 9

2 . 4 9 7

5 . 9

3 5

6 1

2 8 . 8

7 1

2 1 . 9

6 9

5 0 . 6

2 2

– 4 . 0

4 2

– 2 . 3

3 4

4 . 6

8 7

– 0 . 9

4 7

6 . 6

9 8

3 . 4

0 4

S 1 0

3 . 8 6 3

4 . 4

7 8

6 8

2 7 . 6

1 3

2 0 . 7

6 0

5 7 . 8

5 7

– 1 . 6

7 1

6 . 6

9 0

– 1 . 4

9 2

0 . 2

7 4

8 . 3

0 7

–

8 . 2

4 2

S 1 1

2 . 3 8 0

6 . 0

1 1

7 0

2 9 . 1

1 5

2 2 . 2

4 8

4 8 . 5

0 1

0 . 8

2 9

– 5 . 4

6 0

2 . 7

7 7

– 4 . 6

1 4

5 . 8

8 6

2 . 7

2 4

S 1 2

2 . 8 1 1

7 . 8

4 4

5 1

2 4 . 5

9 5

2 3 . 7

2 6

4 7 . 5

5 6

– 4 . 1

3 3

– 8 . 9

4 9

– 1 3 . 3

3

– 0 . 0

6 1

– 0 . 3

0 7

4 . 1

7 9

S 1 3

2 . 9 9 4

8 . 2

8 4

5 2

2 3 . 9

6 7

2 4 . 0

8 1

5 1 . 3

7 5

– 3 . 2

6 6

6 . 9

1 2

– 1 . 9

6 0

0 . 0

5 4

– 0 . 4

5 5

–

0 . 2

4 4

S 1 4

3 . 9 0 4

2 . 7

9 5

1 1 7

3 8 . 2

6 9

1 7 . 4

2 3

5 2 . 7

6 1

– 2 . 7

4 7

9 . 8

3 5

– 2 . 6

3 1

– 1 . 0

8 1

2 . 3

8 8

–

0 . 7

5 1

S 1 5

4 . 4 1 9

3 . 7

7 2

1 2 1

3 4 . 7

8 6

2 0 . 0

0 5

5 5 . 3

1 9

1 . 7

8 0

– 1 0 . 9

6 2

– 0 . 8

3 3

1 2 . 2

5 9

– 1 . 8

5 8

–

3 . 9

0 5

S 1 6

3 . 1 7 6

4 . 0

0 9

7 2

3 1 . 6

5 4

1 9 . 8

8 4

5 5 . 7

3 4

– 2 . 4

6 8

4 . 5

4 3

2 . 7

0 2

1 . 3

2 1

0 . 9

1 6

6 . 1

3 6

S 1 7

2 . 6 0 5

4 . 6

7 6

7 1

2 8 . 2

5 0

2 0 . 3

5 6

5 4 . 6

7 2

3 . 5

0 4

– 6 . 2

9 3

– 1 . 4

2 8

0 . 9

4 6

– 1 . 1

3 9

6 . 4

1 1

S 1 8

2 . 3 9 2

8 . 1

9 6

6 3

2 2 . 3

9 9

2 4 . 0

7 2

4 6 . 1

6 8

– 8 . 7

3 5

6 . 8

5 8

– 3 . 2

7 8

0 . 0

5 8

1 . 2

2 3

–

5 . 1

1 2

S 1 9

4 . 8 8 8

1 . 2

9 9

2 4 8

5 2 . 8

2 1

1 5 . 2

9 8

5 8 . 0

3 4

– 1 . 8

4 8

– 8 . 3

0 0

0 . 8

– 0 . 7

8 0

– 1 6 . 4

2

1

5 . 5

4 3

S 2 0

2 . 9 5 8

2 . 2

9 7

7 8

5 1 . 2

9 7

1 6 . 2

7 7

5 3 . 4

4 6

– 5 . 6

6 1

8 . 0

9 6

8 . 2

3 5

0 . 2

9 9

– 9 . 5

6 1

–

3 . 8

8 0

S 2 1

4 . 1 6 2

1 . 9

6 7

2 4 8

5 2 . 8

6 9

1 3 . 9

8 7

5 5 . 5

4 7

– 1 . 5

3 4

– 9 . 3

0 0

6 . 0

6 0

– 0 . 7

6 1

1 . 8

3 9

5 . 3

2 1

S 2 2

4 . 3 3 4

1 . 3

9 0

2 1 8

4 7 . 6

8 7

1 7 . 5

8 8

5 3 . 9

6 7

– 3 . 1

9 5

0 . 6

5 7

4 . 3

8 5

– 0 . 0

1 6

– 1 0 . 9

2

–

0 . 9

1 2

S 2 3

3 . 0 0 9

4 . 9

5 3

2 6 1

2 8 . 9

7 0

2 0 . 8

0 1

5 6 . 6

6 1

2 . 9

2 9

– 3 . 2

0 2

– 0 . 3

8 4

– 4 . 7

7 7

– 6 . 3

4 2

–

7 . 5

4 3

S 2 4

3 . 4 0 5

4 . 6

9 7

2 7 0

2 9 . 1

0 2

2 0 . 7

6 8

5 7 . 9

0 1

– 0 . 1

6 3

– 2 . 1

1 5

– 0 . 7

4 6

– 8 . 6

7 1

– 1 . 5

5 9

–

9 . 9

0 8

S 2 5

3 . 4 9 1

4 . 6

1 2

2 5 1

2 3 . 2

5 6

2 2 . 2

9 3

5 6 . 2

6 3

– 5 . 8

1 2

– 9 . 8

1 4

– 0 . 4

1 5 . 5

5 4

– 3 . 3

0 5

–

9 . 8

0 3

S 2 6

3 . 6

1 2 3

2 . 0

3 4

2 9 8

2 8 . 8

7 8

1 9 . 8

3 8

5 8 . 0

3 0

4 . 9

3 9

– 1 . 7

1 0

0 . 6

6 6

5 . 8

7 5

– 6 . 4

2 9

–

5 . 7

2 1

S 2 7

3 . 4 3 7

1 . 8

4 6

3 0 4

2 9 . 3

0 2

1 9 . 8

5 5

5 8 . 0

3 1

4 . 5

2 2

– 2 . 5

6 5

1 . 9

3 5

1 . 4

7 2

– 1 . 4

8 9

2 . 5

8 2

S 2 8

3 . 6 5 4

2 . 0

8 9

2 8 3

2 9 . 1

6 7

1 7 . 6

2 6

5 7 . 9

4 8

– 4 . 4

2 0

– 4 . 4

7 5

– 1 . 0

7 1

4 . 2

1 3

0 . 4

0 3

1 0 . 2

6

S 2 9

4 . 8 6 0

1 . 6

6 5

2 9 0

5 1 . 8

7 9

1 3 . 7

8 8

5 8 . 0

5 4 6

0 . 8

0 2

– 4 . 0

7 5

0 . 6

8 4

1 . 3

1 4

0 . 0

8 3

1 3 . 9

6

S 3 0

2 . 5 0 4

8 . 4

9 6

5 1

2 2 . 9

8 3

2 1 . 9

7 3

5 7 . 1

4 2 4

1 0 . 5

4 3

2 . 3

3 7

– 6 . 2

5

0 . 7

0 4

1 . 9

6 1

–

5 . 5

4 9

A v e r a g e

– 0 . 6

6 5

– 2 . 1

8 8

0 . 6

3 9

– 0 . 2

4 2

– 0 . 6

0 9

0 . 1

4 3




7.7.9 Fabric Water Absorbency (FWA)

From Table 7.13, the water absorbency (sec) of bioscoured organic cotton fabricwas noticed better in sample no. 7 which was treated with 6% pectinase, 2%

protease, 0.6% lipase and 0.8% cellualse at 45 min reaction time, 60°C, and pH

8.5. It may be due to higher removal of wax/oil component in the organic cotton

up to 84.2% and presence of cellulase enzyme which improves partial surface

hydrophilic nature in the organic cotton fabric. The highest time in seconds

for water absorbency of the organic cotton fabric was noticed in sample no.

18 which has treated with absence of pectinase and protease enzymes. These

enzymes are playing important role in the fabric water absorbency by removal

of pectin and wax components and also noticed improved water absorbency by

adding the cellulase enzyme in the bioscouring process.

7.7.10 Summary

The process optimization of bioscouring of 100% organic cotton fabric

through enzyme technology has been studied with selective specic mixed

enzymatic system using four enzymes namely alkaline pectinase, protease,

lipase and cellulase. The process variables such as enzyme concentration,temperature and reaction time was optimized to achieve the required water

absorbency and pectin removal during bioscouring process on the organic

cotton fabrics. The alkaline pectinase enzymes are better active and catalyze

the degradation of pectin at temperature range of 55–60°C and time of 45

min to achieve required level of 75–80% pectin degradation. The pH of

the process bath is also a major inuence for better reaction of enzyme to

catalyze the hydrolysis of pectin groups. The higher enzyme concentration at

6% level and higher temperature of 60°C took lesser time to achieve required

pectin hydrolysis. Process variables are optimized using MATLAB 7.0 andit will pave the way to predict the enzyme kinetics at various concentrations,

temperature and reaction time to achieve required degradation of pectin with

minimum error %. This study will be helpful to the organic cotton processors

for the ecofriendly and sustainable textile wet processing using specic mixed

enzymatic system in bioscouring processes.

7.8 Sonication and aerodynamic principles –

enzymatic activity

7.8.1 Ultrasonic treatment

Ultrasonic technique holds a promise in applications in the eld of textiles.

Ultrasonics represents a special branch of general acoustics, the science of




mechanical oscillations of solids, liquids and gaseous media. Ultrasound

can enhance a wide variety of chemical and physical processes, mainly by

generating cavitation in liquid medium. The sonicator used is of 20 kHz

frequency which is found to be suitable for inducing cavitation (Abramov

1998; Kamel et al. 2005). It is well known that cavitation which causes

formation and collapse of micro bubbles are most effective for better bre

opening which enhances water absorbency and dye uptake. This activated

state causes chemical reaction between the fabric and the enzyme by forming

waves and severe shear force capable of breaking chemical bonds. Ultrasound

energy has great potential in industrial processes as it offers reduction in cost,

time, energy and efuents. Ultrasound reduces processing time and energyconsumption, maintains or improves product quality, and reduces the use of

auxiliary chemicals (Yachmenev 2005).

7.8.2 Sonication – basic principle

In a solid, both longitudinal and transverse, waves can be transmitted whereas

in gas and liquids only longitudinal waves can be transmitted. In liquids,

longitudinal vibrations of molecules generate compression and refractions,

i.e., areas of high pressure and low local pressure. The latter gives rise tocavities or bubbles, which expand and nally, during the compression

phase, collapse violently generating shock waves. The phenomena of bubble

formation and collapse (known as cavitations) are generally responsible for

most of ultrasonic effects observed in solid/ liquid or liquid/liquid systems.

7.8.3 Ultrasonic application – textile wet processing

New bio-preparation processes that utilize highly specic enzymes instead of

conventional organic/inorganic chemicals are becoming increasingly popularin the textile industry (Yachmenev et al. 2005). The major shortcoming

of this new technology is that the processing time is much longer than the

conventional method. This limitation was overcome by use of ultrasound

energy in combination with enzyme processing. The combined enzyme/

ultrasound bio-preparation of greige cotton offers signicant advantages such

as less consumption of expensive enzymes, shorter processing time, better

uniformity of treatment and a notable decrease in the amount and toxicity of

the resulting textile wastewater efuents. Sonolysis, enzyme treatment, and acombination of the two processes were tested for the degradation of phenol

in aqueous medium. Degradation of starch followed by ultrasonic desizing

could lead to considerable energy saving as compared to conventional starch

sizing and desizing (Sakakibara et al. 1996). Desizing of the pure cotton




fabric with alpha amylase was conducted by the ultrasonic wave method and

the traditional one respectively. Research on the effect of ultrasonic wave

on the enzyme desizing of the fabric indicated that it can improve desizing

percentage, wettability and whiteness of the fabric, reduce the strength loss,

treatment time and temperature, and save energy. Therefore, it has bright

prospect of application. Introduction of ultrasonic energy during enzymatic

bio-preparation/bio-nishing of cotton textiles signicantly improved

enzyme performance but did not contribute to a decrease in fabric strength.

Experimental data indicate that the maximum benet provided by sonication

of enzyme processing solution occurred at lower enzyme concentrations

(Yachmenev et al. 2005).The hydrolysis of maltoheptaose by α-amylase, andthe resulting reaction was followed by the continuous monitoring of changes

in ultrasonic velocity. As the reaction proceeds, ultrasonic velocity increases

because the hydration level of the product is higher than that of the starting

substrate. It is simple to recalculate the ultrasonic curve to give the time

dependence of the amount of substrate that has been hydrolysed, providing

the kinetic prole of the reaction, and allowing the enzyme’s activity faster

(Fig. 7.23) (Sakakibara et al. 1996).

Fig. 7.23 Hydrolysis of starch by alpha amylase using sonication technique

7.8.4 Effect of sonication on bioscouring of organic

cottonEffect of sonication on bioscouring of organic cotton was studied by

Vigneswaran et al. (2013) using laboratory model OSCAR Ultrasonicator,

Model: Micro clean 103 at 20 kHz which was supplied by M/s. OSCAR




Ultrasonicor (P) limited, Mumbai; and reported that (i) the actual pectin

removal and weight loss of the bioscoured organic cotton fabric was achieved

as 78.40% and 4.80% respectively with the error of 1.218% in case of without

ultrasonic treatment; with ultrasonic treatment, the fabric weight loss is

observed as 5.46% and pectin removal up to 81.46%. Secondly, the sonicator

efciency shows 8–12% higher bioscouring performance on organic cotton

fabric through mixed enzymatic system when compared to without sonication.

7.8.5 Aerodynamic system

Aerodynamic technique has been studied and used for a variety of applications

in liquids, dispersions and polymers (Pinheiro 2000). Limited research works

have been reported to acceleration of enzyme kinetics through aerodynamic

system (air pressure) to improve the reaction of substrate and enzyme binding

to high quality and standardization of process parameters (Xia Yuan-jing

and Li Zhi-yi 2009; Michal Gross and Rainer Jaenicke 1994). Aerodynamic

system of enzyme acceleration has great potential in industrial processes as it

offers reduction in cost, time, energy and efuents.

7.8.6 Effect of air pressure on enzyme activity

Elevated hydrostatic pressure has been used to increase catalytic activity

and thermal stability of enzymes. For increase in pressure at 20°C results

in an exponential acceleration of the hydrolysis rate catalyzed by cellulase

reaching a 6.5-fold increase in activity at 4700 atm (4.7 kbar). Due to a strong

temperature dependence of the enzyme, acceleration effect of high pressure

becomes more pronounced at high temperatures (Fig. 7.24). At 50°C, under a

pressure of 3.6 kbar, cellulase enzyme shows activity which is more than 30

times higher than the activity at normal conditions (20°C, 1 atm). At pressuresof higher than 3.6 kbar, the enzymatic activity is decreased due to a pressure-

induced denaturation (Vadim Mozhaev 1996). Air pressure amplitude serves

as a critical control parameter of periodic pressure solid state fermentation

process. Effects of different air pressure amplitudes on cellulase production

by Trichoderma viride-SL were investigated. The effects of these two factors

on the stability of Rhizomucor miehei lipase have been investigated. The

stability criterion used was residual hydrolytic activity of the lipase (Margarita

and Pere 2004). Experimental and theoretical parameters, obtained by linearregression analysis, were compared with theoretical kinetics in order to

validate the series-type inactivation model. The lipase enzyme was activated

by either thermal or pressure treatment (Feitkenhauer and Meyer 2003).

Moreover conformational studies made by uorescence spectroscopy suggest




that the conformational changes induced by pressure were different from those

induced by temperature. In addition they show that after thermal deactivation

there were less intermolecular hydrogen bonded structures formed than was

the case for deactivation by high pressure. The organic cotton fabric was

treated with mixed enzymatic system using laboratory model breaker dyeing

bath tted with air pump model U9900 which was supplied by M/s BOY U®

(Fig. 7.25).

Fig. 7.24 Enzymatic treatments in (a) laboratory model beaker bath and

(b) air nozzle in beaker

Fig. 7.25 Single stage enzymatic desizing and scouring process – Trial I

7.8.7 Effect of air pressure on bioscouring of organiccotton fabric

The process optimization of bioscouring of 100% organic cotton fabric

through enzyme technology with aerodynamic system have been studied




with selective specic mixed enzymatic system using four enzymes namely

alkaline pectinase, protease, lipase and cellulase. The process variables such

as enzyme concentration, temperature and reaction time have optimized to

achieve the required water absorbency and pectin removal during bioscouring

process by pectinolytic and proteolytic activity on the organic cotton fabrics.

These process variables are selected based on the articial neural network

(ANN) and output of experiment was resulted with fabric physic properties

such as fabric weight loss, water absorbency, wetting area, whiteness index,

yellowness index, and brightness index using MATLAB 7.0 software with

minimum error and also studied with and without aerodynamic treatments.

The test results have analyzed to predict the optimum process parametersto achieve the required bioscouring fabric properties and removal of pectin

degrading rate and compared their results with actual trials. This study will

be helpful to the organic cotton processors for the ecofriendly and sustainable

textile wet processing using specic mixed enzymatic system in bioscouring

processes. From the research study the following conclusions were derived: (i)

The alkaline pectinase enzymes are better active and catalyze the degradation

of pectin at temperature range of 55–60°C and time of 45 min to achieve

required level of 75–80% pectin degradation. The pH of the process bath is

also a major inuence for better reaction of enzyme to catalyze the hydrolysisof pectin group. (ii) The higher enzyme concentration at 6% level and higher

temperature of 60°C took lesser time to achieve required pectin hydrolysis.

Process variables are optimized using MATLAB 7.0 and it will pave the way

to predict the enzyme kinetics at various concentrations, temperature and

reaction time to achieve required degradation of pectin with minimum error

%. (iii) The output result of the software to achieve the desired bioscouring

of organic cotton fabric on their physical properties such as fabric weight

loss, water absorbency, wetting area, whiteness index, yellowness index, and brightness index in the specic enzymatic system, out of which the software

opted best process conditions at 8% alkaline pectinase, 3% protease, 0.8%

lipase and 0.8% cellulase process condition at temperature of 55°C and

reaction time 60 min at pH 8.5 with 1.0% desirability. (iv) From the best opted

test results, the actual pectin and weight loss of the bioscoured organic cotton

fabric was achieved 68.40% and 4.80% respectively with error of 1.218%

in case of without aerodynamic treatment. With aerodynamic treatment, the

fabric weight loss was observed 6.38% and pectin removal up to 76.42%,

and (v) the overall aerodynamic efciency was achieved 9.72%, 24.08% and37.20% treated at 8 kPa, 12 kPa and 16 kPa air pressure levels respectively

on organic cotton fabric through mixed enzymatic system when compared to

without aerodynamic.




7.9 Biopreparation of organic cotton fabric

This part of research work deals with single-stage enzymatic desizing andscouring process and enzymatic scouring and bleaching process with various

approaches for improvement of the organic cotton fabrics characteristics. The

organic cotton fabric properties such fabric weight loss (%), water absorbency,

fabric wetting area, fabric whiteness index, yellowness index, and brightness

index have been studied with various enzymatic process conditions. An attempt

has been made in this research work to combine fabric preparation (desizing,

scouring and bleaching) in a single stage and six different process sequences

were designed to optimize the results. The comparison and performance level

of various enzymatic process trials have been made to analyse the quality

of organic cotton fabrics with chemical scouring method which has been

followed in the industrial practices.

7.9.1 Single-stage enzymatic desizing and scouring

process

The single-stage enzymatic desizing and scouring processes have been carried

out with alpha amylase for biodesizing and specic mixed enzymes such asalkaline pectinase, protease, lipase and cellulase enzymes for bioscouring.

The process parameters of single-stage continuous wet processing of organic

cotton fabrics through chemical method and enzymatic method are given in

Tables 7.15 and 7.16, respectively. The Trials I and II represent the single-

stage enzymatic desizing and scouring process carried out with and without

washing process in the middle of the biotreatment (Figs. 7.26 and 7.27). In

the rst sequence (Trials I and II), was conducted to assess the inuence of

desizing combined with scouring enzymes and also carried out with ultrasonic

and aerodynamic systems.

Fig. 7.26 Single stage enzymatic desizing and scouring process – Trial II




Fig. 7.27 Comparison of Trail I and Trail II on fabric weight loss (%) in biopreparation

of organic cotton fabric

Table 7.15 Process parameters of single-stage continuous wet processing of organic

cotton fabrics – chemical method

Desizing Scouring Bleaching

Hydrochloric

acid (HCl)

1% Sodium hydroxide

(NaOH)

3% Hydrogen

peroxide

(H2O

2)

3%

Wetting agent 0.5%

(owm)

Sodium carbonate

(Na2Co3)

1% Sodium silicate

(Na2Sio3)

2%

MLR 1:20 Wetting agent 0.5% Sodium

hydroxide

(NaOH)

1%

Temperature 50°C MLR 1:20 MLR 1:20

Time 60 min Temperature Boil Temperature 95°C

Time 60 min Time 60 min

Table 7.16 Process parameters of single-stage continuous wet processing of organiccotton fabrics – mixed enzymatic method

Biodesizing Bioscouring Biobleaching

Alpha amylase 3.50% Alkaline

pectinase

8% Peroxidase 2%

Wetting agent 1% Protease 3% MLR 1:20

pH 7.0–7.5 Lipase 0.8% pH 7.0

Temperature 55°C Cellulase 0.8% Temperature 55°C

Time 60 min pH 9.5 Time 15 minMLR 1:20 Temperature 55°C

Time 60 min

MLR 1:20




7.9.1.1 Assessment of single-stage continuous enzymatic

processThe sample produced using above process sequence were assessed using

iodine test for identifying the starch removal on the enzymatic treatment in

the samples and residual pectin assessment using ruthenium red test. Besides

change in weight of the samples, drop absorbency, tensile strength, whiteness,

yellowness, brightness index were assessed and compared with the samples

obtained from the individual treatments. Whiteness of samples was measured

using spectrophotometer and FTIR analysis was carried out for identication

of residual impurities and structural changes in the bre using relevant

functional groups.

7.9.1.2 Iodine test

After enzymatic desizing process, the samples treated with various trials

showed clear yellow color with iodine solution, characteristics to that of dilute

iodine solution, without even faint discoloration that showed the absence of

residual starch or degraded starch in the fabric samples, similar to that of

desizing treatments using combination analysis.

7.9.1.3 Extractable impurities and residual pectin

Higher removal of impurities from the raw cotton bre can be expected in

the combined preparation in presence of cellulase in the reaction, which by

hydrolyzing the surface cellulose layers, could facilitate the access to other

substrates for hydrolysis by the respective enzymes (pectinase, protease

and lipase). However, there was a signicant difference in Trial I values as

compared to those obtained in the protease, pectinase, lipase and cellulasetreated samples by many folds. When the pectinase, protease, lipase and

cellulase were added at regular intervals, one after another removal of pectin

in the bres was less than that of the treatments where the enzymes were

added at once in terms of residual pectin measured by ruthenium red staining.

7.9.1.4 Fabric weight loss (%)

The performance of single-stage enzymatic desizing and scouring of

organic cotton are given in Tables 7.17 and 7.18, respectively. Figure 7.28shows the fabric weight loss (%) after single-stage enzymatic desizing and

scouring process with alpha amylase and mixed enzymatic system in scouring

process. From the test results, there is a signicant differences found in fabric

weight loss (%) between chemical method and normal mixed enzymatic and




ultrasonic treatment at F(2,29)

(8.27) > Fcrit

(4.291) at 95% condence level.

But in the case of aero dynamic system, there is no signicant differences

between chemical and aerodynamic system at F(2,29)

(3.287) > Fcrit

(4.291) at

95% condence level. It may be due to better enzyme kinetics on catalysis

of starch and non cellulosic groups present in the organic cotton fabric in

presence of air pressure which energies the enzymes in reaction. It is also

noticed that Trial II samples were noticed higher fabric weight loss (%) when

compared to Trial I samples in all the cases of enzymatic treatment, it may

be due to washing process introduced between biodesizing and bioscouring

which enrich the enzymatic reaction on the cotton bre.

Fig. 7.28 Comparison of Trail I and Trail II on fabric wax removal (%) in

biopreparation of organic cotton fabric

7.9.1.5 Fabric wax removal (%)

Figure 7.29 shows wax removal percentage on the organic cotton fabric

after single-stage enzymatic desizing and scouring process. It is clear that

% wax removal on the normal mixed enzymatic method, ultrasonic and

aerodynamic system were noticed 56.8%, 60.1% and 63.1% in Trial I and

57.2%, 62.8% and 68.4% in Trial II, respectively. But when compared tochemical alkaline scouring process, it was noticed up to 80.2% wax removal

on the organic cotton fabric. There is signicant differences noticed between

all the enzymatic treatments in Trial I at F(2,29)

> Fcrit

in various combination of

ANOVA multivariant analysis which are given in Table 7.20.




Fig. 7.29 Comparison of Trail I and Trail II on pectin removal (%) in biopreparation of


Table 7.17 Test results of Trial I single-stage enzymatic desizing and scouring

process

Fabric characteristics Normal

mixed

enzymatic

method

Ultrasonic

method

Aerodynamic

method

% change

Ultrasonic Aerodynamic

Fabric

weight loss (%)

12.40 12.72 13.04 2.581 5.161

Wax removal (%) 56.8 60.1 63.1 5.810 11.092

Pectin removal (%) 62 66.5 68.6 7.258 10.645

Fabric wetting area (mm2) 202 219 230 8.416 13.861

Whiteness Index 52.45 55.63 56.82 6.063 8.332

Yellowness Index 21.82 20.68 20.12 –5.225 –7.791Brightness Index 53.65 58.42 60.51 8.891 12.787

Fabric tensile strength

(g/tex)

Warp way 296.55 285.71 283.34 –3.655 –4.454

Weft way 249.49 225.28 219.04 –9.703 –12.204

7.9.1.6 Pectin removal (%)

Figure 7.30 shows the pectin degradation level on the single-stage enzymatic

desizing and scouring process of organic cotton fabric. It was noticed that

alkaline pectinase enzyme catalysis the pectin groups on the organic cotton

fabric was noticed higher up to 79.2% in aerodynamic system in Trial II. It

is interesting that there is no signicant differences between the chemical




method of pectin removal (78.4%) and also noticed that there is signicant

differences found in Trial I and II enzymatic treatments, it may be due to

washing process in the middle of biotreatment. It was also noticed that

washing process removes all the degraded starch after biodesizing with alpha

amylase and improves the better enzymatic reactions in bioscouring process

in next sequence of enzyme treatment in Trial II.

Fig. 7.30 Effect of biopreparation Trial I and II on water absorbency characteristics of


Table 7.18 Test results of Trial II single-stage enzymatic desizing and scouring

process

Fabric characteristics Normalmixed

enzymatic

method

Ultrasonicmethod

Aerodynamicmethod

% change


Fabric weight loss (%) 13.35 13.85 14.28 3.745 6.966

Wax removal (%) 57.2 62.8 68.4 9.790 19.580

Pectin removal (%) 64.2 74.2 79.1 15.576 23.209

Fabric wetting area (mm2) 212 252 270 18.868 27.358

Whiteness Index 54.52 58.45 62.14 7.208 13.977

Yellowness Index 18.41 13.45 13.04 –26.942 –29.169

Brightness Index 60.25 66.38 69.45 10.174 15.270

Fabric tensile strength

(g/tex)

Warp way 292.14 287.52 279.58 –1.581 –4.300

Weft way 238.65 221.85 218.69 –7.039 –8.363




Table 7.19 Performance level of Trial I and II in enzymatic desizing and scouring

process

Fabric characteristics % change

Normal mixed

enzymatic method

Ultrasonic

method

Aerodynamic

method

Fabric weight loss (%) 7.66 8.88 9.51

Wax removal (%) 0.70 4.49 8.40

Pectin removal (%) 3.55 11.58 15.31

Fabric wetting area (mm2) 4.95 15.07 17.39

Whiteness Index 3.95 5.07 9.36

Yellowness Index –15.63 –34.96 –35.19

Brightness Index

Tensile strength (g/tex)

12.30 13.63 14.77

Warp way –1.487 –0.633 –1.327

Weft way –4.344 –1.522 –0.159

7.9.1.7 Fabric water absorbency and wetting

characteristicsFigures 7.31 and 7.32 represent the water absorbency and wetting characteristics

of single-stage enzymatic desizing and scouring process of organic cotton

fabric. From the test results, the aerodynamic system of enzymatic treatment

samples were noticed similar to the chemical scouring fabric characteristics

and having <1 sec water absorbency and noticed >300 mm2 water wetting area.

It was noticed that there is no signicant differences between the chemical and

aerodynamic methods and also ultrasonic treatments, it may be due to higher

pectin and wax component break down in ultrasonic and aerodynamic system.

There is a signicant differences noticed between Trial I and Trial II in case

comparison between various enzymatic treatments as given in Table 7.20.

7.9.1.8 Fabric whiteness, yellowness index, and

brightness

Comparisons of fabric characteristics such as whiteness index, yellowness index

and brightness index of single-stage enzymatic desizing and scouring process

are given in Fig. 7.33. The performance levels of Trial I and II of these sample

characteristics are given in Table 7.17. From the test results, whiteness index

and brightness index were noticed higher up to 5.07%, and 13.63% in case of

ultrasonic treatments and also noticed 9.36% and 14.77% in case of aerodynamic

system. It was noticed that there is signicant differences F(2,29)

> Fcrit

between

Trial I and II enzymatic treatments. It may be due to washing process introduced




in single-stage enzymatic desizing and scouring process. The washing process

improves the fabric scouring characteristics and enzymatic degradation rate of

pectin and wax components in the organic cotton fabric (Tables 7.18 and 7.19),

respectively, and ANOVA multivariant test results are given in Table 7.20. It

was noticed that higher pectin and wax components in the organic cotton fabric

while bioscouring with ultrasonic and aerodynamic system which improves

whiteness and brightness index of the cotton fabric by better endo pectinolytic

and proteinolytic on the cotton bre structure.

Fig. 7.31 Effect of biopreparation Trial I and II on fabric wetting characteristics of


Fig. 7.32 Effect of biopreparation Trial I and II on water absorbency characteristics of





Fig. 7.33 Comparison of tensile characteristics of organic cotton fabric processed

through chemical and enzymatic desizing and scouring methods

7.9.1.9 Fabric tensile characteristics

Figure 7.34 shows the comparison of organic cotton fabric tensile characteristics

(both warp and weft way) of single-stage chemical and enzymatic desizing and

Fig. 7.34 Single-stage enzymatic scouring and bleaching process – Trial III




scouring process. From Table 7.20, the ANOVA test results, there is signicant

differences found between chemical (alkali) and enzymatic treatments at F(2,29)

> Fcrit

(8.521 > 4.182) between Trial I and II at 95% condence values both

ultrasonic and aerodynamic system of treatments. It may be due to higher

fabric strength loss and weight loss in chemical scouring which weakens the

bre molecules at higher concentration of alkali treatments. The performance

levels of Trial I and II of these sample characteristics are given in Table 7.19.

From the test results, there is no signicant difference between Trial I and

Trial II by incorporating washing process between enzymatic desizing and

scouring process, but it improves the fabric properties in terms of whiteness,

and brightness index in concern.

Table 7.20 ANOVA Multivariant Analysis – single-stage enzymatic desizing and

scouring process

Source of Variation SS df MS F Pvalue Fcrit

Fabric weight loss

Between Trials I and II 1750.35 29 60.357 17.52 0.006 4.182

Between normal and ultrasonic methods 354.854 29 12.236 6.527 0.034 4.182

Between normal and aerodynamic

methods

457.127 29 15.763 8.624 0.048 4.182

Wax removal (%)




methods

65.820 29 2.2697 11.457 0.004 4.182

Pectin removal (%)



Between normal and aerodynamicmethods

124.23 29 4.284 7.852 0.012 4.182

Fabric wetting area




methods

1425.36 29 49.150 11.862 0.025 4.182

Whiteness Index


Between normal and ultrasonic methods 36.354 29 1.254 8.132 0.048 4.182Between normal and aerodynamic

methods

34.681 29 1.196 8.562 0.045 4.182

Yellowness Index





Source of Variation SS df MS F Pvalue Fcrit



methods

13.681 29 0.472 8.694 0.281 4.182

Brightness Index




methods

61.425 29 2.118 34.821 0.041 4.182

Fabric tensile characteristics




methods

476.52 29 18.65 0.301 0.638 4.182

7.9.2 Single-stage enzymatic scouring and bleaching

process

The single-stage enzymatic scouring and bleaching process was carried out

with mixed enzymes such as alkaline pectinase, protease, lipase and cellulase

enzymes for bioscouring and hydrogen peroxide for bleaching process which

are optimized in the Chapters 5 and 6 for ultrasonic and aerodynamic system,

respectively. The Trial III to VI represent the single-stage enzymatic scouring

and bleaching process carried out with various combinations of enzymes in

bioscouring process (Figs. 7.35 to 7.38). The fabric properties such as fabric

weight loss, wax removal, pectin removal, water absorbency, wetting area,

whiteness index, yellowness index and brightness index of normal method,

ultrasonic method and aerodynamic method of enzymatic treatments are

compared and test results are given in Tables 9.7–9.9, respectively.

Fig. 7.35 Single-stage enzymatic scouring and bleaching process – Trial IV




Fig. 7.36 Single-stage enzymatic scouring and bleaching process – Trial V

Fig. 7.37 Single-stage enzymatic scouring and bleaching process – Trial VI

Fig. 7.38 Effect of fabric weight loss (%) on combined bioscouring and bleaching

treatment of organic cotton fabric




7.9.2.1 Fabric weight loss (%)

Figure 7.39 shows the fabric weight loss (%) of the organic cotton fabric aftersingle-stage enzymatic scouring and bleaching process carried out normal

method, ultrasonic method and aerodynamic method. From the test results,

the fabric weight losses (%) of the aerodynamic and ultrasonic method of

enzymatic treatment were noticed similar to the alkaline scouring and hydrogen

peroxide bleaching nature. There is no signicant differences noticed when

compared between chemical method and Trial VI at 95% condence level F(2,29)

< Fcrit

(4.38 < 4.081), which may be due to better degradation of pectinolytic

and proteinolytic reaction between alkaline pectinase, lipase and protease

enzymes in the bioscouring process. It is also noticed that the concentration

of cellulase enzyme in the bioscouring imparts higher fabric weight loss

(%) and signicant improvement in the endo-pectinolytic activity on the

cellulosic bre structure. There are signicant differences noticed between

various enzymes combinations of trials at Trial III and IV and Trial V and

VI at various enzymatic process conditions which are shown in Tables 7.23

and 7.24, respectively, for ANOVA multivariant analyses at 95% condence

level. The performance levels of Trial III to VI at various combinations of

enzyme treatments with normal, ultrasonic and aerodynamic system are givenin Tables 7.21 and 7.22.

Fig.7.39 Effect of wax removal (%) on combined bioscouring and bleaching treatment

of organic cotton fabric




Table 7.21 Test results of Trial III single-stage enzymatic scouring and bleaching

process

Fabric

characteristics

Normal

mixed

enzymatic

method

Ultrasonic

method

Aerodynamic

method

% change


Fabric weight loss

(%)

3.42 3.86 4.31 12.865 26.023

Wax removal (%) 68.5 70.5 72.2 2.920 5.401

Pectin removal

(%)

65.9 68.9 69.4 4.552 5.311

Fabric wetting

area (mm2)

184 203 210 10.326 14.130

Whiteness Index 68.63 69.9 71.58 1.851 4.298



Fabric tensile

strength (g/tex)

Warp way 268.22 263.54 262.85 –1.74 –2.00

Weft way 234.52 228.54 229.51 –2.54 –2.31

Table 7.22 Test results of Trial IV single-stage enzymatic scouring and bleaching

process

Fabric

characteristics

Normal

mixed

enzymatic

method

Ultrasonic

method

Aerodynamic

method

% change


Fabric weight loss

(%)

3.73 4.16 4.84 11.528 29.759

Wax removal (%) 70.5 72.5 74.5 2.837 5.674

Pectin removal (%) 68.5 71.8 74.8 4.818 9.197

Fabric wetting area

(mm2)

195 218 232 11.795 18.974

Whiteness Index 71.56 74.08 75.92 3.522 6.093



Fabric Tensilestrength (g/tex)

Warp way 263.52 261.52 260.52 –0.75 –1.13

Weft way 230.52 224.52 222.67 –2.60 –3.40




7.9.2.2 Fabric wax removal (%)

Figure 7.40 shows the comparisons of degradation of low volatile wax and oilcomponents present in the organic cotton fabric after single-stage enzymatic

scouring and bleaching carried out by normal method, ultrasonic method and

aerodynamic method. From the test results, the higher degradation of wax

and oil substances were noticed in Trial VI, it may be because of protease and

lipase enzymes added in rst in bioscouring process and then added alkaline

pectinase and cellulase enzymes in the single-stage bioprocess sequences

which imparts better removal of low volatile fatty molecules on the cotton

bre structure and increases endo pectinolytic activity on the organic cottonfabric. It is also noticed Trial VI of aerodynamic and ultrasonic treatments have

no signicant differences when compared to chemical method of treatments

at F(2,29)

< Fcrit

at 95% condence level of comparison of test results. ANOVA

multivariant analyses were carried out to analyze the signicant differences

of various enzymatic trials between Trial III and VI, and their test results are

given in Tables 7.23 and 7.24.

Fig. 7.40 Effect of pectin removal (%) on combined bioscouring and bleaching





Table 7.23 Test results of Trial V single-stage enzymatic scouring and bleaching

process

Fabric

characteristics

Normal

mixed

enzymatic

method

Ultrasonic

method

Aerodynamic

method

% change


Fabric weight loss

(%)

4.20 4.31 4.45 2.619 5.952

Wax removal (%) 68.4 68.6 71.5 0.292 4.532

Pectin removal (%) 69.5 70.1 72.1 0.863 3.741

Fabric wetting area

(mm2)

222 236 248 6.306 11.712

Whiteness Index 71.56 72.6 74.56 1.453 4.192



Fabric tensile

strength (g/tex)

Warp way 263.96 260.52 258.94 –1.30 –1.91

Weft way 234.04 230.9 226.54 –1.34 –3.20

Table 7.24 Test results of Trial VI single-stage enzymatic scouring and bleaching

process

Fabric

characteristics

Normal

mixed

enzymatic

method

Ultrasonic

method

Aerodynamic

method

% Change


Fabric weight loss

(%)

4.65 4.92 5.27 5.806 13.333

Wax removal (%) 74.5 75.6 82.5 1.477 10.738

Pectin removal (%) 73.5 74.6 78.4 1.497 6.667

Fabric wetting area(mm2)

242 270 291 11.570 20.248

Whiteness Index 75.65 80.25 84.24 6.081 11.355



Fabric tensile

strength (g/tex)

264.51 259.85 258.06 –1.76 –2.43

Warp way

Weft way 234.34 232.86 230.7 –0.63 –1.55

7.9.2.3 Fabric pectin removal (%)

Alkaline pectinase enzyme plays important role in the breakdown of pectin

groups in the organic cotton bre structure. Figure 7.41 shows the fabric




pectin removal (%) of the organic cotton fabric after single-stage enzymatic

scouring and bleaching process carried out normal method, ultrasonic method

and aerodynamic method. The alkaline pectinase enzyme was noticed higher

reaction on the pectin removal (%) treated at ultrasonic and aerodynamic

systems. There is a signicant difference noticed between Trial V and Trial

VI, which may be due to higher fabric weight loss (%) and wax removal on

the organic cotton fabric which are subjected combination of enzymes added

in the bioscouring sequence of process.

Fig. 7.41 Effect of fabric water absorbency characteristics on combined bioscouring

and bleaching treatment of organic cotton fabric

7.9.2.4 Fabric water absorbency and wetting

characteristics

Figures 7.42 and 7.43 show the fabric water absorbency and wetting

characteristics of the organic cotton fabric after single-stage enzymatic

scouring and bleaching process at various combinations of mixed enzymes in

the process sequences at normal method, ultrasonic method and aerodynamic

method. From the test results, ultrasonic and aerodynamic system of mixed

enzymatic treated in Trial VI was noticed water absorbency <1 sec and higher

wetting characteristics when compared to normal mixed enzymatic system.

It is interesting observation that test results are comparable with chemical

alkaline scouring followed in the industrial practices. These improved water

absorbency and wetting characteristics, may be due to higher pectin and wax

component break downs in the organic cotton bre structure and enhanced

enzyme catalysis in the ultrasonic and aerodynamic system.




Fig. 7.42 Effect of fabric wetting characteristics on combined bioscouring and

bleaching treatment of organic cotton fabric

Fig. 7.43 Effect of fabric whiteness index on combined bioscouring and bleaching





7.9.2.5 Fabric whiteness, yellowness and brightness index

Figures 7.44–7.46 show the comparison of single-stage enzymatic scouring and bleaching of organic cotton fabric treated at normal method, ultrasonic method

and aerodynamic method carried out at various enzymatic combinations.

From the test results, higher fabric whiteness and brightness index were

noticed better in ultrasonic and aerodynamic system and comparable results

with chemical alkaline cotton fabric. There is signicant differences noticed

at F(2,29)

> Fcrit

between Trial V and VI at 95% condence level of test analysis,

it may be due to higher pectin degradation up to 78% and 80% in case of

ultrasonic and aerodynamic system in Trial VI. It may be because of protease

and lipase enzymes added in rst in the sequence of bioscouring which breaks

the surface wax and oil substances and then added pectinase and cellulose

which enhances the higher degradation of pectin groups in the enzymatic

treatments. Yellowness index of Trial VI samples were noticed lower and

comparable with chemical alkaline scouring followed in industrial practices.

The test results of various combinations of Trials III to VI and compared with

ANOVA multivariant analysis at 95% condence level and their test results

are given in Tables 7.24 and 7.25.

Table 7.25 Performance level of Trial III and IV in enzymatic scouring and bleaching

process


Normal mixed

enzymatic method

Ultrasonic

method

Aerodynamic

method


Wax removal (%) 2.92 2.84 3.19

Pectin removal (%) 3.95 4.21 7.78




Brightness Index 6.05 7.67 9.48

Fabric tensile strength (g/tex) 1.75229 0.76649 –0.88644

Warp way

Weft way 1.70561 –1.75899 –1.38026




Fig. 7.44 Effect of fabric yellowness index on combined bioscouring and bleaching


Fig. 7.45 Effect of fabric brightness index on combined bioscouring and bleaching





Fig. 7.46 Comparison of tensile characteristics of organic cotton fabric processed

through chemical and enzymatic scouring and bleaching methods.

7.9.2.6 Fabric tensile characteristicsFigure 7.47 shows the fabric tensile characteristics (both warp and weft way) of

organic cotton fabric treated by single-stage chemical and enzymatic desizing

and scouring process. From the test results, there is signicant differences

found between chemical and enzymatic treatments at 95% condent level Fact

> Fcri 6.842 > 4.182. The performance level of Trial III and IV, V and VI of

these sample characteristics are given in Tables 7.25 and 7.26, respectively. It

was also noticed that there is signicant differences found in case of Trial III

and IV, V and VI treatments with normal and aerodynamic system of enzymetreatments which may be due to higher non-cellulosic compounds removal

in aerodynamic system and their ANOVA test results are given in Table 7.27.

Table 7.26 Performance level of Trial V and VI in enzymatic desizing and scouring

process


Normal mixed

enzymatic method

Ultrasonic

method

Aerodynamic

method


Wax removal (%) 8.92 10.20 15.38

Pectin removal (%) 5.76 6.42 8.74


Contd...





Normal mixed

enzymatic method

Ultrasonic

method

Aerodynamic

method



Brightness Index 5.61 7.59 11.94

Fabric tensile strength (g/tex) –0.208 –0.257 –0.339

Warp way

Weft way –0.128 –0.348 –1.836

Fig. 7.47 Waste water efuent after scouring (a) chemical method (b) enzyme method

Table 7.27 ANOVA Multivariant Analysis – single-stage enzymatic scouring and

bleaching process between Trial III and IV

Source of variation SS df MS F Pvalue Fcrit

Fabric weight loss

Between Trials III and IV 1345.21 29 46.38 13.58 0.012 4.182

Between normal and ultrasonic

methods

451.52 29 15.57 5.630 0.031 4.182


methods

368.63 29 12.71 6.924 0.042 4.182

Wax removal (%)



methods

96.25 29 3.319 9.634 0.038 4.182

Contd...

Contd...






methods

56.63 29 1.953 8.637 0.081 4.182

Pectin removal (%)



methods

96.35 29 3.323 11.52 0.127 4.182


methods

124.67 29 4.298 8.637 0.024 4.182

Fabric wetting area



methods

1124.63 29 38.78 6.83 0.098 4.182


methods

1325.67 29 45.71 6.18 0.352 4.182

Whiteness Index


Between normal and ultrasonicmethods

42.860 29 1.478 10.62 0.051 4.182


methods

37.951 29 1.309 14.52 0.042 4.182

Yellowness Index



methods

17.36 29 0.599 8.652 0.351 4.182


methods

9.865 29 0.340 6.124 0.054 4.182

Brightness Index



methods

118.62 29 4.090 9.863 0.241 4.182


methods

92.53 29 3.191 11.53 0.058 4.182


Between Trials III and IV 18.92 29 3.425 0.035 0.870 4.182Between normal and ultrasonic

methods

71.50 29 38.75 2.561 0.094 4.182


methods

87.63 29 42.35 9.254 0.0345 4.182

Contd...




Table 7.28 ANOVA Multivariant Analysis – single-stage enzymatic scouring and

bleaching process between Trial V and VI


Fabric weight loss

Between Trials V and VI 851.24 29 29.35 21.43 0.041 4.182


Between normal and aerodynamic methods 258.63 29 8.918 10.25 0.085 4.182

Wax removal (%)




Pectin removal (%)




Fabric wetting area




Whiteness Index




Yellowness Index




Brightness Index











7.9.2.7 Waste water efuent analysis

The waste water efuent after processed through chemical and enzymaticmethod of organic cotton fabric was analyzed and their comparison test results

are given in Table 7.29. From the analysis the enzymatic method of scouring is

better than chemical scouring in COD and BOD for ecofriendly processing in

concern. From the waste water analysis of enzymatic scouring efuent meets

the standard level of government norms for ecofriendly processing.

Table 7.29 Waste water efuent analysis

Parameters Government

standards

Chemical

method

Enzyme method

pH 6.8–8.5 9–13 7–8.5

BOD 30 mg/lit 300 40–50

COD 100 mg/lit 600 150–220

Suspended solids 100 mg/lit 90 30–40

TDS Approx. 2100 mg/lit 3000–5000 1500–2400

Oil and grease 10 mg/lit 15–30 12–15

Sulphates Nil 1–2 mg/lit Nil

Chlorides 600 mg/lit 1250–1850 150–300

Color (units) 100 units 2000–2400 600–850

Turbidity (fau) 15 180 35–60

Note: Scouring efuent analysis

7.9.2.8 Summary

The organic cotton fabric treated with ultrasonic and aerodynamic system in

the single-stage enzymatic scouring and bleaching treatments were noticed

higher degradation of pectin (above 75%) and wax components (above

80%) in the bioscouring process and also noticed better water absorbency

<1 sec and fabric whiteness (above 80%) and brightness index (above 73%).

These fabric properties are comparable with chemical alkaline scouring and

bleaching followed in the industrial practices. Sonication method improves

enzyme cavitation in the catalysis of enzymes for breakdown of pectin and

wax/oil substances faster than normal method of treatments. Aerodynamic

method improves catalysis of enzymes by boosting/energizing air pressure of

living organisms.




7.10 References

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Mori, T., Sakimoto, M., Kagi, T., and Saki, T., ‘Enzymatic desizing of polyvinyl alcoholfrom cotton fabrics’, J Chemical Tech and Biotech, 1999, 68(2), 151–156.

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Abstract: This chapter is to outline and review the latest developments andadvance in medical textiles and biopolymers for wound management providing

the overview with generalized scope of biotechnology about novelties in medicalproducts and properties. The past decade has seen considerable efforts in the useof polymer nanobres for biomedical and biotechnological applications. Theseinclude tissue engineering, controlled drug release, wound dressings, medicalimplants, dental composites, molecular ltration, biosensors and preservation ofbioactive agents. An overview of these applications has been discussed. Most ofthese applications are still being tested in laboratories worldwide with some atthe infancy stage. Signicant advancements are necessary before clinical usageor commercialization can be realized. More attention should be given to naturalbiopolymers (e.g., chitin, alginate, etc.) so that better biological compatibilityand performance can be realized. In order to advance the biotechnological

and especially biomedical applications of polymer nanobres from perspectiveto commercialized stages, collaborative interdisciplinary researches involvingsurgeons, material scientists, biologists, physiologists, clinicians, and engineersare required. It is believed that continual investments from academia, government,and industry into this eld will not only shorten the distance between laboratoryand practical utilization stages in any of the above reviewed areas but also openup other new range of opportunities for polymer nanobres in biomedical andbiotechnological applications.

Keywords: Biomedical, wound care, drug release, biopolymer, biosensor,polymer nanobre

8.1 Introduction

New generation medical textiles are signicantly growing eld with great

expansion in wound management products. Virtually new products are

coming in the market with signicantly improved properties using advanced

technologies. These new products are in the centre of research which is highly

technical, technological, functional, and effective oriented (Petrulyte 2008).

The qualities of wound care products include that they are bacteriostatic,

anti-viral, fungi static, non-toxic, high absorbent, non-allergic, breathable,

haemostatic, and biocompatible. Many additional advantages over traditional

materials have products modied or blended with biopolymers based on

alginate, chitin/chitosan, and collagen (Verreck et al. 2003). Textile structures

8

Biotechnology and biomaterials for hygienic and

health care textiles



Biotechnology and biomaterials for hygienic and health care textiles 399

used for modern wound dressings are of large variety: sliver, yarn, woven, non-

woven, knitted, crochet, braided, embroidered, composite materials (Pavlova

and Draganova 1993). Wound care also applies to materials like hydrogels,

matrix (tissue engineering), lms, hydrocolloids, and foams. Additives along

with special functions can be introduced in advanced wound dressings with

the aim to absorb odours, provide strong antibacterial properties, smooth pain

and relieve irritation. Because of unique properties as high surface area to

volume ratio, lm thinness, nano scale bre diameter, porosity, light weight,

nanobres are used in wound care (Matsuda et al. 1993).

8.2 Medical textilesMedical textiles are also known as healthcare textiles and most rapidly

expanding sector in the technical textile market. The textile materials for

medical and healthcare products are ranges from simple gauze or bandage

materials to scaffolds for tissue engineering applications (Bense and

Woodhouse 1999). Advanced medical textiles are signicantly developing

area because of their major expansion in such elds like wound healing and

controlled release, bandaging and pressure garments, implantable devices as

well as medical devices, and development of new intelligent textile products(Choi et al. 1999). The basic requirements of textile material for medical

applications are biocompatible; good resistance to alkalis, acids and micro-

organisms; good dimensional stability; elasticity free from contamination or

impurities; absorption/repellency and air permeability.

These medical and healthcare textile products can be classied into

following main areas;

• barrier material (for infection control)

• bandaging and pressure garment

• wound care material

• hygiene material

• implantable material (sutures, articial joints)

• extra corporal devices (articial kidney)

Medical textiles products are mainly used for hygienic and biological

applications primarily for rst aid, clinical and rehabilitation purposes. It

consists of all those textile materials used in health and hygienic applications

in both consumer and medical markets (Matsuda et al. 1992). Today these

medical products are manufactured with new treatment options (textile- based implants instead of scarce donor organs; articial tissues, joints and

ligaments), Speed up recovery after medical treatment (innovative wound

dressings; light, breathable orthoses/protheses) to enhance quality of life of

chronically ill people (functional clothing). Surgeons wear, wound dressings,




bandages, articial ligaments, sutures, articial liver/kidney/lungs, nappies,

sanitary towels, vascular grafts/heart valves, articial joints/bones, eye contact

lenses and articial cornea and the like are some of the examples of medical

textiles (Ruizcardona et al. 1996).

8.2.1 Medical applications

The enzyme inhibition is the important application in the use of antibiotics

in medicine (Matsuda et al. 1992). There are also many necessary co-

enzymes, or co-factors for culturing the tissue and bacterial reactions with

organic non-protein molecules, which either enhance or are necessary for

the enzyme’s activity because enzymes work most effectively within narrow

ranges of temperature and pH. Deviations cause the enzyme to change

shape (denaturation) and to become less effective; it may happen if the

body overheats as a result of physical exertion or when lactic acid produced

by anaerobic respiration lower the pH of body uids (Chekmareva 2002).

Several possibilities exist for producing entirely new bre materials, so called

biopolymers, using biotechnological process routes, naturally occurring

polyester; Polyhydroxybutyrate (PHB) is produced by bacterial fermentation

of a sugar feed stock and commercially available as ‘Biopol’. This polymer isstable under normal conditions but biodegrades completely in any microbial

active environment. These kinds of biopolymers with textile potential include

polylactates and polycaprolactones, which are also applied in medical

applications (Zong et al. 2003).

8.2.1.1 Dressing – types and usage

A dressing is an adjunct used by a person for application to a wound to promote

healing and/or prevent further harm. A dressing is designed to be in directcontact with the wound, which makes it different from a bandage, which is

primarily used to hold a dressing in place. Dressings are frequently used in

rst aid and nursing. A dressing can have a number of purposes, depending on

the type, severity and position of the wound, although all purposes are focused

towards promoting recovery and preventing further harm from the wound.

The main purposes of a dressing are:

• Stem bleeding – helps to seal the wound to expedite the clotting

process;• Absorb exudate – soak up blood, plasma and other uids exuded from

the wound;

• Ease pain – some dressings may have a pain relieving effect, and

placebo effect;




• Debride the wound – the removal of slough and foreign objects from

the wound;

• Protection – from infection and mechanical damage; and

• Promote healing – through granulation and epithelialization.

Types of dressing: Historically, a dressing was usually a piece of material,

cloth, but the use of cobwebs, dung, leaves and honey has also been described.

However, modern dressings include gauzes (which may be impregnated with

an agent designed to help sterility or to speed healing), lms, gels, foams,

hydrocolloids, alginates, hydrogels and polysaccharide pastes, granules and

beads (Jia et al. 2002). Many gauze dressings have a layer of nonstick lm

over the absorbent gauze to prevent the wound from adhering to the dressing.Dressings can be impregnated with antiseptic chemicals, as in boracic lint

or where medicinal castor oil was used in the rst surgical dressings. The

various types of dressings can be used to accomplish different objectives

including (i) controlling the moisture content, so that the wound stays moist

or dry, (ii) protecting the wound from infection, (iii) removing slough, and (iv)

maintaining the optimum pH and temperature to encourage healing (Wang et

al. 2004).

Usage of dressings: Applying a dressing is a rst-aid skill, today almost

all come in a prepackaged sterile wrapping, date coded to ensure sterility andfulll the ‘protection from infection’ aim of a dressing. Applying and changing

dressings is one common task in nursing. An “ideal” wound dressing is one

that is sterile, breathable, and conducive for a moist healing environment.

8.2.1.2 Dressings for wound healing

An ideal dressing is one that can provide an environment at the surface of the

wound in which healing can take place at the maximum rate consistent with

the reproduction of the healing wound with an acceptable cosmetic appearance

(Thomas 1990). Modern wound dressings are developed to serve the purpose

of facilitating wound healing apart from the basic function of covering wounds

from further infection. It has been recognized that ideal dressings should have

the characteristics of (1) haemostatic, (2) efciency as bacterial barrier, (3)

absorption of excess exudates (wound uid), (4) provision and maintenance

of a moist environment, or appropriate water vapor transmission rate, and

provision of adequate gaseous exchange, (5) ability to conform to the contour

of the wound area, (6) functional adhesion, i.e. adherent to healthy tissue butnon-adherent to wound tissue, (7) painless to patient and ease of removal, and

(8) low cost. Current efforts using polymer nanobrous membranes as medical

dressings are still in its early stage. Some major factors such as an increasing

aging population with more chronic wounds and unexpected sufferings of




civilians from terrorist attacks, warfare conicts, and frequent casualties from

trafc accidents, there is an increasing demand for advanced wound care

products. Ionic silver exhibits antimicrobial activity against a broad range of

micro-organisms. As a consequence, silver is included in many commercially

available healthcare products. The use of silver is increasing rapidly in the

eld of wound care, and a wide variety of silver containing dressings are now

available in market (e.g., hydrobre dressing, polyurethane foams and gauzes).

It is proposed that hygiene should be emphasized and targeted towards those

applications that have demonstrable benets in wound care (Wang et al. 2002).

The problem of traditional wound dressings are well known that gauze adheres

tightly to wounds and leads to the occlusion and accumulation of wounddischarge under the dressing, which favors the growth of pathogenic microora

in the wound. In this regard, new technology will dramatically accelerate the

development of innovative dressing materials for wound healing.

8.3 Modern wound dressings

Modern wound dressings are signicantly differ from traditional in both

design and properties. The modern wound dressing will imply both textiles

(gauze, net, and tricot fabric) and other materials such as lms and lm-

forming compositions, sponges, hydrocolloids, gels, powders, pastes, and

combinations thereof (Nazarenko et al. 2002). Numerous publications reveal a

variety of new research direction and show that there are important advantages

and good prospects in this eld. Extensive research was stimulated by a change

in the commonly accepted notions about the optimum conditions of wound

healing. This implies that a dressing must also maintain a certain optimum

microclimate including vapor and air circulation. There is an increase in the

level of other requirements on dressings, such as efcacy of the rst aid and

post-operation repair, and the increased level of aesthetic demands. In orderto meet all these requirements, dressings must ensure good modeling of the

wound surface, be atraumatic, make possible contactless monitoring of the

wound, produce no toxic and local irritant action, admit sterilization, provide

a maximum level of comfort. The main role in performing the aforementioned

functions belongs to the polymer matrix. The large variety of wound dressings

created to the present is explained to a considerable extent by the broad

spectrum of available polymers whose physicochemical properties determine

the properties and functions of each dressing (Hansson 1997).

8.3.1 Polymeric wound dressings

The rst investigations devoted to the creation of polymeric wound dressings

are implicated the preliminary chemical modication of polymers in order to




introduce certain functional groups into macromolecules, which were used

for grafting the polymer chains. Extensive research was devoted to materials

based on the well-known natural polysaccharide – cellulose and its numerous

derivatives. An advantage of cellulose-bre-based materials is the existence

of rich sources of raw materials, the well-developed technological basis for

their processing, and a large variety of their forms, including textiles (gauze).

It is the very low cost that makes traditional cellulose-based bandages still

competitive. On the other hand, the high hygienic, sorption, and mechanical

properties of materials based on cellulose bres allow them to exist together

with the new polymeric wound dressings. Many investigations are still

devoted to the modication of traditional (e.g., gauze) dressings with theaim of eliminating the aforementioned disadvantages and imparting new

useful properties. The rich special features of the chemical structure and

supramolecular structure of biopolymers (polysaccharides, proteins) offer

broad possibilities for new solutions in the creation of wound dressings. Many

of these polymers possess high biocompatibility and intrinsic physiological

activity. This group of polymers includes alginate, which is known to stimulate

repair processes (Doyle et al. 1996).

8.3.2 Wound dressings – Antimicrobial properties

One of the main functions of wound dressings is to protect the wound from

penetration of a pathogenic microora from the environment. The traditional

cotton gauze dressing only provides a reliable initial mechanical protection,

but, absorbing the wound discharge, it becomes a medium favoring the

growth of the pathogenic microora. In order to prevent a wound from

the development of pyoinammatory complications, it is expedient to use

dressings producing a certain antimicrobial activity (Nazarenko et al. 2002;

Fedorov 2000).

8.3.3 Chitosan – Antimicrobial agent

Chitosan is a ß-1,4-linked polymer of glucosamine (2-amino-2-deoxy-ß-

D-glucose) and lesser amounts of N-acetylglucosamine. It is formed by the

deacetylation of chitin (poly-N-acetylglucosamine). Chitosan is antimicrobial

against a wide range of target organisms. Activity varies considerable with

the type of chitosan, the target organism and the environment in which itis applied (Lim and Hudson 2003). The main directions of research aimed

at the development of new wound dressings are considered using chitosan

biopolymer-based products. An important modern trend is the use of

biocompatible natural and synthetic polymers and their compositions as the




bases of wound dressings. The new products have exible design, and possess

combined properties (including antimicrobial activity), which expands their

functions and free of the disadvantages of traditional textile materials.

Important advantages of new dressings are the atraumatic character, effective

curative action, and reduced therapy time. The production of chitosan-based

wound dressings and bandages have become a rapidly developing eld of

polymer-based medical applications (Sashiwa and Aiba 2004).

8.3.3.1 Chitosan-based dressings

Chitosan biopolymer-based wound dressings can be manufactured invarious forms, including bres (Hirano et al. 1999), lms (Wang et al.

2002), asymmetric spongy membranes (Mi et al. 2001), and gels (Ishihara

et al. 2001). The mechanical properties of chitosan wound dressings can

be improved by introducing polymers of a different chemical nature. A

chitosan lm with antimicrobial properties (Kordestani 2001) may contain

up to 20% of another hydrophilic polymer (e.g., polyvinyl Alcohol (PVA),

gelatin, collagen, Polyvinylpyrrolidone (PVP), polyethylene glycol (PEG),

and poly (methacrylic acid) (PMA)), which increases the lm strength and

improves adhesion. Wound dressings based on chitosan and sodium alginatecomplexes were obtained in the form of lms (Wang et al. 2002) and sponges

impregnated with silver sulfadiazine (Kim et al. 1999). The equilibrium

adsorption capacity of the sponge and the release of the drug component can

be controlled by varying conditions of the chitosan – alginic acid complex

formation. A combination of different chitosan-based materials was used

to obtain two-layer dressings (Loke et al. 2000; Mi et al. 2002). A special

group of wound dressings includes those based on chitin and its derivatives,

in particular, chitosan. The unique properties of chitosan as a carrier for biologically active components are related to its chemical nature, as a cationic

biodegradable polymer with intrinsic biological activity (Markin et al. 1994;

Sandford and Steinnes 1991).

8.3.3.2 Chitosan – grafting technique

Chitosan is a water-soluble biocompatible and biodegradable polymer.

Chemical modication of Chitosan is important for the production of

biofunctional materials with a wide practical application in many areas.Among various methods, graft copolymerization is most attractive because

it is a useful technique for modifying the chemical and physical properties of

natural polymers. Grafting of chitosan is a common way to improve chitosan




properties such as increasing chelating (Yang and Yuan 2001) or complexation

properties (Chen and Wang 2001), bacteriostatic effect (Jung et al. 1999)

or enhancing the adsorption properties. Several methods of grafting onto

chitosan are known, such as grafting initiated by free radicals, grafting using

radiation, enzymatic grafting (Jayakumar et al. 2005), graft copolymerization

via polycondensation, graft copolymerization via oxidative coupling, cyclic

monomer graft copolymerization (ring-opening) (Zohuriaan 2005), etc.

The enzymatic grafting is new method of preparing the chiton biomedical

wound dressings. Tatjana Romaškevič et al. (2007) have studied the grating

of chitosan based on maltogenase (MG) enzyme for preparation of biowound

products. Enzyme: Maltogenase (MG) from Bacillus Stearothermophilus (glucan

1,4-α-maltohydrolase, E.C.3.2.1.133) recombinant exo-acting maltogenic

amylase, removes maltose units (through 1,4-α-D-glucosidic linkages) from

the non-reducing chain ends in malt oligosaccharides and polysaccharides,

such as amylase and amylopectin.

Immobilization of maltogenase via covalent binding: The immobilization

of MG was carried out in 0.1 M citrate buffer (pH 6.5). The mixture of 0.25

cm3 (500 IU) of MG, 5 ml of buffer and 3.5 g of wet carrier activated with GA

Chs-g-PEGMEMA (immediately after the synthesis) was stirred at 40°C for 30min and was left overnight at 4°C. The immobilized enzyme was thoroughly

washed with buffer. The efciency of immobilization was dened as the activity

of the immobilized MG in percent values from the activity of the native enzyme

used for the immobilization (Tatjana Romaškevič et al. 2007). The yield of

immobilization was dened as the protein quantity of the immobilized enzyme

in percent values from the quantity of the protein of the native enzyme used

for the immobilization. Figure 8.1 shows the Immobilization of enzyme via

covalent binding onto chitosan graft copolymers.

Fig. 8.1 Immobilization of enzyme via covalent binding onto Chitosan graft copolymers




8.3.4 Alginate and collagen wound dressing

Alginate is capable of forming high viscosity gels whose degree ofstructurization can be controlled by introducing cations (calcium group).

This principle was used for the creation of numerous wound dressings in the

form of sponges and bres (Suzuki et al. 1999; Thomas et al. 2000). In order

to increase the elasticity of alginate-based wound dressings, it is possible to

introduce poly (ethylene oxide). Alginate drapes are recommended for use

in surgery as a hemostatic dressing material. The group of natural polymers,

which are rather frequently used as bases or components of wound dressings,

also includes collagen (Tomihata et al. 1994). The main difculty that hinders

a still wider use of natural collagen is its very low solubility in typical protein

solvents. In most cases, the collagen-based wound dressings have the form of

sponges, which are obtained by lyophilic drying of various collagen containing

compositions (Grzybowski et al. 1997).

8.3.5 Biologically active PVA-based gel dressing

The biologically active PVA-based gel dressing employs a polymer-based

matrix containing a bound antibiotic which is activated only in the case ofwound infection. Proteinases present in exudating wounds hydrolyze the

bonds between the antibiotic and PVA only in the presence of Staphylococcus

aureus or Pseudomonas aeruginosa, after which gentamicin is released into

the wound medium. The kinetics of release of various substances (hydrophilic,

hydrophobic, proteins) from a hydrogel based on human serum albumin and

poly (ethylene glycol) (PEG) was studied (Gayet and Fortier 1996). The

use of biodegradable polymers is another direction in the creation of wound

dressings (Kobayashi et al. 1991).

8.3.6 Polymer nanobres – Biomedical and

biotechnological applications

Research in polymer nanobres has been undergone signicant progress in

the last one decade. One of the main driving forces for this progress is the

increasing use of these polymer nanobres for biomedical and biotechnological

applications (Yanzhong et al. 2005). The latest research and advancement made

in the use of polymer nanobres are focused on the applications such as tissue

engineering, controlled drug release, wound dressings, medical implants, and

nanocomposites for dental restoration, molecular separation, biosensors and

preservation of bioactive agents. Within the association of nanotechnology

and nanostructured materials, a nanobre generally refers to a bre having a

diameter less than 100 nm (Grafe and Graham 2003). Several techniques such




as electrospinning (Reneker and Chun 1996; Huang et al. 2003), melt-blown

(Ward 2001; Gu et al. 2003), phase separation (Ma and Zhang 1999; Yang et

al. 2004), self-assembly (Hartgerink et al. 2001; Cheng 2003), and template

synthesis (Martin 1996; Feng et al. 2002) have been employed to produce

suitable polymer nanobres for different purposes. Amongst, electrospinning

is the most popular and preferred technique to use. It is simple, cost-effective

and able to produce continuous nanobres of various materials from polymers

to ceramics. In addition, electrospinning seems to be the only method which

can be further developed for large scale production of continuous nanobres

for industrial applications.

Polymer nanobre structures usually provide superior hydrophobic property because an effective contact angle increases with decrease in bre

diameter. The use of polymer nanobres for biomedical and biotechnological

applications has some intrinsic advantages. From a biological point of view,

a great variety of natural biomaterials are deposited in brous forms or

structures such as silk, keratin, collagen, viral spike proteins, polysaccharide

cellulose and chitin. The recent uses of polymer nanobres for biomedical

and biotechnological applications (Fig. 8.2) which include tissue engineering,

controlled drug release, dressings for wound healing, medical implants,

nanocomposites for dental applications, molecular separation, biosensors and preservation of bioactive agents (Risbud et al. 2002).

Fig. 8.2 Polymer nanobres in biomedical and biotechnological applications




8.3.6.1 Scaffolds for tissue engineering

Recently, poly (L-lactic acid) (PLLA) polymer nanobres were used asa scaffold onto which nerve stem cells (NSCs) were cultured (Yang et al.

2004). The nanobres were produced following a liquid-liquid phase

separation method (Ma and Zhang 1999). Biodegradable scaffold is generally

recognized as an indispensable element in engineering living tissues. They

are used as temporary templates for cell seeding, invasion, proliferation and

differentiation prior to the regeneration of biologically functional tissue or

natural extracellular matrix (ECM) (Stephens et al. 2003; Fertala et al. 2001).

A pioneer work employing a composite nanobrous scaffold for therapeutic

application in gene delivery was recently reported by Luu et al. (2003).

Scaffold was electro spun by incorporating plasmid DNA into synthetic

biodegradable polymers of PLGA and PLA-PEG. The DNA released from

the scaffold was not only intact, but also capable of cellular transfection, and

had even successfully encoded a protein β-galactosidase. The architecture of

a scaffold and the material used play an important role in modulating tissue

growth and response behavior of the cells which have been cultured onto the

scaffold. Polymer nanobres have been considered for use as scaffolds for

engineering tissues such as cartilages (Fertala et al. 2001; Li et al. 2002), bones(Yoshimoto et al. 2003), arterial blood vessels (Huang et al. 2000; Nagapudi

et al. 2002; Xu et al. 2004), heart (Zong et al. 2003), nerves (Silva et al. 2004),

etc. Biodegradable polymer nanobres of PLA, PLGA, and PEG-PLA were

attempted for use in heart or cardiac tissue constructs (Zong et al. 2003).

8.4 Enzymes in medical applications

Developments of medical applications using enzymes have been reecting

the magnitude of the potential rewards for example; pancreatic enzymeshave been in use since the nineteenth century for the treatment of digestive

disorders. As enzymes are specic biological catalysts, they should make the

most desirable therapeutic agents for the treatment of metabolic diseases.

The variety of enzymes and their potential therapeutic applications are

considerable. Table 8.1 shows the selection of enzymes which have realised

this potential to become important therapeutic agents. At present, the most

successful applications are extracellular: purely topical uses, the removal

of toxic substances and treatment of life-threatening disorders within blood

circulation. In contrast to the industrial use of enzymes, therapeutically useful

enzymes are required in relatively tiny amounts but at a very high degree of

purity and specicity. A major potential therapeutic application of enzymes

is in the treatment of cancer (Buchko et al. 2001). Asparaginase enzyme has




proved to be particularly promising for the treatment of acute lymphocytic

leukaemia. This enzyme action depends upon the fact that tumour cells are

decient in aspartate ammonia lipase activity, which restricts their ability to

synthesis the normally non-essential amino acid L-asparagine. The action of

the asparaginase does not affect the functioning of normal cells which are

able to synthesis enough for their requirements, but reduce the free exogenous

concentration and so induces a state of fatal starvation in the susceptible

tumour cells.

Table 8.1 Therapeutic enzymes in medical applications

Enzyme EC number Reaction UseUricase 1.7.3.3 Urate + O

2 → Allantoin Gout

Rhodanase 2.8.1.1 S2O

32– + CN – → SO

32– + SCN –

Cyanide

poisoning

Ribonuclease 3.1.26.4 RNA hydrolysis Antiviral

Hyaluronidase 3.2.1.35 Hyaluronate hydrolysis Heart attack

Lysozyme 3.2.1.17 Bacterial cell wall hydrolysis Antibiotic

Collagenase 3.4.24.3 Collagen hydrolysis Skin ulcers

Asparaginase 3.5.1.1L-Asparagine H

2

O→ L-aspartate +

NH3 Leukaemia

Glutaminase 3.5.1.2L-Glutamine H

2O→ L-glutamate +

NH3

Leukaemia

Lactamase 3.5.2.6 Penicillin→ PenicilloatePenicillin

allergy

Streptokinase 3.4.22.10 Plasminogen→ Plasmin Blood clots

Trypsin 3.4.21.4 Protein hydrolysis Inammation

Urokinase 3.4.21.31 Plasminogen→ Plasmin Blood clots

8.4.1 Enzymes in medicine

Enzymes are produced by living cells they are substances that act as a catalyst

in living organisms. All enzymes are proteins and therefore have a tertiary

structure. Each enzyme is a specic shape with an active site which is specic

to one substrate molecule. When the substrate combines with the active site

enzyme substrate complexes are formed. The uses of important enzymes in

medicine include (i) killing disease causing micro organisms, (ii) prompting

wound healing, and (iii) diagnosing certain diseases. Enzymes are beingused heavily in medicine as direct pharmaceutical products. The medical and

pharmaceutical application of enzymes covers such a wide range of ideas.

In contrast to industrial uses where production is on a much larger scale,

uses of medical and pharmaceutical enzyme applications generally require




small quantities of highly puried enzymes. Enzymes and enzyme-generated

products are administered to patients in very small doses; to avoid possible

side effects.

Enzymes are being used to detect and measure amounts of glucose in

blood. The amount of glucose in the blood and urine is a crucial indicator

in the diagnosis of diabetes; when there is a deciency of insulin resulting

in high glucose levels in the blood. It is detected using the enzyme glucose

oxidase which is impregnated onto a strip of paper, and a biosensor. This

instrument uses glucose oxidase as its biological system (Gekas and Lopez

1985). The enzyme may catalyse the reaction between glucose and oxygen

to form gluconic acid. The biosensor then uses the amount of gluconic acid produced to indicate the quantity of glucose and oxygen there was in the blood

this is indicated by a colour change. Enzymes are very important when coming

to diagnose disease particularly in the case of a damaged liver. Enzymes that

would be normally found in the liver, leak into the blood stream. By testing

the blood for alternate enzyme activity, liver damage can be conrmed.

Enzymes are vitally important in preventing excessive blood clotting

and reducing the tendency for platelets and red blood cells to ‘clog’, because

enzymes are playing a part in removing metabolic waste and improving

circulation of proteases. For example, Trypsin and chymotrypsin can be usedin brinolysis, this a process that dissolves blood clots. One use is in the case

of thrombosis, this is when blot clots form in damaged blood vessels, if these

clots are carried to an small artery and may become blocked a heart attack

stroke can be caused. This can be treated by enzymes such as trypsin and

protease. Digestion of the insoluble brin clot takes place and because the

enzymes are proteins this results in a conversion to minor acids, consequently

freeing the trapped blood cells and eliminating the clot. This process is called

‘brinolysis’. Opposite to the prevention of clotting; the enzyme protease can be used as a debriding agent they are used to clean the wound and accelerate

the healing process. Enzymes can also be used in drug manufacture where

the synthesis of drugs is difcult therefore enzymes are used to perform the

chemical procedure. Enzymes can also be used to aid digestion where they

are used to supplement amylase, lipase and protease produced mainly by the

pancreas. (Ref. www.enzymes.co.uk/ enzyme technology, date 3.4.2013)

The main enzyme application in medicine is the production of antibiotics

in particular penicillin. The major pharmaceutical; products produced using

enzyme technology are the antibiotic, semi-synthetic penicillin’s. Antibioticsare chemical substances produced by micro organisms which are effective in

dilute solution in preventing the spread of other micro organisms (Khil et al.

2003). Most inhibit growth rather than kill the micro organism on which they

act. One of the best known antibiotics is penicillin – discovered by Alexander




Fleming in 1928. It was found that it acts on growing bacteria, killing them

and preventing their growth. It is believed to compete with paraaminobenzoic

acid for the active site of an enzyme. In this way they do not kill the acteria but

simply stop them from reproducing. New developed drugs should therefore

be used with much more restraint and discrimination and more time should be

used searching for natural antibiotics to the development of new strains using

genetic engineering.

8.4.2 Benets of enzymes for health care

The human body is constituted by trillion cells and function upon thousands of

metabolic enzymes as energy factor and to carry out numberless biochemical

processes. Metabolic enzymes act as the workhorses of the body because

they also regulate and control each and every process that keeps the body

functioning harmoniously. Mixtures of enzymes had a positive effect in

reducing swelling and inammation, enhancing immune function, improving

circulation, reducing pain, strengthening connective tissue, and speeding

recovery from traumatic injury.

8.4.2.1 Proteases

Proteases are a very extensive family of enzymes. There is potential for

several of these agents to be used in the treatment of wounds. Only two of

them, papain and collagenase, have had a substantial and continuous role for

this indication. Briey, a more accepted term for proteases is peptidases, and

the whole group of enzymes can be subdivided into exopeptidases, which

remove amino acids from the ends (either the N- or C-terminus) of proteins,

and endopeptidases (or proteinases), which cleave bonds within protein.

8.4.2.2 Proteolytic enzymes

In recent years, certain proteolytic enzymes such as serratopeptase and

nattokinase have been employed for their brinolysis properties because these

enzymes possess the ability to emulsify and digest arterial plaque. Proteolytic

enzymes act as a natural anti-inammatory and have also demonstrated the

ability to break down plasma proteins and cellular debris into smaller fragments

at the site of an injury helping their dispersal through the lymphatic system.

This is turn relieves swelling and pain. The healing properties have been

found to be excellent therapeutic agents for minor musculoskeletal injuries

and for accelerating recovery in sports injuries, surgery and burns. Proteolytic

enzymes are useful in the ght against bacterial and fungal infections. When




it comes to ghting viruses – enzymes attack and dissolve the isoprin bonds

– the exterior protein shell coating of the virus, rendering it useless. Enzymes

are to modulate immune function by enhancing the production of cytokines,

increasing the phagocytic activity of macrophages and facilitating the break-

up of circulating immune complexes which are the major causes of auto-

immune diseases such as arthritis, rheumatoid arthritis and multiple sclerosis.

8.4.2.3 Serine proteinases

The serine proteinases are well known members in chymotrypsin, trypsin,

plasmin, plasminogen activators, and leukocyte elastases. In general, serine

proteinases are potential enzymes with a broad range of catalytic activity

and are readily available in tissues (Theron et al. 2001). The second group

of proteinases is the cysteine proteinases, and certainly papain is the better

known member of this group and the best studied. The aspartic proteinases

have a number of well-known enzymes, such a rennin and pepsin. The

metalloproteinases (MMPs) are well known in the eld of wound healing

because of their properties of being able to cleave collagens and other

extracellular matrix components.

Many of these peptidases require zinc or calcium cations (or both) fortheir activity and considerable overlap in substrate specicity among the

MMPs. Removal of the necrotic tissue is essential to reduce the bacterial

burden, which in turn decreases the amount of exudate produced. In response

to wounding, keratinocytes migrate from the edge of the wound and assume

a collagenolytic phenotype. Indeed, it has been shown that collagenase

expression is rapidly induced in wound-edge keratinocytes, persists during

healing, and stops at reepithelialization. Moreover, the activity of collagenase

is required for keratinocyte migration on a type I collagen matrix.

A human burn wound indicates that both collagenase and its inhibitor,

tissue inhibitor of metalloproteinases, are articulated during wound repair. The

relationships between proteinase activity and keratinocyte migration appear

to be very tightly regulated, and it is clear that the type of proteinase expressed

is critical. For example, in experimental studies using injured cornea, it was

shown that failure to reepithelialize correlated with the amount of gelatinases,

but not with the increase in collagenase and stromelysin (Min et al. 2004).

The concept of proteolytic enzymes to digest necrotic tissue as an adjunct

in the treatment of complex wounds is probably stems from observing theageless healing techniques of natives in tropical countries. For example,

wound debridements seem to have utilized the papain-rich material obtained

by scratching the skin of the green fruit of the papaw tree (Carica papaya)

and been used for the treatment of several skin conditions. Trypsin directly




hydrolyzes a large number of naturally occurring proteins (Layman et al. 2003).

Chymotrypsin acts upon different bonds in proteins than does trypsin, but its

spectrum of activity is similar. Trypsin and chymotrypsin preparations were

prepared from pancreatic sources (usually beef) but are no longer available for

the treatment of wounds. Papain–urea-based combinations and collagenase

are identied in prominent in terms of their use in chronic wounds.

8.4.2.4 Papain–urea-based combinations

A well-known and widely used enzymatic system is the papain–urea

combination. In this system, papain is used to attack and break down any

protein containing cysteine residues. This property of papain renders the

combination quite nonselective because most proteins, including growth

factors, contain cysteine residues. Collagen contains no cysteine residues

and is thus unaffected by papain. However, urea’s role in this enzymatic

combination is to facilitate the proteolytic action of papain by altering the

three-dimensional structure of proteins and disrupting their hydrogen bonds,

as well as exposing by solvent action of papain. Urea also plays a role in the

reduction of disulde bridges; as the disulde bridges are reduced, cysteine

residues become exposed and are, therefore, more susceptible to the action of papain. It should be noted that the combination of papain and urea is probably

twice as effective in protein digestion as papain alone.

8.4.2.5 Collagenase preparations

Collagenase is another well known and established enzyme preparation used

for debridement. Its development as a debriding agent applications came

to a peak in the early 1970s. The commercially available preparation of

collagenase is derived from bacteria (Clostridium histolyticum). Collagenaseis a water-soluble proteinase that specically attacks and breaks down

collagen. Collagenase is reported to be most effective in a pH range of 6 to

8, It can hydrolyze native collagen and thereby facilitate rapid debridement

and healing of chronic wounds. The mechanism of action of collagenase is

to degrade collagen and convert it to gelatin. An interesting observation is

that the collagenase preparation may be selective for nonviable collagen.

This effect needs to be studied further, but it is thought that viable collagen is

surrounded and protected by mucopolysaccharide sheaths.Collagenase has been found to be remarkably gentle on viable cells.

For example, cell suspensions prepared with collagenase, then stored at low

temperature, were found equal to trypsinized cells in their viability and growth.

Similarly, collagenase can be used as a permanent ingredient of culture media




without loss of cell viability. In more recent work, the addition of collagenase

derived from Clostridium histolyticum to keratinocyte cultures enhanced their

proliferation and migration up to 10-fold. Some potentially underestimated

effects of collagenase, such as angiogenesis and epithelialization; wound

debridement is being accomplished by this enzyme. As is the case with the

papain-based debriding systems, there is considerable published information

detailing the effectiveness of collagenase for wound debridement for all types

of wounds.

8.4.2.6 Enzyme reaction on wound healing

In some cases, chemically modied cellulose acquires intrinsic physiological

activity that imparts to cellulose dressings curative properties even without

the introduction of drugs. An interesting example is viscose bres partly

hydrolyzed by the enzyme cellulase (Tereschenko and Shamolia 1998;

Gusakov et al. 1985). The ability of partly hydrolyzed viscose to absorb

staphylococci, which is 90% higher as compared to the initial bres, decreases

the degree of wound contamination by these microbes. Another cellulose

derivative — carboxymethylcellulose (CMC) containing acid functional

groups which is capable of binding peptides in the wound medium, thusinhibiting the activity of some enzymes such as elastase (Edwards et al. 1999).

Mono-CMC gauze is widely used as a hemostatic dressing. At the same

time, the aforementioned cellulose derivatives are potential matrices for the

physical or chemical immobilization of biologically active substances. In this

respect, an interesting alternative approach is offered by the introduction of

biologically active substances into soluble polymer compositions, followed by

the formation of wound dressings of the desired shape. By selecting polymers

with various ctional groups, it is possible to inuence the character of

interactions between components of the system, thus controlling the kinetics

of desorption of the biologically active substances and the physicochemical

properties of wound dressings (Bisaria and Ghose 1981).

The biological activity of a PVA-based lm containing chlorhexidine

bigluconate and lysozyme was practically completely retained upon radiation

sterilization and subsequent long-term storage. An analysis of the literature

shows evidence of the extensive search for new “ideal dressings” for the

treatment of various wounds. Characteristic trends in the modern stage of

this research are the rejection of traditional textile bases and expansion of thecircle of novel base materials, which provides for the development of dressing

with improved properties and increased functions.




8.4.3 Enzymatic pre-treatment eliminates infectious

bacteriaHospital acquired infections are signicantly a growing problem in the medical

eld. Hospital-acquired infections are one of the leading causes of mortality

and increased morbidity in inpatients and place a heavy burden on the health

system (Percival et al. 2005). Between 3 and 10% of inpatients acquire

an infection during their hospital stay. The mortality rate for nosocomial

infections is 1%, and they contribute to 3% of mortality from other diseases.

As for medical costs, it has been estimated that infections of this type lengthen

hospital stays by between ve and ten days, a statistic that underscores the

economic impact of the problem. Nosocomial infections of endogenous origin

occur mainly as a result of contact with hospital gowns and sheets.

In fact, any type of linen used in a hospital setting can harbour bacteria and

spread infection to patients and medical staff. To address the above problems,

Researchers in the Molecular and Industrial Biotechnology Group of the

Universitat Politècnica de Catalunya BarcelonaTech (UPC), in Barcelona,

Spain (2012), have improved the antimicrobial properties of medical textiles

using an enzymatic pre-treatment combined with simultaneous deposition of

nanoparticles and biopolymers under ultrasonic irradiation (Ilana Perelshtein etal. 2012). The technique can be used to create completely sterile antimicrobial

textiles that can help prevent hospital-acquired infections. The research goal is

to improve the antimicrobial properties of medical textiles by using ultrasonic

irradiation to deposit zinc oxide nanoparticles and biopolymers on the materials.

By applying these enzymes, the researchers have been reported that increased the

durability of the nanoparticles on the fabric remain present even after 70 laundry

cycles. The effectiveness of the antimicrobial treatment has been boosted by

incorporating hybrid materials that combine organic and inorganic components

(zinc and chitosan nanoparticles) within the fabric. In addition to eliminating any

bacteria present, these materials prevent the growth of new microbes.

8.4.4 Biotechnological applications

8.4.4.1 Molecular separation

Polymer nanobres have been used as industrial lters, recognizing their

high ltration efciency, and also potential for protective clothing against

biochemical attacks (Grafe and Graham 2003; Graham et al. 2002). Likewise,

electrospun polymer nanobres with functionalized surface can be extended for

use for high efcient biomolecular or protein separation. The high surface area

to weight ratio makes nanobres an ideal substrate for molecular separation.




The principle of separation is similar to that of afnity chromatography. This

involves utilizing a specic interaction between one kind of solute molecule and

a second molecule or functional group that is immobilized on the nanobrous

membrane (stationary phase). For example, the immobilized molecule may

be an antibody to some specic protein. When the solute containing a mixture

of proteins is passed by the antibody, only the specic protein is reacted and

is bound to the stationary phase (Loscertales et al. 2002; Wnek et al. 2003).

To fulll this molecular separation, proper surface functionalisation of the

nanobrous membrane is important. Structural and material properties of the

nanobrous membranes are also important so that the membranes can withstand

the imposed forces acting on them during the ltration process. At present, polymer nanobrous membrane for molecular separation is still a concept that

needs to be realized (Flemming et al. 1999; Neimark et al. 2003).

8.4.4.2 Biosensor

Biosensors are typically consisting of biofunctional membrane and transducer

(Fig. 8.3), which is widely used for environmental and clinical purposes. The

performance of a biosensor is accessed generally include sensitivity, selectivity,

response time, reproducibility, and aging, all of which are dependent directlyon the property of the sensing membrane used. Among these, sensitivity is

particularly important because there is a strong need for detection of gases and

biological substances at low concentration. Improve the sensitivity will require

using sensor lms with larger surface area to unit mass ratio (Xu et al. 1990).

This provides an opportunity for polymer nanobres to be used as biosensors.

Fig. 8.3 Principle of biosensor




• Some plastics are now referred to as being ‘degradable’, ‘oxy-

degradable’ or ‘UV-degradable’. This means that they break down

when exposed to light or air, but these plastics are still primarily (as

much as 98 per cent) oil based.

• Biopolymers (also called renewable polymers) are produced from

biomass for use in the packaging industry.

• Biomass comes from crops such as sugar beet, potatoes or wheat:

when used to produce biopolymers, these are classied as non food

crops. These can be converted in the following pathways:

Sugar beet > Glyconic acid > Polyglonic acid

Starch > (fermentation) > Lactic acid > Polylactic acid (PLA) Biomass > (fermentation) > Bioethanol > Ethene > Polyethylene

8.5.1 Polylactic acid (PLA)

Poly (lactic acid) or polylactide (PLA) is thermoplastic aliphatic polyester

commonly made from a-hydroxy acids, derived from renewable resources,

such as

• corn starch (in the United States),

• tapioca products (roots, chips or starch mostly in Asia) or• sugarcanes (in the rest of world).

It can biodegrade under certain conditions, such as the presence of

oxygen, and is difcult to recycle.

PLA is not a polyacid (polyelectrolyte) but rather polyester. Bacterial

fermentation is used to produce lactic acid from corn starch or cane sugar.

Two lactic acid molecules undergo a single esterication and then catalytically

cyclized to make a cyclic lactide ester (Fig. 8.5). PLA of high molecular weight

is produced from the dilactate ester by ring-opening polymerization (Tucci et

al. 2001). Polymerization of a racemic mixture of L- and D-lactides usually

leads to the synthesis of poly-DL-lactide (PDLLA) which is amorphous. Table

8.2 shows the properties of polylactic acid, polycaprolactone and nylon 11.

Fig. 8.5 Catalytic and thermolytic ring-opening polymerization of

lactide (left) to polylactide (right)




Table 8.2 General properties of polylactic acid, polycaprolactone and nylon 11

Polymer properties Nylon 11 Polycaprolactone(PCL) PolylacticAcid (PLA)

Density (lb/in3) 0.0361–0.0379 0.0401–0.0434 0.0448–0.0459

Tensile modulus (psi) 8,100 30,000–50,000 5,00,000

Ultimate tensile elongation 20% 300–500% 2.50%

Hardness (Shore’D’) – 80 50

Dielectric strength (V/mil) 650–749 412–429 305–406

Heat capacity (BTU/lb*F) 0.405–0.421 0.468–0.478 0.282–0.289

Coefcient of Friction 0.15–0.25 – 0.16–0.32

Crystallinity – 67% 0–1%

Melt temperature (°F) 347–376 136–145 306

Degradation temperature

(°F)

608 247 518

Glass transition temperature

(°F)

108 –140 140

Viscosity (psi-s) 0.0187–0.0275 – 0.0177–0.0256

Biodegradability

PLA is considered both as biodegradable (e.g., adapted for short-term

packaging) and as biocompatible in contact with living tissues (e.g., for

biomedical applications such as implants, sutures, drug encapsulation, etc.)

(Kenawy et al. 2003). PLA can be degraded by biotic degradation (i.e., simple

hydrolysis of the ester bond without requiring the presence of enzymes to

catalyze it). During the biodegradation process, and only in a second step,

the enzymes degrade the residual oligomers till nal mineralization (biotic

degradation). As long as the basic monomers (lactic acid) are produced from

renewable resources (carbohydrates) by fermentation, PLA complies with the

rising worldwide concept of sustainable development and is classied as an

environmentally friendly material (Choi et al. 1999; Desai 2000).

Applications

PLA is currently used in a number of biomedical applications, such as sutures,

stents, dialysis media and drug delivery devices (Zeng et al. 2003). The total

degradation time of PLA is a few years. It is also being evaluated as a material

for tissue engineering. PLA is a sustainable alternative to petrochemical-derived products, since the lactides from which it is ultimately produced can

be derived from the fermentation of agricultural by-products such as corn

starch or other carbohydrate-rich substances like maize, sugar or wheat. PLA

can be an alternative to high-impact polystyrene by using as much as 1 wt%




non-PLA due to creating co-polymers which can strengthen PLA plastic

(Draye et al. 1998).

8.5.2 Biopolymer stimuli-responsive polymeric system

for textiles

Textile material is considered as unique in nature properties compare among

various kinds of products (Mather 2001). Textile material for clothing is

an example of a material which is personal, comfortable, and used almost

anywhere and anytime. Clothing is considered as an extension to body

physiological characteristics and is very close to the body skin (protection, breathability, sensing). “Smart” textiles are expected to act both as sensors

and actuators, so they should not be confused with other existing high-

performance or multifunctional textiles that are in fact “passive” materials

with advanced properties. In recent studies, an increasing amount of research

is being done on functional nishing of textile materials by incorporating

stimuli-responsive polymeric systems (Liu and Hu 2005; Jocic 2008). Through

this approach, the new value-added textile material can be created containing

bres that maintains advantageous conventional properties (e.g., mechanical

strength, exibility and wear comfort) but with advanced functionalities and/or environmental responsiveness implemented by the modication of a very

thin surface layer of the material.

Currently, the most encouraging option for producing efcient surface

modifying systems comprises the use of hydrogels. This polymeric form

exhibits specic volume phase transition (swelling and shrinking) properties

which can be triggered by various stimuli (temperature, pH, humidity,

etc.) (Fig. 8.6). Hydrogels responsive to temperature and pH have been the

most widely studied systems since these two factors have a physiologicalsignicance (Qu et al. 2001). Versatile dual responsive hydrogels have been

reported mainly for biomedical applications and a number of reviews coming

up in this area in recent times address the latest developments. However,

due to the need for biocompatibility and biodegradability, biopolymer -based

hydrogels are currently of great interest. Such hydrogels can be prepared

by combining a thermo responsive synthetic polymer with a natural based

pH-responsive polymeric component, resulting in dual responsive hydrogel

systems (Prabaharan and Mano 2006). Among the wide choice of natural

polymers, biopolymer chitosan is a good option for combining with syntheticstimuli-responsive polymers. Chitosan is a typical pH-sensitive polymer

which responds to the changes in the pH of the surrounding medium by

protonation/ deprotonation that imparts charges on its amino groups. The pH-

induced phase transition will result in varying dimensions of the hydrogel




(swelling and deswelling). Furthermore, the interesting intrinsic properties of

chitosan are its biodegradability, antibacterial activity and biocompatibility

(Qu et al. 1999).

Fig. 8.6 Different forms of stimuli-responsive polymers and their macroscopic

response [Source: Dragan Jocić 2008]

Microgel is a dispersion of cross linked hydrogel particles whichare swollen by a good solvent. It may also be dened as a disperse phase

of discrete polymeric gel particles with sizes ranging between 1 nm and 1

μm. Microgel particles are insoluble and do not form solutions like linear or

branched polymers, but they may be considered to form colloidal dispersions.

The preparation of microgel can be achieved by different methods such

as: emulsion polymerization; anionic copolymerization; cross linking of

neighbouring polymeric chains; inverse micro-emulsion polymerization or

surfactant-free dispersion polymerization (SFDP). Emulsion polymerization

is a versatile technique which yields narrow particle size distributions.

Conventional emulsion polymerization enables preparation of very small

microgel particles (i.e. particle diameters less than 150 nm) and suffers

from the difculty of completely removing the residual surfactant used for

emulsion stabilization. Surfactant-free dispersion polymerization (SFDP)

yields microgel particles with diameter range between 100 and 1000 nm, and

this method does not suffer from residual surfactant contamination.

Among synthetic polymers, poly (N-isopropylacrylamide) (poly-

NiPAAm) is the most intensively investigated thermo responsive polymerwhich exhibits a volume phase transition (i.e. hydration–dehydration change

due to side-chain re-conguration) in response to even slight temperature

changes. The microgel of poly-NiPAAm and chitosan (PNCS) was prepared

by the surfactant-free dispersion copolymerization method (Lee et al. 2001;




2002). Three kinds of reactions occur in the reaction system of dispersion

copolymerization. In addition, Liu et al. (2005) stated that in the chemical

structure of the grafting copolymers poly-NiPAAm was attached to the C6-

OH reactive group of chitosan and to amino group. In summary, the covalent

bonding between chitosan and poly-NiPAAm can be created from different

chitosan functional groups: the terminal carbonyl groups of its backbone,

its amino groups or its C6-OH reactive groups (Alvarez et al. 2005). The

possibility of anionic persulfate (from the initiator) immobilization inside

degraded chitosan chains through electrostatic attraction has been also

mentioned (Chen et al. 2005). Hence, chitosan plays multiple roles in the

reaction system: in one way it can increase the polymerization rate by servingas a surfactant; on the other hand, the degraded chitosan chain can inhibit free

radicals and slow down the polymerization (Kim et al. 2000).

However, current environmental and human health concerns increasingly

focus on polymers derived from biological precursors or produced by modern

biotechnology, which are also called “biopolymers” (Van Schijndel et al.

1998). Among the different types of biopolymers, polysaccharides are the

second most diverse and complex groups of biopolymers (after proteins).

Since chitosan has a highly reactive primary amino group and also

primary and secondary hydroxyl groups, a high functionality of chitosan can be achieved by simply introducing a specic functional group to the chitosan

structure. Since chitosan has excellent hydrogel forming properties, another

approach is to control the micro- and macrostructure of the chitosan gel itself

without any chemical modication. The structure and properties of chitosan

gels may be varied by the appropriate choice of the preparation method used

(Igarashi et al. 2002), and by blending two or more polymers to obtain the

desired properties.

8.5.3 Chitosan hydrogels

In designing “smart” textile materials, the application of chitosan-based, pH-

sensitive, temperature-sensitive and temperature/pH dual-sensitive hydrogels

is of special interest. In addition to chitosan, other biopolymers or synthetic

polymers are also used as components to produce effective “smart” hydrogels.

Hydrogels are dened as water-swollen three-dimensional networks based on

hydrophilic polymer chains, in which retained water constitutes at least 20%

of weight. They are capable of absorbing a large volume of water or other biological uids (Peppas 1986). Hydrogels swell and shrink in the presence

or absence of water.

Nowadays, considerable efforts have been made to synthesize hydrogels

based on chemical modication of natural polymers in order to use them as




thickeners in food, moisture releasers to plants, uid uptake and retention

in the sanitary area, hydrophilic coatings for textile applications, contact

lenses, and drug-delivery matrices for pharmaceutical applications (Yazdani

et al. 2003). Chitosan hydrogels can be obtained by various mechanisms of

chemical and physical cross linking such as covalent, ionic, hydrogen bonding

and hydrophobic association. Covalently cross-linked chitosan hydrogels are

chemical hydrogels that are formed by irreversible covalent links. Chitosan

hydrogels formed by reversible links (ionic interactions or secondary

interactions) are physical hydrogels. Stable hydrogels can be formed by the

addition of cross linkers (covalently or ionically cross linked hydrogels) or

the direct interaction between polymeric chains without the addition of crosslinkers (complexation with another polymer or aggregation after chitosan

grafting) (Berger et al. 2004). The absorbing capacity of chitosan gels is

highly improved by the presence of synthetic polymers such as polyacrylate.

The gels obtained by self-curing chitosan with acrylic acid (AA) and methyl

acrylate (MA) absorb up to 500 times their dry weight and therefore they can

be classied as super-absorbents (Borzacchiello et al. 2001).

The ability of polymer hydrogels to undergo a volume transition between

swollen and collapsed phases as a function of their environment is one of the

most remarkable and universal properties of these materials. The phenomenonof gel volume transitions, which can be induced by temperature, pH, solvent

composition, ionic strength, electric eld, light, stress or the presence of

specic chemical stimuli, is reversible and has prompted researchers to

explore the potential of gels as actuators, sensors, controllable membranes for

separations and modulators for the delivery of drugs (Goycoolea et al. 2003).

8.5.4 Bioplastics

Conventional plastic derived from petrochemical source takes a long time

to biodegrade when dispersed. Rising concern about shortage of fossil fuel

and long term environmental impact resulted biotechnology research to

develop eco-friendly alternatives (Mo et al. 2004). Bioplastics are produced

by using ‘biopolymers’ which can be produced as secondary metabolites

through fermentation or modication of polymers from renewable sources.

Actively involved in understanding the nature of biological polymers that can

ll up following strategies: (i) Screening and modication of microorganisms

for efcient production of lactic acid through fermentation path and efcientconversion to polylactic acid; (ii) hyper expression and protein engineering

of polyester synthase for production of medical grade nano-material; and

(iii) bio-polylactic acid and plastoGAL series of triglycerides derived from

vegetable source for use in bioplastics industry (Khan et al. 2003).




8.5.5 Bioplastics from microorganisms

Bioplastics are degradable polymers that are naturally degraded by the actionof microorganisms such as bacteria, fungi and algae, such as made from

• Polyhydroxy alkanoates (PHAs): PHB

• Polyactides

• Aliphatic polyesters

• Polysaccharides

• Blends of above

Benets of these bioplastics are 100 % biodegradable, produced from

natural, renewable resources, recycled, composted or burned without

producing toxic byproducts.

8.5.6 Bio-pharmaceuticals

Bio-pharmaceuticals are effectively replacing conventional medicines due to

the advantages of specicity and compatibility. Recently the research work

focuses on healthcare biopolymers that can be used in improving human

life (Matthews et al. 2002). These enzyme technologists focus on the vast

array of dietary enzymes which can supplement human metabolism in the

most compatible manner. Access to methods for evaluating the reactivity and pharmacology of natural ingredients allows pharmaceutical specialists to

choose the best neutraceutical formulations.

8.5.7 Bacterial cellulose

The speciality papers and nonwovens are produced based on bacterially

grown cellulose bres these are extremely ne and resilient and are used

as specialized lters, odour absorbers and reinforcing blends with aramids.

Attempts have been made to transfer certain advantageous textile propertiesinto microorganisms where they can be more readily reproduced by bulk

fermentation processes. The spider DNA is transferred into bacteria with the

air of manufacturing proteins with the strength and resilience of spider silk

for use in bulletproof vests (Jin et al. 2003). This note of caution needs to be

echoed across the whole spectrum of biotechnology developments. Although

biological systems have many attractive possibilities and new approaches to

all sorts of problems and needs, considerable advances are still being made

in conventional technologies, such as, catalysis, chemical synthesis and

physical bre modication which need to be kept in perspective. There is also

still great concern in society about the unbridled advance of biotechnology,

especially with regard to the modication of natural species with possible

unknown long-term consequences.




8.5.8 Nanocomposite for dental application

Polymer nanobres can be used as reinforcement in dental compositeapplications. A dental restorative composite is generally made of some dental

resin such as 2,2’-bis-[4- (methacryloxypropoxy)-phenyl]-propane (bis-

GMA) and tri-ethylene glycol dimethacrylate (TEGDMA) and llers (e.g.,

silica or ceramic particles). The use of polymer nanobres as reinforcement

for engineering composites have so far provided only marginal enhancement

in terms of strength and stiffness properties. Nevertheless, limited research

work along this direction has indicated that polymer nanobres are effective

for improving fracture toughness of the composites (Kim and Reneker

1999; Dzenis and Reneker 2001). Fracture toughness is one of the important

considerations in developing polymer composite dental devices such as an

orthodontic bracket (Teo et al. 2004).

8.5.9 Recycling of biopolymers

The two sides of the sustainability platform that happen to directly oppose

one another are recycling and degrading of polymer materials. As of late, the

balance of the two seems to be heavily weighing toward the biopolymers andsteering away from recycling (Laurencin et al. 1999). Despite the primary

reason for the initiative of biodegradable resins being introduced into common,

everyday uses, such as degradable bags and bottles, the other side of the

sustainability platform is being researched. The recyclability of biodegradable

resins seems to be slightly odd, as the primary reason for the use of these

materials is to dissipate to eliminate the need to recycle. As it turns out, the

products made from degradable resins do see some degree of recycling. But

as with any recycling process it is not cost effective to recycle. Recycling is

an expensive and difcult process that turns used products into new ones. Inaddition, biopolymers offer even more levels of problematic scenarios while

recycling. It is inferred, though from limited resources, that recycling these

resins can produce yield loss from contamination and incompetent critical

mass. This means that when recycled bottles are received to again be recycled,

ones that possess a degree of biodegradable material must be separated because

of the losses associated with them. They can prove to hinder the performance

of the primary material in bottles, PET. It is a suggestion to not include these

materials in applications that do typically see a lot of recycling since they can

augment to the economic burdens even in minimal amounts of contamination

(Curtis and Wilkinson, 2001).

Despite the complexity in recycling, Nature works has announced a plan

for post-consumer PLA bottles that is designed to institute a large-volume




“buy-back” system for the problematic resin. PLA can be sorted from other

plastics using standard near-infrared equipment. The ability to separate PLA

can mechanically or chemically be depolymerized into its monomer. Recycling

of PLA can produce problems for the larger scale recycling of resins like PET

and HDPE, some arbitrary independent studies have “veried” that PLA can

subsist with negligible inuence on the current recycling medium.

8.7 Future trends in medical textiles

The rst revolution began almost two decades ago with the realization that moist

wound healing principles were applicable to the treatment of chronic wounds

(Vincent Falanga, 2002). Since then, development of variety of dressings capable

of providing optimal coverage for wounds in different situations and actually

stimulating wound repair. The second revolution, still ongoing, began about

ten years ago with the successful testing of advanced technological products,

such as topically applied growth factors and bioengineered skin. Finally, the

third revolution began a few years ago with the introduction of the concept

of wound dress preparation, which allows us to break down into individual

components the critical steps involved in optimizing the clinical aspects and

the microenvironment of chronic wounds. Innovative materials are also foundin the eld of medicine and many applications are possible, ranging from

tissue engineering to wound dressings and implants (Vigo 1999). In the eld of

biomedical technology, biologists and textile engineers cooperate closely and

develop biomaterials and implants as well as methods enabling the regeneration

of tissue, for example restorable, three-dimensional, shapeable eeces in which

the cartilage cells can be grown. New opportunities for modern textiles have

also opened up in the treatment of wounds. In view of the growing number of

elderly people and diabetics in modern society, the treatment of problematic

wounds is a major application area of such textiles. Innovative medical textiles

will important role in the treatment of wounds and skin in future. The integration

of therapeutic substances turns textiles into innovative medical products.

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Index 437

C

Candida cylindracea (lipase B), 208Candida rugosa, 208

Carbohydrate polymer, 94

Carbohydrates, 33

Carbon dioxide, 268

Carbonic anhydrase, 135

Carbonization, 54

Carboxyl acid, 8

Carboxyl methyl cellulose, 321

Carboxyl, 190, 196

Carboxylase, 197

Carcinogenic, 283

Cardiovascular disorders, 312

Catalase, 36, 40

Catalytic constant, 12

Cell bundles, 88

Cell disruption, 28

Cell matrix, 94Cellobiase, 36

Celluclast 1.5 L, 80, 82

Cellulase EBT, 80, 81

Cellulase, 23, 35

Cellulomonas, 80

Cellulosic material, 37

Cellusoft L, 80

Centrifugation, 24

Cerrena unicolor, 123

Chain scission, 196

Chelating agents, 65

Chelator, 120

Chelators, 66

Chemical coagulation, 267

Chemical efuents, 23

Chemical properties, 193

Chemical resistance, 190Chitosan hydrogels, 422

Chitosan-based dressings, 404

Chlorhexidinem, 414

Chlorination, 135

Chlorine dioxide, 301

Chromatography, 24, 29, 218

Chronic wounds, 413

Chymotrypsin, 32, 204

Chymotrypsinolysis, 206

Clean technology, 251

Cleaning procedures, 304

Clinical analysis, 16

Clogging, 270

Clostridium, 80

Clothing, 189Coagulation, 272

Collagen wound dressing, 406

Collagenase, 204, 409, 413

Color spectroscopy, 99, 108

Coloration, 265

Colour removal, 271, 276

Comfort characteristics, 200

Comfort, 142

Competitive inhibition, 13Complex pollutants, 278

Compliance, 304

Composite materials, 399

Concentration, 24

Congestion, 306

Contamination, 26

Copolymer, 200, 203

Corn starch, 418Cosmetic enzymes, 300

Cotton, 53

Cotton-lined gloves, 310

Coughing, 306

Crease recovery, 95

Creep resistance, 216

Crochet, 399

Cross-linked polyester, 192

Crude enzyme, 324Crystalline cellulose, 92

Cuticle, 55

Cutinases, 322




D

Debriding agent, 410Decolorization of dye water efuent,

23, 43

Decolouration, 251, 276

Degradation, 196, 205

Degree of polymerization, 59

Degree of whiteness, 73

Degumming, 23, 42, 154

Deinking, 31

Denaturation, 72

Denimax Acid L, 80

Denimax L, 80

Dental application, 425

Depolymerisation, 69

Dermatitis, 308

Desalting, 29

Design-Expert software, 331

Desizing efciency, 74Desizing, 23, 61

Detergents, 23, 31

De-waxing, 156

Dew-retted, 120

Dextran, 204

Dextranase, 204

Dextrin, 64

Digestive, 32

Diisocyanate, 211

Dinitrosalycilic, 227

Dipole, 193

Dissolved oxygen, 265

DNA-modifying enzymes, 33

Down steam processing, 23

Drapability, 41

Drug release, 398

Dry rubbing, 85Dye detoxication, 116

Dye efuents, 251

Dye house, 276

Dye solubility, 273

Dye substantively, 256

Dye waste water, 251

Dyeability of wool, 144

Dyeing efuents, 24

Dyeing, 251

Dyestuff, 146

E

Ecologically sustainable, 289

Eco-mark, 252

Economical competitive, 320

Effect of temperature, 327

Efuent characteristics, 258

Efuent disposals, 320

Efuent treatment, 251

Ehabilitation, 399

Electro chemical process, 270

Electro dialysis, 270

Electrochemical, 261, 272Electrolytic precipitation, 269

Electro-oxidation, 252, 260

Electrophoresis, 29

Electrospinning, 407

Elongation at break, 95

Elongation, 67

Embroidered, 399

Employee training, 301

Endergonic, 6

Endogluconase, 36

Endonuclease, 33

Energy and efuents, 367

Engineering controls, 302

Environment friendly,320

Environmentally safe, 320

Enzymatic desizing, 324

Enzymatic grafting, 405Enzyme activity, 7

Enzyme allergy, 300

Enzyme assay, 324

Enzyme classication, 2



Index 439

Enzyme Commission, 2

Enzyme complex, 9

Enzyme concentration, 8

Enzyme diffusion, 140

Enzyme effect on color, 23

Enzyme exposure, 306

Enzyme handling, 300

Enzyme inhibition, 13

Enzyme kinetics, 12

Enzyme preparation, 307

Enzyme production system, 323Enzyme purication, 29

Enzyme retting, 53

Enzyme safety, 300

Enzyme structure, 2

Enzyme-handling, 299

Enzyme–substrate complex, 37

Epicuticle, 128

Equalization, 267

Eri silk 149Ester bonds, 71

Ethylene glycol, 192

Exergonic, 6

Exhaust ventilation, 303

Exoglucanase, 36

Extraction, 24

Extremozyme, 145

Eye contact, 400

F

Fabric handle, 78

Fabric water absorbency, 334

Factorial design, 280

Fashionable effects, 42

Fats, 65

Fatty acids, 60Fatty substances, 319

Feed backward propagation, 356

Feedback inhibition, 10

Fermentation, 16, 23

Ferric chloride, 267

Ferric sulphate, 267

Ferrous sulphate, 267

Fertilizers, 320

Fiber formation, 192

Fibre neness, 67

Fibrillation, 226

Fibrin, 54

Fibrinolysis, 410Fibroin, 152

Filtration, 24

Finishing of cotton knits, 23, 44

Finishing, 257

Flax ber, 112

Flax retting, 53, 116

Flax rippling, 116

Flax, 53

Fleece, 127Flexural modulus, 216

Flexural rigidity, 67, 96, 99, 103

Floatation,265

Flocculation, 29, 265, 272

Flocculent sludge, 268

Flooding, 310

Fluidity, 73

Fluorescein Isothiocyanate, 136Fluorescence spectroscopy, 367

Fluorescent dye, 138

Foam fractionation, 269

Foams, 401

Food enzymes, 300

Food ingredients, 300

Functional, 190

Fungal biotransformation, 286

Fungal culture, 98Fungal, 63

Fungi static, 398

Fusarium solani, 41




G

Gel dressing, 406Gel ltration chromatography, 218

Gelatin, 159

Gels, 401

Genetic disorders, 312

Genetic engineering, 15

Geotrichum sp., 286

Globular proteins, 1

Glucose Oxidase, 77

Glucose, 323

Glucosidic linkage, 35

Glutaminase, 409

Granular enzyme, 302

Granules, 401

Green chemistry, 252

Group specicity, 12

H

Haemostatic, 401

Hairiness, 67

Hand properties, 189

Handle 142

Hardness, 216

Harmful effects, 274

Hazard Communication, 302

Health care textiles, 398Healthcare dressings, 321

Heart valves, 400

Heat set stability, 190

Hemicellulase, 74

High absorbent, 398

High modulus, 190

Horse radish peroxidase, 278

Host organism, 31

Hyaluronidase, 409

Hydrocolloids, 401

Hydrogel, 204, 401

Hydrogen bonding, 59, 61

Hydrogen peroxide, 321

Hydrolase, 2, 40

Hydrolysate, 26

Hydrolysis, 190

Hydrophilicity, 190

Hydrophobic, 195, 218

Hydrophobicity, 212

Hydroxyhexanoate, 207

Hydroxyl, 192

Hygienic, 189Hygienic, 321, 398

Hypochlorite, 261

I

Imitation, 307

Immobilization, 161, 405

Immune system, 307

Immuno Sorbent Assay, 308Implantable, 399

Indigo pigments, 37

Induced t theory, 6

Industrial applications, 15

Industrial efuents, 273

Industrial enzyme, 25

Industrial hygienists, 299

Industrial pollution, 23

Inhalation, 306

Inhibitors, 9

Initial modulus, 96

Inoculation, 23, 27

Integrated bio-desizing and bio-

scouring, 23

Intermolecular, 215

Intracellular, 27

Iodine solution, 325Iodine test, 331

Ionic silver, 402

Isomerase, 2



Index 441

J

Jute bre, 87Jute, 53

K

Keratin, 125

Keratinisation, 133

Kinetic energy, 7

Kinetic theory, 11

L

Laccase complex, 122

Laccase production, 277

Laccase, 37, 101, 251, 319

Lactamase, 409

Leather and food industries, 23, 32

Lenses, 400

Lepidoptera, 149

Ligase, 2, 33

Light fastness, 190

Lignin degrading, 276

Lignin peroxidase, 98, 277

Lignin, 31, 93

Lignin-cellulose complex, 123

Lime, 267

Linen, 113Linkage specicity, 12

Lipase, 23, 32, 319

Lock-and-key, 5

Long-term safety, 300

Low shrinkage, 190

Lumen wall, 55

Lungs, 400

Lyase, 2, 66

Lyocell, 189

Lysine, 30

Lysozyme, 409

M

Macro-brillar, 141Macro-molecular, 196

Macromolecule, 193

Maltogenase, 405

Management systems, 300

Manganese peroxidase, 98, 277

Manganese pyrochlorophyllide, 284

Mechanical occulation, 267

Mechanical properties, 142, 193

Medical examination, 305

Medical monitoring, 300

Medical personnel , 299

Medical surveillance, 304

Medical textiles, 399

Medicinal products, 24

Membrane fouling, 260

Membrane processes, 252

Membranes, 26Mercerization, 23, 256, 261

Mesophilic proteases, 145

Metabolic, 2

Methotrexate, 13

Methyl glucuronic acid, 89

Methylamine, 195

Michaelis constant, 12

Microbes, 24

Microbial degradation, 276

Microbispora, 237

Microber, 195

Microltration, 273

Microgel, 421

Micronaire, 57

Mineral matter, 254

Mixed enzymatic system, 355

Mixed enzymes, 319Mixed inhibition, 14

Modal, 189

Modern biotechnology, 16




Saccharomyces cerevisae, 63

Safe host systems, 300

Safe processing, 320

Safety precaution, 299

Safety professionals, 299

Safety program, 300

Salt linkages, 134

Salt load, 265

Sand blasting, 40

Sanitary towels, 400

Saponication, 255Saturniidae, 149

Savinase, 141, 158

Scaffolds, 161, 408

Scouring, 23

Screening, 265, 266

Sedimentation, 265, 266

Seed culture, 323

Seed-coat, 82

Sensitization, 300Sericin, 54, 154

Serine proteases, 32, 412

Shrinkage, 95

Shrink-proof, 157

Shrink-proong, 139

Silk degumming, 53

Silk broin, 160, 237

Silk garment, 54Silk, 148

Silk, 53

Silk-like handle, 198

Silkworm, 148

Singeing, 255

Skin irritation, 306

Skin prick test, 314

Sludge digester, 268

Sludge management, 271Smart textiles, 420

Sneezing, 306

Sodium hydroxide, 67

Sodium silicate, 256

Softness, 142

Solid waste, 271

Solvent dyes, 256

Solvent extraction, 67

Sonication, 365

Sonicator efciency, 367

Soy protein, 159

Specic mixed enzyme, 95

Spill cleanup, 304Splashing, 309

Sports, 189

Stabilizers, 256

Staining methods, 70

Static electricity, 131

Steam cleaning, 309

Sterilization, 23

Stimuli-responsive, 420

Stockings, 321Stoichiometry, 207

Strength, 67, 73

Streptokinase, 409

Streptomyces, 80, 237

Substrate concentration, 6, 9

Subtilisin-PEG, 139

Surface brils, 82

Surface modication, 197Surface tension, 65

Surfactants, 27, 66

Surgical clothing’s, 321

Suspended solids, 276

Sustainable planet, 34

Sustainable process, 320

Sutures, 400

Sweeping, 309

Swelling, 58Symptoms, 306

Synthetic bre, 189



Index 445

T

Tassar silk, 149Tegawa scale, 64

Temperature, 201

Tenacity, 96

Tencel, 189

Tensile strength, 202

Tergazyme, 160

Tertiary treatment, 269

Textile auxiliaries, 23, 42

Textile industry, 276

Textile processing, 17

Textile wet processing, 320

Thennomyces lanoginosus, 63

Thermal degradation, 196

Thermal evaporation, 270

Thermal properties, 189, 195

Thermo stability, 31

Thermoactinomyces, 63, 237Thermo-elastic, 239

Thermolabile, 29

Thermonospora,80

Thermoplastic, 60

U

Ultimate cells, 88Ultra ltration, 29, 159, 274

Ultrasonic energy, 366

Ultrasonic irradiation, 415

Ultrasonic, 319

Univoltine, 149

Uricase, 409

Urokinase, 409

V

Vascular grafts, 400

Vibrational energy, 7

Viscose rayon, 189

Viscozyme 120 L, 80

Volatile compounds, 276

W

Wash down effect, 232

Wash fastness, 147

Washing, 23

W t t f t 34 282

bioprocessing of textiles (2014)

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