Christian P. Kubicek is a Professor at the Institute of Chemical Engineering at the Vienna University of Technology, is Head of the Institute’s Research Division of Biotechnology and Microbiology, has authored more than 300 refereed journal articles and more than 20 book chapters, and has served as an editor and editorial board member for several international peer reviewed journals.

“Written by a leading researcher in the field, this book is a valuable tool for researchers, engineers, and industry professionals interested in advancing the development and production of biofuels.”  (Redaktion Landtechnik, 1 March 2012)

• Preface ix1 The Plant Biomass 11.1 The Structure of Plant Cell Wall 11.2 Chemical and Physicochemical Properties of the Major Plant Cell Wall Constituents 31.2.1 Cellulose 31.2.2 Pectin 61.2.3 Hemicelluloses 81.2.4 Lignin 111.3 Abundant Sources of Carbohydrate Polymers and Their Monomer Composition 131.3.1 Agricultural Wastes 131.3.2 Forest Product Residues 141.3.3 Energy Crops 151.3.4 Weedy Lignocellulosic Substrates 151.4 Biosynthesis of Plant Cell Wall Polymers 161.4.1 Cellulose 161.4.2 Hemicellulose Biosynthesis 191.4.3 Pectin Biosynthesis 201.4.4 Lignin Biosynthesis 231.5 Strategies for Manipulating Wall Composition 261.5.1 Manipulation of Plant Cell Wall Polymer Composition 261.5.2 Manipulation of Plant Lignin Content 272 The Actors: Plant Biomass Degradation by Fungi 292.1 Ecological Perspectives 292.2 The Major Three Mechanisms of Lignocellulose Degradation by Fungi 302.2.1 White Rot 312.2.2 Brown Rot Fungi 352.2.3 Soft Rot Fungi 392.3 Plant Cell Wall Degradation by Plant Pathogenic Fungi 402.4 Anaerobic Fungi 413 The Tools—Part 1: Enzymology of Cellulose Degradation 453.1 General Properties and Classification of Enzymes That Hydrolyze Polysaccharides 453.2 Fungal Cellulolytic Enzymes 493.2.1 Cellulose-Binding Domains 523.2.2 Cellobiohydrolases (EC 3.2.1.91) 553.2.3 Endo--1,4-Glucanases (EC 3.2.1.4) 583.2.4 -1,4-Glucosidases 643.3 Nonenzymatic Proteins Involved in Cellulose Hydrolysis 653.3.1 GH61 Proteins 653.3.2 Swollenin 674 The Tools—Part 2: Enzymology of Hemicellulose Degradation 694.1 Xyloglucan Hydrolysis 694.2 Degradation of the Xylan Backbone 724.2.1 GH10 Xylanases 734.2.2 GH11 Xylanase 754.2.3 GH30 Glucuronoxylan Xylanohydrolases 764.2.4 GH3 -Xylosidases 764.2.5 GH43 -Xylosidases 774.2.6 GH54 -Xylosidases 784.3 Degradation of the Galactomannan Backbone 784.4 Degradation of Pectin 804.4.1 Hydrolytic Pectin Degradation 804.4.2 Pectin Degradation by -Elimination 824.5 Accessory Glycoside Hydrolases for Hemicelluloses Degradation 844.5.1 Enzymes that Act on Arabinose-Containing Substituents 854.5.2 Enzymes that Act on Galactose-Containing Substituents 864.5.3 -Xylosidases 894.5.4 -Fucosidases 904.5.5 -Glucuronidases and Glucuronan Lyases 904.5.6 Accessory Hydrolases for Pectin Degradation 914.6 Other Accessory Enzymes 924.6.1 Feruloyl and p-Coumaroyl Esterases 924.6.2 Acetyl- and Methylesterases 934.6.3 Pectin Esterases 954.6.4 Glucuronoyl Esterases 955 The Tools—Part 3: Enzymology of Lignin Degradation 995.1 Lignin Peroxidase 1015.2 Manganese Peroxidase 1055.3 Versatile Peroxidase 1085.4 Dye-Oxidizing Peroxidase 1095.5 Laccases 1105.6 Enzymes Generating Hydrogen Peroxide 1155.6.1 Glyoxal Oxidase 1155.6.2 Other FAD-Dependent Oxidases 1165.7 Cellobiose Dehydrogenase 1165.8 Enzymes Essential for Oxalic Acid Formation 1175.9 Glycopeptides 1186 Catabolic Pathways of Soluble Degradation Products from Plant Biomass 1196.1 Uptake of Mono- and Oligosaccharides 1196.2 Metabolism of D-Glucose and D-Mannose 1216.3 Catabolism of D-Galactose 1226.4 Catabolism of Pentoses 1256.5 Catabolism of Hexuronic Acids 1277 Regulation of Formation of Plant Biomass-Degrading Enzymes in Fungi 1297.1 The Cellulase Inducer Enigma 1297.2 Inducers for Hemicellulases 1337.3 Transcriptional Regulation of Cellulase and Hemicellulase Gene Expression 1347.3.1 Activators of Cellulase and Hemicellulase Gene Expression 1357.3.2 Specific Repressors of Cellulase and Hemicellulase Gene Expression 1387.3.3 Wide Domain Regulators of Cellulase and Hemicellulase Gene Transcription 1387.3.4 Regulation of Cellulase and Hemicellulase Transcription at the Level of Genome Accessability 1447.3.5 Signal Transduction to Cellulase and Hemicellulase Gene Expression 1457.4 Regulation of Ligninase Gene Expression 1468 The Fungal Secretory Pathways and Their Relation to Lignocellulose Degradation 1498.1 The Fungal Secretory Pathway 1498.1.1 It Starts at the Endoplasmic Reticulum 1498.1.2 Quality Control in the ER 1528.1.3 Golgi Apparatus 1548.1.4 The Plasma Membrane 1568.1.5 Vacuoles 1588.2 Protein Glycosylation 1598.2.1 N-Glycosylation 1598.2.2 O-Glycan Synthesis 1618.3 Strategies for Improvement of the Fungal Secretory Pathway 1618.3.1 Folding and UPR 1638.3.2 Engineering of Protein Glycosylation 1639 Production of Cellulases and Hemicellulases by Fungi 1659.1 Fungal Producer Strains 1659.1.1 Trichoderma Reesei 1659.1.2 Other Fungal Producer Strains 1679.1.3 Thermophilic Fungi 1699.2 Strain Improvement 1709.2.1 Strain Breeding by Classical Mutagenesis 1709.2.2 Strain Improvement by Targeted Gene Manipulation 1719.2.3 Strain Improvement by Complementation with Heterologous Enzymes 1769.2.4 Strain Improvement by Protein Engineering 1799.3 Cellulase Production 1809.3.1 Cellulase Fermentation 1809.3.2 Cellulase Downstream Processing 18210 Production of Fermentable Sugars from Lignocelluloses 18510.1 Pretreatment Technologies 18610.1.1 Physical Pretreatment 18610.1.2 Chemical Pretreatment 18610.1.3 Physicothermal Pretreatment 18910.1.4 Solvent Treatment 19110.1.5 Biological Pretreatment 19310.1.6 Summarizing Considerations on Pretreatment 19510.2 Hydrolysis 19510.2.1 Enzymatic Hydrolysis 19510.2.2 Chemical Hydrolysis 20011 Lignocellulose Biorefinery 20111.1 Ethanol 20211.1.1 Yeast Strain Improvement 20311.1.2 Alternatives to S. cerevisiae as an Ethanol Producer 21011.1.3 Process Improvement 21211.2 n-Butanol 21211.3 Advanced Biofuel Alcohols 21311.3.1 Isobutanol 21311.3.2 Syngas 21411.3.3 Fuels from Isoprenoid and Fatty Acid Pathways 21511.4 Lactic Acid 21511.5 Succinic Acid 21711.6 Xylitol 22211.7 1,3-Propanediol 22211.8 Polyhydroxyalkanoate 22311.9 Other Products 22311.10 Refinement by Chemical Processes 22511.10.1 Furfural 22511.10.2 Levulinic Acid 22511.10.3 Uses of Lignin 22511.10.4 Extraction of Chemicals from Lignocelluloses Biomass 226Acknowledgments 229References 231Index 285

Christian P. Kubicek is a Professor at the Institute of Chemical Engineering at the Vienna University of Technology, is Head of the Institute’s Research Division of Biotechnology and Microbiology, has authored more than 300 refereed journal articles and more than 20 book chapters, and has served as an editor and editorial board member for several international peer reviewed journals.

“Written by a leading researcher in the field, this book is a valuable tool for researchers, engineers, and industry professionals interested in advancing the development and production of biofuels.”  (Redaktion Landtechnik, 1 March 2012)

• Preface ix1 The Plant Biomass 11.1 The Structure of Plant Cell Wall 11.2 Chemical and Physicochemical Properties of the Major Plant Cell Wall Constituents 31.2.1 Cellulose 31.2.2 Pectin 61.2.3 Hemicelluloses 81.2.4 Lignin 111.3 Abundant Sources of Carbohydrate Polymers and Their Monomer Composition 131.3.1 Agricultural Wastes 131.3.2 Forest Product Residues 141.3.3 Energy Crops 151.3.4 Weedy Lignocellulosic Substrates 151.4 Biosynthesis of Plant Cell Wall Polymers 161.4.1 Cellulose 161.4.2 Hemicellulose Biosynthesis 191.4.3 Pectin Biosynthesis 201.4.4 Lignin Biosynthesis 231.5 Strategies for Manipulating Wall Composition 261.5.1 Manipulation of Plant Cell Wall Polymer Composition 261.5.2 Manipulation of Plant Lignin Content 272 The Actors: Plant Biomass Degradation by Fungi 292.1 Ecological Perspectives 292.2 The Major Three Mechanisms of Lignocellulose Degradation by Fungi 302.2.1 White Rot 312.2.2 Brown Rot Fungi 352.2.3 Soft Rot Fungi 392.3 Plant Cell Wall Degradation by Plant Pathogenic Fungi 402.4 Anaerobic Fungi 413 The Tools—Part 1: Enzymology of Cellulose Degradation 453.1 General Properties and Classification of Enzymes That Hydrolyze Polysaccharides 453.2 Fungal Cellulolytic Enzymes 493.2.1 Cellulose-Binding Domains 523.2.2 Cellobiohydrolases (EC 3.2.1.91) 553.2.3 Endo--1,4-Glucanases (EC 3.2.1.4) 583.2.4 -1,4-Glucosidases 643.3 Nonenzymatic Proteins Involved in Cellulose Hydrolysis 653.3.1 GH61 Proteins 653.3.2 Swollenin 674 The Tools—Part 2: Enzymology of Hemicellulose Degradation 694.1 Xyloglucan Hydrolysis 694.2 Degradation of the Xylan Backbone 724.2.1 GH10 Xylanases 734.2.2 GH11 Xylanase 754.2.3 GH30 Glucuronoxylan Xylanohydrolases 764.2.4 GH3 -Xylosidases 764.2.5 GH43 -Xylosidases 774.2.6 GH54 -Xylosidases 784.3 Degradation of the Galactomannan Backbone 784.4 Degradation of Pectin 804.4.1 Hydrolytic Pectin Degradation 804.4.2 Pectin Degradation by -Elimination 824.5 Accessory Glycoside Hydrolases for Hemicelluloses Degradation 844.5.1 Enzymes that Act on Arabinose-Containing Substituents 854.5.2 Enzymes that Act on Galactose-Containing Substituents 864.5.3 -Xylosidases 894.5.4 -Fucosidases 904.5.5 -Glucuronidases and Glucuronan Lyases 904.5.6 Accessory Hydrolases for Pectin Degradation 914.6 Other Accessory Enzymes 924.6.1 Feruloyl and p-Coumaroyl Esterases 924.6.2 Acetyl- and Methylesterases 934.6.3 Pectin Esterases 954.6.4 Glucuronoyl Esterases 955 The Tools—Part 3: Enzymology of Lignin Degradation 995.1 Lignin Peroxidase 1015.2 Manganese Peroxidase 1055.3 Versatile Peroxidase 1085.4 Dye-Oxidizing Peroxidase 1095.5 Laccases 1105.6 Enzymes Generating Hydrogen Peroxide 1155.6.1 Glyoxal Oxidase 1155.6.2 Other FAD-Dependent Oxidases 1165.7 Cellobiose Dehydrogenase 1165.8 Enzymes Essential for Oxalic Acid Formation 1175.9 Glycopeptides 1186 Catabolic Pathways of Soluble Degradation Products from Plant Biomass 1196.1 Uptake of Mono- and Oligosaccharides 1196.2 Metabolism of D-Glucose and D-Mannose 1216.3 Catabolism of D-Galactose 1226.4 Catabolism of Pentoses 1256.5 Catabolism of Hexuronic Acids 1277 Regulation of Formation of Plant Biomass-Degrading Enzymes in Fungi 1297.1 The Cellulase Inducer Enigma 1297.2 Inducers for Hemicellulases 1337.3 Transcriptional Regulation of Cellulase and Hemicellulase Gene Expression 1347.3.1 Activators of Cellulase and Hemicellulase Gene Expression 1357.3.2 Specific Repressors of Cellulase and Hemicellulase Gene Expression 1387.3.3 Wide Domain Regulators of Cellulase and Hemicellulase Gene Transcription 1387.3.4 Regulation of Cellulase and Hemicellulase Transcription at the Level of Genome Accessability 1447.3.5 Signal Transduction to Cellulase and Hemicellulase Gene Expression 1457.4 Regulation of Ligninase Gene Expression 1468 The Fungal Secretory Pathways and Their Relation to Lignocellulose Degradation 1498.1 The Fungal Secretory Pathway 1498.1.1 It Starts at the Endoplasmic Reticulum 1498.1.2 Quality Control in the ER 1528.1.3 Golgi Apparatus 1548.1.4 The Plasma Membrane 1568.1.5 Vacuoles 1588.2 Protein Glycosylation 1598.2.1 N-Glycosylation 1598.2.2 O-Glycan Synthesis 1618.3 Strategies for Improvement of the Fungal Secretory Pathway 1618.3.1 Folding and UPR 1638.3.2 Engineering of Protein Glycosylation 1639 Production of Cellulases and Hemicellulases by Fungi 1659.1 Fungal Producer Strains 1659.1.1 Trichoderma Reesei 1659.1.2 Other Fungal Producer Strains 1679.1.3 Thermophilic Fungi 1699.2 Strain Improvement 1709.2.1 Strain Breeding by Classical Mutagenesis 1709.2.2 Strain Improvement by Targeted Gene Manipulation 1719.2.3 Strain Improvement by Complementation with Heterologous Enzymes 1769.2.4 Strain Improvement by Protein Engineering 1799.3 Cellulase Production 1809.3.1 Cellulase Fermentation 1809.3.2 Cellulase Downstream Processing 18210 Production of Fermentable Sugars from Lignocelluloses 18510.1 Pretreatment Technologies 18610.1.1 Physical Pretreatment 18610.1.2 Chemical Pretreatment 18610.1.3 Physicothermal Pretreatment 18910.1.4 Solvent Treatment 19110.1.5 Biological Pretreatment 19310.1.6 Summarizing Considerations on Pretreatment 19510.2 Hydrolysis 19510.2.1 Enzymatic Hydrolysis 19510.2.2 Chemical Hydrolysis 20011 Lignocellulose Biorefinery 20111.1 Ethanol 20211.1.1 Yeast Strain Improvement 20311.1.2 Alternatives to S. cerevisiae as an Ethanol Producer 21011.1.3 Process Improvement 21211.2 n-Butanol 21211.3 Advanced Biofuel Alcohols 21311.3.1 Isobutanol 21311.3.2 Syngas 21411.3.3 Fuels from Isoprenoid and Fatty Acid Pathways 21511.4 Lactic Acid 21511.5 Succinic Acid 21711.6 Xylitol 22211.7 1,3-Propanediol 22211.8 Polyhydroxyalkanoate 22311.9 Other Products 22311.10 Refinement by Chemical Processes 22511.10.1 Furfural 22511.10.2 Levulinic Acid 22511.10.3 Uses of Lignin 22511.10.4 Extraction of Chemicals from Lignocelluloses Biomass 226Acknowledgments 229References 231Index 285

Christian P. Kubicek is a Professor at the Institute of Chemical Engineering at the Vienna University of Technology, is Head of the Institute’s Research Division of Biotechnology and Microbiology, has authored more than 300 refereed journal articles and more than 20 book chapters, and has served as an editor and editorial board member for several international peer reviewed journals.

“Written by a leading researcher in the field, this book is a valuable tool for researchers, engineers, and industry professionals interested in advancing the development and production of biofuels.”  (Redaktion Landtechnik, 1 March 2012)

• Preface ix1 The Plant Biomass 11.1 The Structure of Plant Cell Wall 11.2 Chemical and Physicochemical Properties of the Major Plant Cell Wall Constituents 31.2.1 Cellulose 31.2.2 Pectin 61.2.3 Hemicelluloses 81.2.4 Lignin 111.3 Abundant Sources of Carbohydrate Polymers and Their Monomer Composition 131.3.1 Agricultural Wastes 131.3.2 Forest Product Residues 141.3.3 Energy Crops 151.3.4 Weedy Lignocellulosic Substrates 151.4 Biosynthesis of Plant Cell Wall Polymers 161.4.1 Cellulose 161.4.2 Hemicellulose Biosynthesis 191.4.3 Pectin Biosynthesis 201.4.4 Lignin Biosynthesis 231.5 Strategies for Manipulating Wall Composition 261.5.1 Manipulation of Plant Cell Wall Polymer Composition 261.5.2 Manipulation of Plant Lignin Content 272 The Actors: Plant Biomass Degradation by Fungi 292.1 Ecological Perspectives 292.2 The Major Three Mechanisms of Lignocellulose Degradation by Fungi 302.2.1 White Rot 312.2.2 Brown Rot Fungi 352.2.3 Soft Rot Fungi 392.3 Plant Cell Wall Degradation by Plant Pathogenic Fungi 402.4 Anaerobic Fungi 413 The Tools—Part 1: Enzymology of Cellulose Degradation 453.1 General Properties and Classification of Enzymes That Hydrolyze Polysaccharides 453.2 Fungal Cellulolytic Enzymes 493.2.1 Cellulose-Binding Domains 523.2.2 Cellobiohydrolases (EC 3.2.1.91) 553.2.3 Endo--1,4-Glucanases (EC 3.2.1.4) 583.2.4 -1,4-Glucosidases 643.3 Nonenzymatic Proteins Involved in Cellulose Hydrolysis 653.3.1 GH61 Proteins 653.3.2 Swollenin 674 The Tools—Part 2: Enzymology of Hemicellulose Degradation 694.1 Xyloglucan Hydrolysis 694.2 Degradation of the Xylan Backbone 724.2.1 GH10 Xylanases 734.2.2 GH11 Xylanase 754.2.3 GH30 Glucuronoxylan Xylanohydrolases 764.2.4 GH3 -Xylosidases 764.2.5 GH43 -Xylosidases 774.2.6 GH54 -Xylosidases 784.3 Degradation of the Galactomannan Backbone 784.4 Degradation of Pectin 804.4.1 Hydrolytic Pectin Degradation 804.4.2 Pectin Degradation by -Elimination 824.5 Accessory Glycoside Hydrolases for Hemicelluloses Degradation 844.5.1 Enzymes that Act on Arabinose-Containing Substituents 854.5.2 Enzymes that Act on Galactose-Containing Substituents 864.5.3 -Xylosidases 894.5.4 -Fucosidases 904.5.5 -Glucuronidases and Glucuronan Lyases 904.5.6 Accessory Hydrolases for Pectin Degradation 914.6 Other Accessory Enzymes 924.6.1 Feruloyl and p-Coumaroyl Esterases 924.6.2 Acetyl- and Methylesterases 934.6.3 Pectin Esterases 954.6.4 Glucuronoyl Esterases 955 The Tools—Part 3: Enzymology of Lignin Degradation 995.1 Lignin Peroxidase 1015.2 Manganese Peroxidase 1055.3 Versatile Peroxidase 1085.4 Dye-Oxidizing Peroxidase 1095.5 Laccases 1105.6 Enzymes Generating Hydrogen Peroxide 1155.6.1 Glyoxal Oxidase 1155.6.2 Other FAD-Dependent Oxidases 1165.7 Cellobiose Dehydrogenase 1165.8 Enzymes Essential for Oxalic Acid Formation 1175.9 Glycopeptides 1186 Catabolic Pathways of Soluble Degradation Products from Plant Biomass 1196.1 Uptake of Mono- and Oligosaccharides 1196.2 Metabolism of D-Glucose and D-Mannose 1216.3 Catabolism of D-Galactose 1226.4 Catabolism of Pentoses 1256.5 Catabolism of Hexuronic Acids 1277 Regulation of Formation of Plant Biomass-Degrading Enzymes in Fungi 1297.1 The Cellulase Inducer Enigma 1297.2 Inducers for Hemicellulases 1337.3 Transcriptional Regulation of Cellulase and Hemicellulase Gene Expression 1347.3.1 Activators of Cellulase and Hemicellulase Gene Expression 1357.3.2 Specific Repressors of Cellulase and Hemicellulase Gene Expression 1387.3.3 Wide Domain Regulators of Cellulase and Hemicellulase Gene Transcription 1387.3.4 Regulation of Cellulase and Hemicellulase Transcription at the Level of Genome Accessability 1447.3.5 Signal Transduction to Cellulase and Hemicellulase Gene Expression 1457.4 Regulation of Ligninase Gene Expression 1468 The Fungal Secretory Pathways and Their Relation to Lignocellulose Degradation 1498.1 The Fungal Secretory Pathway 1498.1.1 It Starts at the Endoplasmic Reticulum 1498.1.2 Quality Control in the ER 1528.1.3 Golgi Apparatus 1548.1.4 The Plasma Membrane 1568.1.5 Vacuoles 1588.2 Protein Glycosylation 1598.2.1 N-Glycosylation 1598.2.2 O-Glycan Synthesis 1618.3 Strategies for Improvement of the Fungal Secretory Pathway 1618.3.1 Folding and UPR 1638.3.2 Engineering of Protein Glycosylation 1639 Production of Cellulases and Hemicellulases by Fungi 1659.1 Fungal Producer Strains 1659.1.1 Trichoderma Reesei 1659.1.2 Other Fungal Producer Strains 1679.1.3 Thermophilic Fungi 1699.2 Strain Improvement 1709.2.1 Strain Breeding by Classical Mutagenesis 1709.2.2 Strain Improvement by Targeted Gene Manipulation 1719.2.3 Strain Improvement by Complementation with Heterologous Enzymes 1769.2.4 Strain Improvement by Protein Engineering 1799.3 Cellulase Production 1809.3.1 Cellulase Fermentation 1809.3.2 Cellulase Downstream Processing 18210 Production of Fermentable Sugars from Lignocelluloses 18510.1 Pretreatment Technologies 18610.1.1 Physical Pretreatment 18610.1.2 Chemical Pretreatment 18610.1.3 Physicothermal Pretreatment 18910.1.4 Solvent Treatment 19110.1.5 Biological Pretreatment 19310.1.6 Summarizing Considerations on Pretreatment 19510.2 Hydrolysis 19510.2.1 Enzymatic Hydrolysis 19510.2.2 Chemical Hydrolysis 20011 Lignocellulose Biorefinery 20111.1 Ethanol 20211.1.1 Yeast Strain Improvement 20311.1.2 Alternatives to S. cerevisiae as an Ethanol Producer 21011.1.3 Process Improvement 21211.2 n-Butanol 21211.3 Advanced Biofuel Alcohols 21311.3.1 Isobutanol 21311.3.2 Syngas 21411.3.3 Fuels from Isoprenoid and Fatty Acid Pathways 21511.4 Lactic Acid 21511.5 Succinic Acid 21711.6 Xylitol 22211.7 1,3-Propanediol 22211.8 Polyhydroxyalkanoate 22311.9 Other Products 22311.10 Refinement by Chemical Processes 22511.10.1 Furfural 22511.10.2 Levulinic Acid 22511.10.3 Uses of Lignin 22511.10.4 Extraction of Chemicals from Lignocelluloses Biomass 226Acknowledgments 229References 231Index 285