Universiti Sains Malaysia - DEVELOPMENT OF SOLID STATE...
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DEVELOPMENT OF SOLID STATE FERMENTATION SYSTEM FOR ENZYME PRODUCTION AND ITS USAGE IN A PILOT SCALE DEINKING OF PRINTED WASTE PAPER by LEE CREE KEONG Thesis submitted in fulfIllment of the requirements for the degree of Doctor of Philosophy DECEMBER 2010
Transcript of Universiti Sains Malaysia - DEVELOPMENT OF SOLID STATE...
DEVELOPMENT OF SOLID STATE FERMENTATION SYSTEM FOR ENZYME
PRODUCTION AND ITS USAGE IN A PILOT SCALE DEINKING OF PRINTED WASTE PAPER
by
LEE CREE KEONG
Thesis submitted in fulfIllment of the requirements for the degree
of Doctor of Philosophy
DECEMBER 2010
8504£·7 (6
~ T1) ISO; L4-T1 "Y() \0
Acknowledgements
I would like to express my greatest appreciation and gratitude to my project main
supervisor, Professor Dr. Darah Ibrahim and Co-supervisor Professor Dr. Ibrahim
Che Omar and Professor Dr. Wan RosH Wan Daud for their unlimited advice,
invaluable help, support and guidance through this PhD project. Without them this
thesis would not be possible.
Also, special thanks to School of Industrial Technology for allowing me to use the
equipment and apparatus without any problem. Also thanks to Encik Azizan, Encik
AzH, Encik Raja and Encik Abu for their helps and allow me to borrow apparatus
that I need. I also would like to express my special thanks to School of Biological
Science for allowing me to use the facilities available throughout my research study
without any problem.
Furthermore, special thanks and greatest appreciation to Ministry of Science,
Technology And Innovation, and also Ministry of Finance Malaysia for flnanced my
research work and Universiti Sains Malaysia for given me USM Fellowship.
Last but not least to my fellow lab-mates and my entire friends who have directly and
indirectly given me their support and encouragement. Finally, exceptionally thanks to
my family for their love, moral support and encouragement.
Lee Chee Keong
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TABLE OF CONTENT
PAGE
Acknowledgements ................................................................. 11
Table of Contents... .............. . .. . .. .... ... ....... ..... ... ... ... ................ 111
List of Tables ................................................................. ......... XlI
List of Figures ..................................................... ".................... xiii
List of Plates ................................................................. .......... XVlI
List of Abbreviations ............................................................... XlX
List of Symbols .................................................................. ..... XXIV
Abstrak ................................................................................ XXV
Abstract ............................................................................... xxviii
CHAPTER ONE: INTRODUCTION.......................................... 1
1.1 Paper Industries in Malaysia and the Need for Paper Recycling... 1
1.2 Economical and Industrial Advantages of Solid State
Fermentation.................................................................... 5
1.3 Benefits of Enzyme Application in Paper Recycling Industries .... 8
1.4 Research Scope and Objectives ............................................ 10
CHAPTER TWO: LITERATURE REVIEW............................. .... 13
2.1 SOLID STATE FERMENTATION ...................................... 13
2.1.1 General Considerations ............................................. 13
2.1.1.1 DefInition and History of Solid State Fermentation .. , 13
2.1.1.2 Selection of Microorganisms ............................. 18
2.1.1.3 Substrates and Particles size .............................. 21
2.1.1.4 Comparison of Characteristics Between Solid-State
Fermentation (SSF) and Submerged Fermentation
(SmF)......................................................... 25
2.1.1.5 Research Practices Versus Industrial Applications.. . 32
2.1.2 Factors That Influence Solid State Fermentation Processes 35
2.1.2.1 Temperature and Heat Transfer ......... ................. 35
2.1.2.2 pH and Risks of Contamination.......................... 38
2.1.2.3 Moisture Content and Water Activity .. ................. 40
111
2.1.2.4 Aeration and Agitation. .... .... ........ . . ..... ... ... .... .. 43
2.2 PRODUCTION OF ENZYMES VIA SOLID STATE
FERMENTATION PROCESS ............................................ 47
2.2.1 Cellulases.............................................................. 47
2.2.1.1 Basic Mode of Action .,. .................................. 48
2.2.1.2 Production of Cellulases .................................. 49
2.2.1.3 Characteristic and Properties of Cellulases ............. 52
2.2.2 Xylanase... ... ............ . .. ...... ............ ..... . ............ ...... 55
2.2.2.1 Basic Mode of Action...... ...... ......... ................ 56
2.2.2.2 Production ofXylanase ................................... 57
2.2.2.3 Characteristic and Properties ofXylanase ............. 61
2.3 SOLID STATE FERMENTATION BIOREACTORS ............... 63
2.3.1 Fundamentals and Overview ...................................... 63
2.3 .1.1 Bioreactors Selection and Design ........................ 63
2.3.1.2 Overview of Bioreactor Types ........................... 68
2.3.2 Classification and Design of SSF Bioreactors .................. 70
2.3.2.1 Unaerated and Unmixed Bioreactor ..................... 70
2.3.2.2 Force Aerated Without Mixing Bioreactor ............ 74
2.3 .2.3 Rotating Drum and Stirred Drum Bioreactor ......... 79
2.3.2.4 Intermittently Mixed Force Aerated Bioreactor ...... 82
2.3.2.5 Continuously Mixed Force Aerated Bioreactor ...... 85
2.4 APPLICATION OF ENZYMES IN PULP AND PAPER
INDUSTRIES ............................. , ..... . ..... .... . . ... ... ........ .... 89
2.4.1 Biobleaching of Kraft Pulp...... ......... ... ... .. .... . ..... ... ...... 90
2.4.2 Biopulping and Biomechanical Pulping ........................... 91
2.4.3 Pitch Control ...................... , .............................. , ..... 93
2.5 RECYCLING OF PAPER .................................................. 94
2.5.1 Recycling Process ..................................................... 94
2.5.2 Benefits of Recycled Waste Paper .................................. 96
2.5.3 Contribution of recycling to Malaysia's Economy............... 96
2.5.4 Paper Consumption and Waste Paper Generation in Malaysia 97
2.5.5 Deinking Process ................ , ............... , .... .. ........... ... 98
2.5.5.1 Deinking Principles ............ ............................ 98
IV
2.5.5.2 Chemical Deinking ........................................ 100
2.5.5.3 Enzymatic Deinking ... .............................. ...... 102
2.5.6 Quality of Recycled Papers ........................................ 105
CHAPTER THREE: DEVELOPMENT AND THE STUDY OF A
SOLID STATE FERMENTATION SYSTEM 106
3.1 INTRODUCTION ........................................................... 106
3.2 MATERIALS AND METHODS ......................................... 107
3.2.1 Development and Fabrication of Solid-State Fermenter .... 107
3.2.2 Production of Cellulases and Xylanase via SSF Process
UsingFERMSOSTAT® ............................................ 107
3.2.2.1 Microorganism .............................................. 107
3.2.2.2 Solid State Fennentation Condition ..................... 108
3.2.2.2.1 Fungal Cultivation for Spore Production
and Inoculum Preparation .................. 108
3.2.2.2.2 Substrates Preparation and Sterilization .... 109
3.2.2.2.3 Fennentation Medium Composition ....... 110
3.2.2.2.4 Inoculation Process........................... 11 0
3.2.2.2.5 Sampling Process ..................... ......... 110
3.2.2.2.6 Enzymes Extraction....................... .... 11 0
3.2.3 Determination Of Enzyme Activities ............................ 111
3.2.3.1 Determination ofCMC-ase Activity ............ ........ 111
3.2.3.2 Determination of FPase Activity ......................... 112
3.2.3.3 Determination ofXylanase Activity ................... , 112
3.2.4 Analysis ............................................................... , 113
3.2.4.1 Detennination of Fungal Growth ............ ............ 113
3.2.4.2 Determination of Moisture Content ........ ..... ........ 113
3.2.4.3 Online Monitoring of Temperature ...................... 114
3.2.5 Factors Affecting the Production of Cellulases and
Xylanase Using FERMSOSTAT® ............................... 114
3.2.5.1 Effect of Amount of Substrate ..................... ...... 114
3.2.5.2 Effect of Moisture Content ............................... 115
3.2.5.3 Effect ofIncubation Temperature ....................... 115
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3.2.5.4 Effect of Aeration Rate................................ .... 115
3.2.5.5 Effect of Aeration Time............................... .... 115
3.2.5.6 Effect of Mixing Rate ...................................... 115
3.2.5.7 Effect of Mixing Intensity .............................. ... 116
3.2.6 Comparison of Enzymes Production Using Trichoderma
reseei..................................................................... 116
3.2.7 Statistical Method ...... ................................. ...... ...... 116
3.3 RESULTS AND DISCUSSIONS .......... .............. ............ ...... 116
3.3.1 Development and Fabrication of Solid-State Fermenter .... 116
3.3.1.1 FennenterVessel........................................... 117
3.3.1.2 Sterilization System ..................... .................. 120
3.3.1.3 Mixing System ......... .................. .................. 121
3.3.1.4 Aeration System ....................................... ..... 122
3.3.1.5 Inoculation System ......................................... 124
3.3.1.6 Substrate Port and Sampling Process.................. 125
3.3.2 Comparison of FERMSOSTAT® with Other Known
Fermenters Design ................................................... 126
3.3.3 Activities of Cellulases and Xylanase ........................... 128
3.3.3.1 Cellulases Activities .. , ... ... .... ........ .... .......... .... 128
3.3.3.2 Xylanase Activity .......................................... 129
3.3.4 Factors Affecting the Production of Cellulases and
Xylanase UsingFERMSOSTAT® ............................... 129
3.3.4.1 Effect of Amount of Substrates ........................... 129
3.3.4.2 Effect of Moisture Content................................ 136
3.3.4.3 Effect of Incubation Temperature ....................... 142
3.3.4.4 Effect of Aeration Rate ........................... ......... 148
3.3.4.5 Effect of Aeration Time ................................... 153
3.3.4.6 Effect of Mixing Rate ................................. ..... 158
3.3.4.7 Effect of Mixing Intensity ............ ............... ...... 162
3.3.5 Comparison of Enzymes Production Using Trichoderma
reseei................................................ ..................... 167
3.4 CONCLUSIONS.............................................................. 173
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CHAPTER FOUR: DEVELOPMENT OF A PILOT SCALE
EI'.fZYMATIC DEINKING SYSTEM ... ......... 175
4.1 INTRODUCTION......................................................... ... 175
4.2 MATERIALS AND METHODS...... .................................... 176
4.2.1 Fabrication of an Enzymatic Deinking System... ... .. . .. . ... 176
4.2.2 Comparison of Developed Enzymatic Deinking System
with Standard Waste Paper Recycling Process............... 177
4.3 RESULTS AND DISCUSSION .......................................... 178
4.3.1 Fabrication of An Enzymatic Deinking System... .. . .... ..... 182
4.3.1.1 Components of the Enzymatic Hydrolysis System 182
4.3.1.1.1 Enzymes Storage Vessel.................... 182
4.3.1.1.2 Bioreactor ...................................... 182
4.3.1.2 Components of Flotation System .............. , .. . . ... 184
4.3.1.2.1 Flotation Vessel...... ..... ................... 184
4.3.1.2.2 Foam Scraping and Ink Reservoir Unit ... 186
4.3.1.2.3 Blower and Nozzle Unit ..................... 187
4.3.1.3 Components of Pulp Collecting Vessel ......... .... . .. 188
4.3.1.3.1 Pulp Collecting Vessel...... ...... ....... ... 188
4.3.1.4 Supporting Components ................................. 189
4.3.1.4.1 WaterlEnzyme Recycling Tank ............ 190
4.3.1.4.2 Compressor and Vacuum Pump .......... 191
4.3 .1.4.3 Diaphragm and Peristaltic Pumps ......... 192
4.3.1.4.4 Heater, pH Probe and Thermocouple '" 193
4.3.2 Operation of an Enzymatic Deinking System. ... .. .. . . . . ... ... 194
4.3.2.1 Control Panel .......... ........................ .......... .... 194
4.3.2.2 Enzymatic Hydrolysis System ... ........................ 196
4.3.2.3 Flotation System ............... ..................... ...... 197
4.3.2.4 Pulp Collecting Vessel.................................... 197
4.3.3 Comparison of Developed Enzymatic Deinking System
with Standard Waste Paper Recycling Process ............... 198
4.3.3.1 Standard Plant Concept for Newsprint and Improved
Paper Grades ....... ......................................... 198
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4.3.32 Standard Pla.nt Concept for Deinked Pulp from
Mixed Office Waste Paper ................................ 200
4.3.3.3 Comparisons with Standard Waste Paper Recycling
Process ...................................................... 202
4.4 CONCLUSIONS.............................................................. 206
CHAPTER FIVE: ENZYMATIC DEINKING USING MIXED
OFFICE WASTE PAPER AND OLD
NEWSPAPERS....................................... ... 207
5.1 INTRODUCTION........................................................ .... 207
5.2 MATERIALS AND METHODS.......... ................................ 209
5.2.1 Selection and Preparation of MOW and ONP ................ 209
5.2.2 Sources of Enzymes ................................................. 211
5.2.3 Enzymatic Deinking Process .................................... ... 211
5.2.4 Effect of PUlping Process .......................................... 213
5.2.4.1 Pulping Time ................................................ 213
5.2.4.2 Pulping Consistency.............................. ......... 213
5.2.5 Effect of Enzymatic Hydrolysis Process ........................ 213
5.2.5.1 Effect of Hydrolysis Temperature ............... ....... 214
5.2.52 Effect of pH ................................................. 214
5.2.5.3 Effect of Enzyme Concentration ................... ...... 214
5.2.5.4 Effect of Hydrolysis Time ...... ........................... 214
5.2.6 Effect of the Flotation Process .................................... 215
5.2.6.1 Effect of Flotation pH .............................. ....... 215
5.2.62 Effect of Surfactant Concentration ............... ... .... 215
5.2.6.3 Effect of Flotation Time ................................... 215
5.2.7 Evaluation of Enzymatic Deinking Process ..................... 216
5.2.7.1 Preparation of Handsheet for Physical Test of Pulp 216
5.2.7.2 Conditioning of the Handsheet .. , ... ............. ........ 220
5.2.7.3 Determination of De-inked Paper Brightness ... ... ... 220
5.2.7.4 Total Reducing Sugar Produced from Enzymatic
Hydrolysis ................................................ ... 221
5.2.8 Comparison with Chemical Deinking Method. . . ... . . . . .. . . ... 221
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5.2.9 Statistical Method ................................................... 222
5.3 RESULTS AND DISCUSSION........................................... 222
5.3.1 Selection and Preparati~n of MOW and ONP ................ 222
5.3.2 Preparation of Enzymes ............................................ 223
5.3.3 Effect of Pulping Process ........................................... 223
5.3.3.1 Pulping Consistency............ ................ ........ ... 224
5.3.3.2 Pulping Time............................................ .... 227
5.3.4 Effect of Enzymatic Hydrolysis Process ........................ 230
5.3.4.l Effect of Temperature ................................. .... 231
5.3.4.2 Effect of pH ................................................. 234
5.3.4.3 Effect of Enzyme Concentration ......................... 237
5.3.4.4 Effect oflncubation Time............................. .... 242
5.3.5 5.3.5 Effect ofthe Flotation Process... . ............... . . .. . . . .. 246
5.3.5.1 Effect of Flotation pH ..................................... 246
5.3.5.2 Effect of Surfactant Concentration ...................... 249
5.3.5.3 Effect of Flotation Time .................................. , 252
5.3.6 Comparison with Chemical Deinking Method ........ , ........ 255
5.4 CONCLUSIONS.............................................................. 258
CHAPTER SIX: COMPARATIVE PROPERTIES OF DEINKED
PAPER AND EFFLUENT
CHARACTERISTICS FROM ENZYMATIC
AND CHEMICAL DEINKING PROCESS........ 261
6.1 INTRODUCTION........................................................ .... 261
6.2 MATERIALS AND METHODS.......................................... 263
6.2.1 Evaluation of Enzymatic and Chemical Deinking Process ., 263
6.2.1.1 Preparation of Handsheet for Physical Test of Pulp .. , 263
6.2.1.2 Conditioning of the Handsheet ........................... 263
6.2.2 Determination of De-inked Paper Properties .. , ... .. . . . . ... . . . 263
6.2.2.1 Optical Properties of Paper .................... , ....... , 264
6.2.2.1.1 Brightness of Paper ........................... 264
6.2.2.1.2 Opacity of Paper .................. " ... . . . . . . . 264
6.2.2.2 Mechanical Properties of Paper .................. . . .... 265
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6.2.2.2.1 Tensile Strength .,. ............... ........... ... 265
6.2.2.2.2 Internal Tearing Resistance.................. 266
6.2.2.2.3 Bursting Strength....... ........... ........... 267
6.2.2.3 Pulp Properties... . . . ... .................. ... ...... ........ 269
6.2.2.3.1 Freeness of Pulp .............................. 269
6.2.2.3.2 Residual ink of pulp .......................... 271
6.2.3 Emuent Characteristics after Deinking Process ............... 272
6.2.3.1 Biochemical Oxygen Demand ........................... 272
6.2.3.2 Chemical Oxygen Demand ............................... 273
6.3 RESULTS AND DISCUSSION ........................................... 274
6.3.1 Evaluation of Enzymatic and Chemical Deinking Process 274
6.3.2 Optical Properties of Paper....................................... 275
6.3.2.1 Brightness of paper ........................................ 275
6.3.2.2 Opacity of paper ........................................... 278
6.3.3 Mechanical Properties of Paper .................................. 281
6.3.3.1 Tensile Strength ............................................ 281
6.3.3.2 Internal Tearing Resistance .............................. 284
6.3.3.3 Bursting Strength .......................................... 287
6.3.4 Pulp Properties ....................................................... 291
6.3.4.1 Freeness of Pulp ............................................ 291
6.3.4.2 Residual Ink of pulp ....................................... 295
6.3.5 Emuent characteristics after deinking process ................ 299
6.3.5.1 BODs and COD ............................................. 299
6.4 CONCLUSIONS.............................................................. 302
CHAPTER SEVEN: GENERAL DISCUSSION............................. 305
7.1 Production of Cellulases and Xylanase Enzyme via SSF using
PKC and Sugarcane Baggase .............................................. 305
7.2 Cellulases Synthesis and Regulation..................................... 308
7.3 Mechanism of Cellulose Hydrolysis...................................... 309
7.4 Xylanase Synthesis and Regulation...................................... 310
7.5 Mechanism ofXylan Hydrolysis .......................................... 312
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7.6 Possible Mechanisms of Enzymatic Deinking of\Vaste Paper ...... 313
7.7 Effect of Cellulases to Xylanase Ratio on Dcinking Efficiency.. .... 314
7.8 Effect of Enzymes on Mechanical Strength of Deinked Paper... ... 315
7.9 Pulp Properties after Deinking Process .................................. 316
CHAPTER EIGHT: GENERAL CONCLUSIONS AND
RECOMMENDATIONS FOR FUTURE
RESEARCH.............................................. 318
8.1 General Conclusions... ....... .. . ..... ...... .. .. .... ........ . ... ... ...... 318
8.2 Recommendations for Future Research ..... , . .... . ..... .... ... . .. 323
REFERENCES ...................................................................... 326
APPENDIXES 1-10
LIST OF PUBLICATIONS
COMPETITIONS
AWARDS
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Table 2.1
Table 2.2
Table 2.3
Table 2.4
Table 2.5
Table 2.6
Table 2.7
Table 2.8
Table 2.9
Table 3.1
Table 3.2
Table 4.1
Table 5.1
Table 5.2
Table 5.3
Table 5.4
Table 6.1
LIST OF TABLES
SSF process can be defined In terms of the following properties
Typical examples of SSF process in traditional fermentations
Major groups of microorganisms involved in SSF processes
Agro-industrial crops and residues that have potential to be used as substrates
Common substrates pre-treatment steps
Gomparison of characteristics between SSF and SmF fermentation
Comparison of advantages and disadvantages of SSF process
Hemicellulase enzymes
Properties of recycled paper
Initials Conditions used/set for the SSF process
Summary of optimum conditions for production of cellulases and xylanases enzymes by A. niger USM AI 1
Comparison of main process stages involved in both chemical and enzymatic deinking process
Initial conditions set for the enzymatic hydrolysis of pulp (MOW and ONP)
Initial conditions set for the flotation process
Summary of selected optimum enzymatic hydrolysis of MOW andONP
Summary of selected optimum enzymatic deinking of MOW andONP
Summary of pulp and paper properties after deinking process.
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PAGE 14
14
19
22
24
26
27
56
105
114
173
204
214
215
245
259
303
LIST OF FIGURES
PAGE
Figure 2.1 Basic features of various SSF bioreactors and their 69 classification based on different agitation and aeration system
Figure 2.2 Basic design features of tray bioreactor and possible design 71 variations
Figure 2.3 Basic design features of packed bed bioreactors and 74 possible design variations
Figure 2.4 Stirred Drum Bioreactor (horizontal paddle mixer) used in 79 the Wageningen University of Agriculture
Figure 2.5 Continuously Mixed Force Aerated Bioreactor 86
Figure 2.6 Washing and flotation process used in deinking process 99
Figure 3.1 Schematic diagram of FERMSOSTAT® with complete 119 fennentation system
Figure 3.2 Effect of different amount of substrate on CMCase activity 130 and production
Figure 3.3 Effect of different amount of substrate on xylanase activity 132 and production
Figure 3.4 Effect of different amount substrate on FPase activity and 134 production
Figure 3.5 Effect of different moisture content on CMCase activity l37 and production
Figure 3.6 Effect of different moisture content on xylanase activity 139 and production
Figure 3.7 Effect of different moisture content on FPase activity and 141 production
Figure 3.8 Effect of different incubation temperature on CMCase 143 activity and production
Figure 3.9 Effect of different incubation temperature on xylanase 145 activity and production
Figure 3.10 Effect of different incubation temperature on FPase 146 activity and production
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Figure 3.11 Effect of different aeration rate on CMCase activity and 149 production
Figure 3.12 Effect of different aeration rate on xylanase activity and ISO production
Figure 3.13 Effect of different aeration rate on FPase activity and 151 production
Figure 3.14 Effect of different aeration time on CMCase activity and 154 production
Figure 3.15 Effect of different aeration time on xylanase activity and 155 production
Figure 3.16 Effect of different aeration time on FPase activity and 157 production
Figure 3.17 Effect of different mixing rate on CMCase activity and 159 production
Figure 3.18 Effect of different mixing rate on xylanase activity and 159 production
Figure 3.19 Effect of different mixing rate on FPase activity and 161 production
Figure 3.20 Effect of different mixing intensity on CMCase activity 163 and production
Figure 3.21 Effect of different mixing intensity on xylanase activity 165 and production
Figure 3.22 Effect of different mixing intensity on FPase activity and 166 production
Figure 3.23 Production ofCMCase enzyme by A. niger USM AI 1 and 168 T. reesei under optimized fermentation conditions
Figure 3.24 Production of xylanase enzyme by A. niger USM AI 1 and 170 T. reesei under optimized fermentation conditions
Figure 3.25 Production of FPase enzyme by A. niger USM AI 1 and T. 171 reesei under optimized fermentation conditions
Figure 4.1 Schematic diagram of developed pilot scale of enzymatic 179 de inking system
Figure 4.2 Schematic diagram of diaphragm pump used in the 192
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deinking process.
Figure 4.3 Circulation of temperature control water around bioreactor 194 and heater (i) and Heater used in deinking process (ii).
Figure 4.4 Control panel for the operational of enzymatic de inking 195 system.
Figure 4.5 Plant concept for newsprint and improved paper grades 199
Figure 4.6 Plant concept for DIP from mixed office wastepaper 201
Figure 4.7 Overview of enzymatic deinking process used in present 203 work
Figure 5.1 Effect of different pulping consistency on ink removal 225 using MOW
Figure 5.2 Effect of different pulping consistency on ink removal 225 usingONP
Figure 5.3 Effect of different pulping time on ink removal usmg 228 MOW
Figure 5.4 Effect of different pulping time on ink removal using ONP 228
Figure 5.5 Effect of hydrolysis temperature on ink removal using 232 MOW
Figure 5.6 Effect of hydrolysis temperature on ink removal usmg 232 ONP
Figure 5.7 Effect of hydrolysis pH on ink removal using MOW 235
Figure 5.8 Effect of hydrolysis pH on ink removal using ONP 235
Figure 5.9 Effect of different enzyme concentration on ink removal 239 using MOW
Figure 5.10 Effect of different enzyme concentration on ink removal 239 usingONP
Figure 5.11 Effect of different enzymatic hydrolysis time on ink 243 removal using MOW
Figure 5.12 Effect of different enzymatic hydrolysis time on ink 243 removal using ONP
Figure 5.13 Effect of flotation pH on ink removal using MOW and 247 ONP
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Figure 5.14 Effect of Tween 80 concentration on ink removal using 250 MOW
Figure 5.15 Effect of Tween 80 concentration on ink removal using 250 ONP
Figure 5.16 Effect of flotation time on ink removal using MOW and 253 ONP
Figure 5.17 Comparison of enzymatic deinking process with chemical 256 deinking process on ink removal using MOW (A) and ONP (B)
Figure 6.1 Brightness of the enzymatic and chemical deinking 276 processes of MOW (A) and ONP (B)
Figure 6.2 Opacity of the enzymatic and chemical de inking processes 279 of MOW (A) and ONP(B)
Figure 6.3 Tensile index of the enzymatic and chemical deinking 282 processes of MOW (A) and ONP (B)
Figure 6.4 Tear index of the enzymatic and chemical deinking 285 processes of MOW (A) and ONP (B)
Figure 6.5 Burst index of the enzymatic and chemical deinking 289 processes of MOW (A) and ONP (B)
Figure 6.6 Freeness of the enzymatic and chemical deinking processes 292 of MOW (A) and ONP (B)
Figure 6.7 Residual ink after enzymatic and chemical deinking 296 processes of MOW (A) and ONP (B)
Figure 6.8 BOD and COD of the enzymatic and chemical deinking 300 processes of MOW (A) and ONP (B)
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Plate 3.1
LIST OF PLATES
Aspergillus niger USM AI 1 after 3-4 days grown on PDA agar
PAGE
108
Plate 3.2 Aspergillus niger USM AI 1 after 4-5 days grown on slant 109 PDA in medium bottle
Plate 3.3 A combination of PKC and sugarcane bagasse at 1:1 ratio 109 was used as substrate in SSF process
Plate 3.4 Developed solid state fermenter: FERMSOSTAT® 118
Plate 3.5 FERMSOSTAT® with complete fermentation system 119
Plate 3.6 Mixing system provided in FERMSOSTAT® 122
Plate 3.7 Substrate port (A) and Sampling port (B) provided m 125 FERMSOSTAT®
Plate 4.1 Overview of developed pilot scale of enzymatic de inking 180 system
Plate 4.2 Developed pilot scale of enzymatic deinking system; (i) 181 Front view, (ii) Left view and (iii) Right view
Plate 4.3 Enzyme storage vessel; Front view (i) and Top view (ii) 183
Plate 4.4 Bioreactor equipped with mixer; Front view (i) and Top 183 view (ii)
Plate 4.5 Flotation vessel; Front view (i) and Top view (ii) 185
Plate 4.6 Foam scraping unit used in flotation process 187
Plate 4.7 Blower (i), single nozzle (ii) and 12 Nozzles (iii) used in the 188 flotation system
Plate 4.8 Pulp collecting vessel; Front view (i) and Top view (ii) 189
Plate 4.9 Water/enzyme recycling tank that used in deinking process 190
Plate 4.10 Compressor (i) and Vacuum pump (ii) used in the de inking 191 process
Plate 4.11 Peristaltic pump used in the deinking process 193
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Plate 5.1 MOW used in enzymatic deinking process 210
Plate 5.2 ONP used in enzymatic deinking process 210
Plate 5.3 Sheet making machine, press and couch roll that was used to 217 prepare hand sheet
Plate 5.4 Standard disintegrator 217
Plate 5.5 Drying rings that were used to dry the wet handsheet 219
Plate 5.6 Brightness and opacity tester 221
Plate 6.1 Tensile strength tester 266
Plate 6.2 Electronic Elmendorf Tearing Tester 267
Plate 6.3 Bursting strength tester 268
Plate 6.4 Freeness tester 270
Plate 6.5 Spec scan 2000 software version V.2.3.30 271
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A. niger
APHA
Aw
BC
Bhd
BOD
BP
C/N
em
CMC
CMCase
CMFAB
Co
C02
CoCh·6H20
COD
COOH
CSF
DNA
DNS
DO
E.C.
e.g.
etc
LIST OF ABBREVIATIONS
Aspergillus niger
American Public Health Association
Water activity
Before Century
Berhad
Biological Oxygen Demand
Before Present
Carbon per nitrogen
Centimeter
Carboxymethyl cellulose
Carboxymethyl cellulase
Continuously mixed force aerated bioreactor
Corporation
Carbon dioxide
Cobaltous Chloride Hexahydrate
Chemical Oxygen Dissolve
Carboxyl group
Canadian Standard Freeness
Deoxyribonucleic acid
Dinitrosalieylic acid
Dissolved oxygen
Enzyme Commision
Example
et cetera
xix
FAS
FeS04-7H20
Fig.
FPU
g
G. sepiarium
g/cm3
gIL
g/m2
h
HC
HCI
HW
IBRL
RvIFAB
Inc
K 2Cr20 7
KCI
kDa
Kg
KH2P04
KLa
Km
kPa
kPam2/g
Ferrous ammonium sulfate
Ferrous sulfate heptahydrate
Figure
Filter Paper Activity Units
Gram
Gloeophyllum sepiarium
Gram per centimeter cubic
Gram per liter
Gram per meter square
Hour
High consistency
Hydrochloric Acid
Heavy weight
Industrial Biotechnology Research Laboratory
Intermittently mixed, forcefully aerated bioreactors
Incorporated
Potassium dichromate
Potassium chloride
Kilo Dalton
Kilogram
Potassium dihydrogen phosphate
Volumetric oxygen transfer coefficient
Michaelis-Menten constant
Kilo pascal
Kilo pascal meter square per gram
xx
Ks Affinity constant biomass/substrate
kWh Kilo watt hour
L Liter
Llheg Liter per hour per gram
LlKgem Liter Per kilogram per meter
Llmin Liter per minute
LC Low consistency
liP Lignin peroxidase
LTD Limited
LW Light weight
M Molar
m2 Meter square
m2jcm3 Meter square per centimeter cubic
m2jg Meter square per gram
m3 Meter cubic
MC Medium consistency
Met Methionine
mglml Milligram per milliliter
MgS04-7H20 Magnesium sulfate heptahydrate
mm Minute
ml Milliliter
mm Millimeter
mM Millimolar
mm2 Millimeter square
mmol Millimole
mNm2jg Milli Newton meter square per gram
xxi
MNI Malaysian Newsprint Industries
MnP Manganese-dependent peroxidase
MnS04-4H20 Mangan (II) Sulfate Tetrahydrate
MOW Mixed Office Wastepaper
N Normality
NaCI Natrium chloride
NaOH Sodium hydroxide
NfuN03 Ammonium nitrate
nm Nanometer
Nmfg Newton meter per gram
02 Oxygen
OD Optical density
ONP Old newspaper
PKC Palm kernel cake
PM Paper machine
ppm Part per million
pSI Pounds per square inch
RD Rotating drum
RH Relative humidity
RM Ringgit Malaysia
rpm Rotation per minute
RT Room Temperature
s Second
SD Stirred drum
Sdn Sendirian
xxii
SDS-PAGE
SEM
SmF
sp.
SSF
T. reesei
T80
TAPPI
TM
Tris-HeI
Jlm
Jlmollmg-min
U/mgo
U/g
UK
USA
USD
USM
UV
v/v
v/w
Vmax
w/v
w/w
Sodium dodecyl sulphate polyacrylamide gel electrophoresis
Scanning Electron Microscope
Submerged Fermentation
Species
Solid State Fermentation
Trichoderma reesei
Tween 80
Technical Association of the Pulp and Paper Industry
Trademark
Trimethylpyridine hydrochloric
Micrometer
Micromole per milligram per minute
Unit Activity per milligram
Unit Activity per gram
United Kingdom
United State of America
US Dollar
Universiti Sains Malaysia
Ultraviolet
Volume per volume
Volume per weight
Maximum velocity
Weight per volume
Weight per weight
Multiply
Zinc Sulfate heptahydrate
xxiii
LIST OF SYMBOLS f3 Beta
% Percentage
& And
P Para
°c Degree Celsius
a Alpha
± Plus minus
= Equal
+ Plus
Minus
$ Dollar
fl Specific growth rate
flmax Maximum Specific Growth Rate
> More than
< Less than
41 Doubling time
P< Probability less than
P> Probability more than
® Register
xxiv
PEMBANGUNAN SISTEM FERMENTASI KEADAAN PEPEJAL UNTUK
PENGHASILAN ENZIM nAN KEGUNAANY A DALAM SKALA PERINTIS
PENYAHD~ATANKERTASBUANGANBERCETAK
ABSTRAK
Kajian ini memberi tumpuan kepada pembangunan fermenter keadaan pepejal untuk
penghasilan enzim dan pembangunan sistem penyahdakwatan secara enzimatik
berskala rintis. Fermenter yang bam dibangunkan itu dikenali sebagai
FERMSOSTAT®. Fermentor keadaan pepejal yang bam dibangunkan itu digunakan
dalam kajian penghasilan enzim selulase dan xilanase dengan menggunakan pencilan
tempatan, Aspergillus niger DSM All. Pengoptimuman untuk penghasilan enzim
telah dijalankan dan didapati keadaan optimum penghasilan enzim adalah 0.5 kg
substrat (gabungan PKC dan hampas tebu, nisbah 1: 1); 70% (bib) kandungan
kelembapan; pengeraman pada suhu 30°C; pengudaraan pada 4.0 Lljeg substrat
pemfermentasian selama 5 minit dan pengadukan pada 0.5 psm selama 5 minit. Di
bawah keadaan optimum pemfermentasian keadaan pepejal, aktiviti CMCase,
xilanase dan FPase yang diperolehi masing-masing adalah sebanyak 62.6 U/g, 390.8
Dig dan 3.4 Dig. Ini mewakili kira-kira 9.6, 7.8 dan 3.4 kali ganda peningkatan
dalam aktiviti masing-masing selepas proses pengoptimuman berbanding dengan
sebelum proses pengoptimunan. Apabila kajian perbandingan penghasilan enzim
dijalankan dengan menggunakan Trichoderma reesei, aktiviti CMCase, xilanase dan
FPase yang diperolehi masing-masing adalah 57.3 Dig, 563.0 Dig dan 2.2 Dig.
Enzim yang dihasilkan digunakan dalam kajian penyahdakwatan campuran kertas
terpakai pejabat (CKTP) dan surat khabar lama (SKL) dengan menggunakan sistem
xxv
penyahdakwatan berenzim pada skala rintis yang telah dibangunkan. Kaj ian
pengoptimuman proses penyahdakwatan berenzim mendapati bahawa keadaan
optimum CKTP adalah pempulpaan pada konsistensi 2% selama 60 min; hidrolisis
berenzim pada suhu 50°C, pH 5.5, 0.8 U CMCase aktiviti dan 4.0 U xilanase aktiviti
bagi 1 gram pulp a kering dan hidrolisis selama 60 minit dan proses pengapungan
pada pH 8.0, 0.20% (bib) Tween 80 dan masa pengapungan selama 5 minit.
Manakala, keadaan optimum penyahdakwatan SKL yang perolehi adalah
pempulpaan pada konsistensi 3% selama 45 minit; hidrolisis berenzim pada suhu
50°C, pH 5.5, 0.4 U CMCase aktiviti dan 2.0 U xilanase aktiviti bagi 1 gram pulpa
kering dan hidrolisis selama 45 minit dan proses pengapungan pada pH 8.0, 0.55%
(bib) Tween 80 and masa pengapungan selama 20 min. Tahap keeekapan
penyahdakwatan yang diperolehi oleh CKTP dan SKL di bawah keadaan optimum
masing-masing adalah kira-kira 6.0% and 6.3%. Manakala, tahap keeekapan
penyahdakwatan seeara kimia bagi CKTP dan SKL masing-masing adalah kira-kira
2.9% dan 3.5%. Kedua-dua proses penyahdakwatan berenzim dan kimia bukan
sahaja mempengaruhi dengan ketara eiri optikal dan mekanikal kertas terpakai yang
dikaji tetapi juga eiri pulpa dan air buangan yang dihasilkan. Penyahdakwatan
berenzim CKTP menunjukkan peningkatan dalam keeerahan (4.7 unit), indeks tensil
(14.1%), indeks peeah (3.4%), kebebasan (2.0%) dan penyingkiran sisa dakwat
(44.5%) tetapi penurunan dalam kelegapan (2.6%) dan indeks koyak (9.6%).
Manakala, penyahdakwatan berenzim SKL menunjukkan peningkatan dalam
keeerahan (2.5 unit), indeks tensil (10.2%), indeks peeah (3.8%), kebebasan (2.9%)
dan penyingkiran sisa dakwat (51.15%) tetapi penurunan dalam kelegapan (0.4%)
dan indeks koyak (3.9%). Berbeza dengan penyahdakwatan berenzim,
penyahdakwatan CKTP seeara kimia menunjukkan peningkatan dalam keeerahan
XXVI
(2.3 units), indeks tensil (1.1%), indeks pecah (1.2%), kebebasan (1.9%) dan
penyingkiran sisa dakwat (3 L 1 %) tetapi penurunan dalam kelegapan (1.4%) dan
indeks koyak (1.9%). Manakala, penyahdakwatan SKL secara kimia menunjukkan
bedakunya peningkatan kecerahan (1.4 unit), indeks tensil (6.8%), indeks pecah
(3.0%), kebebasan (2.3%) dan penyingkiran sisa dakwat (49.8%) tetapi penurunan
dalam kelegapan (0.1%) dan indeks koyak (1.1%). Nilai kepeduan oksigen kimia
(COD) yang diperolehi daripada process penyahdakwatan berenzim ke atas CKTP
dan SKL masing-masing adalah 33.9% and 33.8% lebih rendah berbanding dengan
proses penyahdakwatan secara kimia. Manakala, nilai keperluan oksigen biokimia
(BOD5) yang diperolehi daripada process penyahdakwatan berenzim ke atas CKTP
dan SKL masing-masing adalah 47.1 % and 39.3% lebih rendah berbanding dengan
proses penyahdakwatan secara kimia. Ini secara langsung akan mengurangkan kos
rawatan air buangan. Tambahan lagi, berdasarkan Akta Persekitaran Malaysia 1974,
rawatan tidak perlu dijalankan ke atas air buangan daripada penyahdakwatan CKTP
kerana air buangan tersebut berada di bawah paras yang dibenarkan untuk dibuang ke
persekitaran. Hasil yang diperolehi daripada kajian ini mencadangkan bahawa
penyahdakwatan berenzim boleh menjadi satu alternatif yang berpotensi tinggi
kepada kaedah kimia, yang menunjukkan beberapa masalah dan keburukan
berbanding dengan penyahdakwatan berenzim dalam pengitaran semula kertas
buangan.
xxvii
DEVELOPMENT OF SOLID STATE FERMENTATION SYSTEM FOR
ENZYME PRODUCTION AND ITS USAGE IN A PILOT SCALE DEINKING
OF PRINTED WASTE PAPER
ABSTRACT
The present work deals with the development of solid state fermenter for enzymes
production and the development of a pilot scale of enzymatic deinking system. The
newly developed solid state fermenter, was named FERMSOSTAT®, which was
used for the production of cellulases and xylanase using a local isolate; Aspergillus
niger USM AI 1. Optimization of enzymes production was carried out and the
optimum conditions obtained were 0.5 kg substrate (a combination of PKC and
sugarcane baggase at 1:1 ratio); 70% (w/w) moisture content; incubated at 30°C;
aeration at 4.0 L/heg fermented substrate for 5 min and mixing at 0.5 rpm for 5 min.
Under the optimum SSF conditions, the CMCase, xylanase and FPase activities
obtained were 62.6 U/g, 390.8 U/g and 3.4 U/g, respectively. This represents about
9.6, 7.8 and 3.4 folds increased in their respective enzymes activities after the
optimization process. When comparative study on the enzymes production was
performed using Trichoderma reesei, the CMCase, xylanase and FPase activities
obtained were about 57.3 U/g, 563.0 U/g and 2.2 U/g, respectively. The enzymes
produced were used in the deinking studies of mixed office waste paper (MOW) and
old newspaper (ONP) using newly developed pilot scale enzymatic deinking system.
Optimization of enzymatic deinking process was carried out and the optimum
conditions obtained for MOW were pulping at 2% consistency for 60 min; enzymatic
hydrolysis at 50°C, pH 5.5, 0.8 U of CMCase and 4.0 U of xylanase per gram of air
XXVlll
dry weight of pulp and 60 min of hydrolysis time and flotation process at pH 8.0,
0.20% (w/w) of Tween 80 and 5 min of flotation time. Meanwhile, the optimum
conditions obtained for the de inking of ONP were pulping at 3% consistency for 45
min; enzymatic hydrolysis at 50°C, pH 5.5, 0.4 U ofCMCase and 2.0 U ofxylanase
per gram of air dry weight of pulp and 45 min of· hydrolysis time and flotation
process at pH 8.0, 0.55% (w/w) of Tween 80 and 20 min of flotation time. The
deinking efficiency obtained for MOW and ONP under their respective optimum
conditions were about 6.0% and 6.3%, respectively. The deinking efficiency detected
by chemical deinking of MOW and ONP were about 2.9% and 3.5%, respectively.
Both enzymatic and chemical deinking processes not only significantly influence
optical and mechanical properties of the deinked paper but also the pulp properties
and the effluent generated. Enzymatic deinking of MOW showed improvement in
brightness (4.7 units), tensile index (14.1%), burst index (3.4%), freeness (2.0%) and
residual ink removal (44.5%) but decreased in opacity (2.6%) and tear index (9.6%).
Meanwhile, enzymatic deinlcing of ONP showed improvement in brightness (2.5
units), tensile index (10.2%), burst index (3.8%), freeness (2.9%) and residual ink
removal (51.15%) but decreased in opacity (0.4%) and tear index (3.9%). Unlike
enzymatic deinking, chemical deinking of MOW demonstrated increased in the
brightness (2.3 units), tensile index (1.1 %), burst index (1.2%), freeness (1.9%) and
residual ink removal (31.1%) but loss in opacity (1.4%) and tear index (0.1%).
Meanwhile, chemical de inking of ONP showed enhanced in brightness (1.4 units),
tensile index (6.8%), burst index (3.0%), freeness (2.3%) and residual ink removal
(49.8%) but decreased in opacity (0.1%) and tear index (1.1%). Biological oxygen
demand (BODs) and chemical oxygen demand (COD) analysis indicated that,
effluent produced from the enzymatic deinking process was lower compared to
XXIX
chemical deinking process. About 33.9% and 33.8% lower in COD values were
obtained after enzymatic deinking ofONP and MOW compared to chemical deinking
process, respectively. Meanwhile, BODs obtained from enzymatic deinking of MOW
and ONP were 47.1% and 39.3% lower compared to chemical deinking process,
respectively. This directly will reduce the wastewater treatment cost. In addition,
based on the Malaysia Environmental Act 1974; no wastewater treatment was
required on the effluent obtained from deinking of MOW, where the effluents
obtained were within the pennitted value for safety discharged into the environment.
The results obtained from this work suggested that the enzymatic deinking can be a
highly potential alternative to the chemical method, which showed several problems
and disadvantages compared to enzymatic deinking in the recycling of waste paper.
xxx
CHAPTER O~~: INTRODUCTION
1.1 Paper Industries in Malaysia and the Need for Paper Recycling
The paper and paper products industries are essentially part of the larger wood-based
products sector. Meanwhile, the paper making industry has a close correlation to the
packaging and writing industry. The paper industry continues to grow even though
the current drive in many nations is to go "paperless" including Malaysia. Indeed,
according to Food and Agriculture Organization, the global demand for paper would
increase by about 3.1 % annually and it is predicted to reach about 440 million metric
tons by the year 2010 (Daily Express newspaper, 2005). Therefore, the Malaysian
government has identified this industry as one of the priority areas for investment in
the second Industrial Master Plan (Center for International Forest Research, 2006).
The strategy is to achieve self sufficiency, to reduce import and encourage foreign
capital. With that, the government had given duty-free for importation of industrial
paper machines and also the pulp and waste papers recovered.
Malaysia has extensive forest resources with forest covering close to 60% of its land
area, but remained to be a major importer of paper. The country's pulp and paper
industry is not as well developed as those in other ·countries. It is crowded and highly
fragmented, which made up more than 300 companies with more than 5000 workers.
Meanwhile, the capital investment in pulp and paper industry is more than RM 250
millions (Ministry of International Trade and Industry, 2005). Basically the industry
included pulp/paper mills, paper converter and producers of paper
containers/corrugated carton boxes. Out of 300 companies, less than 30% are pulp
and paper mills. The paper mills in Malaysia are small by the standard of the industry
in the developed countries. Only 6 out of 20 mills producing more than 100, 000
1
metric tones and up to 300, 000 metric tones per annum of papers. Unfortunately,
none of these companies reach the critical level of 500, 000 metric tons per year,
which is often seen as the standard for a minimum competitive level in tenns of
world competition (Center for International Forest Research, 2006). Local production
has a total capacity of more than 1.3 million metric tones per annum in the year 2005.
However, it was not able to meet the total consumption of 115 kg per capital or more
than 2.2 million metric tones per annum in year 2005 (World Resources Institute,
2005).
Malaysia needs to import pulp, newsprint, printing/writing papers and industrial
papers. According to Department of Statistics (2003), Malaysia spent more than RM
3.0 billion importing 1.40 million metric tones of all types of paper. Out ofRM 3.0
billion, more than RM I billion was spent on importing printing/writing paper. The
import is inevitable, since local consumption is more than 380, 000 metric tons per
year (The Star Newspaper, 2009). Meanwhile there is only one mill in Malaysia
(Sabah Forest) produces printing/writing paper with the capacity of 165,000 metric
tones per year. On the other hand, Malaysia produces 280,000 metric tons of
newsprint per annum with almost 99% of it are produced by Malaysian Newsprint
Industries Sdn Bhd. However, that did not meet the local demand of about 320,000
metric tons of newsprint per annum (Malaysian Newsprint Industries Sdn Bhd,
2007).
The pulp and paper industry in Malaysia is heavily dependant on imported fiber
particularly virgin pulp due to the lack of domestically sourced of fresh fibers. In
addition, the industry is also facing the need to fmd new sources of fiber to
2
strengthen and retain the quality of secondary fibers as the use of recycled paper is
growing in Malaysia (Center for International Forest Research, 2006). This was due
to the fact that waste paper constitutes 95% of raw materials in Malaysian paper
mills. Moreover, for the year 2002, import of pulp and paper was 7 times greater than
its export (Department of Statistics, 2003). Therefore, there is a need to increase the
waste paper recovery rates in Malaysia, which only hovers around 60% (Malaysian
Newsprint Industries Sdn Bhd, 2007). However, these present a challenge for pulp
and paper mill with current chemical deinking process and technologies, which is
less effective in de inking of mixed office waste paper (MOW). MOW is a fast
growing source of materials for recycling and it is the most difficult raw material to
be de-inked (Prasad, 1993; Gubitz et ai., 1998a). Nevertheless, it has to be borne in
mind that MOW is a large, virtually untapped source of high quality fiber that could
be used for high quality grade papers and many other products only if the current
deinking process could be improved.
A major difficulty in dealing with secondary fiber is the removal of ink through the
process of deinking. The degree of difficulty in ink removal depends primarily on the
ink types, printing process and fiber type. Currently, Malaysian paper mills used
chemical approach to de-ink the waste paper, which generally is considered to be
more efficient with respect to the ink detachment. However, it requires the use of
large amount of chemical agent and not environmental friendly (prasad et ai., 1993;
Woodword et ai., 1994). In addition, deinking in an alkaline environment induces a
smeared pulp or low brightness. Furthermore, this chemical method resulted in a
significant increase in the level and concentration of COD in the effluent water
caused by the dissolution of carbohydrates and: organic additives present in the
3
fibrous materials. Besides that, the efr1uent will also contain high impurities of
chemicals used in the deinking process. Ultimately, this results in a highly
environmentally damaging and costly wastewater treatment to meet the
environmental regulations (prasad et ai., 1992).
Due to significant number of disadvantages resulted from the conventional chemical
method; an alternative method for deinking process must be sought. Alternatively,
the use of enzymes has been reported to be a potentially efficient solution to
overcome the problem encountered by commonly employed deinking techniques
(prasad et ai., 1992; Putz et ai., 1994). The potential of enzymatic deinking has been
assessed and proven successful using a number of different types of enzymes (Gubitz
et ai., 1998a). For example, the removal of oil carrier-based inks can be facilitated by
the addition of lipases and esterases. Meanwhile, hemicellulases and cellulases can
be used to release ink from fiber surface by partially hydrolysis of carbohydrate
molecules on the fiber surface.
To date, few studies on the effect of enzymes on deinking of MOW and ONP have
been carried out using enzyme preparations containing mixed activities including
both cellulases and hemicellulases (prasad et ai., 1993; Jeffries et al., 1994; Pala et
ai., 2004). However, none of these studies has been performed in Malaysia.
Therefore, the need to explore the possibility of using enzymes in the deinking of
MOW and ONP with the aim to recycle the waste paper must be given priority as an
environmental friendly approach in the Malaysia paper industries.
4
1.2 Economical and Industrial Advantages of Solid State Fermentation
Solid state Fennentation (SSF) has gained importance in recent years III
biotechnology industries due to its applications in the production of biologically
active secondary metabolites. There are several industrially and economically
important advantages, in which SSF can offers over submerged fermentation
(SmF). The fennenters used in SSF are simple in design, constrict and small reactor
and do not have much automation in the control system. In SSF, slow mixing is
performed with no foam generation, in which nonnally found in SmF by using
antifoam sensor. Additional benefit includes substrate volume used is higher due to
low moisture content and less water is used during the fennentation. Therefore,
smaller volume of bioreactor is required and smaller working space is needed, which
led to lower capital investment (pandey et al., 2008b). On the other hand, the
substrates use in SSF are generally agro-industrial residues with minimal or no
pretreatment and thus is relatively simple, uses less energy than SmF (Wang and
Yang, 2007). Besides that, the substrate itself contains most of the necessary nutrient
required by the microbial to grow and this makes the fennentation media simpler
(Cannel and Moo-Young, 1980a; Steinkraus, 1984; Kumar and Lonsane, 1990;
Raimbault, 1998; Perez-Guerra"et al., 2003).
The products produced by SSF process achieved higher yields, relatively high
concentration and higher product titers than those obtained in SmF. This is because
small volumes of solvent or water are required to extract the desired product if
necessary. Less water is needed in up-stream processing resulting in lesser
wastewater generation in the down stream processing and lower recovery cost, which
fonn some of the advantages point of SSF processes. Additional benefits include
5
easy and simple disposal or treatment of the fermented residue due to the low water
content of the fermented residue. Furthermore~ the fermented residue can be dried
and stored at room temperature without any significant loss in the activity. On
the other hand, the fermented substrates can be used directly as fertilizer or animal
feed (Kumar and Lonsane, 1990; Perez-Guerra et al., 2003) without the need to
isolate the product. Another example is the production of glucoamylse using wheat
bran mixed with corn flour. After fermentation the fermented substrates can be used
directly for the hydrolysis of cassava flour to produce fermentable sugars (Wang and
Yang, 2007).
Cost is another factor that makes SSF favorable over SmF process. About 40% of the
industrial enzymes are produced using SmF. However, compared to SmF, SSF shows
lower cost and high productivity. It has been reported that the production cost of
crude cellulases in SmF is approximately USD20Ikg, which was about 100 fold more
expensive compared to SSF (Tengerdy, 1996; Pandey et al., 2000a, 2008a). The low
production cost by SSF method may due to low cost of the agro-industrial residues,
which is used as substrates, minimum sterility and low equipment requirement. On
the other hand, SSF can be an economical alternative for phytase enzyme production
due to high enzyme concentration and activities. Furthermore, the fermented
substrates containing phytase can mixed with animal feed as a value added
supplement. Thus, reduces the animal feed cost (Bogar et al., 2003)
SSF possesses a unique characteristics which normally is not done in SmF is the
employment of mixed microbial cultures. It has been reported that, mixed culture can
be used in production of broad range of enzymes (Koroleva et al., 2002; Stepanova et
6
al., 2003; Yang et al., 2004) with different productivity (Gutierrez-Correa et al.,
1999; Massadeh et al., 2001). Meanwhile. mixed cultures used in food industry are
important for food flavour production. For example, the production of various aroma
active components was reported by Nout and Aidoo (2002). Whereas, 70 volatile
compounds can be produced when balnboo sprouts was processed by undefined
mixed cultures (Fu et al., 2002).
Global demand for citric acid is large and still growing all over the world. It is one of
the world's largest fermentation products with an estimated annual production of I
million tons (Soccol et al., 2004). At present, it is mainly produced using the SmF.
However, the production by SSF using low cost agro-industrial residues has attracted
attention. The substrates that can be used include bagasse (prado et al., 2004), apple
pomace (Shojaosadati and Babaeipour, 2002) coffee husk, fruits waste (Kumar et al.,
2003a), sugar cane bagasse (Kumar et al., 2003b) and cassava bagasse
(Vandenberghe et al., 2000). Higher productivity of lactic acid from SSF was
obtained compared to SmF (Soccol et al., 1994a, 1994b). In addition, the production
of lactic acid using SSF was able to overcome some problems faced in SmF such as
difficulty in mixing and aeration using the fungal fermenta~ion. This also indicates
that SSF is highly potential for economic production of lactic acid.
Biological control agent such as fungal spores can be produced by both SSF and
SmF. However, spores production is the only process where SSF dominates over
SmF in all aspects such as better yield, abundant, robust, morphology, healthy and
high stability (Holker et al., 2004; Ramachandran et al., 2007; Wang and Yang,
2007). This may due to the SSF conditions used are close to natural habitat of the
7
fungi. Spore of Coniothyrium minitans can be used as biopesticides against the plant
pathogen Sclerotinia sclerotiorum. Meanwhile, C. minitans spores produced by SSF
are of better quality, with greater resistance to UV-irradiation and desiccation during
recovery procedures and survive longer after storage (McQuilken and Whipps, 1995;
McQuilken et al., 1997; Oostra et al., 2000; Vrije et oz., 2001; Wang and Yang,
2007).
In conclusions, for the past few years, development in SSF technology has been
significant and it is on the way for economical commercialization. It has also bcen
found economically viable for various processes such as secondary metabolites,
enzymes, biotechnology and pharmaceutical products.
1.3 Benefits of Enzyme Application in Paper Recycling Industries
The use of enzyme in de inking of various type of waste paper has been assessed and
reported successful using various type of enzymes and mixture of enzymes
preparations (Gubitz et al., 1998a). Enzymatic deinking has been described to be able
to deink high quality waste paper such as MOW, in which its reuse is usually limited
by the high content of toners (Jeffries et al., 1994). MOW is the most difficult raw
materials to be deinked by conventional deinking process. This is because the toners
contain thermoplastic binders, which polymerize and fuse onto the paper fibers
during the high temperature printing process. They will remain as flat, large and
rigid particles and when treated with chemical are poorly separated from the fiber
during flotation process (Quick and Hodgson, 1986; Shrinath et ai., 1991; Jeffries et
al., 1994, 1995; Viesturs et ai., 1999). Longer mechanical action will degrade the
8
fiber but not efficient to break down the toner particles to size which is favorable to
be removed by flotation process (Shrinath et al., 1991).
Conventional chemical de inking process is carried out under alkaline environment,
which is generally considered more efficiency. However, it requires the use of large
amounts of chemical agents such as sodium hydroxide, sodium carbonate, sodium
silicate, hydrogen peroxide and surfactants (prasad et al., 1993; Woodword et al.,
1994). This resulted in significant increase in the level and concentration of COD in
the effluent water. Alternatively, the use of enzymes has been reported to be a
potentially less polluting solution to overcome the problem encountered by chemical
deinking techniques (Prasad et al., 1992; Putz et al., 1994). Furthermore, enzymatic
deinking avoids the alkaline environment commonly requires in the traditional
deinking which cause yellowing of the recycled pulp. In addition enzymatic deinking
reduces chemical usage means lower waste treatment costs and less impact to the
environment (putz et al., 1994). Besides the ink removal, during enzymatic deinking
cellulases acts directly on fines and microfibrils protruding out from the surfaces.
This action would remove fmes content and improved the interfibrillar bonding,
which may result in better paper strength properties (Jeffries et al., 1994; Gubitz et
al., 1998b; Lee and Eom, 1999). In addition, enzymatic deinking enhances the
brightness and cleanliness of the pulp. Additional benefits include the improved
operation of thickeners due to better drainage. Improved drainage may results in
faster machine speed which yields significant savings in energy and thus the overall
cost (Heise et al., 1996).
9
The use of bleach chemicals is usually lower for enzymatic deinking than for
conventional chemical deinking. The reduction of chemical use would result in lower
waste treatment costs while minimizes the impact on the environment. Lower
bleaching costs and less pollution can also be achieved, since enzymatic deinked
pulps have been proved to be easier to bleach which consequently require less
bleaching chemicals than the pulps de inked by chemical methods (pratima and
Pramod, 1998).
In addition, successful deVelopment of enzymatic de inking may directly increase the
recycling of waste paper, which indirectly may increase the economic of paper
recycling industry. This is because cheaper operational cost of enzymatic de inking
compared to current chemical deinking process and less pollution. Thus, this will
encourage more capital investment in the industry and more working opportunity for
the local residents. In addition, it may also help in the enzymes industry by
increasing the enzyme production at a cheaper cost via solid state fermentation
process using agro-industrial residual as valuable substrates. Furthermore, the use of
recycled fiber reduces the need for virgin pulp. This may result in great savings on
the water and energy required for pulping, bleaching and refining, which would also
eventually reduce pollution problems (Pratima and Pramod, 1998). Additional
benefits include reduction in the amount of waste paper into landfill which will
reduce associated disposal cost.
1.4 Research Scope and Objectives
In this study, solid state fermenter, which named FERMSOSTAT® was designed and
developed for laboratory use. It was fabricated by a local fabricator based on the
10
findings obtained by Pang et al., (2005) using tray SSF system. The developed solid
state fem1enter was able to monitor and control the parameters that may affect the
solid state fermentation process. FERMSOSTAT® was used for the production of
cellulases and xylanase enzymes using Aspergillus niger USM AI 1 (pang et al.,
2005) grown on palm kernel cake a.~d sugarcane baggase as substrates. Parameters
that affect the enzymes production were optimized in order to increase the enzymes
productivities. The parameters that were optimized included; amount of substrates,
moisture content, incubation temperature, aeration rate and aeration time, mixing rate
and mixing intensity. The production of cellulases and xylanase by A. niger USM AI
1 was compared using T reesei grown on the same substrates under optimized
conditions. The enzymes produced by A. niger USM AI 1 were used in the deinking
of mixed office waste paper and old newspaper.
A pilot scale of enzymatic deinking system was designed and developed. It was
fabricated based on the data obtained by Lee et a1., (2007) using laboratory scale
enzymatic deinking system. The strategy for the development of the deinking system
was determined. The enzymatic deinking system developed was used throughout this
study for de inking of MOW and ~NP. Parameters that affected the deinking process
include pulping process, enzymatic hydrolysis and flotation process. Optimization of
pUlping process was carried out involving the pulping consistency and pulping time.
The optimization of enzymatic hydrolysis of MOW and ONP were performed in
order to determine the most effective conditions for ink removal. The conditions that
were optimized include the temperature, pH, enzymes concentration and hydrolysis
time. The efficiency of the deinking process was greatly influenced by flotation
process. Therefore, the parameters that were involved in flotation process were
11
optimized in order to improve the deinking efficiency and brightness. The parameters
that were optimized in the flotation process include the flotation pH, surfactant
concentration and flotation time. Conventional chemical de inking process was
carried out in order to compare the effectiveness of enzymatic deinking of MOW and
ONP.
After optimization of enzymatic de inking process, the quality of the de-inked paper
was determined and compared with chemical deinking process using the standard
T APPI tests methods. The properties examined include not only the optical
properties of the paper such as brightness and opacity but also mechanical properties
of the paper like burst strength, tensile strength and tear resistance of paper.
Furthermore, pulp property such as pulp freeness was also determined. In addition,
the residual ink remaining on the deinked paper after deinking process was examined . using Spec Scan 2000 software. The wastewater effluent generated from both
enzymatic and chemical deinking processes were examined by determining the
BODs and COD.
The objectives of the current research are as follows:
.:. To design and develop a laboratory scale solid state fermenter .
• :. To optimize cellulases and xylanase production via solid state fermentation
process using FERMSOSTAT® .
• :. To design and develop a pilot scale enzymatic deinking system and optimize
the deinking process .
• :. To compare the efficiency of enzymatic deinking process and quality of the
deinked paper with chemical deinking process .
• :. To analyze the wastewater effluent generated from the deinking process.
12
CHAPTER TWO: LITERATURE REVIE\V
2.1 SOLID STATE FERMENTATION
2.1.1 General Considerations
2.1.1.1 Definition and History of Solid State Fermentation
Solid state fermentation (SSF) involves the growth of microorganisms (mainly fungi)
on moist solid substrate in the absence or near absence of free flowing water
(Stredansky and Cont~ 1999; Adinarayana et ai., 2003; Kumar et ai., 2003b;
Kashyap et aI., 2003; Pandey et ai., 2008b). In a wider definition, SSF can be seen as
including processes during which microorganisms are cultivated in the presence of a
liquid phase at maximum substrate concentrations (Mitchell et ai., 2000a) or on inert
support (Ooijkaas et ai., 2000). On the other hand, SSF process can also be defmed
based on its properties as stated in Table 2.1. The basic principle of SSF is the "solid
substrate bed". This bed contains moist substrates, which are polymeric in nature and
insoluble in water act as a source of carbon, nitrogen, minerals, water and other
nutrients to support the growth and metabolism of microorganism. SSF has been
conventionally more applicable for filamentous fungi that grow on the surface of the
solid substrates particle and penetrate through the inter particle spaces into the depth
of the solid bed (Pandey, 2003). In addition, SSF simulates the fermentation reactions
occurring close to the natural environment in which microorganisms are adapted and
has been credited to be responsible for the beginning of the fermentation technique in
ancient time (Pandey et ai., 2000a). Therefore, it is not surprising that almost all the
fermentation processes used in ancient time were based on the principles of SSF.
The history and development of SSF have been reviewed by several authors from
time to time. Enzymes production and food fermentation are the areas where SSF
13
originated. Typical examples of SSF processes in traditional fennentations are
summarized 1..'1. Table 2.2. SSF is the state of art technology that is used in many
applications in the food industry in Asia (Udo and Jurgen, 2005).
Table 2.1: SSF process can be defmed in tenns of the following properties (Raimbault, 1998)
No Condition 1 Solid porous matrix either can be biodegradable or not but with a large surface area
per unit volume (103 to 106 m2 fcm3) for ready microbial growth.
2 The matrix should possess water absorption capacity one or several times its dry weight with a relatively high water activity on the solid/gas interface in order to allow high rates of biochemical processes.
3 The support/carrier should be able to absorb or contain available nutrients/foodstuffs to support the growth of microbial such as water, carbohydrates, nitrogen sources and mineral salts.
4 The solid support should not be contaminated by inhibitors of microbial activities.
5 The solid matrix should be able to stand compression or gentle stirring as required for a given fermentation process. This requires small granular or fibrous particles that do not tend to break or stick to each other.
6 Air mixture of oxygen with other gases and aerosols should flow under a relatively low pressure and mix the fermenting mash.
7 The solid/gas interface should be a good environment for the fast development of specific cultures of fungus, moulds, yeasts or bacteria, either in single or mixed cultures.
Table 2.2: Typical examples of SSF process in traditional fennentations (Raimbault, 1998; Udo and Jurgen, 2005; Pandey, et al., 2008b)
No SSF Fermentation 1 "Blue cheese" making in French, which uses perforated fresh cheese as substrate and
Penicillium roquefortii as inoculum.
2 Enzyme rich soy sauce "koji" which uses steamed rice as solid substrate and inoculated with Aspergillus oryzae as an enzymatic starter for different hydrolytic processes.
3 Indonesian "tempeh" or Indian "ragi" which use steamed and cracked legume seeds as solid substrate and a variety of non toxic moulds such as Rhizopus sp. as microbial seed.
4 Composting of lignocellulosic fibers, naturally contaminated by a large variety of organisms including cellulolytic bacteria, moulds and Streptomyces sp.
5 Saccharification of rice used for the production of alcoholic beverages such as "sake".
6 Production of "angkak", which is rice that is colored red by Monascus purpurea metabolites.
14
For example SSF had been used in cheese making by Penicillium rouquefortii before
the birth of Christ. In 2000 BC, SSF process was used to make bread as were
reported by Egyptians. SSF is used in the production of enzyme rich soy sauce Koji
in China was reported in the years 1000 BP. In 17th century, the koji process was
migrated from China to Japan by the Buddhist priests. During this period, several
fermented food such as "tempeh" or Indian "ragi", miso were mentioned in many
South East Asian countries. During the 18th century, SSF was used for the
production of vinegar from the apple pomace. While in late 19th century, SSF
processes were used in compo sting and solid waste treatment. For the first time the
production of primary metabolites such as enzymes and organic acids using SSF
were reported in the beginning of 20th century (Raimbault, 1998; Udo and Jurgen,
2005; Pandey et a1., 2008b).
SSF processes were nearly completely ignored in western countries in the period of
1940's due to dramatic discovery and development of penicillin using submerged
fermentation (SmF) technology. Since the development of penicillin took place in
SmF and due to enormous importance of penicillin during the world war, SmF
became a role model technology for the production of any compound. Subsequently,
researchers at that time put their entire attention and focus on development of SmF
and somehow or other, knowingly or unknowingly, SSF got totally neglected. During
the period of 1950-60, reports were published describing steroid transformation using
fungal cultures. SSF attained another milestone achievement during 1960-70 when
mycotoxins production by SSF was reported successful using fungal cultures that
resulted a significant impact on cancer research. During this period, production of
protein enriched cattle feed (single cell protein) was another important application
15
oriented fmding of SSF research. Since then enormous work has been carried out and
SSF regained a fresh attention from researchers and industries allover the world
(pandey et at., 1999; Classen et at., 2000; Medeiros et at., 2000a; Haddadin et ai.,
2001; Han et at., 2001; Ogbonna et al., 2001; Yang et al., 2001; Pandey et ai.,
2008b).
SSF offers numerous potential in processing of agro-industrial residues. This is
mainly due to SSF processes required lower energy consumption, produce lesser
wastewater, biomass energy conservation and are environmental friendly as they
resolve the problem of solid wastes disposal. Substrates that have been traditionally
used in SSF include wide variety of agricultural products or residues such as rice,
cassava, wheat, millet, barley, grains, sugarcane, beans, com, canola meal, soybeans
and etc. However, non-traditional substrates such as abundant supply of agricultural,
forest and food-processing wastes are also be of interest in industrial process
development (perez-Guerra et at., 2003). A closer examination of SSF processes in
several research centers throughout the world has led to the realization of numerous
economical and practical advantages ofSSF (Steinkraus, 1984; Lonsane et al., 1985).
Research on SSF has led to a wide range of applications at the laboratory scale
(Gupte and Madamwar, 1997; Gutierrez-Correa and Tengerdy, 1998; Hang and
Woodams, 1998).
In addition, during 1991-2006, apart from special issue of journals and books more
than 1400 publications have appeared in various journals, proceedings and books
(pandey et ai., 2008b). Current decade has witnessed much promise in the
development of several bioprocesses and products (Smits et ai., 1996) such as
16
bioremediation and biodegradation of hazardous compounds, biotransformation of
crops and crop-residues for nutritional enrichment, biopulping, and biological
detoxification of agro-industrial residues and production of value added products
such as antibiotics, enzymes and organic acids. In recent years, the increasing
demand for the natural products in the food industry has encouraged the development
of biotechnological processes for the production of flavor compounds. The use of
SSF as a means to improve economical feasibility would be of potential benefit
(Longo and Sanromffn, 2006; Pandey et al., 2008b). These have led to the
development of bioreactor, which was suitable for SSF process. However, SSF up
scaling, neces.sary for use on an industrial scale, raises severe engineering problems
due to the build up of temperature, pH, 02, moisture and temperature gradients.
Mitchell et al., (2003) reported that mathematical models have been considered as
important tools for optimizing the design and operation of SSF bioreactors. The
models must be able to describe the kinetics of microbial growth, how this may affect
by the environmental conditions and vice versa. This can be done in two levels of
sophistication. Moreover, simple empirical equations are used in many bioreactors to
describe the models of kinetics (Mitchell et al., 2004). Sensors development and
measurement in fermentation processes are important developments in SSF. Bellon
Maurel et aI., (2003) discussed current online methods and applications of the
methods with a potential to measure process variables in SSF that are not easily
accessible and measurable.
17
2.1.1.2 Selection of Microorganisms
Fungi, yeast and bacteria can grow on solid substrates and find their application in
SSF processes. Some examples of SSF processes for each category of
microorganisms are summarized and presented in Table 2.3. However, based upon
the type of microorganisms used, SSF processes can be generally classified into t\;vo
main groups, indigenous or natural and individual culture of SSF. The former
normally involved natural micro-flora and the latter mainly using pure culture or
mixed-culture in the SSF process. Composting and ensiling are the two typical
examples of SSF processes that involve natural micro-flora mainly bacteria
(Pandey et al., 2008b). Bacteria also have been used for some food processes such as
sausages, Japanese natto, fermented soybean paste, Chinese vinegar (Ramcsh and
Lonsane, 1991; Prabhu and Chandrasekaran, 1995). On the other hand, yeasts can be
used for ethanol, food and feed production (Saucedo-Castaneda et al., 1992a, 1992b).
However, filamentous fungi are the most important group of microorganisms used
and dominate in SSF research works. Koji process is one of the most important
applications of SSF with filamentous fungi and one such unique example of that
involving pure culture of Aspergillus oryzae. Generally pure cultures are used in
industrial SSF processes since it helps in increased productivity or optimum substrate
utilization for the production of desired product. Bioconversion of agro-industrial
residues such as wheat straw for protein enrichment by Candida utilis and
Chaetomium cellulolyticum is one of the good examples of SSF processes involving
mixed cultures (Pandey, 1992; Raimbault, 1998).
Selection of suitable microorganisms for a particular application is one of the most
important criteria in SSF process. The wide varieties of wild type microorganisms are
18
Table 2.3: Major groups of microorganisms involved in SSF processes (Raimbault, 1998)
Microorganisms Fungi Aspergillus niger Aspergillus oryzae Penicillium notatum Phanerochaete chrysosporium Rhizopus oligosporus Mucorsp. Rhizopus sp.
Yeast Endomicopsis burtonii Saccharomyces cerevisiae Schwanniomyces castelli
Bacteria Bacillus sp. Pseudomonas sp.
SSF process
~ Feed, Amylase, Citric Acid, Protein ~ Koji, Food, Citric Acid ~ Penicillin, Cheese ~ Composting, Lignin Degradation ~ Tempeh, Soybean, Amylase, Lipase ~ Composting, Food, Enzymes ~ Composting, Food, Enzymes, Organic Acid
~ Tape, Cassava, Rice ~ Food, Ethanol ~ Ethanol, Amylase
~ Composting, Natto, Amylase ~ Composting
unable to meet commercial requirement in terms of productivity and yield of the end
product. The importance of the microorganism can be seen from the fact that
Aspergillus niger itself can produce at least 21 types of different enzymes such as
cellulase, xylanases, lipase, protease and etc. On the other hand, one enzyme alone
can be produced by several different microorganisms in different quantities. For
example, a-amylase can be produced by as many as 64 different strains of
microorganisms which include Aspergillus sp., Bacillus sp., anaerobic bacterium and
etc (pandey, 1992; Pandey et ai., 2008b).
Several groups of microorganisms are capable to grow on solid substrates; however
theoretical classification based on water activity showed that only fungi and yeast are
considered as suitable microorganisms for SSF process. Bacterial cultures might not
be suitable for SSF owing to high water activity requirement. However, some authors
reported that bacterial cultures can be well managed and manipulated for SSF
processes (Pandey, 1992; Nampoothiri and Pandey, 1996; Selvakumar and Pandey,
19
1999; Pandey et al., 2000a). Cultivation of the filamentous fungi in SSF processes
have been carried out since long time ago for different applications either in
laboratory or in pilot scale. SSF process can be used for koji and tempeh production
(Raimbault, 1998), for mycotoxins production (Bhumiratna, 1980), for lignocellulose
fermentation (Matteau and Bone, 1980) and for fungal spores production (Lotong
and Suwarnarit, 1983). Three main classes of filamentous fungi that most commonly
been used in SSF process include Ascomycetes (Aspergillus and Penicillium),
Basidiomycetes (White rot fungi, Polyporus) and Phycomycetes (Mucor and
Rhizopus). Several yeasts have been used for ethanol fermentation or protein
enrichment. Protein production by Sporotrichum pulverulentum was reported by
Smith et al., (1986), while Saccharomyces cerevisiae has most commonly been used
for ethanol production (Gibbons et al., 1984; Kargi et al., 1985).
SSF research works have received increased attention and focus due to vast variety of
interesting compounds that can be efficiently produced, which includes gibberellic
acid (Kumar and Lonsane, 1987), penicillin (Barrios-Gonzalez et al., 1993), enzymes
(Solis-Pereira et al., 1993) and citric acid (Hang et al., 1987). About 50% of the
microorganisms used in applications of SSF belong to the class of filamentous fungi,
30% to yeast, 15% to actinomycetes and 5% to bacteria (Gutierrez-Rojas et ai., 1995).
The remarkable preference of filamentous fungi for the SSF process is due to the low
water activity requirements, their efficient way to adherence, colonize on suitable
substrate surfaces, penetrate into the substrate and their ability to assimilate mixtures
of different polysaccharides used (Mitchell et al., 1991).
20
In addition, filamentous fungi are the best adapted to grow due to their physiological,
enzymological and biochemical properties. The hyphal mode of fungal growth gives
a major advantage to filamentous fungi over unicellular microorganisms in the
colonization of solid substrates and the utilization of available nutrients. Moreover,
their ability to grow at low water activity (Aw) and hig..1. osmotic pressure conditions
(high nutrient concentration) makes fungi efficient (Mitchell and Lonsane, 1992). In
addition, the hyphal mode of growth gives the filamentous fungi the power to
penetrate into the substrates particles. Hyphal extension provides an alternative to
motility, enabling hyphae to extend toward new sources of food (nutrient), branching
provides a means to optimize substrate utilization and colonization which ensures a
firm and solid structure. The hydrolytic enzymes are excreted in high quantity at the
hyphal tip with minimum dilution makes the action of the enzymes very efficient in
hydrolysis and allows further penetration into most substrates particles. Penetration
will increase the accessibility of all available nutrients within the substrate particles.
The resulting contact catalysis is very efficient and the converted simple products are
in close contact, enter through the mycelium across the cell membrane to promote
biosynthesis and fungal metabolic activities (Raimbault, 1998).
2.1.1.3 Substrates and Particles Size
Selection of suitable substrate is another important key aspect in SSF process. It
seems that two terms, solid state fermentation and solid substrate fermentation have
often been equivocally used. It would be logical to distinguish these two terms. Solid
substrate fermentation should only be used to define those processes in which the
substrate itself acts as a carbon or energy source, occurring in the absence or near
absence of free water. But solid state fermentation should be defined as any
21
fermentation process occur employing a natural substrate as above or an inert
substrate used as solid support in the absence or near absence of free water (pandey
et ai., 2008b). The substrate not only must possess enough moisture, all needed
nutrients to support growth and metabolism of microorganism, but also serves as an
anchorage for the cells. However, it must keep in mind that some nutrient may not be
enough or even present in the particular substrates. In such case, it is necessary to
supplement them additionally.
The selection of a substrate for SSF process depends upon several factors, mainly
related with the cost, availability and consistency of the raw materials. Research on
the selection of a suitable substrate for SSF process has mainly centered around
tropical origin due to the agro-industrial crops and residues offer potential advantages
for their application as substrates (pandey et at., 2000c; Pandey et at., 2008b). The
agro-industrial crops and residues that have potential have been used as substrates in
SSF process are presented in Table 2.4. Among all the potential substrate, wheat bran
has been the good choice. A lot of SSF research work has been developed using this
substrate for the production of bulk chemicals and value added fme products.
Table 2.4: Agro-industrial crops and residues that have potential to be used as substrates (Pandey et at., 2000c, 2008a, 2008b; Hoogschagen et at., 2001; Peralta-Perez et at., 2001)
Crops or Residues Crop
Residues Bran, straw Hull Bagasse Pulp, husk, spent ground pomace Wastes Coconut cake, soybean cake, peanut cake, canola meal
Example ~ Cassava, soybean, sugar beet, sweet potato,
potato, sweet sorghum
~ Wheat, rice ~ Soy, com, rice ~ Sugarcane, cassava ~ Coffee processing ~ Fruit processing (Apple, grape) ~ Pineapple and carrot processing ~ Oil processing mill
22
Among the several other factors, substrate particle size and moisture level or water
activity are the most important critical for the microbial growth and activity.
Generally, reduction in substrate particle size through milling provides smaller
substrate particles that would provide larger surface area for the microbial attack and
growth. However, at the same time, very tiny substrate particles may result in
substrate agglomeration, matrix compaction in most of the cases, which may interfere
with microbial respiration and aeration, increasing mass and heat transfer problems.
Thus, these may result in poor microbial growth and low productivity of the desired
end products. On the other hand, larger substrate particles size provides better
respiration and aeration efficiency owing to increase substrate inter-particle space,
but provides limited surface area for the microbial attack. Thus, it must be remarked
that particle size could show an optimum range and necessary to come through at a
compromised particle size for a particular process (pandey et al., 2000a; Pandey et
al., 2008b).
Generally when dealing with agro-industrial natural materials, it has been a common
practice to prepare and pre-treat some substrates to convert them to a more suitable
and easily accessible for microbial attack (pandey et al., 2008b). Table 2.5 shows the
common substrates pre-treatment steps before its being used in SSF process. Size
reduction of substrate by chopping, grinding or grating is one of the most common
substrates pre-treatment steps. It must be noted that the substrate particle size in SSF
processes does not remain constant and tends to diminish through out the
fermentation process. Mycelium formation is the main cause for void fraction
variation. This is particularly true in fermentation which involves fungi.
23
Table 2.5: Common substrates pre-treatment steps (Mitchell et al., 1992; Raimbault, 1998)
No Steps 1 );- Size reduction by chopping, grinding, sifting or grating
2 );- Damage to outer substrate layers by grinding, pearling or cracking.
3 );- Chemical or enzymatic hydrolysis of polymers to improve substrate accessible by the fungus.
4 );- Supplementation with nutrients, determine the moisture content through a mineral solution.
5 );- Setting the pH growth medium.
6 );- Cooking or vapor treatment of substrate (macromolecular structure) as pre-degradation and elimination of major contaminants.
Solid substrate material, which are polymeric in nature and insoluble in water, acts as
both physical support (sugarcane bagasse) and source of nutrients. The solid
substrate can be a naturally occurring agricultural crops, residues, inert support or
synthetic materials (pandey, 1992; Pandey et aZ., 2000b; Pandey et aZ., 2000c;
Pandey et aZ., 2001a). In term of selection of suitable substrate, two major
considerations must take into account; first is the specific substrate that requires
suitable value-addition and/or disposal. The second could be related with the goal of
producing a desired product from a suitable substrate.
Based on the nature of the solid substrate used/involved, two types of SSF process
can be distinguished. First is the solid substrate serves as a support and a nutrient
source, while the second is an inert support. The former utilized substrates that are
heterogeneous and water insoluble materials from agro-industrial or by-products
from food industry which have lignocellulosic nature (pandey, 1992). The latter is
saturated with liquid medium, which contains all the needed nutrients. However, this
process is less used and presents economical disadvantages (Ooijkaas et al., 2000).
24
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