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Lignocellulosic Biomass

Production and Industrial

Applications

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Lignocellulosic Biomass

Production and Industrial

Applications

Edited by

Arindam Kuila and Vinay Sharma

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Names: Kuila, Arindam, editor. | Sharma, Vinay, editor.

Title: Lignocellulosic biomass production and industrial applications /

edited by Arindam Kuila and Vinay Sharma.

Description: Beverly, MA : Scrivener Publishing ; Hoboken, NJ : John Wiley &

Sons, 2017. | Includes index. |

Identifiers: LCCN 2017010812 (print) | LCCN 2017013368 (ebook) |

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Subjects: LCSH: Lignocellulose--Biotechnology. | Biomass--Industrial applications.

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v

Contents

Preface xv

1 Valorization of Lignocellulosic Materials toPolyhydroxyalkanoates (PHAs) 1

Arpan Das1.1 Introduction 1

1.1.1 What is PHA? 31.1.2 Mechanism of PHA Production 3

1.2 Lignocellulose: An Abundant Carbon Source for PHAProduction 51.2.1 Cellulose 51.2.2 Hemicelluloses 71.2.3 Lignin 71.2.4 Pectin 8

1.3 Lignocellulosic Pretreatment Techniques 81.3.1 Physical Pretreatment Techniques 8

1.3.1.1 Milling 81.3.1.2 Irradiation 10

1.3.2 Chemical Pretreatment 101.3.2.1 Acid Hydrolysis Pretreatment 101.3.2.2 Alkaline Hydrolysis 111.3.2.3 Oxidative Delignification by Peroxide 111.3.2.4 Organosolv Process 111.3.2.5 Ozonolysis Pretreatment 121.3.2.6 Ionic Liquids Pretreatment 12

1.3.3 Physico-Chemical Pretreatment 121.3.3.1 Liquid Hot-Water Pretreatment 131.3.3.2 Steam Explosion 131.3.3.3 Ammonia Fiber Explosion (AFEX) 13

1.3.4 Bological Pretreatment 14

vi Contents

1.4 Hydrolysis of Lingocellulose 141.5 Lignocellulose Biomass as Substrate for PHA Production 161.6 Conclusion 19References 19

2 Biological Gaseous Energy Recovery from Lignocellulosic Biomass 27

Shantonu Roy2.1 Introduction 272.2 Simple Sugars as Feedstock 282.3 Complex Substrates as Feedstock 322.4 Biomass Feedstock 32

2.4.1 Energy Crop 332.4.1.1 Miscanthus sp. 342.4.1.2 Sweet Sorghum Extract 342.4.1.3 Sugar Beet Juice 34

2.4.2 Algal Biomass 352.5 Waste as Feedstock 36

2.5.1 Municipal Solid Waste (MSW) 372.5.2 Food Waste 38

2.6 Industrial Wastewater 382.6.1 Dairy Industry Wastewater 382.6.2 Distillery Wastewater 392.6.3 Chemical Wastewaters 392.6.4 Glycerol 402.6.5 Palm Oil Mill Effluent 40

2.7 Conclusion 40Acknowledgments 41References 41

3 Alkali Treatment to Improve Physical, Mechanical andChemical Properties of Lignocellulosic Natural Fibers for Use in Various Applications 47

Suvendu Manna, Prosenjit Saha, Sukanya Chowdhury

and Sabu Thomas3.1 Introduction 48

3.1.1 Composition of Natural Fibers 493.1.2 Properties of Natural Fibers 51

3.2 Alkali Treatment 523.2.1 General Processing 523.2.2 Steam Treatment 543.2.3 Alkali-Steam Treatment 54

Contents vii

3.3 Application of the Alkali-Steam-Treated Fibers 553.3.1 Biocomposite 55

3.3.1.1 Green Biocomposite 553.3.1.2 Bionanocomposites 56

3.3.2 Water Treatment 573.3.2.1 Fluoride Removal 573.3.2.2 Dye Removal 583.3.2.3 Heavy Metal Removal 59

3.4 Summary 59References 60

4 Biodiesel Production from Lignocellulosic BiomassUsing Oleaginous Microbes 65

S.P. Jeevan Kumar, Lohit K. Srinivas Gujjala,

Archana Dash, Bitasta Talukdar and Rintu Banerjee4.1 Introduction 664.2 Lignocellulosics Distribution, Availability and Diversity 67

4.2.1 Forest Trees and Residues 674.2.2 Food Crops 684.2.3 Non-Food/Energy Crops 684.2.4 Tree-Based Oils 694.2.5 Industrial Process Residues 70

4.3 Prospective Oleaginous Microbes for Lipid Production 704.3.1 Oleaginous Algae 70

4.3.1.1 Green Algae 704.3.1.2 Blue-Green Algae 714.3.1.3 Golden Algae 724.3.1.4 Red and Brown Algae 724.3.1.5 Diatoms 72

4.3.2 Oleaginous Yeast and Mold 724.3.3 Metabolic Engineering Approaches for

LCB Utilization 734.3.3.1 Metabolic Engineering of Xylose and

Co-Utilization of Substrate 744.4 Technical Know-How for Biodiesel Production from LCBs 774.5 Fermentation 78

4.5.1 Solid-State Fermentation (SSF) 794.5.2 Submerged Fermentation (SF) 79

4.6 Transesterification for Biodiesel Production 804.6.1 Biodiesel 804.6.2 Transesterification 80

viii Contents

4.6.3 Acid/Base Transesterification 814.6.4 Enzymatic Transesterification 81

4.6.4.1 Lipases 824.6.5 In-Situ Transesterification 82

4.7 Characteristics of Fatty Acid Methyl Esters 834.8 Conclusion 83References 84

5 Biopulping of Lignocellulose 93

Arijit Jana, Debashish Ghosh, Diptarka Dasgupta,

Pradeep Kumar Das Mohapatra and

Keshab Chandra Mondal5.1 Introduction 935.2 Composition of Lignocellulosic Biomass 955.3 Pulping and its Various Processes 975.4 Biopulping – Process Overview 98

5.4.1 Role of Rot Fungi and its Effect in Wood Biomass 1005.4.2 Role of Enzymes in Biopulping 102

5.5 Advantages and Disadvantages of Biopulping 1045.6 Future Prospects 105Acknowledgment 105References 106

6 Second Generation Bioethanol Production from Residual Biomass of the Rice Processing Industry 111

Luciana Luft, Juliana R. F. da Silva, Raquel C. Kuhn

and Marcio A. Mazutti6.1 Introduction 1126.2 Residual Biomass 1126.3 Rice and Processing 1136.4 Pretreatment Techniques 115

6.4.1 Physical Pretreatment 1166.4.1.1 Mechanical 1166.4.1.2 Microwave 1176.4.1.3 Pyrolysis 118

6.4.2 Physicochemical Pretreatment 1186.4.2.1 Steam Explosion 1196.4.2.2 Wet Oxidation 1196.4.2.3 Ultrasound 1206.4.2.4 Supercritical CO

2 Explosion 120

6.4.2.5 Ammonia Fiber Expansion 121

Contents ix

6.4.3 Chemical Pretreatment 1216.4.3.1 Ozonolysis 1216.4.3.2 Acid Treatment 1226.4.3.3 Alkaline Treatment 1236.4.3.4 Organosolv Process 1236.4.3.5 Ionic Liquids 123

6.4.4 Biological Pretreatment 1246.5 Hydrolysis 1246.6 Fermentation 1256.7 Bioethanol Production 1276.8 Concluding Remarks 127Acknowledgments 128References 128

7 Microbial Enzymes and Lignocellulosic Fuel Production 135

Avanthi Althuri, Anjani Devi Chintagunta,

Knawang Chhunji Sherpa, Rajiv Chandra Rajak,

Debajyoti Kundu, Jagriti Singh, Akanksha Rastogi

and Rintu Banerjee7.1 Introduction 136

7.1.1 Enzymes for Lignocellulosic Biomass-BasedBiofuel Production 136

7.2 Lignocellulosic Biomass as Sustainable Alternativefor Fuel Production 1377.2.1 Constituents of Lignocelluloses: Cellulose,

Hemicellulose, Lignin and Other Biomolecules 1387.3 Enzymes and Their Sources for Biofuel Generation 1397.4 Microbial Enzymes towards Lignocellulosic Biomass

Degradation 1427.4.1 Ligninases 146

7.4.1.1 Lignin Peroxidase 1467.4.1.2 Manganese Peroxidase 1477.4.1.3 Hybrid Peroxidase 1477.4.1.4 Phenol Oxidases 1487.4.1.5 Other Lignin-Degrading Enzymes 149

7.4.2 Carbohydratases 1507.4.2.1 Cellulase 1507.4.2.2 Auxiliary Cellulose-Degrading Enzymes 1517.4.2.3 Hemicellulase 1527.4.2.4 Expansins and Swollenins 154

x Contents

7.4.2.5 Carboxyl Esterases 1567.4.2.6 Zymase 157

7.5 Applications in Biofuel Production 1597.5.1 Bioethanol 1597.5.2 Biomethane and Biomanure 161

7.6 Conclusion 162References 163

8 Sugarcane: A Potential Agricultural Crop forBioeconomy through Biorefinery 171

Knawang Chhunji Sherpa, Rajiv Chandra Rajak and

Rintu Banerjee8.1 Introduction 1718.2 Present Status of Sugarcane Production

and its Availability 1738.3 Morphology of Sugarcane 1748.4 Factors Involved in Sugarcane Production 175

8.4.1 Climatic Conditions 1758.4.1.1 Temperature 1758.4.1.2 Rainfall and Relative Humidity 1758.4.1.3 Sunlight 175

8.4.2 Soil Quality 1768.4.3 Varieties of Sugarcane 1768.4.4 Land Requirement 1778.4.5 Propagation 1778.4.6 Nutrient Management 1828.4.7 Water Management 1828.4.8 Weed Management 1838.4.9 Biotic Factors: Pests and Pathogens 183

8.4.10 Crop Rotation 1838.4.11 Ratooning 1838.4.12 Intercropping 185

8.5 Major Limitations of Sugarcane Production 1858.6 An Overview of Biotechnological Developments

for Sugarcane Improvement 1868.7 By-Products of Sugarcane Processing 188

8.7.1 Bagasse 1888.7.2 Molasses 1898.7.3 Vinasse 189

8.8 Applications of Sugarcane for Biorefinery Concept 189

Contents xi

8.9 Utilization of Sugarcane Residue forBioethanol Production 190


9 Lignocellulosic Biomass Availability Map: A GIS-Based Approach for Assessing Production Statistics of Lignocellulosics and its Application in Biorefinery 197

Sanjeev Kumar, G. Lohit Kumar Srinivas and Rintu Banerjee9.1 Introduction 1989.2 Geographical Information System (GIS) 1999.3 Application of GIS in Mapping Lignocellulosic Biomass 2029.4 Biofuels from Lignocellulosics 2099.5 Conclusion 211References 212

10 Lignocellulosic Biomass Utilization for the Production of Sustainable Chemicals and Polymers 215

Gunjan Mukherjee, Gourav Dhiman and Nadeem Akhtar10.1 Introduction 21610.2 Lignocellulosic Biomass 21610.3 Pretreatment Strategies 219

10.3.1 Physical Pretreatment 21910.3.1.1 Physical Comminution and Extrusion 21910.3.1.2 Pyrolysis, Irradiation and

Pulsed Electric Field 21910.3.2 Chemical Pretreatment 220

10.3.2.1 Acid and Alkali Pretreatment 22010.3.4 Thermophysical Pretreatments 22110.3.5 Thermochemical Pretreatments 222

10.3.5.1 Oxidation 22210.3.6 Biological Pretreatment 223

10.4 Value-Added Chemicals from Lignocellulosic Biomass 22410.4.1 Lignocellulose-Derived Sugars 22410.4.2 Lignin-Derived Chemicals 225

10.4.2.1 Vanillin 22610.4.2.2 Vanillin-Based Resins 22610.4.2.3 Cyanate Ester Resins 22610.4.2.4 Epoxide Resins 22710.4.2.5 Benaoxazine Resins 22710.4.2.6 Polyester 22710.4.2.7 Polyurethanes 227

xii Contents

10.5 Sustainable Polymers from Lignocellulosic Biomass 22810.5.1 Sugar-Containing Polymers 228

10.5.1.1 1,4-Diacid-Based Polymers 228 10.5.1.2 5-(Hydroxymethyl) Furfural

(HMF)- and 2,5-Furandicarboxylic Acid (FDCA)-Based Polymers 229

10.5.1.3 3-HPA (3-Hydroxy Propionic Acid)Platform-Based Polymers 229

10.5.1.4 Aspartic Acid Platform-BasedPolymers 230

10.5.1.5 Glutamic Acid Platform-BasedPolymers 230

10.5.1.6 Glucaric Acid-Based Polymers 230 10.5.1.7 Itaconic Acid (ITA)

Platform-Based Polymers 231 10.5.1.8 Levulinic Acid Platform-Based

Polymer 231 10.5.1.9 3-Hydroxy-Butyrolactone (3-HBL)

Platform-Based Polymer 23210.5.1.10 Sorbitol-Based Polymers 23210.5.1.11 Glycerol-Based Polymers 23210.5.1.12 Lactic Acid-Based Platform 23310.5.1.13 Acetone-Butanol-

Ethanol-Based Polymer 23310.5.1.14 Xylose/Furfural/Arabinitol

Platform-Based Polymer 23310.5.1.15 Polyhydroxyalkanoate (PHA) 23410.5.1.16 Rubber Polymers 23410.5.1.17 Other Lignocelluolse-Derived

Polymers 23410.6 Potential Challenges for a Sustainable Biorefinery 23410.7 Environmental Effects of Biorefineries 23510.8 Future Perspectives of Biorefineries and Their Products 23610.9 Conclusion 236References 237

Contents xiii

11 Utilization of Lignocellulosic Biomass for Biobutanol Production 247

Anand Prakash, Vinay Sharma, Deepak Kumar,

Arindam Kuila and Arun Kumar Sharma11.1 Introduction 24711.2 Bioconversion of Lignocellulosic Biomass to Biobutanol 24811.3 Composition of Lignocellulosic Biomass 24811.4 Structure of Lignocellulosic Biomass 24811.5 Biobutanol Production from Lignocellulosic Biomass 249

11.5.1 Pretreatment 25011.5.2 Hydrolysis 250

11.5.2.1 Cellulases and Xylanases 25111.5.2.2 The Cellulase of Trichoderma

reesei RUT-C30 25311.5.3 Fermentation 255

11.5.3.1 Development of New Fermentation Technologies 257


12 Application of Lignocellulosic Biomass in the Paper Industry 265

Mainak Mukhopadhyay and Debalina Bhattacharya12.1 Introduction 26512.2 Major Raw Materials Used in the Paper Industry 266

12.2.1 Agricultural Residues 266 12.2.1.1 Sugarcane Bagasse 266 12.2.1.2 Corn Stalks 267 12.2.1.3 Rice Straw, Wheat Straw and

Cereal Straw 267 12.2.1.4 Bamboo 267 12.2.1.5 Sabai Grass 267 12.2.1.6 Jute 268 12.2.1.7 Ramie 268 12.2.1.8 Leaf Fibers 268 12.2.1.9 Cotton Fibers 268

12.2.1.10 Cotton Rags 26812.3 Pulp and Papermaking Process 269

12.3.1 Pulping Process 269 12.3.1.1 Mechanical Pulping 269 12.3.1.2 Chemical Pulping 270

xiv Contents

12.3.2 Bleaching Process 270 12.3.2.1 Chlorine Bleaching 271 12.3.2.2 Elemental Chlorine Free Bleaching

(ECF Bleaching) 271 12.3.2.3 Total Chlorine Free Bleaching

(TCF Bleaching) 271 12.3.2.4 Hydrogen Peroxide (H

2O

2)

Brightening 27212.4 Waste Generation 272

12.4.1 Wastewater 27212.4.2 Rejects 27212.4.3 Green Liquor Sludge, Dregs and Lime Mud 27212.4.4 Wastewater Treatment Sludge 27312.4.5 Primary Sludge 27312.4.6 Secondary or Biological Sludge 27312.4.7 Organic Pollutants and Suspended Solids 27312.4.8 Organochlorine Compounds 27312.4.9 Inorganic Chemicals 274

12.4.10 Chlorophenolics 27412.4.11 Dioxins and Furans 274

12.5 Waste to Value-Added Products 27412.5.1 Biogas 27412.5.2 C5 and C6 Sugar Fermentation 27512.5.3 Value-Added Compounds from Lignins 27512.5.4 Organic Solutions 275


Index 279

Preface

Lignocellulosic materials such as agricultural residues (e.g., wheat straw, sugarcane bagasse, corn stover), forest products (hardwood and soft-wood), and crops such as switchgrass and salix, are becoming a potentsource for generating different valuable products. Lignocellulosic biomass is mainly composed of cellulose, hemicellulose and lignin, along withsmaller amounts of pectin, protein and extractives (soluble nonstructuralmaterials such as nonstructural sugars, nitrogenous material, chlorophyll and waxes). Cellulose and hemicellulose are the main constituents of lig-nocellulosic biomass, occupying a major portion of the fibrous structure of plant cell walls. This book, entitled Lignocellulosic Biomass Production and Industrial Applications, describes the utilization of lignocellulosic bio-mass for different possible applications. Although there have been numer-ous reports on lignocellulosic biomass for biofuel application, there have been very few other applications reported for lignocellulosic biomass-based chemicals, polymers, etc. Therefore, this book covers all of the pos-sible lignocellulosic biomass applications. It describes the different types of biofuel production, such as bioethanol, biobutanol, biodiesel and biogas,from lignocellulosic biomass. Also presented are various other lignocel-lulosic biomass biorefinery applications for the production of chemicals, polymers, paper and bioplastics. In addition, there is a discussion of the major benefits, limitations and future prospects of the use of lignocellu-losic biomass.

Arindam KuilaVinay Sharma

Banasthali, IndiaFebruary 2017

xv

1

Arindam Kuila and Vinay Sharma (eds.) Lignocellulosic Biomass Production and Industrial

Applications, (1–26) © 2017 Scrivener Publishing LLC

1

Valorization of Lignocellulosic Materials to Polyhydroxyalkanoates (PHAs)

Arpan Das

Department of Microbiology, Maulana Azad College, Kolkata,

West Bengal, India

AbstractBiobased products have generated great interest since sustainable development

policies are expanding along with decreasing fossil fuel reserves and growing

environmental concerns. Among the petrochemical products, synthetic plastic

plays an important role in human daily life, but its recalcitrant properties cause

pervasive environmental pollution. In this regard, Polyhydroxyalkanoates (PHAs)

are very encouraging resources that might serve as an eco-friendly alternative to

petrochemical plastics. But the main obstacle is the cost of that polymer material,

which is used as a carbon source during the production of PHAs. Lignocellulosic

biomasses represent a very promising substrate for PHA production as they are

cheap, abundant and do not compete with the human food chain. Lignocellulosic

hydrolysates with a wide range of sugars and organic acids can extensively influ-

ence the overall yield of PHAs. This chapter provides a glimpse into the current

research focusing on the production of PHAs using lignocellulosic materials as

main carbon source.

Keywords: Polyhydroxyalkanoates, agrowastes, lignocellulose, cellulose,

hemicellulose, lignin

1.1 Introduction

The accumulation of petrochemical polymers in our surroundings and growing awareness of environmental pollution throughout the world has

Corresponding author: [email protected]

2 Lignocellulosic Biomass Production and Industrial Applications

triggered the search for new biocompatible products for a safe environ-ment. Currently, most polymer products are designed and prepared syn-thetically and very limited consideration is given to their ultimate disposal. However, these nondegradable plastics are building up in the environment at the rate of 25 million tons per year, which may persist for hundreds of years. Under these circumstances it is worth designing and developing appropriate biodegradable materials whose disposal ensures a better envi-ronment and ecosystem. Polyhydroxyalkanoates (PHAs) are biodegradable and biocompatible plastics that have been identified as an alternative to petroleum-based synthetic plastics. This type of polyester polymer is pro-duced by many bacteria, archaea as well as some fungi. It accumulates as discrete granules to levels as high as 90% of cell dry weight as a response toenvironmental stress and nutrient imbalance (when the carbon substrate is in excess of other nutrients such as nitrogen, sulfur, phosphorus or oxygen [1]), and plays a role as a sink for intracellular energy and carbon storage. These water insoluble storage polymers are biodegradable, exhibit thermo-plastic properties and can be produced from different renewable carbon sources. PHAs are high molecular mass polymers with properties similarto conventional plastics such as polypropylene. Therefore, they have a wide range of applications such as in the manufacture of bottles, packaging mate-rials, films for agriculture and also in medical applications [2, 3]. The mainadvantage is that the biodegradable polymers are completely degraded towater, carbon dioxide and methane by anaerobic microorganisms in vari-ous environments such as soil, sea, lake water and sewage and, hence, are easily disposable without harm to the environment. However, the high cost of PHA production compared to cheap petrochemical polymers, prevents their use on an industrial scale. Continuous efforts are being made and several studies are going on to develop a cost-effective strategy by using inexpensive substrates as a carbon source, which can significantly affect the production of PHA and has become an important objective for the commercialization of bioplastics. Since about 45% of the total cost of PHAproduction are attributed to carbon sources, such as refined glucose or sucrose [4], cheap wastes from agriculture and the food industry are used as inexpensive carbon substrates, thus improving the economic feasibility of PHA production. Moreover, lignocellulosic biomasses are considered to be very promising renewable sources for the biotechnological produc-tion of fuels and chemicals, including PHA. Lignocellulose hydrolysate isa potentially inexpensive and renewable feedstock that can be processedthrough different physical, chemical or enzymatic processes to ferment-able sugars such as glucose, galactose, xylose, and mannose. However, during the process to produce fermentable sugars, other by-products like

Valorization of Lignocellulosic Materials 3

acetic acid, 5-hydroxymethyl furfural, formic acid, phenolic compounds,etc., are released during the treatment of hemicellulose and lignin. Thesecompounds are exceedingly toxic to microorganisms during subse-quent fermentation processes. In order to increase the fermentability of the hydrolysate, a number of detoxifcation methods are also required to remove potential inhibitors. Overliming [5], activated charcoal [6], mem-brane filtration [7], ion exchange resins [8], and biological treatments [9] are among the most frequently used treatments.

1.1.1 What is PHA?

Polyhydroxyalkanoates (PHAs) are storage compounds that are widely produced by many microorganisms under nutrient-limited growth condi-tions, such as nitrogen, phosphorous or oxygen starvation, and when an excess of carbon source is present [10]. These storage materials serve as the carbon and energy reserves of the producing microorganisms. Generally, PHAs are considered as an alternative to petrochemical-based syntheticpolymers. Based on the chain length of the fatty acid monomers, PHAs canbe classified into three categories: short-chain-length (scl) PHAs with 3 to5 carbon atoms, medium-chain-length (mcl) PHAs with 6 to 14 carbon atoms and long-chain-length (lcl) PHAs with more than 14 carbon atoms [11]. The difference in length and/or chemical structure of the alkyl side chain of the PHAs influences the material properties of the polymers to a great extent [12]. In general, the scl-PHAs are more crystalline than themcl-PHAs. As such, scl-PHAs usually exhibit thermoplastic-like proper-ties, while mcl-PHAs and lcl-PHAs behave like elastomers or adhesives.Due to their physical characteristics, scl-PHAs can be used for manufac-turing items for packaging or everyday plastics commodities. However, PHAs are disadvantaged due to their significantly higher production costs, while a major portion of the final cost is represented by the price of carbon substrate (28–50%). Therefore, research has focused on inexpensive fer-mentable raw materials as substrates for biotechnological PHA production.

1.1.2 Mechanism of PHA Production

Polyhydroxybutyrate is the intracellular granule, synthesized by bacteria,and acts as an energy storage facility. In some Bacillus sp., it provides energy for sporulation [13]. The low molecular weight P(3HB) is a part of bacte-rial Ca2+ channels [14]. These granules are synthesized by the microorgan-isms in a limited concentration of O, N, P, S, or trace elements, e.g., Mg,Ca, Fe and high carbon concentration in the medium [15]. Generally these


nutrient sources are used for the synthesis of proteins essential for the growth in bacteria. But, nitrogen source depletion leads to the cessationof protein synthesis, which in turn leads to the inhibition of tricarbox-ylic acid cycle (TCA cycle) enzymes, such as citrate synthase and isocitrate dehydrogenase, and consequently slows down the TCA cycle [16]. As aresult, the acetyl-CoA routes to P(3HB) biosynthesis. Both the shorten-ing of external nutrients and internal sources, such as RNA or enzymes, facilitate the PHA synthesis. Figure 1.1 is a schematic representation of glucose and xylose metabolism for PHA production. Xylose is assimilated in bacteria by the pentose phosphate pathway through isomerization to d-xylulose by xylose isomerase, followed by a phosphorylation by xylu-lokinase that produces d-xylulose 5-phosphate, finally yielding glucose 6-phosphate. It has been noticed that in some bacteria like Pseudomonas

Figure 1.1 Flow diagram of polyhydroxyalkanoates (PHA) production from

lignocellulose precursor molecules.

Xylose

Xylulose

Xylulose-5P

Fructose-6P

Glyceraldehyde-3P

Pyruvate2 KDPG

Glucose

Glucose-6P

Acetyl CoA Malonyl CoA

R-3-Hydroxyacyl ACP

R-3-Hydroxyacyl COA

PHAs

TCA cycle


putida, the enzymes responsible for converting xylose to the entry inter-mediate xylulose 5-phosphate of PP pathway are missing. By introducing the relevant enzymes XylA and XylB, P. putida KT2440 was able to utilize xylose [11].

From Figure 1.1 it can also be seen that PHB formation and the TCA cycle share the same precursor, acetyl-coenzyme A (acetyl-coA), indicat-ing that when synthesizing PHAs using aerobic bacteria, the role of oxy-gen becomes crucial. It is also reported that when dissolved oxygen (DO) is limited to a certain degree (30–60%), the PHA production quantity changes. The best DO level for optimal PHA production has been found to be 30%. The mechanism behind this is that, under limited DO conditions, an influx of acetyl-coA will move towards PHA production and away from the TCA cycle [17].

1.2 Lignocellulose: An Abundant Carbon Sourcefor PHA Production

Chemically, lignocellulose, the most abundant raw material on earth, is com-posed of two linear polymers, cellulose and hemicellulose with a nonlinear lignin polymer [18]. In addition, small amounts of other materials, such as ash, protein, pectin, etc., are present in different degrees based on the source. Lignocellulose is physically hard, dense and recalcitrant towards degrada-tion. However, it is an extremely rich and abundant source of carbon andchemical energy, therefore, the recycling of carbon involving lignocelluloses is essential to maintain the global carbon cycle. Although the composition of lignocellulose strongly depends on the type and origin of the particu-lar plant biomass (Table 1.1), the average proportions (w/w) are as follows: 35–50% cellulose, 20–40% hemicellulose, and 5–30% lignin [19, 20].

1.2.1 Cellulose

Cellulose, the most widespread organic material in the world, is the pri-mary product of photosynthesis in terrestrial environments. Its regen-eration occurs rapidly, and it does not represent a direct food resource for humans [21]. Cellulose naturally occurs in wood, hemp and other plant-based materials and serves as the dominant reinforcing materialin plant structures. This biopolymer is also synthesized by algae, tuni-cates, some fungi, invertebrates and certain bacteria belonging to thegenera Acetobacter, Agrobacterium, Alcaligenes, Pseudomonas, Rhizobiumor Sarcina. Even some amoeba (protozoa, for example, Dictyostelium


discoideum) can synthesize cellulose [22, 23]. Since its discovery in 1838 by Payen, the chemical and physical properties of cellulose have been extensively investigated. A number of efforts of scientists from very dif-ffferent fields have been dedicated to understanding and controlling its biosynthesis, assembly and structural features. It is a linear condensation polymer consisting of D-anhydroglucopyranose joined together by β-1,4-glycosidic bonds with a degree of polymerization (DP) from 100 to 20,000 [24]. It also has a technical name, 1,4-β-polyanhydroglucopyranose. Every d-glucose unit is corkscrewed at 180° with respect to its neighbors, and the repeated segment is frequently treated as a dimer of glucose, known as cel-lobiose. Each cellulose chain possesses a directional chemical asymmetry with respect to the terminus of its molecular axis: one end is a chemical reducing functionality (hemiacetal unit) and the other is a hydroxyl group,known as the non-reducing end. Coupling of adjacent cellulose chains and sheets of cellulose by hydrogen bonds and van der Waals forces results in a parallel alignment and a crystalline structure with straight, stable supramo-lecular fibers of great tensile strength and low accessibility, which is known as cellulose microfibril. Due to the fact that cellulose possesses a substan-tial degree of crystallinity, it functions as a rigid, load-bearing component of the cell wall. The individual chains in these fibrils are associated in vari-ous degrees of parallelism. Regions containing highly oriented chains are called crystallites; those in which the chains are more randomly oriented are termed amorphous. In naturally occurring cellulose, the degree of crys-tallinity varies between 40% and 90% and the rest of the cellulose is amor-phous. The amorphous regions are the target site for enzymatic hydrolysis

Table 1.1 Variations in cellulose, hemicellulose and lignin composition in differ-

ent lignocellulosic materials.

Lignocellulose Cellulose Hemicellulose Lignin Ref.

Sugar cane bagasse 42 25 20 [66]

Rice straw 32.1 24 18 [67]

Wheat straw 29–35 26–32 16–21 [68]

Newspaper 40–55 25–40 18–30 [69]

Agricultural residues 37–50 25–50 5–15 [70]

Hardwood 45–47 25–40 20–25 [71]

Softwood 40–45 25–29 30–60 [71]

Grasses 25–40 35–50 – [70]


and these regions facilitate the penetration and adsorption of enzyme. The resistance of celluloses to enzymatic breakdown is a function of theirdegree of crystallinity. Furthermore, the rigidity of the cellulose microfibril is strengthened within a matrix of hemicellulose lignin and pectin.

1.2.2 Hemicelluloses

Hemicellulose is the second most abundant component of lignocellulosic biomass. The dominant sugars in hemicelluloses are mannose in softwoods and xylose in hardwoods and agriculture residues. Furthermore, these heteropolymers contain galactose, glucose, arabinose, and small amountsof rhamnose, glucuronic acid, methyl glucuronic acid, and galacturonic acid [25]. The average degree of polymerization of hemicelluloses is in therange of 80–200. They are usually associated with various other cell wall components such as cellulose, cell wall proteins, lignin, and other phenolic compounds by covalent and hydrogen bonding, and by ionic and hydro-phobic interactions [26]. In contrast to cellulose, which is crystalline andstrong, hemicellulose have a random, amorphous, and branched structurewith little resistance to hydrolysis, and they are more easily hydrolyzed by acids to their monomer components. Composition of hemicelluloses is very variable in nature and depends on the plant source.

1.2.3 Lignin

Lignin, the third main heterogeneous polymer in lignocellulosic residues, is a very complex molecule constructed of aromatic alcohols, includ-ing coniferyl alcohol, sinapyl and p-coumaryl units linked in a three-dimensional structure [27]. It is present in the middle lamella and acts as cement between the plant cells. It is also located in the layers of the cell walls, forming, together with hemicelluloses, an amorphous matrix in which cellulose fibrils are embedded and protected against biodegra-dation. Lignin acts as a binder of the lignocellulosic constituents, giving the plant structural support, impermeability, and resistance against micro-bial attack and oxidative stress. Not surprisingly, lignin is the most recal-citrant component of the plant cell wall, and the higher the proportion of lignin, the higher the resistance to chemical and enzymatic degradation[28]. Generally, softwoods contain more lignin than hardwoods and most of the agriculture residues. There are chemical bonds between lignin and hemicellulose and even cellulose. Lignin is one of the drawbacks of usinglignocellulosic materials in fermentation, as it makes lignocellulose resis-tant to chemical and biological degradation.


1.2.4 Pectin

Pectins are polymers of d-galactopyranosyluronic acids joined by α-d-(1→4) glycosidic linkages. The main chain can be modified in various ways (ramification with neutral sugars, esterification, acetylation) [29]. Pectin isan acidic cell wall polysaccharide that functions as a sol-like matrix, pro-viding water and ion retention, support and facilitation of cell wall modi-fying enzymes, cell wall porosity, cell-to-cell adhesion, cell expansion, cellsignaling, developmental regulation, and defense [30].

1.3 Lignocellulosic Pretreatment Techniques

The structure of cellulose imparts tightly packed arrangements that are water insoluble and resistant to depolymerization [31]. Thus, it is impera-tive that a pretreatment regime alter the structure of biomass to make thecellulose more accessible to hydrolysis. A glimpse of different pretreatmentprocesses is shown in Table 1.2. An effective pretreatment must meet the following requirements: (1) increase the accessible cellulose surface area,(2) disrupt the lignin barrier as well as cellulose crystallinity to allow proper enzymatic attack, (3) limit the formation of toxic degradation products that are inhibitory towards the enzymes or fermentative microorganisms, (4) reduce the loss of sugar components (cellulose and hemicellulose) and (5) minimize the capital and operating costs. Wide spectrums of pretreat-ment protocols have been investigated for hydrolysis and a few of themhave been developed sufficiently to be called technologies. Pretreatmentapproaches can be broadly classified into four categories: (1) physical; (2) chemical; (3) physicochemical and (4) biological.

1.3.1 Physical Pretreatment Techniques

Physical methods of pretreatment like milling and steam treatment will reduce particle sizes thereby increasing the available surface area for enzy-matic attack. Steam explosion loosens the crystalline complex and alsoremoves the pentose while increasing the surface area. However, the draw-back of the process is that steam treatment may generate certain cellulaseinhibitors that can interfere with the enzymatic hydrolysis of the cellulosic substrate.

1.3.1.1 Milling

Milling can be employed to alter the inherent ultrastructure of lignocellu-loses and degree of crystallinity, and consequently make it more accessible

Valorization of Lignocellulosic Materials 9T

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to enzymatic degradation. Milling and particle size reduction have been applied prior to enzymatic hydrolysis, or even other pretreatment pro-cesses with dilute acid, steam or ammonia, on several lignocellulosic waste materials [31, 32]. Among the milling processes, colloid mill, fibrillator and dissolver are suitable only for wet materials, while the extruder, roller mill, cryogenic mill and hammer mill are usually used for dry materials. The ball mill can be used for both dry and wet materials. Grinding with hammer milling of waste paper is a favorable method [33]. Milling can improve susceptibility to enzymatic hydrolysis by reducing the particle size and degree of crystallinity of lignocelluloses, which improves enzymatic degradation of these materials.

1.3.1.2 Irradiation

Irradiation by gamma rays, electron beam and microwaves can improveenzymatic hydrolysis of lignocelluloses. The combination of the pre-radiation and other methods, such as acid treatment, can further acceler-ate degradation of cellulose into glucose. The cellulose component of the lignocellulose materials can be degraded by irradiation to fragile fibers and low molecular weight oligosaccharides and even cellobiose, that could be due to preferential dissociation of the glucoside bonds of the cellulose chains by irradiation in the presence of lignin. But a very high irradiation can lead to the decomposition of oligosaccharides and the glucose ring structure [34].

1.3.2 Chemical Pretreatment

In general, chemical pretreatment processes selectivity degrades the bio-mass component, but they involve relatively harsh reaction conditions, which may not be ideal in a biosaccharification scheme due to adverse effects on downstream biological processing. Different chemical pretreat-ments that are generally practiced include acid, alkaline, ozonolysis, oxi-dative H

2O

2 delignification, organosolv, etc. [31, 35]. Besides these, their

combinational effects have also been found suitable. However, utilization of various chemicals in the pretreatment procedures is a major drawback and affects the total economy of the bioconversion of the lignocellulosic biomass.

1.3.2.1 Acid Hydrolysis Pretreatment

Both concentrated and diluted acids such as H2SO

4, HCl and perchloric acids

have been used to treat lignocellulosic materials. Pretreatment with acid


hydrolysis can result in improvement of enzymatic hydrolysis of lignocel-lulosic biomasses to release fermentable sugars. Although they are powerfulagents for cellulose hydrolysis, concentrated acids are toxic, corrosive, haz-ardous, and thus require corrosion resistant reactors, which makes the pre-treatment process very expensive. In addition, the concentrated acid must be recovered after hydrolysis to make the process economically feasible [36].

1.3.2.2 Alkaline Hydrolysis

The effect of alkaline pretreatment depends on the lignin content of thelignocellulosic materials. Alkali pretreatment processes can be effective at lower temperatures and pressures than many other pretreatment technolo-gies, but it requires longer times on the order of hours or days. Compared with acid, alkaline pretreatments cause less sugar degradation, and many of the caustic salts can be recovered and/or regenerated [37]. Sodium, cal-cium, potassium, and ammonium hydroxides are widely used alkaline pre-treatment agents. Out of these, sodium hydroxide has been studied themost. However, calcium hydroxide (slake lime) also has been shown to bean effective pretreatment agent and is the least expensive.

1.3.2.3 Oxidative Delignification by Peroxide

Lignin biodegradation has been reported to be catalyzed in the presenceof H

2O

2. The pretreatment of cane bagasse with hydrogen peroxide greatly

enhanced its susceptibility to enzymatic hydrolysis [38]. About 50% of thelignin and most of the hemicellulose were solubilized by 2% H

2O

2 at 30 °C

within 8 h, and 95% efficiency of glucose production from cellulose was achieved in the subsequent saccharification by cellulase at 45 °C for 24 h. Wet oxidation combined with base addition readily oxidizes lignin fromwheat straw, thus making the polysaccharides more susceptible to enzy-matic hydrolysis. Furfural and hydroxymethylfurfural, known inhibitors of microbial growth when other pretreatment systems are applied, werenot observed following the wet oxidation treatment.

1.3.2.4 Organosolv Process

The organosolvation method is a promising pretreatment strategy, and it has attracted much attention and has proven potential for utilization in lignocellulosic pretreatment. In this process, an organic or aqueous organic solvent mixture with inorganic acid catalysts (HCl or H

2SO

4) is

used to break the internal lignin and hemicellulose bonds [39]. The com-monly used solvents in the process are methanol, ethanol, acetone, ethylene


glycol, triethylene glycol, and tetrahydrofurfuryl alcohol. Other organic acids like oxalic, acetylsalicylic, and salicylic acids can also be used as cata-lysts in the organosolvation process. Treatment of lignocellulosic materials with these organosolvs at temperatures ranging from 140 to 220 °C causes lignin breakdown into fragments which are quite soluble in the solvent system [40]. This technique yields three separate fractions: dry lignin, an aqueous hemicellulose stream, and a relatively pure cellulose fraction.

1.3.2.5 Ozonolysis Pretreatment

Ozone pretreatment is one way of reducing the lignin content of lignocellu-losic wastes which results in an increase of the in-vitro enzymatic digestibil-ity of the treated material, and unlike other chemical treatments, it does notyield toxic products [41]. Although ozone can be used to degrade lignin and hemicellulose in many lignocellulosic materials, such as wheat straw, ricestraw, bagasse, peanut, pine, cotton straw, sawdust, etc., the degradation is mainly limited to lignin. In this process hemicellulose portions are slightly affected, but cellulose is not. Ozonolysis pretreatment has an advantage inthat the reactions are carried out at room temperature and under normal pressure. Furthermore, after pretreatment ozone can be easily decomposed by using a catalytic bed or increasing the temperature to minimize environ-mental pollution. A drawback of ozone pretreatment is that a large amount of ozone is required, which can make the process expensive [42].

1.3.2.6 6 Ionic Liquids Pretreatment

Another technology for lignocellulose fractionation is using ionic liquids. Ionic liquids (ILs) are organic salts which exist as liquids at low tempera-tures; often well below 100 °C. They have negligible (or very low) vaporpressures, generally good thermal stability and there is a variety of com-binations of anions and cations that can be used to synthesize ILs [43]. Recent studies have showed that cellulose and lignin both can be dissolved in a variety of ILs and can be easily regenerated from these solutions by means of addition of a non-solvent [44]. Dadi et al. [45] used 1-n-butyl-3-methylimidazolium chloride to dissolve cellulose. The regenerated cellulosehad an amorphous structure allowing a greater number of sites for enzyme adsorption and improving the enzymatic hydrolysis rate by 50-fold.

1.3.3 Physico-Chemical Pretreatment

Physico-chemical pretreatment is a combination of different processes for chemical and physical treatments. In these procedures, milder chemical

lignocellulosic biomass - startseite€¦ · 6.4.3.4 organosolv process 123 6.4.3.5 ionic liquids...

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