ABSTRACT
The production and characterization of protease and pectinase from bacteria isolated from market solid waste and the bio-scouring of cotton using the enzymes was investigated. Using morphological, cultural and biochemical properties, the isolate was confirmed as Bacillus subtilis. Protease and pectinase were produced by Bacillus subtilis using submerged fermentation in gelatin broth and pectin broth respectively. The enzymes were purified using ammonium sulphate precipitation, dialysis and ion-exchange chromatography. Glucose as a carbon source and peptone as a nitrogen source gave optimum activity for both enzymes. Purified protease exhibited maximum activity of 8.72U/ml at 40oC while pectinase exhibited maximum activity of 8.98U/ml at 50oC. Application of the enzymes on bio-scouring of cotton fabrics was tested. Pectinase was more effective than protease under optimized conditions. Optimum scouring temperature for both enzymes was between 40oC and 50oC with pectinase bio-scoured fabric showing 15.5% weight loss while protease bio-scoured fabric had 14.3% weight loss. Optimum pH for pectinase scouring was pH 9 with 14.8% weight loss in fabric while optimum pH for protease scoured fabric was pH 7 with 12.3% weight loss in fabric. After 120 minutes of bio-scouring, 15.4% weight loss was recorded for pectinase treated fabric while protease treated fabric showed 10.9% weight loss. The application of protease and pectinase for the scouring of cotton fabric revealed that they can be used as bio-scouring agents for the treatment of textile materials.
TABLE OF
CONTENTS
Title Page i
Declaration ii
Certification iii
Dedication iv
Acknowledgements v
Table of Contents vi
List of Tables x
List of Figures xi
Abstract xii
CHAPTER 1: INTRODUCTION
1.1 Background
of the Study 1
1.2 Justification 3
1.3 Main
Objective 3
1.3.1
Specific objectives 4
CHAPTER 2: LITERATURE
REVIEW 5
2.1 Enzymes 5
2.2 Protease 7
2.3 Pectinase 8
2.4 Bacillus subtilis 8
2.5 Fermentation 10
2.5.1 Submerged
fermentation 11
2.5.2 Solid
state fermentation 12
2.6 Cotton 13
2.7 Scouring
and Bio-Scouring 15
CHAPTER 3: MATERIALS AND
METHODS 17
3.1 Sample
Collection 17
3.2 Isolation
of Bacillus subtilis 17
3.2.1 Gram’s
staining 17
3.2.2
Endospore staining 18
3.2.3 Capsule
staining 18
3.2.4 Catalase
test 18
3.2.5 Oxidase
test 19
3.2.6 Indole
test 19
3.2.7 Citrate
utilization test 19
3.2.8 Starch
hydrolysis test 19
3.2.9 Nitrate
reduction test 20
3.2.10 Sugar
fermentation 20
3.2.1
Methyl
red test 20
3.2.1 2 Voges-Proskauer
test 21
3.2.13 Urease
test 21
3.2.14 Gelatinase
test 21
3.2.15 storage
of isolate 22
3.3 Screening
for Enzyme Activity 22
3.3.1 Screening
for protease activity 22
3.3.2 Screening
for pectinase activity 22
3.4 Protease
Production 22
3.5 Purification
of Crude Protease Extract 23
3.5.1.
Ammonium sulphate precipitation 23
3.5.2 Dialysis
for protease 23
3.5.3 Diethylaminoethyl (DEAE) column
chromatography for protease 23
3.6 Protease Assay 24
3.6.1 Characterization
of protease enzyme 24
3.7 Pectinase
Production 25
3.8 Purification of Pectinase 25
3.8.1 Ammonium sulphate precipitation 25
3.8.2 Dialysis for pectinase 25
3.8.3 Diethylaminoethyl (DEAE) column
chromatography for pectinase 26
3.9 Pectinase Assay 26
3.9.1 Characterization of pectinase 26
3.10 Scouring 27
3.10.1 Conventional
scouring 27
3.10.2 Bio-scouring 27
3.10.3 Optimization of different parameters for
bio-scouring 27
3.10.4 Weight loss determination 28
CHAPTER 4: RESULTS AND DISCUSSION 29
4.1 Results 29
4.1.1 Screening and
identification of protease producing organisms 29
4.1.2 Screening and
identification of pectinase producing organisms 29
4.1.3 Morphological
and biochemical properties of isolate 29
4.1.4 Protease
activity 32
4.1.5 Pectinase
activity 35
4.1.6 Optimization
of production media 38
4.1.7 Bio-scouring 43
4.2 Discussion 49
CHAPTER 5: CONCLUSION AND
RECOMMENDATIONS 51
5.1 Conclusion 51
5.2 Recommendations 51
References 53
Appendices 53
LIST
OF TABLES
PAGES
4.1: Morphological characteristics of isolate 30
4.2: Biochemical properties of isolate 31
LIST
OF FIGURES
PAGES
4.1: Effect of temperature on protease activity
33
4.2: Effect pH on protease activity 34
4.3: Effect of temperature on pectinase
activity 36
4.4: Effect of pH on pectinase activity 37
4.5: Protease activity for various carbon
sources 39
4.6: Pectinase activity for various carbon
sources 40
4.7: Protease activity for nitrogen sources 41
4.8: Pectinase activity for nitrogen sources 42
4.9: Effect of alkali, pectinase and protease
on cotton scouring 45
4.10: Effect of temperature on pectinase and
protease bio-scouring of cotton 46
4.11: Effect of time on protease and pectinase
bio-scouring of cotton 47
4.12: Effect of pH on protease and pectinase
bio-scouring of cotton 48
CHAPTER 1
INTRODUCTION
1.1 BACKGROUND OF THE STUDY
Enzymes are high molecular weight proteins
which catalyse biochemical reactions. The use of microorganisms for the production
of extracellular enzymes for industrial use is a preferable option to the use of plants and animal sources. Enzymes such
as lipase, pectinase, amylase and protease can be produced from bacteria and
fungi using different fermentation techniques such as Solid State Fermentation
and Submerged fermentation under optimized conditions (Machado et al., 2004). Proteases are enzymes
which catalyse the hydrolysis of proteins and are of different kinds such as
serine, alkaline, acidic, neutral and carboxyl proteases (Ahuja et al., 2004). They are found in all
living organisms and are produced extracellularly by bacteria and fungi (Rao et al., 1998). Pectinase is a high
molecular weight enzyme that catalyses the breakdown of pectin, a
polysaccharide found in plant tissues into smaller molecules. Protease is of
industrial importance as it is used in leather processing, food industries,
textiles and pharmaceuticals while pectinase is utilized in the food industry
and textiles. Bacillus subtilis is
one of the widely used microorganisms for the industrial production of both
pectinase and protease. They are Gram-positive, mesophilic organisms found
commonly in soil and the underneath of vegetation. Culturing and growth of Bacillus subtilis is easy, making it a
choice microbe for industrial use (Bandow, 2002). Waste products generated in markets
usually consist of both plant and animal material. Animal waste products from meat, poultry and
fish can supply a large amount of protein rich material for bioconversion to
recoverable products of importance in various industries (Dalev, 1994). These
animal waste consists of portions of an animal that cannot be sold as meat or
used in meat-products. Such waste includes tendons, bones, and the contents of
the gastro-intestinal tract, blood and organs and these vary with each type of
animal (Udandi et al., 2009). Remains
of vegetables, cassava, yam and other plant crops make up plant related market
waste. Research has been carried out on many other related sources for enzyme
production such as slaughter house waste, dairy effluent and sewage waste
(Vasantha and Subramanian, 2012). These wastes produced in market places are
inexpensive sources for bioconversion processes into products such as enzymes.
Cotton is the most valuable natural
source of cellulose and its use is constantly increasing. It is highly
hydrophobic and slightly coloured (Boylston and Herbert, 1995). To prepare
cotton fibres for further treatment and use, pre-treatment processes are needed
and these include scouring and bleaching.
In these treatments large amounts of auxiliary agents are added. Due to
the high working temperatures, a lot of energy is consumed. Large amounts of
water are used to rinse and deactivate the alkaline-scoured and bleached
fabrics. Conventional scouring of cotton entails the use of alkali (soda ash
and caustic ash) and detergents for the removal of waxes, fats, pectin, mineral
salts and other impurities from raw cotton fabrics (Mosjov, 2012). This process
effectively removes impurities but has a high demand for energy, water and
auxiliary agents. The effluent generated is also ecologically undesirable.
These chemicals also attack cellulose leading to heavy strength loss and weight
loss in the fabric. These setbacks has led to alternative processes such as
bio-scouring being considered. Bio-scouring is a novel process based on the use
of specific enzymes for the removal of impurities from textile materials
(Mangesh and Tesfaye, 2016).
Consequently, the textile industry is
considered to be one of the biggest water, energy, and chemical consumers. To
comply with the increasingly more rigorous environmental regulations and to
save water and energy, biotechnology based alternatives such as the application
of enzymes have been introduced in the the textile sector. Bio-scouring with
enzymes such as pectinase and protease is an alternative to alkali scouring in
the removal of non-cellulose substances from the cotton-fibre surface.
The process has the capacity to occur at
moderate temperatures in a slightly acidic or alkaline medium which is
dependent on the type and nature of enzyme used. The use of enzymes in the
scouring of textile materials such as cotton is gaining global recognition due
to their non-toxic nature. Enzymes have been tested in every step of wet
textile treatment starting from desizing to scouring. Microorganisms are the
best sources of industrial enzymes as their production is economically viable
with low resource consumption and low emissions (Adivarekar et al., 2007). Enzymatic treatment of textiles is of great
interest as it is effective under mild treatment conditions (Deng et al,
2010).
1.2 JUSTIFICATION
Conventional scouring of cotton fibre is
essential for the removal of impurities such as wax and fats. However, it has a
high energy demand and its effluent is non-biodegradable hence making it toxic
ecologically. It also leads to reduction in strength and loss of fabric weight.
Therefore, the use of enzymes such as protease and pectinase presents an
ecologically responsive option as they are easily biodegradable. It also leaves
the cellulose structure of the cotton fabric almost intact, preventing
excessive weight and strength loss.
1.3 MAIN OBJECTIVE
The main objective is to produce pectinase
and protease and compare their effects on bio-scouring of cotton fibres.
1.3.1 Specific objectives
v
Isolation
of Bacillus subtilis from market solid waste.
v
To
produce and purify protease enzyme.
v
To
perform the assay of protease enzyme.
v
To
characterize the protease enzyme produced.
v
To
produce and purify pectinase enzyme.
v
To
perform the assay of pectinase.
v
To
characterize the pectinase enzyme produced.
v
To
compare conventional scouring of raw cotton fabric and bio-scouring using
enzymes.
v
To
determine and compare the scouring potentials of pectinase and protease
application on cotton.
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