LIST OF CONTENTS

LIST OF ABBREVIATIONS......................................................................................... I

LIST OF TABLES......................................................................................................... Ill

LIST OF FIGURES........................................................................................................ V

Chapter: 1 Current insights on microbial xylanases 01

1.1. Introduction 02
1.2. Xylanases and related accessory enzymes 04
1.2.1. Endo-xylanases (E.C. 3.2.1.8) 04
1.2.1.1. Endo-xylanases: Classification and families 04
1.2.1.2. Endo-xylanases: Multiplicity 05
1.2.2. P-xylosidases (E.C. 3.2.1.37) 07
1.2.3. a-L-arabinofuranosidase (E.C. 3.2.1.55) 07
1.2.4. a-D-glucuronidase (E.C. 3.2.1.1) 08
1.2.5. Acetylxylan esterases (E.C. 3.1.1.6) 08
1.3. Xylanase production: Substrates and major microbial producers 08
1.3.1. Xylan - The substrate: Biochemistry, natural sources and its
importance 08
1.3.1.1. Abundance and biochemistry ofxylan 08
1.3.1.2. Pure xylan: Impact on xylanase production and alternative resources 11
1.3.2. Major microorganisms used for xylanase production 11
1.3.2.1. Bacterial xylanases 12
1.3.2.2. Fungal xylanases 13
1.4. Bioprocess techniques for xylanase production 14
1.4:1. Submergedfermentation technique 14
1.4.2. Solid state fermentation technique 15
1.5. Optimization of xylanase production: Traditional v/s Statistical
approach 16
1.6. Regulatory mechanism of xylanase biosynthesis 19
1.7. Purification strategies and characterization of microbial 20
xylanases
1.8. Xylanases - Important agents for biotechnological applications 23
1.8.1. Xylanases: Important agents in pulp and paper sectors and in
deinking studies 25
1.8.1.1. Native woodfibers to pulp: General processing 25

1.8.1.2. Choice ofxylanases for pre-bleaching and their mode ofaction on 26

pulp
1.8.1.3. Deinking of the waste papers using xylanases 27
1.8.2. Xylanases: Important agents for feed, food andjuice/drink 28
improvement
1.8.2.1. Pre-digestion ofanimal feed employing xylanases 28
I.8.2.2. Dough improvement using xylanases 29
1.8.2.3. Clarity improvements in juices and drinks using xylanases 29
1.8.3. Xylanases: Important agents in lignocellulose bioconversion to
value added products 30
1.8.3.1. Saccharification and solvent production ability ofxylanases 30
1.8.3.2. Production ofxylo-oligosaccharides using xylanases 31
1.8.4. Xylanases: Importance in miscellaneous applications 31
1.9. Future developments and concluding remarks 32
1.10. Relevance of the presented research 33
1.11. Objectives of the present study 33
1.12. References 34
Chapter: 2 Production and improvement of Bacillus altitudinis DHN8
xylanase using traditional and statistical approaches 47
2.1. Introduction 48
2.2. Materials and Methods 49
2.2.1. Processing of agro-residues, chemicals and medium components 49
2.2.2. Isolation, screening and identification ofxylanase producing strain 49
2.2.2.1. Primary screening 49
2.2.2.2. Secondary screening 50
2.2.3. Bacterial identification 50
2.2.4. Selection of agro-residues for cost-effective xylanase production 50
2.2.5. Assessment ofprocess parameters for xylanase production by
OFAT approach 51
2.2.5.1. Optimization of cultural and environmental parameters

2.2.S.2. Optimization ofnutritional parameters

2.2.6. Improvement in xylanase production by employing statistical
methodology
2.2.6.1. Box-Behnken experimental design

IO
2.2.6.2. Software, statistical analysis and data interpretation

to
2.2.7. Laboratory level scale up of Bacillus altitudinis DHN8 xylanase
production
2.2.8.

IT
Analytical methods

)
U)
2.3. Results and Discussion
2.3.1. Isolation, screening and identification ofxylanase producing
microorganisms
2.3.2. Selection of agro-residues for enhanced cellulase-free xylanase

IT)
production
2.3.3. Effect of inoculum size, inoculum age and incubation period on
xylanase production

-o
2.3.4. Effect of pH, temperature and agitation speed on xylanase

Os
production
2.3.5. Influence of various sugars and polysaccharides on xylanase
Os
synthesis
2.3.6. Influence of organic and inorganic nitrogen sources on xylanase
Os

synthesis
to

2.3.7. Improvement in xylanase production by employing Box-Behnken

Os

design
CO
Os

2.3.8. Graphical interpretation of the model

2.3.9. Validation of the experimental model
2.3.10. Laboratory level scale up of B. altitudinis DHN8 xylanase
production
C
(N

2.4. Conclusions
**
-

2.5. References
Chapter:, 3 Purificatiohfcand characterization of cellulase-free xylanase*
iSiflili

zmt'MA4
fermentation
3.1. Introduction 78

3.2. Materials and Methods 79

3.2.1. Production and extraction ofxylanase 79

3.2.2. Purification strategy ofxylanase produced by B. altitudinis DHN8 79

3.2.3. SDS-PAGE and zymogram analysis ofxylanase 80

3.2.4. Biochemical characterization ofxylanase 80

3.2.4.1. Temperature dependent activity and stability ofpartially purified and

purified xylanase 80
3.2.4.2. pH dependent activity and stability ofpartially purified and purified
xylanase 80
3.2.4.3. Effect of metal ions, modulators and organic solvents on purified
xylanase activity 81
3.2.4.4. Substrate specificity of crude and purified xylanase, and kinetic
parameters 81
3.2.5. Analytical methods 81
3.3. Results and Discussion 81
3.3.1. Purification ofxylanase produced from B. altitudinis DHN8 82
3.3.2. SDS-PAGE and zymogram analysis ofpurified xylanase 83
3.3.3. Effect of temperature on partially purified and purified xylanase
activity and stability 84
3.3.4. Effect ofpH on partially purified and purified xylanase activity and
stability 86
3.3.5. Effect of metal compounds, modulators and organic solvents on
purified xylanase activity 87
3.3.6. Substrate specificity of crude and purified xylanase, and kinetic
parameters 90
3.4. Conclusions 90
3.5. References 91
Chapter: 4 Biotechnological applications of Bacillus altitudinis DHN8
cellulase-free xylanase produced iin^^^submerge'd;

: j.-
■■Pllll
4.1. Introduction 96
4.2. Materials and Methods 97
4.2.1. Production of cellulase-free, thermo-alkali-solvent-stable xylanase 97
4.2.2. Application: 1 Potential ofxylanase in biobleaching ofpulp 97
4.2.2.1. Collection ofpulp, chemicals and media 97
4.22.2. Biobleaching ofpulp 97
42.2.3. Optimization of biobleaching conditions using cellulase-free xylanase 98
42.2.4. Chemical bleaching of untreated and xylanase pre-treated pulp 98
4.22.5. Physical analysis ofpulp and pulp filtrate analysis 99
4.2.3. Application: 2 Potential of xylanase in de-inking of waste hand­
written papers 99
42.3.1. Pulp preparation and optimization ofde-inking process 99
42.3.2. Chemical de-inking process 99
42.3.3. Pulp and filtrate analysis 100
4.2.4. Application: 3 Potential ofxylanase in pre-digestion of chick feed 100
42.4.1. Chick feed procurement and improvement in pre-digestion of chick
feed 100
4.2.42. Analytical methods 100
4.3. Results and Discussion 100
4.3.1. Potential of B. altitudinis DHN8 xylanase in biobleaching 100
4.3.1.1. Effect of incubation time on biobleaching 100
4.3.12. Effect ofpH and enzyme charge on biobleaching 101
4.3.1.3. Effect of temperature and agitation speed on biobleaching 103
4.3.1.4. Chemical bleaching of untreated and xylanase pre-treated pulp 105
4.3.1.5. Scanning electron microscopy ofcarton pulp samples 107
4.3.2. Potential of B. altitudinis DHN8 xylanase in bio-deinking 108
4.32.1. Effect of treatment period on bio-deinking of waste papers 109
4.32.2. Optimization ofpulp consistencyfor effective bio-deinking 110
4.32.3. Optimization ofenzyme charge for effective bio-deinking 110
4.32.4. Optimization of treatment temperature for effective bio-deinking 111
4.32.5. Chemical deinking of un-deinked and xylanase deinked pulp samples 112
4.3.3. Potential of B. altitudinis DHN8 xylanase in pre-digestion of chick
feed 113
4.3.3.1. Optimization of chickfeed to xylanase ratio 114
 4.33.2. Optimization ofchickfeed pre-digestion period 114
4.33.3. Optimization ofpre-digestion temperature 115
4.4. Conclusions 116
4.5. References 117
Chaptei•: 5 Improvements in upstream bioprocess parameters for - (
*

Aspergillus tubingensis FDHN1 mediated xylanase production

| through sequential optimization - 122
5.1. Introduction 123
5.2. Materials and Methods 124
5.2.1. Agro-residues preparation, chemicals and medium components 124
5.2.2. Fungal isolation, identification and inoculum preparation 124
5.2.3. Xylanase production using agro-residues and upstream process
optimization 124
5.2.4. Optimization and validation of important upstream process
variables using response surface methodology (RSM) 125
5.2.5. Software and statistical analysis 126
5.2.6. Enzyme extraction from fermented biomass 126
5.2.7. Enzyme assay and protein estimation 127
5.3. Results and Discussion 127
5.3.1. Fungal isolation and identification 127
5.3.2. Selection of agro-residues for xylanase production by A.
tubingensis FDHN1 129
5.3.3. Evaluation of upstream bioprocess parameters for xylanase
production by A. tubingensis FDHN1 through OFAT approach 130
533.1. Effect of incubation period and substrate to moisture ratio 130
53.3.2. Ejfect ofpH and temperature 132
53.3.3. Influence ofcarbon sources and its concentration optimization 134
53.3.4. Influence oforganic and inorganic nitrogen sources 135
5.33.5. Influence ofmetal additives and modulators 136
5.3.4. Optimization and validation of important upstream process
variables using Box-Behnken design (BBD) 137
5.3.5. Validation of the experimental model 144
5.4. Conclusions 144
5.5. References 145
Chapter: 6 Biochemical characterization and downstream processing of
xylanase from Aspergillus tubingensis FDHN1 produced under
solid state fermentation 149
6.1. Introduction 150
6.2. Materials and Methods 151
6.2.1. Scaled up xylanase production by A. tubingensis FDHN1 under
SSF 151
6.2.2. Partial purification ofxylanase produced by A. tubingensis FDHN1 152
6.2.3. Molecular weight determination and activity staining ofxylanase 152
6.2.4. pH and temperature dependent activity and stability ofxylanase 152
6.2.5. Metals, modulators and organic solvent dependent activity of
xylanase 153
6.2.6. Substrate specificity of crude and partially purified xylanase 153
6.2.7. Downstream processing ofA. tubingensis FDHN1 xylanase 154
6.2.7.1. Selection ofan appropriate extraction solvent for xylanase recovery 154
6.2.7.2. Primary assessment ofxylanase recovery parameters 154
6.2.7.3. Determination ofrecovery efficiency of optimized recovery conditions 154
6.2.7.4. 24 factorial design for consolidation ofxylanase recovery parameters 154
6.2.7.5. Software, interpretation and statistical analysis 155
6.2.8. Enzyme assay and protein determination 155
6.3. Results and Discussion 156
6.3.1. Scaled up xylanase production by A. tubingensis FDHN1 under
SSF 156
6.3.2. Molecular weight determination and xylan zymography ofxylanase 156
6.3.3. Effect ofpH on activity and stability of crude and partially purified
xylanase 158
6.3.4. Effect of temperature on activity and stability of crude and partially
purified xylanase 159
6.3.5. Effect of metals, modulators and solvents on partially purified
xylanase activity 161
6.3.6. Substrate specificity of crude and partially purified xylanase 163
6.3.7. Evaluation of downstream process parameters for xylanase
recovery 164
6.3.7.1. Selection ofan appropriate extraction solvent for xylanase recovery 164
6.3.7.2. Effect of extractant volume and extraction time on xylanase recovery 165
63.7.3. Effect of agitation speed and extraction temperature 166
63.7.4. Determination ofxylanase recovery efficiency (%) 167
6.3.8. Consolidation and validation ofxylanase recovery conditions 168
63.8.1. 2* factorial design for xylanase recovery improvement 168
63.8.2. Graphical interpretation ofthe model 171
63.8.3. Validation and optimization ofexperimental model 172
6.4. Conclusions 173
6.5. References 174
Chapter : 7 Potential of Aspergillus tubingensis FDHN1 xylanase in
clarification of various juices and xylitol production from 178
saccharified sugars
7.1. Introduction 179
1.2. Materials and Methods 180
7.2.1. Collection offruits and agro-residues 180
7.2.2. Production of cellulase-poor xylanase by Aspergillus tubingensis FDHN1
under SSF
181
7.2.3. Application: 1 Clarification of apple and tomato juices using cellulase-
poor xylanase
181
7.2.3.I. Preparation offruit juices 181
7.2.3.2. Optimization ofapple and tomato juice clarification 181
7.2.3.3. Determination offndt juice clarity, density and reducing sugar content 182
7.2.4. Application: 2 Xylitol production from saccharified sorghum straw
hydrolysate
182
7.2.4.1. Chemical pre-treatment ofvarious agro-residues 182
7.2.4.2. Enzymatic saccharification of various agro-residues using crude
xylanase 182
7.2A.3. Optimization of various saccharification parameters and combined
effect of crude xylanase and commercial cellulose on sugar release 183
7.2.4.4. Xylitol producing yeast culture and inoculum preparation 183
 7.2.4.5. Medium for xylitol production 183
7.2A.6. Response surface methodology involving Box-Behnken design for
xylitol production 184
7.2.4.7. Software, statistical analysis and data interpretation 185
7.2.4.8. Analytical methods 185
7.3. Results and Discussion 185
7.3.1. Clarification of apple and tomato juice using xylanase 185
7.3.1.1. Effect of enzyme dose on fruit juice clarification 185
7.3.1.2. Effect f incubation time on fruit juice clarification 187
7.3.1.3. Effect of temperature on fruit juice clarification 188
7.3.2. Production ofxylitol from saccharified sugars 190
7.3.2.1. Enzymatic saccharification of various agro-residues 190
7.3.2.2. Effect of hydrogen peroxide concentration on sorghum straw
saccharification 192
73.2.3. Effect of temperature and time on enzymatic saccharification 193
73.2.4. Effect of enzyme dose on enzymatic saccharification 194
73.2.5. Effect ofpH and modulators on enzymatic saccharification 194
73.2.6. Effect of different combinations ofxylanase and commercial cellulose 196
73.2.7. Xylitol production by response surface methodology involving Box-
Behnken design 196
73.2.8. Graphical interpretation of the model 198
73.2.9. Validation ofthe experimental model 200
7.4. Conclusions 200
7.5. References 201
Chapter: 8 Summary and Conclusions , 204
LIST OF PRESENTATIONS AND CONI'ERENCES ATTENDED Mil.
USTW WORKSHOi^AND TRAININGS ATTENDED X”214-’’
<• ’

LIST OF PUBLICATIONS AND COMMUNICATIONS 215