Biotechnology in Biopolymers : Developments, Applications & Challenging Areas [1 ed.] 1847355420, 1847355447, 9781847355423, 9781847355447

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas

Edited by Atul Tiwari and Ravi B. Srivastava

Biotechnology in Biopolymers Developments: Applications & Challenging Areas Edited by Atul Tiwari and Ravi B. Srivastava

A Smithers Group Company Shawbury, Shrewsbury, Shropshire, SY4 4NR, United Kingdom Telephone: +44 (0)1939 250383 Fax: +44 (0)1939 251118 http://www.polymer-books.com

First Published in 2012 by

Smithers Rapra Technology Ltd Shawbury, Shrewsbury, Shropshire, SY4 4NR, UK

©2012, Smithers Rapra Technology Ltd

All rights reserved. Except as permitted under current legislation no part of this publication may be photocopied, reproduced or distributed in any form or by any means or stored in a database or retrieval system, without the prior permission from the copyright holder.

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ISBN: 978-1-84735-542-3 (Hardback) 978-1-84735-543-0 (Softback) 978-1-84735-544-7 (ebook)

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D

edication

1944–2007 Dedicated to Prof. Suresh K. Nema, an eminent materials scientist from India

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas

iv

P

reface

The vast majority of polymers and polymeric composite products that are presently used for various applications are made from petroleum-based synthetic polymers, which do not degrade in a landill or in a compost-like environment. An environmentally conscious alternative is to design/synthesise biopolymers that are biodegradable. In recent times, biotechnological approaches have been increasingly recognised as a key to developing low-cost biopolymers and better biodegradables. Scientists and engineers have been working together to use the inherent strength and performance of biodegradable polymers and natural ibre-reinforced polymers to produce a new class of biobased composites, due to increasing environmental concerns and considerably low price reinforcements in biobased composites. Recent interest in developing sustainable composites, or biocomposites, capable of being recycled and biodegraded, has been growing in many countries, mainly in those where environmental legislation is stricter. The microstructure of biopolymer composites is known to inluence their functional properties, particularly in matrices that serve as carriers of functional components such as lavours or antioxidants. The natural ibres have limited thermal stability and, therefore, thermal degradation may take place during composite processing at high temperatures. The use of lignin in developing polymer blends and composites has shown encouraging results for various commercial, environmental and strategic applications. Similarly, biocomposite foams comprising polyvinyl alcohol, sodium alginate and bioactive glass as illers have been synthesised in order to provide effective physical and biological properties. Biopolymer technology is emerging rapidly, especially in the chemical technology sector. However, other industrial sectors are not moving at the same pace towards biopolymer-based processes and manufacturing strategies. The combination of functional properties, such as thermal, electrical, conducting, optical and biological, has led to the development of a wide range of biofunctional materials, which have provided different biological compatible strategies for the development of ecofriendly technologies. It is envisaged that biopolymers at some point in the future may become the common basis of scaffolding systems for a tissue-engineering approach in order to treat various diseases. Discoveries in modern biotechnology have provided

v

Biotechnology in Biopolymers Developments, Applications & Challenging Areas several effective tools to investigate both the molecular design of silk ibre as well as bioengineering of natural silks in the lab using established recombinant DNA techniques. The production of ibre materials with custom-engineered mechanical properties has been a potential outcome of this technology. Biopolymers and biopolymeric composites nowadays are used as ibres, ilms, sheets, pipes and tubes, woven fabrics, nettings, mouldings, bags and in a variety of other applications. It is increasingly being realised that the use of long-lasting polymers for short-lived applications is not entirely justiied and bioplastics or biodegradable polymers need to be developed from renewable sources for future applications, particularly for agriculture and food-related areas. There is a need to increase the use of food grade and biodegradable polymers that have a direct effect on human health and the environment. This book is about the role of biotechnology in biopolymers and is intended to provide an overview of the development, applications and challenging areas in biopolymers. The book comprises 14 chapters. Chapter 1 provides an overview of biopolymers and Chapter 2 covers spectroscopic analysis of biopolymers. In Chapter 3, thermal analyses of biopolymers are discussed. Chapter 4 highlights the mechanical properties of biopolymers. Chapter 5 presents natural ibres and their uses. Chapter 6 covers various applications of lignin as natural biopolymers. Chapter 7 presents biodegradable polymers and polymeric composites. Chapter 8 discusses the applications of smart chitosan matrices. Chapter 9 is dedicated to biopolymeric scaffolds for tissue engineering. Biopolymer composite artiicial muscles are discussed in Chapter 10. The role of silicon polymers in biomedical engineering is described in Chapter 11 while the use of biotechnology in the development of silk protein is detailed in Chapter 12. The role of biodegradable polymers in agriculture and food safety is explained in Chapter 13. Finally, Chapter 14 provides an overall picture and new insights into the adoption of biopolymers from an international perspective. Each chapter in this book has been written to provide in-depth understanding about the subject to the younger generation of researchers and to serve as a good reference for advanced learners. We are sure that this book will be useful to researchers working in the progressive interdisciplinary ield of biotechnology and materials science. Atul Tiwari, Ph.D., CChem Ravi B. Srivastava, Ph.D.

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C

ontents

1

Biopolymers: An Indispensable Tool for Biotechnology ............... 1 1.1

Introduction ...................................................................... 1

1.2

Spectroscopy Analysis of Biopolymers ............................... 1

1.3

Thermal Analysis of Biopolymers ...................................... 2

1.4

Mechanical Analysis of Biopolymers ................................. 3

1.5

Natural Fibres ................................................................... 5

1.6

Lignin for Various Applications ......................................... 6

1.7

Biodegradable Polymers and Composites ........................... 7

1.8

Engineering of Chitosan as a Smart Biopolymer ................ 8

1.9

Biopolymers for Tissue Engineering ................................... 9

1.10 Biopolymeric Ionic Composites ....................................... 10 1.11 Silicones in Biomedical Engineering ................................. 12 1.12 Biotechnology of Silk Proteins ......................................... 12 1.13 Plasticulture for Agriculture and Food ............................. 13 1.14 Adoption of Biopolymers for Various Applications.......... 15 1.15 Commercially Available Biopolymers............................... 16 2

Spectroscopic Analysis of Biopolymers ...................................... 17 2.1

Introduction .................................................................... 17

2.2

Characterisation by FTIR Analysis .................................. 18

2.3

Characterisation by Raman Spectroscopy ........................ 22

2.4

Characterisation by NMR Spectroscopy .......................... 24

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas

3

2.5

Characterisation by X-ray Diffraction Analysis ............... 27

2.6

Conclusions and Future Prospects ................................... 30

Thermal Analysis of Biopolymer Materials for High-performance Applications ................................................. 37 3.1

Introduction .................................................................... 37

3.2

Description of Methods ................................................... 38

3.3

3.2.1

Thermogravimetric Analysis ................................. 39

3.2.2

Studies in Thermogravimetric Analysis ................. 42

Differential Scanning Calorimetry ................................... 44 3.3.1

3.4

Differential Thermal Analysis .......................................... 46 3.4.1

3.5

3.7 4

viii

Studies in Differential Thermal Analysis ............... 47

Dynamic Mechanical Analysis ......................................... 48 3.5.1

3.6

Studies in Differential Scanning Calorimetric Analysis ........................................... 45

Studies in Dynamic Mechanical Thermal Analysis ................................................. 49

Factors Affecting the Thermal Stability of Biopolymers ... 50 3.6.1

Chemical Constituents of Biopolymer Formulation ......................................................... 50

3.6.2

Molecular Weight and Chain Length .................... 50

3.6.3

Nature of Chemical Bonding in the Material ........ 51

3.6.4

Presence of Fillers and Additives .......................... 51

Conclusion and Future Prospects ..................................... 51

Mechanical Properties of Biopolymers ....................................... 55 4.1

Introduction .................................................................... 55

4.2

Classiication of Polymers ................................................ 57

4.3

Mechanical Response of Polymers ................................... 59

4.4

Theory of Mechanical Behaviour ..................................... 61

Contents

4.5

Nonlinear Rheology of Cross-linked Biopolymer Gels ..... 63

4.6

Mechano-chemical Response of Biopolymers................... 65

4.7

4.8

5

4.6.1

Mechano-chemical Response of Biopolymers at Micro Level ...................................................... 66

4.6.2

Mechano-chemical Response of Biopolymers at Macro Level ......................................................... 69

4.6.3

Deformation Mechanism Map ............................. 70

Some Factors Affecting Mechanical Behaviour of Biopolymers ................................................................ 71 4.7.1

Physical Ageing .................................................... 71

4.7.2

Crystallinity ......................................................... 72

4.7.3

pH of the Medium ............................................... 74

4.7.4

Effect of Composition .......................................... 76

Conclusions and Future Prospects ................................... 76 4.8.1

Actuator, Sensor and Micro-electro-mechanical System Devices ..................................................... 76

4.8.2

In Tissue Engineering ........................................... 77

4.8.3

In Drug Delivery .................................................. 78

Natural Fibres and Their Use in the Production of Biocomposites............................................................................ 89 5.1

Introduction .................................................................... 89

5.2

Biocomposite Materials ................................................... 90

5.3

Natural Fibres ................................................................. 92

5.4

Surface Modiication – Does it Work? ............................. 93

5.5

Thermal and Mechanical Behaviour of Natural Fibres .... 95

5.6

Natural Fibres Classiication According to Their Source .. 96 5.6.1

Vegetable Fibres ................................................... 97

5.6.2

Sisal Fibre from Agave Plants ............................... 98

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas

5.6.3

Coconut (or Coir) Fibre ..................................... 100

5.6.4

Jute Natural Fibre .............................................. 102

5.6.5

Sugar Cane Fibre ................................................ 103

5.6.6

Abaca Fibre ........................................................ 106

5.6.7

Cotton Fibre ....................................................... 107

5.6.8

Kenaf Fibre ........................................................ 109

5.6.9

Bamboo Fibre ..................................................... 110

5.6.10 Açai Fibre ........................................................... 111 5.6.11 Curauá Fibre ...................................................... 112 5.6.12 Banana Fibre ...................................................... 114 5.6.13 Licuri Fibre ........................................................ 116 5.6.14 Ramie (Boehmeria Nivea)................................... 116 5.7 6

Conclusion and Future Prospects ................................... 117

Lignin: Amorphous Natural Biopolymer-based Blends and Composites for Various Applications ................................ 123 6.1

Introduction .................................................................. 123

6.2

Lignin: Origin and Chemistry ........................................ 124

6.3

Toxicity of Lignin .......................................................... 127

6.4

Biodegradation of Lignin ............................................... 128

6.5

Chemical Modiication of Lignin ................................... 130

6.6

Lignin-based Blends ....................................................... 132

6.7

Lignin-based Composites ............................................... 136

6.8

Other Applications of Lignin ......................................... 139

6.9

Developments in a Nutshell ........................................... 143

6.10 Conclusion and Future Prospects ................................... 145 7

Biodegradable Polymers and Polymeric Composites ................ 153 7.1

x

Introduction .................................................................. 153

Contents

7.2

Polymer Composites ...................................................... 155 7.2.1

7.3

Polymer Nanocomposites ................................... 155

Degradation Mechanism ................................................ 156 7.3.1

Abiotic Involvement ........................................... 157 7.3.1.1

Mechanical Degradation...................... 157

7.3.1.2

Photodegradation ................................ 157

7.3.1.3

Thermal Degradation .......................... 158

7.3.1.4

Chemical Degradation ......................... 159

7.3.1.5

Hydrolysis Reaction ............................ 159

7.3.1.6

Ultrasonic Degradation........................ 160

7.3.1.7

Biodeterioration................................... 161

7.3.1.8

Physical Activity .................................. 161

7.3.1.9

Chemical Activity ............................... 161

7.3.1.10 Enzymatic Activity............................... 161 7.3.1.11 Biofragmentation ................................. 162 7.3.1.12 Assimilation......................................... 162 7.3.1.13 Mineralisation ..................................... 162 7.4

Factors Affecting Degradation ....................................... 163 7.4.1

Temperature of Medium..................................... 164

7.4.2

pH of Medium ................................................... 164

7.4.3

Availability of Nutrients ..................................... 164

7.4.4

Chemical Composition ....................................... 164

7.4.5

Crystallinity/Amorphous .................................... 165

7.4.6

Microbial Consortium ........................................ 165

7.5

In Vivo Degradation of Implantable Devices.................. 166

7.6

Mechanisms Responsible for Alteration in the Rate of Polymer Degradation ........................................ 168 7.6.1

Nanocomposites/Blends ..................................... 168 xi

Biotechnology in Biopolymers Developments, Applications & Challenging Areas

7.6.2

Copolymerisation/Cross-linking ......................... 169

7.6.3

Incorporation of Additives ................................. 169

7.6.4

Photoinitiators ................................................... 170

7.6.5

Thermal Initiators .............................................. 170 7.6.5.1

Development of Genetically Modiied Microorganisms ................... 170

7.6.5.2

Synthesis of Artiicial Biopolymers....... 171

7.7

Measurement of Degradation ........................................ 171

7.8

Techniques Used to Assess Degradation ......................... 172

7.9

Applications .................................................................. 175 7.9.1

Agriculture ......................................................... 175

7.9.2

Plasticulture ....................................................... 175

7.9.3

Medicines ........................................................... 176

7.9.4

Polymers for Drug Delivery ................................ 177

7.9.5

Construction Industry ........................................ 177

7.9.6

Packaging Bioplastics: Recycling of Plastics ........ 177

7.10 Wastewater Treatment – Secondary Efluent .................. 181 7.11 Standardisation and Certiication .................................. 183 7.12 Conclusion and Future Prospects ................................... 183 8

xii

Smart Chitosan Matrices for Application to Cholesterol Biosensors ............................................................. 193 8.1

Introduction .................................................................. 193

8.2

Biosensors: Biorecognition Devices ................................ 196

8.3

Matrices Fabrication Methodology ................................ 202 8.3.1

Chemical Oxidative Method .............................. 202

8.3.2

Electrochemical Method ..................................... 203

8.4

Physico-chemical Blending ............................................. 206

8.5

Cholesterol Bioelectrodes ............................................... 212

Contents

8.6

8.7 9

Characterisations ........................................................... 213 8.6.1

Redox Behaviour ................................................ 213

8.6.2

Electrocatalytic Properties .................................. 217

8.6.3

Electrochemical Response .................................. 218

8.6.4

Photometric Response ........................................ 225

Conclusion and Future Perspectives ............................... 226

Biopolymeric Scaffolds for Tissue Engineering ......................... 233 9.1

Introduction .................................................................. 233

9.2

Biopolymers ................................................................... 234

9.3

Types of Biopolymers..................................................... 235 9.3.1

Polysaccharides .................................................. 235

9.3.2

Polypeptides ....................................................... 238

9.3.3

Polynucleotides .................................................. 239

9.3.4

Polyhydroxyalkanoates ...................................... 240

9.4

Chemistry of Biopolymers ............................................. 241

9.5

Polymeric Scaffolds ........................................................ 241

9.6

9.5.1

Conventional Polymeric Scaffolds ...................... 244

9.5.2

Supermacroporous Polymeric Scaffolds .............. 245

9.5.3

DNA Scaffolds ................................................... 247

Biomedical Applications of Biopolymers ........................ 249 9.6.1

Past and Present ................................................. 249

9.6.2

Tissue Engineering Applications ......................... 251 9.6.2.1

Bone tissue engineering ........................ 252

9.6.2.2

Cartilage tissue engineering ................. 254

9.6.2.3

Neural tissue engineering ..................... 256

9.6.2.4

Cardiac Tissue Engineering .................. 259

9.6.2.5

Skin Tissue Engineering ...................... 262

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas

9.6.2.6

10

Corneal Tissue Engineering ................. 266

9.7

Immune Response .......................................................... 267

9.8

Conclusion and Future Prospects ................................... 269

Biopolymer/Ionic Polymer Composite Artiicial Muscles ......... 287 10.1 Introduction .................................................................. 287 10.2 The Family of Biopolymer and Biomimetic Ionic Polymers ............................................................... 287 10.3 Chemical Modelling of Electroactive Polymeric Networks ....................................................................... 289 10.4 Three-dimensional Fabrication of Ionic Polymers/Biopolymers (IPMCs/Chitosan Sol-gels).......... 290 10.5 Modelling and Simulation ............................................. 296 10.6 Continuum Modelling of Charge Transport in Ionic Biopolymers .......................................................... 300 10.6.1 Basic Governing Equation on Charge Transport. 300 10.6.2 Constitutive Equations of Nernst–Planck ........... 300 10.6.3 Actuation Mechanism ........................................ 301 10.6.4 Sensing Mechanism ............................................ 301 10.6.5 Charge Continuity Equation .............................. 302 10.6.6 Charge Equilibrium Equation (Nernst–Planck Equilibrium Equation) ........................................ 302 10.6.7 Poisson’s Equation.............................................. 303 10.6.8 Poisson–Nernst–Planck (PNP) Equation for Charge Dynamic ................................................ 304 10.6.9 Reducing the Problem to One-dimensional Form 304 10.7 Conclusions and Future Prospects ................................. 306

11

Silicone Polymers in Biomedical Engineering Applications....... 309 11.1 Introduction .................................................................. 309

xiv

Contents

11.2 Chemistry of Silicones ................................................... 310 11.2.1 Chemical Structure of Silicone Polymers............. 311 11.2.2 Nomenclature of Silicone Polymers .................... 312 11.2.3 Synthesis of Silicone Polymers ............................ 314 11.2.3.1 Curing of Silicone Polymers ................. 317 11.2.4 Physico-chemical Properties of Silicone Polymers 322 11.3 Biomaterials ................................................................... 323 11.3.1 History of Biomaterial Development .................. 324 11.3.2 Polymers as Biomaterials .................................... 325 11.3.3 Silicone Polymers as Biomaterials ....................... 326 11.3.4 Surface and Bulk Modiication of Silicone Polymers for Medical Applications ..................... 326 11.3.4.1 Physical Modiication of the Silicone Polymer Surface ...................... 327 11.3.4.2 Plasma Pretreatments........................... 327 11.3.4.3 Corona Discharge ................................ 329 11.3.4.4 Laser Treatments ................................. 329 11.3.4.5 Chemical Modiication of the Silicone Polymer Surface ................................... 330 11.3.4.6 Surface Grafting .................................. 330 11.3.4.7 Radiation-induced Graft Polymerisation ..................................... 331 11.3.4.8 Laser-induced Grafting ........................ 331 11.3.4.9 Plasma-induced Grafting ..................... 332 11.3.4.10 Ozone-induced Graft Polymerisation . 334 11.3.4.11 Miscellaneous Surface Modiication Techniques ........................................... 335 11.3.5 Biocompatibility of Silicone Elastomers .............. 336 11.3.6 Biodurability of Silicone Elastomers ................... 339

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas

11.4 Biomedical Engineering Applications of Silicone Elastomers ........................................................ 341 11.4.1 Orthopaedic Applications .................................. 341 11.4.2 Artiicial Cardiac Valves ..................................... 342 11.4.3 Catheters, Stents and Drains .............................. 344 11.4.4 Aesthetic Implants .............................................. 347 11.4.4.1 Silicone Breast Implants ....................... 348 11.4.4.2 Facial Implants .................................... 349 11.4.5 Sensor Applications ............................................ 350 11.4.6 Tissue Engineering.............................................. 351 11.4.7 Medical Instruments ........................................... 354 11.4.7.1 Medical Devices................................... 354 11.4.8 Drug Delivery Systems ....................................... 355 11.4.9 Ophthalmologic Applications ............................. 357 11.5 Conclusions and Future Prospects ................................. 357 12

Biotechnology of the Silk Proteins: Challenges, Approaches and Applications .................................................. 371 12.1 Introduction .................................................................. 371 12.1.1 Silk Sources in Nature ........................................ 371 12.1.1.1 Silkworm Silk ...................................... 372 12.1.1.2 Spider Silk ........................................... 372 12.1.2 Silkworm Silk versus Spider Silk Source ............. 373 12.1.3 Natural Synthesis of Silk .................................... 373 12.1.3.1 Silk Gland............................................ 374 12.1.3.2 Silk Cocoons........................................ 375 12.1.3.3 Spider Web .......................................... 375 12.1.4 Structural Organisation of Silk Proteins ............. 376 12.1.4.1 Silkworm Silk Proteins......................... 376

xvi

Contents

12.1.4.2 Spider-silk Fibroin ............................... 377 12.2 Challenges of Natural Silks ............................................ 378 12.3 Biotechnological Approaches for Modiication for Native Silk Biopolymer ............................................ 379 12.3.1 Recombinant Silkworm Silk ............................... 379 12.3.2 Recombinant Spider Silk .................................... 381 12.4 Applications of Recombinant Silk .................................. 382 12.4.1 Films .................................................................. 382 12.4.2 Scaffolds ............................................................. 383 12.4.3 Hydrogels ........................................................... 383 12.4.4 Fibres ................................................................. 384 12.4.5 Microcapsules and Microspheres ....................... 384 12.5 Limitations .................................................................... 385 12.6 Conclusion and Future Prospects ................................... 385 13

Plasticulture for Agriculture and Food Security ....................... 395 13.1 Introduction .................................................................. 395 13.2 Biodegradability of Polymers ......................................... 396 13.3 Biotechnological Interventions ....................................... 396 13.4 Biodegradable Polymers of Bio-origin ............................ 401 13.5 Applications in the Agriculture and Food Sectors .......... 403 13.5.1 Protected Cultivation ......................................... 404 13.5.2 Biocontainers ..................................................... 407 13.5.3 Mulching ............................................................ 408 13.5.4 Irrigation System ................................................ 410 13.5.5 Controlled-release Fertilisers .............................. 412 13.5.6 Packaging ........................................................... 414 13.6 Climate Change and Food Security ................................ 415

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas

13.7 Biodegradable Polymers and Eco-management .............. 416 13.8 Conclusion and Future Prospects ................................... 418 14

Adoption of Biopolymers: An Analysis Based on American, Japanese and European Patent Data ........................................ 425 14.1 Introduction .................................................................. 425 14.2 Biopolymers ................................................................... 426 14.2.1 Deinition and Classiication .............................. 426 14.2.2 Production ......................................................... 427 14.2.3 Drivers ............................................................... 428 14.3 Patent Analysis .............................................................. 428 14.3.1 Methods ............................................................. 430 14.4 Discussion ..................................................................... 431 14.4.1 Biopolymer Patent Activity ................................. 431 14.4.2 Biopolymers by Industrial Sector ........................ 434 14.4.3 Assignee Analysis ............................................... 437 14.5 Conclusion and Future Prospects ................................... 441

Annexure .......................................................................................... 447 Abbreviations .................................................................................... 451 Index ............................................................................................... 459

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C

ontributors

Atul Tiwari Department of Mechanical Engineering, University of Hawaii at Manoa, Honolulu, 96822, HI, USA

Ravi B. Srivastava, Ashish Yadav Defence Institute of High Altitude Research (DIHAR), Defence Research & Development Organisation (DRDO), Leh-Ladakh, 194101, J&K, India

Anil K. Bajpai, Rajesh K. Saini, Sandeep K. Shukla Bose Memorial Research Laboratory, Government Model Science College (Autonomous), Jabalpur, 482001, MP, India

Lucia H. Innocentini Mei, André L. F. M. Giraldi Department of Polymer Technology, School of Chemical Engineering, State University of Campinas-UNICAMP, P.O. Box 6066, Campinas, 13083-970, SP, Brazil

Shivani B. Mishra Department of Chemical Technology, University of Johannesburg, PO Box 17011, Doornfontein, 2028, Johannesburg, South Africa

Tanushree Vishnoi Department of Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur, 208016, UP, India

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas

Ashutosh Tiwari Biosensors and Bioelectronics, IFM-Linköpings Universitet, 581 83 Linköping, Sweden

Ashok Kumar Department of Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur, 208016, UP, India

Anuj Tripathi Nuclear Agriculture and Biotechnology Division, FIPLY, Lab No 79, Bhabha Atomic Research Center, Trombay, Mumbai, 400 085, India

Mohsen Shahinpoor Biomedical Engineering Laboratory, Mechanical Engineering Department, University of Maine, Orono, 04469, ME, USA

Golok B. Nando, A.G. Jineesh Rubber Technology Center, Indian Institute of Technology, Kharagpur, 721302, India

Sunita Nayak, Nandana Bhardwaj, Sarmistha Talukdar, Banani Kundu, Subia Bano, S.C. Kundu Department of Biotechnology, Indian Institute of Technology, Kharagpur, 7213 02, India

Avrath Chadha Department of Management, Technology, and Economics, ETH Zurich, Kreuzplatz 5, CH-8032 Zurich, Switzerland

x

1

Biopolymers: An Indispensable Tool for Biotechnology

Atul Tiwari, Ravi B. Srivastava, Rajesh K. Saini, Anil K. Bajpai, Lucia H. Innocentini Mei, Shivani B. Mishra, Ashutosh Tiwari, Ashok Kumar, Mohsen Shahinpoor, Golok B. Nando, Subash C. Kundu, and Avrath Chadha

1.1 Introduction The vast majority of polymer and polymeric composites products that are presently used for various applications are made from petroleum-based synthetic polymers that do not degrade in a landill or in a compost-like environment. An environmentally conscious alternative is to design/synthesise biopolymers that are biodegradable. The presence of hydrolysable or oxidisable linkage in the polymer main chain, the presence of suitable substituents, correct stereoconiguration, balance of hydrophobicity and hydrophilicity and conformation lexibility contribute to the biodegradation of hydrolysable polymers, which proceeds in a diffuse manner, with the amorphous regions degrading prior to the degradation of the crystalline and cross-linked regions. In this context, the biotechnological approaches are being increasingly recognised as a key to developing better biodegradables and low-cost biopolymers.

1.2 Spectroscopy Analysis of Biopolymers Spectroscopic techniques are used to understand the development of novel materials. Techniques such as FTIR or Raman spectroscopy could be used to monitor the reactions occurring during the synthesis of new materials. Similarly, the degradation of materials in certain environmental conditions could be studied with these techniques. Other techniques such as X-ray diffraction and X-ray photoelectron spectroscopy may provide in-depth information about the properties or bonding mechanism in the bulk of the material. In Chapter 2 the authors have reviewed the studies that have been conducted on biopolymers using spectroscopic techniques.

1

Biotechnology in Biopolymers Developments, Applications & Challenging Areas

1.3 Thermal Analysis of Biopolymers The thermotropic phase behaviours and associated changes in biopolymers can be studied by variable temperature FTIR and Raman spectroscopy (Figure 1.1). A thermodynamic approach known as differential scanning calorimetry (DSC) has also been used to study the biopolymers. The investigation of thermal properties of biopolymers is crucial for numerous purposes including the development of PEGylated lipid-based drug and substances delivery vehicles. Dynamic Mechanical Analysis (DMA) is a thermal analysis technique that measures the properties of materials as they deform under periodic stress. The DMA technique allows the detection of very weak transitions as well as fast determinations of modulus as a function of temperature and frequency, damping, viscosity and compliance. It is commonly used to study polymer melts as well as solid polymers. It has the advantage of allowing testing on a wide variety of materials, and both liquids and solids can be handled in the same instrument, making it extremely useful for the study related to curing systems. In Chapter 3 the author has mentioned various thermal analysis techniques used in analysing transitions occurring in biopolymers.

0.10

0.05

in )

3000 Wa ven 2500 um ber 2000 (cm 1500 -1) 1000

m

3500

e(

0.00

70.0 60.0 50.0 40.0 30.0 20.0

Ti m

Absorbanc

e (abs)

0.15

10.0

Figure 1.1 3-D FTIR spectra of gases obtained during thermogravimetric decomposition of silicone

2

Biopolymers: An Indispensable Tool for Biotechnology

1.4 Mechanical Analysis of Biopolymers The mechanical properties of biopolymeric networks depend on the constitutive relations of ilaments and branch points, as well as the network architecture (Figure 1.2). Aqueous polymer solutions, especially those of natural biopolymers, are very important materials because they exhibit high stability level and good compatibility and are also biodegradable. It is known that in macromolecular systems, the interaction between polymers inluences the overall mechanical properties of the polymer. Release kinetics of an entrapped hydrophilic compound from a proteinbased matrix has been controlled by the mechanical properties of the matrix such as gel strength and elastic modulus. The microstructure of biopolymer composites is known to inluence their functional properties, particularly in matrices that serve as carriers of functional components such as lavours or antioxidants. Thermal, chemical or mechanical processes that lead to network breakdown or reformation of the matrix may play a signiicant role in migration and release of hydrophilic/ hydrophobic compounds of the matrix. It has been observed that several medical applications also depend on the mechanical stability of polymers and composites such as biopolymers in the synoidal luid for joint lubrication, dental resin composites, etc. Mechanical degradation of biopolymers such as those of starches and proteins is also relevant to the food processing industry. In recent years, scientists and engineers have been working together to use the inherent strength and performance of the biodegradable polymers and natural ibre-reinforced polymers to produce a new class of biobased composites because of increasing environmental concerns and the considerably low price of reinforcements of biobased composites. The mechanical properties of biopolymeric materials are discussed in Chapter 4.

3

Biotechnology in Biopolymers Developments, Applications & Challenging Areas

1.4

Spider

1.2 27 mm s-1

Stress (GPa)

1.0

20 mm s-1

0.8

13 mm s-1

0.6 Cocoon

4 mm s-1

0.4 0.2 0 0

0.05

0.15 Strain

0.25

0.35

Figure 1.2 Stress-strain curves of washed and degummed single-ilament silkworm silk (motor-reeled at 25 °C at the indicated speeds), Nephila spider dragline silk (20 mm s–1 at 25 °C) and standard, degummed commercial silk from a cocoon spun by the animal in the natural ‘igure of eight’ at speeds oscillating between 4 and 15 mm s–1 at 20 °C. The area under the stress-strain curve represents the energy that a ibre can take up before breaking, and thus indicates its toughness. Scale bar, 10 m. Immobilised silkworms (n = 4) were forcibly silked, each providing 3–6 single ilaments, which were tested in a stretching rig (force resolution, 30 N; time resolution, > η, for which the stress relaxation is given by:

(4.12)

Equations (4.11) and (4.12) together describe the rheological behaviour of this model. Equation (4.1) can also be understood as a nonlinear generalisation of the Kelvin–Voigt model in which a dashpot is placed parallel to a nonlinear spring, while Equation (4.12) describes a Newtonian liquid-like stress relaxation. Equating the stresses represented in Equations (4.11) and (4.12) amounts to the assumption that the strain of the system has two contributions with additive compliance.

4.6 Mechano-chemical Response of Biopolymers The ability to predict the behaviour and limitations of polymers in response to mechanical stress is important for determining their performance in a variety of applications. In elastic polymers such as natural rubbers, where mastication is used to reach the desired rheological properties [58–60], it is necessary to know the limits of elasticity before the material stretches to failure. The carbon-carbon bonds and

65

Biotechnology in Biopolymers Developments, Applications & Challenging Areas thioether and polysulfide cross-links can reversibly cleave, leading to self-healing the damage. Several medical applications also depend on the mechanical stability of polymers and composites such as biopolymers in the synoidal fluid for joint lubrication [61] and dental resin composites [62]. Mechanical degradation of biopolymers such as starches and proteins is also relevant to the food processing industry [63–65] and controlled degradation of other natural polymers such as chitosan (deacylated chitin) is important in biomedical applications such as drug delivery and bioadhesives [66].

4.6.1 Mechano-chemical Response of Biopolymers at Micro Level Many biological systems are mechano-responsive to their environment in which a mechanical stimulus is transduced into an electrical signal [67] because most of the eukaryotic cells have the capability to detect mechanical stress, which allows for touch sensitivity, hearing and detecting the flow of blood and urine [68]. In some specialised sensory cells, trans-membrane ion channels are forced open when a complex arrangement of proteins is subjected to mechanical stress [69,70]. The coupling of molecules to the active sites of enzymes results in mechanical deformation of the bound molecule, causing distortions as it coordinates to the active site geometry defined by the tertiary protein structure, and thus leading to a variety of efficient chemical reactions [71,72]. The response of polymeric materials to mechanical stress was published by Staudinger, who observed a decrease in the molecular weight of polymers in response to mastication [73–75]. It was suggested that the molecular weight reduction resulted from homolytic carbon-carbon bond cleavage due to mechanical force [76]. More recent work has demonstrated that cleavage occurs more easily for certain chemical bonds than for others. Encina and co-workers [77] reported that ultrasonic chain scission of polyvinylpyrrolidone with randomly incorporated peroxide linkages occurred ten times faster than that of neat polyvinylpyrrolidone. More recently, an attempt to mimic biological mechano-chemical transduction, targeted cleavage or rearrangement of bonds has been demonstrated in polymer-bound small molecules called mechanophores [78]. Mechanophores (Figure 4.3) possess strategically weakened bonds that undergo useful reactions when force is transferred to the mechanophore from the polymer chain segments. The importance of this configuration (polymer chains linked on either side of the mechanophore) for force transfer has also been demonstrated using control

66

Mechanical Properties of Biopolymers polymers in which the mechanophore is located at the chain ends or in the centre without spanning the force activated portion of the mechanophore [79].

(a)

(b) (c)

Figure 4.3 Schematics of a bifunctional mechanophore (a), control molecules containing a mechanophore linkage at the polymer chain end (b) or in the centre of the chain (c). The mechanophore is marked in the figure

Recently, Smith and Chu directly observed the stretching of individual polymers in a spatially homogeneous velocity gradient through the use of fluorescent labelled DNA molecules. Even at the highest strain rates, distinct conformational shapes (dumbbell, kinked, half dumbbell or folded) were observed (Figure 4.4) [80]. They concluded that the rate of stretching of individual DNA molecules in an elongational flow field is highly variable and depends on the molecular conformation that develops during stretching. The variability is due to a dependence of the dynamics on the initial, random equilibrium conformation of the polymer coil. If a coiled chain starts out at equilibrium with both of its ends on the same side of the centre of mass with respect to a plane perpendicular to the stretching axis, a large fold is likely to develop. If the same initial shape is rotated by 90°, it is likely to develop a dumbbell, a half dumbbell or a kinked shape. Given the variety of conformations that have been observed in the extensional flow studies, it remains poorly understood in what polymer conformation selective midpoint scission occurs.

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas

Dumbbell Mechanical Stretching

Half Dumbbell Coiled Folded

Biopolymer Gel

Kinked

Figure 4.4 Schematics of different possible polymer conformations during the act of stretching. The midpoint of each polymer chain has been indicated in black with a gradient to include the central 15% of the chain where bond scission has been shown to occur

It should be noted that experiments for polymer chain scission in an elongational flow field require specialised devices and are relatively difficult to perform [81]. Perhaps an even greater limitation in these experiments is the need for very high-molecularweight polymers (106 Da) of low polydispersities, which is relatively difficult to achieve in general [82]. Polymer segments in the high-gradient shear field near the collapsing bubble move at a higher velocity than those segments further away from the collapsing cavity. This velocity gradient causes the polymer chain to become elongated, and tension develops along the backbone of the polymer, which finally leads to chain scission. Similar to the chain scission of the polymer in an elongational flow field, an isolated flexible-chain molecule in an acoustic flow field is expected to undergo a coil-stretch transition. A coiled polymer experiences a strong hydrodynamic force generated by the collapse of the bubble. However, the polymer chain does not have to be fully elongated; a coiled polymer with various conformational shapes (dumbbell, kinked, half dumbbell) experiences chain scission [83–86]. The best model to date comes from the work of van der Hoff and Gall [87–89]. They found that the chain scission of polystyrene in THF could be modelled well when it was assumed that the probability of chain cleavage was distributed in a Gaussian manner within the middle 15% of the chain. This centre cleavage model is also consistent with the ‘stretching and breakage’ mechanism, since the solvodynamic forces are predicted to be the greatest in the centre of the polymer chain.

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Mechanical Properties of Biopolymers

4.6.2 Mechano-chemical Response of Biopolymers at Macro Level In this emerging area of mechano-chemistry, macroscopic loads applied to the bulk polymer cause selective chemical transformations at the atomic level and forceinduced chemical reactions in bulk polymers (Figure 4.5). The mechano-chemical change is often highly localised to regions of high stress concentration in the bulk polymer. Rigorous demonstration of the inferred chemical change requires carefully designed controls. However, the necessary controls have not been performed for all of the examples cited below, leaving open questions about the mechano-chemical activation. At the bulk level, where there are more atoms, non-covalent forces begin to shape the characteristics of a polymer at both the local and the global level. Such responses include chain slippage and the disruption of non-covalent interstrand interactions (e.g. H-bonding), chain segment alignment and conformational changes. Some of these interactions are inherent to the structure of the material while others have been designed to impart specific mechanical responses or enhance properties already present in the material. When discussing these effects, both the cessation of interactions and the inducement of interactions must be considered [90,91].

Supermolecular level

Microscopic level

Slippage

Atomistic level Bond Changes

Crazing

Figure 4.5 Diagram depicting the hierarchical levels of mechano-chemical change at the atomistic level, where chemical bond changes and conformational changes occur. On a supramolecular level, chain slippage occurs as a response to force and deformation. At the microscopic level, voids, cavitation, yield or crazing and crack formation take place along with large-scale viscoelastic deformation

69

Biotechnology in Biopolymers Developments, Applications & Challenging Areas On a bulk scale, various polymer responses indicate that structural damage has occurred, thus signalling that repair is necessary to prevent complete failure of the material. Changes in a polymer’s colour upon application of mechanical force can be useful as a damage sensor for the visual indication of stress. When the chemical identity, intermolecular interactions or orientation of chromophores are altered using mechanical stress, the appearance or change of colour can be used to identify a strained or damaged polymer [79,92–95]. This indication is relevant for any polymerbased material subjected to mechanical damage. Mechanical stress might also lead to the initiation of self-repair by the formation of new interchain interactions or new covalent bonds [96–98].

4.6.3 Deformation Mechanism Map Many studies on synthetic polymers show that the competition between shear deformation and cracking/crazing is strongly dependent on the entanglement density of a polymer network and polymers with less entanglement density could deform by crazing while the high entanglement density counterpart deforms by shear [99]. The deformation mechanism map of gelatin films with different amounts of structural order and ageing enthalpy is shown in Figure 4.6. It is clear from the figure that at a testing environment of 20 °C and 50% relative humidity (RH), gelatin without any ageing enthalpy deforms by brittle cracking or crazing at low crystallinity and shear bending at higher crystallinity. The transition occurs at crystallinity of ≈5–10 J/g. During a drying process, the mechanical integrity of gelatin is enhanced due to formation of a fringed micelle crystal structure, which serves as a physical crosslinking agent in the network [100]. Therefore, a transition from cracking/crazing to shear deformation is found for gelatin as the crystallinity increases. A close analogy is thus found between synthetic polymers and our biological gelatin material. At an intermediate crystallinity (∆Hcrystal ≈10 J/g), there is a transition from shear to cracking failure. For many synthetic polymers, physical ageing is known to raise the shear yielding stress but has a lesser effect on the crazing stress. Therefore, it is not surprising that for gelatin with similar crystallinity, i.e. similar entanglement density, there is a transition from shear to cracking/crazing as its ageing enthalpy increases. However, from the limited data presented here, the transition may be a straight line or a curved line. Typically, there may be a transition zone between crazing and shear yielding. Such a transition has been observed for many synthetic polymers [99]. At the highest crystallinity (∆Hcrystal ≈15 J/g), the entanglement density of gelatin is high enough that there is no transition from shear failure to cracking/crazing failure. However, at the highest crystallinity of 15 J/g, as the ageing enthalpy increases all gelatins deform by shear, and the ductility of gelatin films decreases [101].

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Mechanical Properties of Biopolymers

8 transition zone

Ageing Enthalpy (J/g)

brittle cracking shear cracking

6

4

2

0 0

5

10 Structural Gelatin (J/g)

15

20

Figure 4.6 Failure mechanism map for gelatin with different amount of structural order and ageing enthalpy. The symbols used in this figure are gelatin samples dried at 50 °C and 50% RH (▲), at 35 °C and 50% RH (∆), at 30 °C and 50% RH ( ) and at 20 °C and 50% RH (•). Reproduced with permission from E.J. Kramer, L.L. Berger and H.H. Kausch, Eds., Advances in Polymer Science, Springer-Verlag, New York, 1989, p.5. ©1989, Springer

4.7 Some Factors Affecting Mechanical Behaviour of Biopolymers

4.7.1 Physical Ageing The mechanical and fracture behaviours of gelatin are issues of technological importance. In order to determine the strength and endurance of gelatin, it is necessary to understand the microscopic deformation and fracture mechanisms of the gelatin. It is well known that during the drying process, gelatin chains may re-associate with each other to form partially denatured collagen-like structures as the solution is cooled

71

Biotechnology in Biopolymers Developments, Applications & Challenging Areas below its coil helix transition temperature, which is very similar to the crystallisation phenomena in synthetic polymers [102–104]. As the solution is further cooled below its glass transition temperature, the glassy amorphous gelatin may also undergo a secondary enthalpic relaxation process often known as physical ageing [105]. The process of physical aging is manifested by the change of thermal and mechanical properties including specific volume, enthalpy, modulus, loss tangent or refractive index for many synthetic polymers [105]. The effect of physical ageing on mechanical behaviour is illustrated in Figure 4.7. When a polymer is cooled and reheated near its Tg, a hysteresis in its enthalpy is observed as shown in Figure 4.7a. The enthalpy of polymer glass transition decreases toward its equilibrium value as it is aged below its Tg. The highly relaxed material does not reach the equilibrium state during reheating through Tg until a relatively high temperature as shown in Figure 4.7b. A classical Tg overshooting peak due to the ageing process is accordingly demonstrated in the DSC measurement. Therefore, the area underneath this endothermic peak at the Tg can be used to quantify the extent of the enthalpy relaxation [101].

4.7.2 Crystallinity At low temperatures, the polymeric material is rigid like glass and the chains are frozen into a particular configuration and their response is reversible and elastic to external stresses and similar to that of a spring. At temperature T < Tg the polymer behaves as a glassy material and at T > Tg the polymer behaves like rubber due to its viscoelastic nature. In this region the modulus of elasticity is low and is a function of time depending on the viscosity of the medium. The molecular segments slide past each other and try to straighten out. With an increase in temperature, the viscoelastic modulus (Eve) further decreases until the polymer becomes a liquid when it vanishes. In a crystalline polymer the rubber-like region is not present. Crystallites inhibit viscous flow. Natural rubber and gutta-percha have the same composition but different molecular structure. Gutta-percha, which is crystalline, is hard and brittle. Rubber is soft and can easily be elongated. Crystallisation takes place during such a stretching. But with further extensions, when maximum crystallisation has been produced, rubber attains high mechanical strength. Some textile fibres show a linear relationship between stress and strain for moderate stresses. But, beyond a certain point, the elongation increases rapidly. In the case of nylon, such a relationship is almost entirely linear. This is due to the fact that the polycrystalline structure does not change in this case on the application of stress [106]. Figure 4.8 illustrates the deformation-induced crystallisation of the polymer chains.

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Mechanical Properties of Biopolymers

enthalpy, H

rubbery state

glassy state physical ageing

Tg (a)

temperature

ageing enthalpy

heat capacity, C = p

dH dt

Tg overshooting peak

rubbery state

glassy state

(b)

temperature

Figure 4.7 Schematic illustrations of (a) enthalpy change and (b) heat capacity change for an amorphous glass undergoing physical ageing process. Reproduced with permission from C.A. Dai and M.W. Liu, Materials Science Engineering A, 2006, 423, 121. ©2006, Elsevier

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Polymer Sample

F

crystalline region

amorphous region

Figure 4.8 As a polymer is subjected to tension, a tear begins to propagate through the material. At the crack tip, deformation induces crystallisation of the polymer chains, leading to slowing of crack growth

It is found that natural rubber or its synthetic analogue shows strain-induced crystallisation (SIC) [107–109]. An explanation is that as a polymer is stretched, the chains align and the overall entropy of the region decreases. The change in entropy associated with crystallisation decreases with respect to the undeformed polymer, thus increasing the rate of crystallisation and the crystallisation rate initially increases as more strain is applied [110]. It was found that the amount of SIC depends upon the cross-link density. At high cross-link densities in natural rubber, the maximum crystallite size is limited. In contrast, at low cross-link densities, fewer crystal nucleation sites are present [111].

4.7.3 pH of the Medium It is observed that when aqueous chitosan (CS) is dialysed against deionised water (DI) water using a dialysis tube, a precipitate is observed at pH > 6.2, while aqueous

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Mechanical Properties of Biopolymers N-palmitoyl chitosan (NPCS) remained in a solution state at pH 6.5 and changed into a massive hydrogel at a higher pH due to self-assembling of NPCS into micelles. The micelles formation takes place due to the hydrophobic interaction between the conjugated palmitoyl groups and as a result aqueous NPCS produces a viscous solution [112], which can act as physical cross-links between NPCS polymers. Figure 4.9 displays the elastic (G′) and viscous (G″) moduli of aqueous CS (pH > 6.2) and NPCS (pH > 6.5) measured at a constant frequency of 0.1 Hz as a function of pH. For aqueous CS, G″ was greater than G′ (P < 0.05), exhibiting a viscous nature over the entire range of pH values investigated. In the case of aqueous NPCS, G′ was found to be approximately an order of magnitude larger than G″ (P < 0.05), indicative of an elastic rather than viscous material. It is known that the charged state and physico-chemical properties of CS are substantially influenced by its environmental pH. At low pH, the free amine groups on CS were protonated (-NH3+), thus limiting the physical contact between CS molecules due to charge repulsion (Figure 4.3b). With increasing pH, the amine groups on CS tended to be deprotonated (-NH2) and hydrogen bonds between the hydroxyl groups and the uncharged amine groups were formed. Therefore, chain entanglements were developed via a simple topological interaction of CS polymers and their dynamic rheological response reflected a viscous nature over a pH range of 3.0–6.2 [113]. 103 10

2

CS NPCS

G′ G′

3.0

4.0

G′′ G′′

G′, G′′(Pa)

101 100 10–1 10–2 10–3 5.0 pH Values

6.0

7.0

Figure 4.9 Elastic (G′) and viscous (G″) moduli of aqueous CS (1% w/v) and aqueous NPCS (1% w/v) measured at a constant frequency of 0.1 Hz as a function of pH (n = 5). Reproduced with permission from Y.L. Chiu, S.C. Chen, C.J. Su, C.W. Hsiao, Y.M. Chen, H.L. Chen and H.W. Sung, Biomaterials, 2009, 30, 4877. ©2009, Elsevier 75

Biotechnology in Biopolymers Developments, Applications & Challenging Areas

4.7.4 Effect of Composition The effect of chemical composition and applied strain on the mechanical properties of gelled gelatin-rich micro-particles resulting from phase-separated gelatin/pullulan mixtures has been investigated. The mechanical properties of microparticles (20–120 μm) were measured using a micromanipulation technique. The compressrelease tests revealed that at a low deformation (up to 0% strain) particles are fully elastic with Young’s modulus proportional to the concentration of gelatin and at the higher deformation (up to 50–80%) particles are viscoelastic. Even at very high load resulting in 50–60% deformation, no fracture of particles was observed and after the load was removed, particles recovered to a fully spherical shape. The viscoelastic behaviour was investigated by a stress-relaxation method, where force relaxation at constant deformation was measured as a function of time. The experimental results were analysed using a standard linear model of viscoelastic solids and the parameters of this model were related to the composition of gelled particles [114].

4.8 Conclusions and Future Prospects In the last decades biopolymers prepared from renewable resources have attracted great attention in both the academic and industrial worlds because polymeric materials reinforced with lignocellulosic fibres offer an answer to maintaining sustainable development of economically and ecologically attractive materials with respect to ultimate disposability and raw materials use. In recent years, scientists and engineers have been working together to use the inherent strength and performance of the biodegradable polymers and natural fibre-reinforced polymers to produce a new class of biobased composites, because of increasing environmental concerns, and to considerably lower the price of reinforcements of biobased composites [115]. Further advantages of natural over synthetic fibres are good specific mechanical properties, reduced tool wear, enhanced energy recovery, biodegradability, etc. Besides this, the natural fibres can also affect the mechanical properties of biomatrices.

4.8.1 Actuator, Sensor and Micro-electro-mechanical System Devices A sensor is a valuable measuring tool device that converts a physical phenomenon into an electrical signal. By using accurate and sensitive sensors the presence of chemical threats in food for human consumption, our environment and the public setting may be minimised if not eliminated. Sensors are also used in the medical field for detecting various chemicals in the human body as well as for monitoring different diseases and illnesses [116].

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Mechanical Properties of Biopolymers In an effort to preserve the environment, scientists, engineers, biologists and others search for other materials to fabricate sensors and other devices. In recent years, biopolymers have been studied as promising materials for several reasons. Biopolymers, for example deoxyribonucleic acid (DNA), show optical and electromagnetic properties that can be used in electronic and photonic devices [117]. These properties include low and tuneable resistivity, and ultralow optical and microwave loss [118–119]. Additionally, biopolymers are inexpensive, biodegradable and easy to integrate into electronic and photonic components and devices. As a comparison, most synthetic plastic or polymer materials are not biodegradable and are derived from non-renewable resources [120]. The fabrication techniques of biopolymers are inexpensive and simple to manipulate. Some examples using biopolymers in devices include a DNA complex used as an electron blocking layer in a light emitting diode (LED) [121], a polypyrrole-doped DNA electrode biosensor [122], doped DNA used as a bottom gate layer in a field effect transistor [123] and a DNA layer fabricated into nanoscale wells in a silicon substrate to demonstrate crystal photonic waveguides [124].

4.8.2 In Tissue Engineering The biomedical burden of treating diseased or injured organs continues to increase in parallel with expanding populations. One of the most frequent, expensive and serious problems facing human health care is the loss or failure of organs or tissues. The medical need for tissue and organ substitutes can arise from trauma, infectious, inherited or age-related diseases, or organ failure. As a result, there is a need for new and innovative technologies in the field of regenerative medicine in order to tackle these problems [125–127]. To overcome these problems, the common approach in tissue engineering is growing cells in three-dimensional scaffolds, either in vivo or in vitro [128]. When applying the third approach, a substrate material should exhibit good biocompatibility, meaning that it should not evoke an unresolved inflammatory response, nor demonstrate extreme immunogenicity or cytotoxicity. In addition, the mechanical properties of the scaffold must be sufficient. The mechanical strength required depends to a great extent on the site of the defect [129]. A variety of different biomaterials are currently being used as scaffolds for reconstruction of soft (such as adipose tissue or skin) or hard (bone) defects. Synthetic and natural polymers, however, are an attractive alternative and are versatile in their applications to the growth of most tissues. Aliphatic polyesters such as polyglycolic acid (PGA), polylactic acid (PLA) and polycaprolactone (PCL) are the most commonly used synthetic polymers, functioning as scaffolds for tissue engineering [130–132]. Naturally derived protein or carbohydrate polymers have been used as carriers for the growth of several tissue types [133,134]. Collagen, in this regard, is the most often used natural polymer [135–137]. Hyaluronan is also used to treat osteoarthritis of the knee [138]. It has 77

Biotechnology in Biopolymers Developments, Applications & Challenging Areas also been suggested that hyaluronan has positive biochemical effects on cartilage cells [139]. Due to its biocompatibility and its presence in the extracellular matrix, hyaluronan is often used as a scaffold in tissue engineering research [140]. Gelatin has long been used in the food industry as a clarification agent, stabiliser and protective coating material [141]. In addition to single-component scaffolds, composites of various naturally derived polymers are also often used in the field of tissue engineering. It is commonly accepted that composite materials show an excellent balance between strengths and weaknesses, and overall exhibit improved characteristics compared with individual components [142]. In order to eliminate undesired characteristics and to exploit the advantages of the individual polymers, various composite scaffolds comprising two or more polymers have often been considered. For example, the stability of polymers that are mechanically weaker can be reinforced by fabricating composites containing mechanically stronger polymers. Similarly, polymers that possess poor cell proliferation or migration properties can be remedied with the incorporation of a more bioactive polymeric phase [143]. Bone deficiency in oral surgery and periodontology is one of the most common challenges that the clinicians have to face and bone grafting procedures have become increasingly important. At present engineered materials including bioceramics, biopolymers, metals and composites are used in bone tissue in order to enhance the regeneration of new bone [144]. For example, anorganic bovine bone (ABB) is widely used for implanto-prosthetic rehabilitation both in the maxilla and in the mandible [145–147]. Bone morphogenetic proteins (BMP) have become particularly interesting in orthopaedic and dentistry surgery [148]. There are several natural polymers that may be used as carriers for BMP delivery.

4.8.3 In Drug Delivery The primary goal of bioadhesive-controlled drug delivery is to localise a delivery device within the body to enhance the drug absorption process in a site-specific manner. Recently, the most promising studies have shown that a number of mucoadhesive carriers can be used for protein delivery [149]. For example, Mathiowitz and co-workers [150] showed that certain biologically adhesive engineered polymer microspheres prepared from biologically erodible polymers, which displayed strong interactions with the mucosa of the gastrointestinal tract, could be ‘developed as delivery systems to transfer biologically active molecules’ (among which insulin and plasmid DNA are mentioned) to the circulation.

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5

Natural Fibres and Their Use in the Production of Biocomposites

Lucia H. Innocentini Mei and André L.F.M. Giraldi

5.1 Introduction Every year, farmers around the world harvest approximately 35 million tons of natural ibres from a wide range of animals such as sheep, rabbits, goats, camels and alpacas and plants like cotton bolls, abaca, sisal leaves, coconut husks, stalks of jute, hemp, lax and ramie [1–3]. Such ibres are transformed into fabrics, ropes and twines that have been fundamental to society since the dawn of civilisation. However, with the advance of synthetics procedures that use fossil sources, natural ibres have been replaced by man-made ibres like those based on acrylic, nylon, polyester and polypropylene. The facile acceptance of the synthetic ibres is credited to their lower cost and to their competitive properties such as uniform strengths, lengths and colours and their customisability. The ierce competition that has arisen due to synthetic products and the economic crisis is causing insecurity among the community that is dependent on natural ibre production and processing. Recently, the International Year of Natural Fibres 2009 was announced to raise awareness related to natural ibres. Natural ibres are part of the family of polymers produced by plants and animals, with long chains and high molecular weights that can be grouped together to form tangled wires giving rise to cords, cables, fabrics and different knitting textiles. Like agriculture, textiles have been a constant companion of the human race for thousands of years, as shown by the fragments of cotton found from about 5000 BC in excavations in Mexico and Pakistan. History also reports the existence of silk since the twenty-seventh century BC, while the oldest wool textile was found in Denmark from 1500 BC, and the oldest wool carpet found in Siberia from 500 BC. Other ibres, like jute and coir, have also been cultivated since ancient times. Although the methods used to make fabrics have been modernised over the years, its uses remain almost the same: still today, most natural ibres are used mainly in clothing and decorative accessories, furniture, upholstery for the home, among other uses. More recently in history, traditional textiles have been used for industrial purposes, as well as in

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas medical implants, and geo-textiles and agro-composite material components for different applications. The following section presents an overview of biocomposite materials and the potential use in manufacturing some natural ibres.

5.2 Biocomposite Materials With the strong emphasis on environmental awareness inherited from the twentieth century, researchers around the globe are motivated to develop recyclable and ecologically sustainable composite materials. However, they face the challenge of developing appropriate methods of waste treatment. For example, to decompose waste by different chemical processes, and employing less energy, the methods are unproitable and undesirable, due to the production of harmful gases during the process. As a consequence, both environmental legislation and consumers in many countries are demanding that materials manufacturers consider the environmental impact of their products, from their conception and recycling to inal disposal. In many cases, this is done with a life cycle assessment of the goods produced. It is, therefore, of interest to develop sustainable composites, or biocomposites, capable of being recycled and biodegraded. Serious efforts towards such developments are now being made in many countries, mainly those where the environmental legislation is stricter. It is worth mentioning that biocomposites can be employed in health areas, speciically in the ield of tissue engineering where these are degraded over time as a result of enzymatic activities in the human body. They can also be reabsorbed, while the neo-tissues will be grown to recover their function simultaneously. These materials must be capable of being used in or on the human body without eliciting an adverse response to surrounding tissues at the implantation site [4,5]. More recently, several researchers reported their efforts towards the development of durable cement-based composites reinforced with natural ibres. However, the longterm durability of such materials may be limited due to the elevated permeability and poor resistance to crack growth. In alkaline environments, such as in cement matrices, ibres from vegetable sources could deteriorate or suffer mineralisation. The degradation process in such a case could be accelerated by the presence of water stored inside their porous structure that limits the use of vegetable ibres in highperformance applications [4,6]. In order to promote the use of vegetable ibres it is necessary to investigate the crack/microstructure interaction by studying the interfacial transition zone between ibres and the cementitious matrix. According to Savastano Jr and co-workers [5] these properties cannot be optimised simply by the design alteration that improves the strength, unless a more balanced approach is chosen to optimise the strength and fracture toughness/resistance, fatigue resistance and

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Natural Fibres and Their Use in the Production of Biocomposites reduced permeability. Challenging approaches need to be chosen for the production of economical material with good properties using renewable raw material. Dedicated efforts towards the design of new materials for automotive industries, packaging, furniture and construction, based on green composites, have already started [6]. There is an increasing demand within automotive companies for materials that have noise reduction capabilities and could reduce weight for fuel eficiency. Several automotive companies are now adopting natural ibres, due to their excellent sound absorbing eficiency and shatter resistance, along with better energy management characteristics compared to glass ibre reinforced composites. Such natural ibre composites reduce the mass of the automotive parts and decrease the consumption of energy during the production stage. Due to this fact, automotive and other companies are developing ‘green’ composites based on natural lignocellulose ibres instead of synthetic ibres. However, such composites show low load-bearing capabilities compared to synthetic ibre (e.g. glass) reinforced thermoplastics. Moreover, it is common to ind variations in the properties of natural ibres that need to be considered during the design of a particular vehicle component. Malkapuram and co-workers [7] mentioned that demand for natural ibres in plastic composites is predicted to grow at 15–20% annually with a growth rate of 15–20% in automotive applications, and 50% or more in selected building applications. Similarly, Martins and co-workers [8] commented on the potential of natural ibre-based composites as reinforcement in polymers and rubber matrices. The authors emphasise the economical and ecological aspects, and the excellent speciic properties of the natural-ibre-based composites. The open technical literature and business magazines have demonstrated that the ield for natural ibre reinforced biocomposites is full of opportunities for those working with conventional composites such as glass ibres and/or those made of synthetic non-biodegradable polymeric matrices. A wide variety of natural ibres is available and more are being explored along with novel biodegradable polymer matrices [4–8]. Recently Ticoalu and co-workers [9] showed interesting results related to the Young’s modulus of various natural ibres as shown in Figure 5.1. The variation of modulus for some ibres represented in this graphic as a function of density is evident, as is the low density of coir ibre compared to the others, justifying its use in special weightless composites as padding for seats in vehicles like trucks, cars, buses, etc. In such applications the lower density of natural ibres compared to other materials reduces the weight of transport and decreases the fuel consumption. Other applications of such biobased composites are gardening/agriculture equipment, tropical housing and as support/holding structure for some fruits, such as passion fruit and grapes. They are also used in cars, planes, ships and trains especially as disposable devices.

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas

50

Young’s Modulus (GPa)

Ramie Flax Hemp Jute Sisal Cotton Coir 0 1,50

1 Density g/cm3

Figure 5.1 Young’s modulus and density of several natural ibres. Adapted from T.C. Ticoalu and F. Aravinthan, Southern Region Engineering Conference, Toowoomba, Australia, 2010

Similar to other polymer-ibre composites, the natural ibres could be processed by different techniques, such as extrusion and/or injection, centrifugation in the presence of ibres and resin to cure in situ, ilament winding, compression moulding, pultrusion, and so on. The natural ibres may suffer premature degradation due to the high temperatures required during the melting of the polymer matrix, which in general are higher than the temperature of the ibre degradation. Therefore, regulatory measures need to be exercised before the processing of the composites so that ibre is not damaged and the inal properties of the composites are achieved.

5.3 Natural Fibres Many natural ibres, which have been widely researched and used to obtain reinforced polymer composites, can be also employed to obtain biocomposites for engineering applications [10,11]. The use of electrospinning technique in some natural polymers

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Natural Fibres and Their Use in the Production of Biocomposites could be used to produce natural ibres and nanoibres that ind wide applications in biotechnology, including health and safety. This section describes the most commonly used natural ibres to produce reinforced composites, with the same potential to produce eco-friendly composites made of biodegradable matrices. According to FAO (Food and Agriculture Organization), approximately 30 million tons of ibres are farmed every year from animals and plants, all over the globe. Exploring new applications for this amount of natural ibres has been the challenge for many scientists and business owners.

5.4 Surface Modification – Does it Work? Interface properties are unique for each polymer-ibre pair, which explains the various studies to improve the properties of interfaces [12]. Therefore, surface modiication of materials has been the subject of many studies as many composites are made with different polarity components. Despite the fact that natural ibres offer many advantages to composites, their highly polar character gives low compatibility with non-polar polymer matrices, such as polypropylene (PP) and polyethylene (PE). The incompatibility may cause problems in the material properties and composite processing. Hydrogen bonds can form between the hydrophilic ibres, causing their agglomeration and non-homogeneous distribution throughout the non-polar polymeric matrix during the fabrication of composites. Besides, the inadequate wettability of ibres by the non-polar polymer matrix may result in weak interfacial adhesion and consequently the stress transfer eficiency from the matrix to the reinforcing ibres is reduced. However, the incompatibility may not be an issue when using polar polymers such as epoxy resin and unsaturated polyester (UP) as matrices. The resulting composites, like other composites with non-polar matrices, will undergo the risk of being spoiled by microorganisms due to the moisture of the environment. The moisture absorption of the natural ibres may cause dimensional changes of the resulting composites and weaken the interfacial adhesion. Further, the natural ibres have limited thermal stability and, therefore, thermal degradation may take place during composite processing at high temperatures, especially in the case of hot compression processes and thermal extrusion. The performance of ibres is critical to obtain the improved physical and mechanical properties of the resulting composites. Thus, treatment of natural ibres is an important factor to promote interfacial adhesion of the matrix-ibre, improving the surface wettability of the natural ibre by polymers (mainly non-polar) and also improving the water resistance of ibres. Physical treatments (e.g. electronic discharge in the different media such as corona technologies and plasma) may create a hydrophobic or hydrophilic surface by changing the surface energy of the ibre, consequently

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas increasing its compatibility with the polymer matrices. These surface treatments only modify the thin surface layer and thus do not change the hygroscopic characteristics of ibres [12]. Among several methods available to treat natural ibres, the chemical modiication could permanently alter the chemical nature of the ibre surface. It is common to use coupling agents that function at the interface to create a chemical bridge between the matrix and reinforcement. It improves the interfacial adhesion when one end of the molecule is tethered to the reinforcement surface, while the functionality at the other end reacts with the polymer phase. One general simpliied reaction of silane coupling agent with a natural ibre may be simpliied as shown in Equation (5.1):

(5.1)

Other techniques, such as graphitisation, can also be used for chemical modiication of natural ibres to make them more resistant against fungal decay. In such case, reduction of water sorption is necessary, which in turn reduces the dynamic strength, such as impact strength, due to the material embrittlement, as suggested by Xie and co-workers [13]. Some of the many physical and/or chemical methods available in the literature for ibre treatments, obtained from Kalia and co-workers [14], are summarised in Table 5.1. Although modiication of natural ibre is beneicial for certain properties of the composite, such as mechanical properties, it could reduce the thermal properties, as discussed in the following section.

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Natural Fibres and Their Use in the Production of Biocomposites

Table 5.1 Common methods and agents used for natural fibre modification Methods of fibre modification

Agents used for fibre modification

Mercerisation

NaOH

Esteriication (e.g. acetylation)

Acetic anhydride

Etheriication

NaOH followed by nucleophilic attack of the intermediate species

Peroxidation

Peroxides (commonly benzoyl or dicumyl peroxide)

Grafting

Vinyl monomers and other unsaturated molecules

Silanisation

Silanes as coupling agents

Isocyanate treatment

Isocyanates

Bleaching

Sodium chlorite

Corona or cold plasma

Ionised gas

Table reproduced with permission from S. Kalia, B.S. Kaith and I. Kaur, Polymer Engineering Science, 2009, 49, 7, 1253. ©2009, Wiley

5.5 Thermal and Mechanical Behaviour of Natural Fibres A limiting factor while using natural ibres as reinforcement in a polymeric matrix is their thermal degradation during their incorporation in the molten polymer matrix. For each polymer there is a range of processing temperatures that may contribute to the ibres degradation, which in turn compromises the properties of the inal product. It is therefore the properties of these composites that need to be veriied before their use. For example, wood plastic composites can be processed in an extruder with a large processing window, while carefully avoiding the ibre degradation. According to Tajvidi and co-workers [15] the degradation of natural ibres is mainly due to the high-temperature processing, which could have a negative effect on the mechanical properties of the composites due to the production of volatile components as a result of material degradation that causes microvoids across the interface. As a consequence, these microvoids may cause defects that will compromise the mechanical properties and the performance of the material under service. It is worth mentioning that most natural ibres have low degradation temperatures of approximately 200 °C, which restricts the production of thermosets that require high curing temperatures, as mentioned by Sgriccia and co-workers [16]. The authors call attention to other characteristics of natural ibres that must be taken into consideration, such as large variability of mechanical properties, lower ultimate strength, lower elongation,

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas problems with nozzle low in injection moulding machines, appearance of bubbles in the product and poor resistance to weathering. The resistance to temperature can be circumvented by chemical modiications to the ibres. However, such a procedure does not necessarily produce positive results. On the contrary, there are studies that report a decrease in heat resistance of the treated ibres [17]. In other words, the type of treatment of any particular natural ibre should be carefully investigated before its use in composites to avoid bad results. As will be commented later, there are some natural ibres, like Açai, that may resist the high temperatures used to process some polymer matrices, without ibre treatment. Regarding the mechanical properties of natural ibres, Kalia and co-workers [14] reviewed the effect of pretreatment of ibres on tensile strength, Young’s modulus and elongation at break. Here we compare the properties of several natural and synthetic ibres. It has been observed that physical properties such as density, tensile strength, tensile modulus and elongation at break between several natural (jute, lax, hemp, ramie, sisal, palm, cotton, coir) and conventional man-made (E-glass, S-glass, Aramid and carbon) ibres [18,19] varies according to the origin of the ibre. For example, the superiority of coir ibre for elongation at break was the highest among all the cited ibres, which varied in the range of 15–40%. Similarly, the Young’s modulus for Ramie varied in the range of 61.4–128 GPa, the highest value among the cited natural ibres and even better than Aramid, S-glass and E-glass if the value of 128 GPa is considered. In case of tensile strength, the best results were obtained by all man-made ibres (2000–4570 MPa). However, high values were also obtained in palm and lax natural ibres, considering their upper limit of 1627 and 1100 MPa, respectively. In some cases, such as coir ibre, the ibre diameter varied in a wide range and comparison with the other ibres was not possible since their diameters were not published by the authors. Additionally, the density values published for natural ibres were clearly lower than the man-made ibres, except for S-glass ibres [17,18].

5.6 Natural Fibres Classification According to Their Source There are three primary sources of natural ibres as presented in Figure 5.2 [15]. In this chapter, only the most used natural ibres obtained from vegetable sources will be discussed, such as coconut, sisal, jute, lax, cotton, kenaf, curauá, bamboo, açai, etc. However, it is important to note that the market of ibres from several animals (sheep, goat cashmere, angora goat, llama, alpaca, guanaco, vicuna, camel, angora rabbit, etc.) is well established, with the textile market being fairly representative [20-23]. Asbestos is a mineral ibre, now disused due to its carcinogenic properties.

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NATURAL FIBRES Main Sources

VEGETABLE Stem, Leaf, Seed and Fruit

Hair Filaments

MINERAL Asbestos replaced by silica fibres

Figure 5.2 The three main sources of natural ibres

Natural ibres can be extracted from different parts of the plants. Fibres obtained from abaca, curauá, pineapple, banana and sisal, for example, are extracted from the leaves of these plants. Others, such as coconut ibres, come from fruit, whereas cotton and açai ibres are extracted from the seeds of their fruits. Similarly, ibres like ramie, lax, jute and hemp come from the bast while others like bamboo and sugar cane bagasse are extracted from the grass [24]. The ibres to be used in reinforcing composites must belong to the family of hard ibres, which are usually extracted from the leaf and bark. While the uses of some ibres, such as jute, coir, sisal, ramie, hemp, are described in the literature, many others are being discovered and their potential to act as reinforcements in composites is being exploited. The characteristics of the ibres of vegetable origin will be presented in this chapter.

5.6.1 Vegetable Fibres Vegetable ibres are mainly composed of hemicelluloses, cellulose, pectins and lignin along with small amounts of extractives [25]. The ibre constituents may vary, however, depending on their source and origin. In comparison to conventional inorganic illers such as carbon and glass ibres, natural ibres could provide advantages, as listed below: (1) low density 97

Biotechnology in Biopolymers Developments, Applications & Challenging Areas (2) biodegradability (3) low cost and abundance (4) desirable ibre aspect ratio (5) relatively lexural modulus and high tensile (6) reduced health hazards (7) lexibility during processing. The biodegradable characteristics of such ibres could facilitate their ultimate disposal by composting or incineration as these options are not possible with most industrial ibres. The ibres also contain sequestered atmospheric carbon dioxide in their structure and are invariably of lower embodied energy compared to industrially produced ibres. There are several vegetable ibres available to produce eco-friendly composites. Varieties of such ibres belong to certain regions and have been discovered recently. The following subsection describes some of the traditional and new ibres, comparing their properties.

5.6.2 Sisal Fibre from Agave Plants Sisal [15] is a leaf ibre from drought-tolerant native plants of the North American continent that was used by the early inhabitants of Mexico as a food source and to manufacture utensils (Figure 5.3). It is an important crop in China, Africa (Kenya, Tanzania and Madagascar) and Latin America (Brazil, Mexico, Haiti, Venezuela and Cuba). World production of sisal and a similar agave ibre, henequen, is estimated to be approximately 300,000 tons and valued at approximately U$ 75 million. The major producers are Brazil (120,000 tons), Tanzania (30,000) and Kenya (25,000). Brazil exports around 100,000 tons of raw ibre and manufactured goods, particularly to the USA. Similarly, Kenya and Tanzania export approximately 20,000 tons and 15,000 tons respectively. Traditionally, sisal has been used in ropes for cordage and sacking, carpets, furniture and making paper pulp. The use of sisal ibres as a reinforcement in composites has gained interest and expectations among materials scientists and engineers. Recently, there has been an increased interest towards inding new applications for sisal-ibrereinforced composites due to its low cost and density, high speciic strength and modulus, availability in tropical countries and renewability. Since it is a ibre that displays a high resistance to traction, it is also being used in plastic composites for

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Natural Fibres and Their Use in the Production of Biocomposites automotive components and as reinforcement in cements. The ibre inds use in brake pads, as a substitute for asbestos, due to its excellent properties. Some uses of sisal are shown in Figure 5.3.

Figure 5.3 Sisal plant and composite. Reproduced with permission from G.I. Williams and R.P. Wool, Applied Composite Materials, 2000, 7, 421. ©2000 Springer

Rong and co-workers [24] investigated the effect of ibre treatment on the mechanical properties of unidirectional sisal-reinforced epoxy composites. Treatments including alkali, acetylation, cyanoethylation, silanisation with coupling agent and heating were carried out to modify the ibre surface and its internal structure. When the treated ibres were incorporated into an epoxy matrix, the mechanical characterisation of the laminates revealed the importance of two types of interface: one between the ibre bundles and the matrix, and the other between the surface of ibres and the matrix. In general, the ibre treatment signiicantly improved the adhesion at the former interface and led the penetration of the matrix resin into the ibres, thereby reducing the ibre pullout. The benzoyl esteriication of sisal ibre enhanced the tensile properties of the short sisal ibre-reinforced polystyrene composites due to the improved interfacial adhesion between the polystyrene matrix and lignocellulosic ibres, as proposed by Manikandan and co-workers [25]. The effect of pre-treatment of sisal ibres in polyhydroxybutyrate (PHB) composites was studied by Cardoso [26], who showed that the mechanical properties such as izod impact and maximum strength were improved with the addition of ibre and ibre treatment.

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas There are several emerging markets for sisal such as speciality paper, ilters, geotextiles, mattresses, carpets and wall coverings. Moreover, the by-product from sisal extraction could be used to make biogas, pharmaceutical ingredients and building material. Joseph and co-workers [27] mentioned the inluence of ibre treatment and orientation in composites made of polyester/sisal. The authors obtained better properties with an increase of the ibre length up to the critical value of 35–45 mm while keeping the original values of modulus and elongation. According to these authors, amounts of ibres less than 20% and over 50% may compromise the properties of the composites. In addition, the ibre orientation in the same direction gave the best results of properties compared to the results made in the transversal direction, and even three times higher compared to other composites with random orientation of ibres. Similar studies with banana ibres were also carried out by Pothan and co-workers [28] and Joseph and co-workers [29]. Success in achieving good properties in composites made of natural ibres therefore depends on the length and orientation of ibres in the composites, and features such as afinity between the ibre and the matrix, which ensures good interfacial interaction.

5.6.3 Coconut (or Coir) Fibre Coconut ibre is a fruit ibre extracted from cocos cifera obtained in several Asian countries like India, Sri Lanka, etc., which has vast applications such as manufacturing of ropes, mats, car mats and seats, geotextiles, articles of clothing, carpets, bags, pillows, mattresses, brushes, mops, runners, rope liner, cork insulation and animal bedding (see Figure 5.4). According to Cardoso [26], coconut ibres can be used in a similar way to sisal, to produce cheaper and ecological bricks. This new application for coconut ibre that resulted from the pioneering work of Cardoso has increased the number of green alternatives to the construction market. Figure 5.4 shows a few important applications of coir ibre [16,30,31].

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Figure 5.4 Some uses of coir ibres [32]. Reproduced with permission from J. Rout, M. Misra, S.S. Tripathy, S.K. Nayak and A.K. Mohanty, Composites Science and Technology, 2001, 61, 1303. ©2001, Elsevier

Rout and co-workers [33] studied the performance of coir as reinforcement in polymer composites and found it unsatisfactory and not comparable to other natural ibres, due to the low cellulose coir (36–43%) and high lignin (41–45%) contents. Surface modiied coir ibres are used as reinforcing agents in general-purpose polyester resin matrix. The mechanical properties of such ibre reinforced composites were found to increase as a result of surface modiication. Among all modiied ibres, bleached coir-polyester composites showed better lexural strength (61.6 MPa) and 2% alkalitreated coir/polyester composites showed higher tensile strength (26.80 MPa). The authors studied the interface of untreated and modiied coir surface-polyester and compared it with glass reinforced coir-polyester composites. Similarly, a work of Cardoso [26] on coir and polyhydroxybutyrate composites (Figure 5.5) demonstrated the importance of improving the interface between ibre and matrix to obtain good properties.

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A

B

Figure 5.5 SEM of tensile fractured surfaces of: (a) coconut ibre polyhydroxybutyrate (PHB) composites; (b) sisal ibre polyhydroxybutyrate (PHB) composites (Magniication 800×). Reproduced with permission from R. Cardoso de Jesus, Processing and Characterisation of Biocomposites of PHB with Acetylated and No-acetylate Natural Fibres of Coconut and Sisal. ©2010, State University of Campinas

5.6.4 Jute Natural Fibre Jute ibres are mainly extracted from the bark of the white jute plant (Corchorus capsularis) and are one of the most common agricultural fibres that exhibit moderate to high mechanical properties. This important tropical crop is cultivated in Bangladesh, India, China, Nepal, Indonesia, Thailand and the Amazon region due to the high temperature and water content. The total annual world production of jute is approximately 2,500 tons. Several reports are available about the use of jute as reinforcing ibres with thermosetting and thermoplastics polymers [4,33,34]. Similar to other ibres, treatment and interfacial modiication are sometimes necessary to improve the properties of short or long jute ibre reinforced composites, as demonstrated by Liu and co-workers [34] (See Figure 5.6). The authors showed that the strength and stiffness of the composites generally increased with treatment of the ibres under isometric conditions. However, it was found that ibre impact damping was distinctly affected by the shrinkage state of the ibres during the alkali treatment. In Brazil, jute is widely used to manufacture disposable bags.

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A

B

Figure 5.6 SEM micrographs of: (a) untreated jute (5000×); (b) 5% NaOH treated jute (5000×). Reproduced with permission from X.Y. Liu and G.C. Dai, eXPRESS Polymer Letters, 2007, 1, 299. ©2007, Budapest University

5.6.5 Sugar Cane Fibre As per Goldemberg [35], the sugar cane bagasse is composed of cellulose, lignin and hemicellulose. Several researchers are currently working to expand the use of sugar cane bagasse for several purposes, including the production of engineering composites, to leverage the huge amount of biomass generated each year in leading countries like Brazil. The ibres from sugar cane are obtained from the stalk or stem of the plant (See Figure 5.7), although sugar cane as a grass has stems that contain bundles of ibres not yet classiied as bast ibres. Nowadays several varieties of sugar cane are available in agriculture. The sugar cane plant grows well in subtropical/tropical regions and each type of sugar cane ibre contains different cell arrangements and spatial conigurations. Sugar cane waste ibre is mainly composed of cellulose (65%), which is a polymer of great commercial interest. Sugar cane ibre consists of chains ordered differently that show strong resistance to climatic changes and to biological action. These waste ibres are found in large quantities in Brazil and draw huge interest in inding a new application. The sugarcane bagasse is the ibrous residual material of sugarcane stems that is left after the sugar extraction process in sugar mills. It contains cellulose (46.0%), hemicellulose (24.5%), lignin (19.95%), fat and waxes (3.5%),

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas ash (2.4%), silica (2.0%) and other elements (1.7%) (Figure 5.7). The contribution percentage of each of these components varies according to the variety, maturity, method of harvesting and the eficiency of the crushing plant. It is estimated that worldwide annual production of the bagasse ibre is approximately 75 million metric tons [36].

Figure 5.7 Sugarcane stem and ibre

Nowadays bagasse is mainly used for burning raw material in the sugar cane mill furnaces. The low caloric power of bagasse makes this an eficient process. Also, the sugar cane mill management encounters problems regarding regulations of ‘clean air’ from the Environmental Protection Agency (EPA), due to the quality of the smoke released in the atmosphere. Currently, 85% of bagasse production is used for burning as a fuel; however, efforts are being made to use sugar cane bagasse in the manufacturing of composites [37]. Bilba and co-workers [38] studied the inluence of the botanical components (water extractives, hemicellulose, cellulose and lignin) of sugar cane plant in composites made of bagasse/cement, for building construction materials. Paiva and co-workers [39] extracted lignin from sugarcane plant to replace phenol in thermosetting resin loaded sugar cane bagasse short ibres. As expected, the authors reported a decrease in thermal stability of the composites with an increase in ibre content. However, the viability of replacing phenol by lignin to produce ecofriendly composites was highlighted. Stael and co-workers [40] studied the three principal natural ibres found in the sugarcane waste material and the effect of their

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Natural Fibres and Their Use in the Production of Biocomposites mixtures in thermoplastic composites. This solid-state nuclear magnetic resonance (NMR) technique was used to provide information on chemical structure and sample homogeneity. Both NMR and mechanical property measurements conirmed that a mixture of these natural ibres could be effectively used in composites. Manzur and co-workers [41] studied the effect of the growth of phanerochaete chrysosporium in a blend of low-density polyethylene (LDPE) and sugar cane bagasse. Similarly, Raj and co-workers [42] studied polyethylene as a binding material to improve the mechanical properties and dimensional stabilities of steam exploded bagasse composites. Other important works with composites based on bagasse and thermosetting matrices such as phenolic [43,44], melamine [45] and thermoplastics such as polyamide 6 [46] and polyvinyl alcohol [47] have also been reported. Composites made of recycled polymer matrixes and ibres are also reported in the literature. Lei and co-workers [48] studied composites based on recycled highdensity polyethylene (RHDPE) and natural ibres prepared by melt blending and the compression moulding technique. They studied the effects of the ibres (i.e. wood and bagasse), and the type and concentration of the coupling agent on the composite properties. It was found that modiied polyethylene, i.e. the maleated and carboxylated derivatives, as well as a titanium-derived mixture improved the compatibility between the bagasse ibre and RHDPE. As a consequence, the mechanical properties of the composites were improved compared to the virgin high-density polyethylene (HDPE) composites. The modulus and impact strength of the composites reached the maximum values when the content of maleated polyethylene was increased. An interesting report from Ventura [49] commented on asphalt paving using cellulose ibres from sugar cane bagasse. Besides being simple and inexpensive, the measure allows the reuse of leftovers from the manufacturing process of sugar and alcohol. Similarly, Lei [48] reported that this type of asphalt can be used in the construction of roads with heavy trafic, airports, loading and unloading areas, bus stops, parking and other structures. The importance of ibre and/or matrix modiication is also reported for sugar cane bagasse composites. It was observed, by Vasquez and co-workers [50] that the tensile strength and the elongation at break of the polypropylene matrix composite decreased with the incorporation of untreated bagasse ibre. However, isocyanate and mercerisation treatments enhanced the tensile properties of composites advocating the need for natural ibre treatment for increasing the chemical compatibility between ibres and matrices.

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5.6.6 Abaca Fibre Cellulosic bast ibres from Abaca are obtained from the pseudo-stem of banana plant (Musa sepientum) in tropical countries [51] (Figure 5.8). The ibres from waste that comes from banana plantations may be used industrially without much expense.

Figure 5.8 Picture of abaca plant (left); ibres (right). Reproduced with permission from M.A Maleque, F.Y. Belal and S.M. Sapuan, The Arabian Journal for Science and Engineering, 2007, 32, 2B, 359. ©2007, Springer

The Philippines is the world’s leading producer of abaca and its production involves a dedicated effort of approximately 90,000 farmers. In the past few years, the export of abaca from the Philippines is more inclined towards the pulp of abaca rather than abaca ibres. Daimler Chrysler proposed an innovative use of this ibre by introducing the material in the protection of the underloor of passenger cars, as ibre resists moisture and stone strikes. The composites of polypropylene (PP) with abaca ibres were patented by this company and are manufactured by Rieter automotive. According to Bledzki and co-workers [51], the ibre of abaca exhibits a high tensile strength and has a speciic lexural strength similar to glass ibres, as well as good resistance against rotting. These authors studied abaca ibre reinforced PP composites with proportions of ibre between 20 and 50 wt.% and results obtained are shown in Figure 5.9. It can be seen in Figure 5.9 that the addition of a coupling agent (maleated polypropylene – MAPP) to the system improved the tensile properties of the resultant composites.

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Natural Fibres and Their Use in the Production of Biocomposites 90

Without MAH-PP With MAH-PP

Strength (MPa)

80 70

Flexural

60 50 Tensile

40 30 20 15

25

35 Fibre Load (%)

45

55

Figure 5.9 Inluence of ibre loading, with and without chemical modiication, on mechanical properties of abaca ibre-PP composites. Reproduced with permission from A.K. Bledzki, A.A. Mamun and O. Faruk, eXPRESS Polymer Letters, 2007, 1, 11, 755. ©2007, Budapest University

5.6.7 Cotton Fibre The word ‘cotton’ (Gossypium hirsutum L.) has Arabian roots as it was introduced into European countries by the Arabic merchants, who named it ‘al-quTum’. Cotton (Gossypium hirsutum L.) is a perennial plant that interestingly grows and reproduces at the same time.

Figure 5.10 Images of cotton plant and ibre

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas The cotton ibres, as shown in Figure 5.10, grow on the seed of a variety of cotton plants of the genus Gossypium. Cotton has a wide variety of applications such as in home furnishings as draperies, bedspreads, window blinds, sheets, pillowcases, towels, washcloths, etc. [52]. The cultivation of this plant is widespread and is estimated to occupy approximately 2.5% of the land area available for plantation on Earth. An interesting statistic was released by the United Nations Conference on Trade and Development that shows the increase in production and projected production of cotton since 1980 until 2013 (Figure 5.11).

30 25

Others Brazil Uzbekistan Pakistan

20 15

India

10

United States

5 China 2012/13

2010/11

0

Figure 5.11 World cotton production (million tons, by main countries, 1980/81– 2012/13). Reproduced after UNCTAD secretariat, based on International Cotton Advisory Committee (ICAC) statistics, 2011 [53].

Hashmi and co-workers [52] studied cotton ibre and reported their performance in reinforcing polymer matrix composites in terms of physical, mechanical and thermal properties. The authors observed that merely adding 27.5% (vol.) of cotton to unsaturated polyester resin increased the lexural strength (from 101.8 to 142 MPa) and the modulus of elasticity at bending (from 2.4 to 4.2 GPa). Similarly, a signiicant increase in impact strength (from 61 to 971 N m/s2 per unit width) was observed. This high surge in value of impact strength achieved by these authors has suggested new applications of cotton ibres. Jiang and co-workers [54] studied biodegradable composites consisting of lax (FF) and cotton (CT) ibre mats with polyester amide (PEA) ilms that were hot-pressed using

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Natural Fibres and Their Use in the Production of Biocomposites the ilm stacking process. The process parameters of pressure, pressing temperature and drying conditions of ibre and matrix were optimised to improve the mechanical properties as well as the visual quality of the composites. The authors found that pressing temperature was an important parameter that controlled the properties of the inal composite. The results suggested that the biodegradable PEA/CT and PEA/ FF composites had superior mechanical properties compared to PP/FF composites.

5.6.8 Kenaf Fibre Kenaf (Hibiscus cannabinus) is a plant (Figure 5.12) that belongs to the jute family and is mainly produced in China and India.

Figure 5.12 Kenaf ibre and plant

The kenaf ibre is composed of cellulose, lignin and hemicellulose with lengths greater than 1 metre (Figure 5.12). Since the exploration of kenaf in the USA in 1940 as twine these ibres are currently being used in the manufacturing of rope and bagging to produce carpet backing, in packaging materials, to obtain various grades of paper and cardboard and as fencing of residences and farms. Kenaf ibres are also used as litter (e.g. horse) and as an absorbent material in oil and chemical spills. These ibres have good properties to attract those who aim to obtain concrete composites loaded with natural ibres to enhance the performance of the inal product. Studies of Feng and co-workers [55] showed the improvement in mechanical properties, such as tensile and impact strength, after using maleated polypropylene

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas (MAPP) in PP loaded with kenaf ibres. The authors observed that the impact strengths were enhanced on addition of the coupling agent in the system due to an effective stress transfer from matrix to ibres. Similarly, Sanadi and co-workers [56] carried out experiments using kenaf polypropylene loaded with varying percentages (up to 60%) of ibres in the composite. The authors used maleated polypropylene (MAPP) to improve the interface between matrix and ibre. In some cases, the results from these composites displayed behaviour that was similar to that obtained from glass ibre-PP injection-moulded composite. This suggested that kenaf ibres could be used as a viable substitute for glass ibres in composites where moisture absorption is not a limiting factor.

5.6.9 Bamboo Fibre Bamboo is an abundant natural resource in Asia and South America (Figure 5.13). Brazil has the largest number of native species and the largest natural forest of bamboo. Bambu-trepador (Chusquea capituliflora) is the commonly known bamboo that is related to Vasconcellos [57]. This plant provides highly versatile material that could be used in applications such as recycling of contaminated water from rivers and lakes. Bamboo is used as a substitute for wood in building construction that prevents the cutting of other trees that are essential to the natural balance of the environment. Due to its excellent properties, bamboo ibres are recognised as an adequate substitute for glass ibres [57]. However, good bamboo composite is obtained by combining the knowledge about the processing parameters and ibre properties.

Figure 5.13 A type of bamboo plant

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Natural Fibres and Their Use in the Production of Biocomposites In comparison with other ibres, bamboo ibre is brittle as it is covered with lignin that provides rigidity to the trunks of trees. Okubo and co-workers [58] studied the effect of bamboo steam-exploded ibre in ecological composites made of PP matrices, and their resulting mechanical properties. They observed an increase of 15% and 30% in tensile strength and modulus of PP, respectively, after the addition of ibres treated by stem explosion. The authors proposed that the elevation in mechanical properties was probably due to the impregnation of ibre within the PP matrix that reduced the number of defects that exist during the mechanical extraction of the ibres. Thwe and Liao [59] added a coupling agent based on polypropylene maleic anhydride in their research with polypropylene (PP) combined with bamboo ibre and hybrid composites of PP with bamboo and ibreglass. It was shown that the presence of a coupling agent and hybridisation with synthetic ibres is a viable approach to improve the mechanical properties such as tensile and lexural strength.

5.6.10 Açai Fibre Açai (Euterpe oleracea) is a palm plant (Figure 5.14) widely found and cultivated in Amazon regions and Para, a state of Brazil. The pulp of its fruit is widely consumed (approximately 180 tons/year) in beverages and other food items.

Figure 5.14 Açai berry trees

Açai fruit is rich in protein, ibre and lipids. Its palm leaves are used for the production of woven products (bags, nets, etc.). The plant serves as the roofs of houses due to its environmental stability. The leaves of this plant may harbour the protozoan

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas trypanosoma cruzi that causes Chagas disease. The açai fruit must be pasteurised before consumption. In Brazil, the açai palm is now widely used by the food industry and by the craft industry, which uses the core of fruit to decorate their products [60]. Similar to other ibres, açai ibre density is low and estimated at 1.308 g/cm3. Low ibre density has generated enormous interest for its use in composites of high performance along with the annual production of huge amounts of biomass [61]. The waste generated by açai after industrialisation is approximately 100,000 tons, which causes serious problems to public health and the environment. This problem has drawn the attention of several researchers in the direction of adding value to açai waste. Martins and co-workers [8] worked on composites based on natural rubber and açai ibres. The resistance to heat is an important characteristic of açai ibre, which was commented on by Embrapa, who registered its thermogravimetric (TG) proile [8]. The ibres showed good thermal stability up to about 230 °C, with a degradation process in three stages: the irst peak at about 280 °C related to the depolymerisation of hemicellulose; the second peak at 345 °C was attributed to decomposition of cellulose and lignin; inally, the third small peak at about 610 °C was attributed to the residue degradation. This thermal behaviour shown by açai ibres is similar to that of major natural ibres already used industrially, such as sisal [62] and coconut [63], and has opened new perspectives for its use in the development of new materials. Castro and co-workers [64] worked with recycled matrices of high-impact polystyrene (HIPS) and polypropylene (PP), moulded by hot compression in the presence of açai ibres. The process conditions adopted were suficient to produce composites without ibre degradation. The authors found that the impact resistance of both polymer composites was increased by incorporating the ibre (Figure 5.15), suggesting the use of ibre for the reinforcement of thermoplastic material.

5.6.11 Curauá Fibre Fibre curauá (Figure 5.16) is found in the Brazilian Amazon and has been reported as one of the most promising ibres for agriculture. The cultivation of curauá can protect rainforest against inappropriate exploitation.

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Tensile strength (MPa) Compression resistance (MPa) Impact resistance kJ/m2

70

1

PP – 1 PPA – 2 HIPS – 3 HIPSA – 4

3 2 2

35

4

4 1

3

1 2

3 4

0 Tensile strength (MPa)

Compression resistance (MPa)

Impact resistance (kJ/m2)

Figure 5.15 Comparison of mechanical trial results: polypropylene (PP), polystyrene (HIPS), polypropylene/açai ibre composite (PPA) and polystyrene/ açai ibre composite (HIPSA). Reproduced after C.D.P.C. Castro, C.G.B.T. Dias and J.A.F. Faria, Materials Research, 2010, 13, 159. ©2010, Materials Research Society [64]

Figure 5.16 Curauá plant

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas Companies like Volkswagen Corp. and Mercedes-Benz Corp. created research centres to interact with the local people near curauá plantations, based on a programme named POEMA (Poverty and Environment in Amazon), aiming to create jobs and new opportunities [65]. In the past few years the curauá ibre has gained importance in the Brazilian economy. The low production cost and good mechanical properties are two important characteristics that contribute to the design of curauá ibre composites with high performance. For example, Volkswagen Corp. and others used the curauá ibre left over from the textile industries with a polypropylene matrix to use as a roof in vehicles [65]. The number of scientific reports from Brazilian work on Curauá is growing signiicantly. Mano and co-workers [66] showed the potential of curauá-loaded plastic composites obtained by injection moulding that have a wide range of applications in the automotive and electronics industries, as well as in the manufacturing of consumer goods. A few other examples of applications are coating recorders, mobile phones and power tools. Unlike glass ibre composites, these ibre composites can be recycled, which opens new perspectives for their future applications as biodegradable composites. Lopes [67] highlighted the properties of the curauá ibre compared to other natural ibres such as bush, banana, sugarcane bagasse-cane, hemp, ramie, sisal, jute, malva and wood. Gonçalves and co-workers [68] treated curauá ibres with ionised air to improve their adhesion to a phenolic matrix and showed that the thermal stability of the ibres remained the same. In addition, the authors registered an increased impact strength that was a function of the time of the ibre treatment. The hydrophilic character of the ibres decreased, suggesting their enhanced afinity for hydrophobic matrices.

5.6.12 Banana Fibre Banana ibres are obtained from the dried pseudo stem of banana trees as part of the biomass generated during cultivation. Balzer and co-workers [69] investigated these long ibres in composites made of polyvinyl chloride and ibres obtained from the pseudo stem of a banana tree (Figure 5.17). The authors concluded that composites obtained from rigid PVC produced an increase in the tensile and impact strength when the ibre content was approximately 10%. Therefore it is possible to work with these ibres to improve their performance in several polymer matrices and to impart good mechanical properties to the inal composites. It is well known that natural ibres such as banana ibres have low thermal stability and high moisture afinity, which may interfere in the mechanical properties of the composites and decrease their performance. The treatment of these ibres is recommended to make them more compatible with the polymer matrices as mentioned earlier.

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Figure 5.17 Long ibres and composite from the pseudo stem of a banana tree. Reproduced with permission from A.L.S. Pereira, presented in part at the Federal University of Ceará, Fortaleza, 2010

Paul and co-workers [71] studied the composites of polypropylene ibre and short banana ibre. After using several types of ibre treatments, they found better tensile and lexural behaviour of the composites that supports the results of other authors who worked with other natural ibres to achieve improvements in the inal properties of composites. Maleque and co-workers [72] reported the superior properties of banana ibre reinforced epoxy composites. The authors obtained favourable results for ultimate strength and lexural strength of composites with the addition of the ibres. Moreover, it was speculated that impact strength of the composites may increase with the addition of ibres. Banana ibres may be used for other engineering applications such as automotive, as reported by Joseph and co-workers [27]. In another similar work, Corbiere-Nicollier and co-workers [73] commented on the good lexural strength values in banana ibre-cement composites. These authors proposed an excellent work on life cycle assessment that focused on the replacement of glass ibres by natural ibres in plastics composites. Similarly, Padilha and co-workers [74] studied the mechanical and rheological behaviour of polypropylene composites loaded with banana tree ibres. They showed that the elastic modulus of polypropylene composites might increase to about 20% when the ibre content was increased from 0 to 15%. Additionally, these authors suggested that the treatment of the ibre surface could be an alternative to improve the tensile strength of reinforced composites.

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5.6.13 Licuri Fibre The Licuri tree (Syagrus coronata) is a native tree from Bahia, a state in Brazil, and is an essential plant in arid regions. The seeds and fruits of this tree are an important edible item and food base for many wild birds. The ibre from this tree is used for ishing nets and its use in composites needs to be explored. Ricl and co-workers [75] reported the composition of Licuri’s ibre and compared it with other natural ibres. It was interesting to note that the hemicellulose content of Licuri ibres was similar to curauá, while its cellulose content was comparable to hemp, lax, jute, sisal and ramie. Additionally, the ibre’s lignin content was similar to sugar cane bagasse, wood and bamboo. Further investigations are under way that may exploit the potential of this ibre as a reinforcement in polymer composites.

5.6.14 Ramie (Boehmeria Nivea) This perennial plant is native to Asia and was grown primarily to produce ibre. It is one of the oldest sources of ibre known by man. Among more than 30 varieties of ramie, the most common type comes from China and Japan. Its leaves are thick and wide, dark green on the upper side and white and woolly beneath. Another common variety of this plant is found in the tropics [76]. The ramie ibre belongs to the family of long ibres such as lax, jute, sisal and hemp, with an average length of 150 to 200 mm. It has a high environmental resistance that is three times more than hemp, four and eight times more than lax and cotton, respectively. Figure 5.18 shows a picture of a ramie plant and a bundle of ramie ibre.

Figure 5.18 Ramie plant and ibres

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Natural Fibres and Their Use in the Production of Biocomposites Ramie may be used in various sectors such as manufacturing of textiles, rope and string. It can also be used to generate the production of cellulose for currency paper, due to its high environmental stability. Moreover, it can be employed as an internal reinforcement in the manufacturing of hoses, tires, wires, parachutes and others. It was introduced in Brazil in 1939, in the southern state of Sao Paulo. Brazil is now the third biggest producer of ramie in the world after China and the Philippines [76].

5.7 Conclusion and Future Prospects According to the authors of this chapter, although a lot of natural ibres have already been explored by many researchers, much remains to be done to expand the knowledge in this ield. The large amount of biomass generated by the whole planet, which can be converted into consumer goods, is still being explored. Thus, the search for new types of ibres for use in composites has been the goal of many researchers engaged in adding value to biomass. Besides being cheaper, natural ibres have low density compared to other materials and do not damage the processing equipment or the environment [77,78]. However, as seen in this chapter, some important parameters should be tightly controlled by those who intend to enter this fascinating ield of research, to ensure reproducibility of important properties of composites, especially those that are thermo-mechanical. To achieve this, a parameter that deserves attention is the moisture content of ibre, which in many cases is high and compromises the inal properties of a product designed for high performance. Other factors no less important are the thermal stability of the ibres, during processing of composites, and their afinity to the polymer matrix. In general, natural ibres are hydrophilic, while polymer matrices tend to be hydrophobic, resulting in a low afinity for each other and very poor mechanical properties. However, if one could treat these ibres properly, they could achieve thermo-mechanical properties as good as, or even higher than in some cases, the conventional synthetic ibres. The surface treatment of natural ibres includes chemical and physical methods, or a combination of both. The choice, however, is based on both cost and environmental impact.

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6

Lignin: Amorphous Natural Biopolymerbased Blends and Composites for Various Applications

Shivani B. Mishra

6.1 Introduction Wood composite, one of the strongest composites existing in nature, is a culmination of highly crystalline and highly amorphous biopolymers. Wood has the ultimate composite properties that make it sustainable through ever-changing climate conditions. The wood composite existing on Earth for billions of years poses a challenge for scientists to imitate the same properties artiicially, despite having modern methodologies and equipments. The perfection of Mother Nature is beyond imagination and we as scientists wonder what it takes to generate composite with the inest synthesis and ultimate properties. This question will remain unanswered and it will take many years to understand the genesis of perfect creativity. Wood is required for many essential domestic and industrial purposes. Most of the applications include wood as a composite material e.g. domestic and ofice furniture, household items like doors and windows, looring, rooing, cupboards, decorative sculptures, sports and recreational equipment. A major part of wood is used in the pulp and paper industry where the wood composite is separated into cellulose, hemicelluloses and lignin for making paper. During the paper-making process the cellulose and hemicellulose parts are used and the lignin is discharged into the pulp and paper mill industrial wastewater. Existence of lignin in wastewater increases substantially the chemical oxygen demand and the biochemical oxygen demand of the water. It resists UV light penetration and thereby indirectly affects the growth of aquatic life by inducing mutagenesis. Lignin and its derivatives are also toxic compounds, like furans and dioxins, in wastewater, which also play a large role in pollution. Lack of recovery of lignin from the wastewater, as well as limited biodegradability and applications, renders it one of the major pollutants that has created a worldwide challenge. Measures have been taken to improve its utility to an extent but it still holds a potential for undiscovered applications. This chapter deals with the details of lignin as a biomolecule, its origin and chemistry, toxic behaviour, biodegradation and chemical modiications. Various applications of lignin, especially

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas its use in blends and composites are discussed extensively in this chapter. Some other applications of lignin such as its use as an antioxidant and a dispersant, its source of energy and its ability to produce nanomaterials have been also mentioned. In the summary of the chapter there is an overview of the future scope of this biomolecule to highlight the important aspects and issues that need to be addressed. Being polyphenolic, the biomolecule holds the opportunity for innovative and novel research ideas to be implemented in such a way as to establish a clean environment with green technology. Waste minimisation through the best possible commercial and domestic utility should be put forward as soon as possible to save nature from further pollution for generations to come.

6.2 Lignin: Origin and Chemistry Lignin is natural, amorphous and the only aromatic biopolymer present in the cell walls of pith, roots, fruit, buds, bark and cork of lycopods, ferns, gymnosperms and angiosperms. It is absent in non-vascular plants such as fungi and algae. It is concentrated in the middle lamella and functions as a iller or cementing agent to impart rigidity to wood tissue. The chemical structure of protolignin (lignin in its native form) from conifer or gymnosperm woods is given in Figure 6.1. The structure and biochemical elucidation interprets lignin to have formed by coupling of phenoxy radicals of p-hydroxyl phenylpropanols. It has been reported that the origin of lignin is due to enzyme-initiated dehydrogenative random polymerisation of coniferyl alcohol, sinapyl alcohol and p-coumaryl alcohol presented in Figure 6.2. This enzyme-initiated dehydrogenation is the irst and the most important step as it generates mesomorphs of the precursor alcohols. The radicals of the mesomorphs can couple in many ways in a random manner. This is accompanied by the addition of water or phenolic or aliphatic hydroxyl groups to intermediate quinonemethide structure shown in Figure 6.3. The lignin has been roughly classiied as hardwood lignin, softwood lignin and grass lignin. Different authors have concluded a proposed structure of this biopolymer but uncertainty in this regard still exists. This is because of the fact that some of the fundamental queries remain biogenetically unsolved e.g. dibenzyl-tetrahydrofuran structures, α- and β-bonds, α-aldehyde groups, etc.

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Figure 6.1 Chemical structure of protolignin

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Coniferyl Alcohol

Coumaryl Alcohol

Sinapyl Alcohol

Figure 6.2 Monomers of protolignin

Ligniication strengthens the cell wall by forming a ramiied network throughout the matrix, anchoring microibrils more irmly and protecting from chemical, physical and biological attack [1]. Lignin and lignans are assembled from same phenolic precursors. On the other hand, lignin and its derivative impart an offensive colour to water that not only is aesthetically unacceptable but also inhibits the natural process of photosynthesis in streams due to absorbance of sunlight. This leads to a chain of adverse effects on the aquatic ecosystem as the growth of primary consumers as well as secondary and tertiary consumers is adversely affected. Lignin is not easily degradable when exposed to biological process [2]. The other physical properties of lignin include its amorphous appearance. The colour of the lignin is brown and the molecular weight of natural lignin is 1,000 to 12,000. However, fractioned lignin sulfonates range from 300 to 140,000. Another property of lignin is its capacity to absorb ultraviolet light at 2800 Å and for lignosulfonates it is 12.5 Å. Lignin is insoluble in most solvents, which reduces its material applications.

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Figure 6.3 Dehydrogenation of coniferyl or sinapyl alcohols to a highly mesomeric free radical

6.3 Toxicity of Lignin The toxicity of a chemical depends upon its ability to affect the metabolic activities of living systems, causing severe changes that cause disease. It is important to monitor the status of a chemical compound and its derivatives affecting the environment in large quantities. Pollution in its worst form is a threat to living bodies and the ecosystem. It is interesting to note that the amorphous biopolymer in pristine form is the cementing agent of cellulose microibrils in plants. Lignin in its pure form is not a challenge for biological activities of plants. Many applications, either domestic or commercial, that make use of wood composite have been successfully implemented for some time and no reports have been found where a composite in its pure form was the cause of extinction of any living body. However, when lignin itself is released from the cellulose network during the paper-making process, it becomes a major factor that inluences the aquatic system. Paper-industry discharge, the combined efluent commonly known as black liquor, contains persistent organic pollutants.

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas These are mainly wood extractives such as lignin, lignin derivatives, stillibines and resin acids. These are xenobiotic compounds present along with some more harmful compounds like dioxins, furans and non-process elements like sodium, potassium, calcium and silica [3]. Lignin and lignin derivatives, chlorinated lignin and sulfonated lignin are the major components of black liquor. When discharged into water, these wood extractives primarily reduce the light transmittance, thereby inluencing the organic productivity resulting in mutagenecity [4]. Earlier in some reports, lignin and lignin derivatives were declared as biologically inert compounds due to high molecular weight. These reports suggest that the compound with a high molecular weight cannot be transported to the living cells [5]. Recently, some researchers investigated the biological effects of lignin and lignin derivatives and concluded that these compounds displayed toxicity when measured by a rapid screening method commonly called RET assay [6]. However, lignin in its puriied form has also been studied for toxicity. These lowmolecular-weight phenolics were found to have antimicrobial activity [7]. The bactericidal effect of puriied lignin was able to control the intestinal pathogens and this probably will help the safe delivery of livestock products. Some lignin derivatives have also shown prebiotic effects that assist beneicial bacteria in monogastric animals [8].

6.4 Biodegradation of Lignin The biodegradation of lignin has been an area of interest across the world as it provides a safe platform for structural modiication. Lignin biodegradation has been investigated by various researchers using different microbes such as fungi, bacteria and enzymes. In this section we will discuss the research work done for lignin biodegradation. Tuomela and co-workers [9] described in their review article the biodegradation of lignin in compost environment. They concluded that microbial fungi eficiently carried out lignin degradation in compost and the synergistic effect with other soil microbes enhances the biodegradations. The lignin fragments were found to give rise to humic acid both in soil and in compost. Jeffries [10], in his review for microbial degradation of lignin, concluded that xylanase and other lignin-degrading enzymes were usually found in bacteria and fungi. However, it is very dificult to understand their multiciplity and substrate speciicity. Interestingly, no enzyme has been discovered to date that has successfully broken the bond between the carbohydrate and the lignin part of the wood composite. In a different study, Lentinula edodes, a white rot fungus, degraded wheat lignin. Based on the results obtained, it was hypothesised that the aromatic chains of lignin were oxidised into oxalic acid and formic acid types of products [11]. Using sulfate reducing conditions, high-molecular-weight lignin was poorly degraded

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Lignin: Amorphous Natural Biopolymer-based Blends and Composites for Various Applications at the rate of 3.5 mg/l/day. The inoculums were received from a simulated landill columns reactor. Under anaerobic conditions, the high-molecular-weight lignin was degraded into low-molecular-weight aromatic compounds like hydrocinnamic acid [12]. Another white rot fungus Trametes elegans was applied to solubilised lignin in spent black liquor of pulp and paper mill industrial wastewater. Polymerisation and depolymerisation of lignin were observed during degradation and the researchers recommended a selective shift of cultural functioning if only depolymerisation had to be chosen for the degradation [13]. Harvey and co-workers [14] studied the oxidative capacity of ligninase enzyme present in Phanerochaete chrysosporium. The ligninase was able to degrade lignin directly and indirectly in the presence of a mediator (vertryl alcohol). In a unique study, researchers added chlorine oxide to the autoclaved suspension of the lignin with mycelium of Phanerochaete chrysosporium. The result showed that the reactive chlorine played a signiicant role in enhancing the degradation, probably due to lignin oxidation and fragmentation [15]. Four different cultures, Streptomyces viridosporus NRR 2414 white-rot fungus, Coriolus versicolor IRRL 6 102, Phanerochaete chrysosporium IRRL 6359 and Phanerochaete chrysosporium NRRL 6361, were applied onto the sugar cane bagasse. Of these four strains, Phanerochaete chrysosporium NRRL 6361was found to be the most actively degrading microbe that released maximum reducing sugars. The results obtained showed that all the four microbes had degraded cellulose more when compared to lignin [16]. Another white rot fungus Irpex lacteus CD2 degraded corn stover lignin. This biodegradation resulted in an increase in conjugated carbonyl groups and a high amount of methyl in methylphenyl ether. The authors reported three modes of biodegradation with the production of benzoic acid due to oxidative cleavage of propyl side chains. The second mode of degradation involved modiication of side chain structure of lignin and the last mode was oxidative ring opening of the aromatic nuclei [17]. In another study by Martinez and co-workers [18], analytical pyrolysis was adopted to investigate the degradation of lignin by Pleurotus species of white rot fungi. In this type of degradation, aromatic ring substitution patterns of lignin units were sustained. Some of the degraded products were found to enter into lignin-polysaccharide complexes. Leonowicz and co-workers [19] wrote a short review about the lignin degradation by white rot fungi. In this review, they discussed the work done in understanding the biochemistry of lignin degradation that occurs due to the synergistic effect of the small molecules, enzymes and radicals. These fungi have multienzyme systems that simultaneously degrade lignin and cellulose in wood composites. Rhus vernicifera laccase enzyme was isolate from lacquer tree sap and was used to degrade lignophenols into aliphatic chains with no carbonyl functional groups present. The researchers also observed that if phenol derivatives are introduced into lignin, it

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas becomes easy to control the degradation process in such a way that the industrially important chemicals can be obtained [20]. Zimmermann [21] discussed the bacteria-based lignin degradation in his mini review. He reported that the basic mineralisation of lignin involves oxidation, demethylation and aromatic ring cleavages. Pseudomonas species and Gram-positive actynomycetes were the most used bacteria for lignin degradation. Table 6.1 provides an overall view of this section dedicated to the biodegradation behaviour of lignin and its derivatives.

Table 6.1 Lignin biodegradation Lignin/lignin derivatives

Source of degradation

Reference

Lignin in paper

Compost

Tomela and co-workers (2000)

Wheat lignin

Lentinula edodes

Crestini and co-workers (1998)

Organosolv lignin

Sulfate reducing condition

Ko and co-workers (2009)

Black liquor lignin

Trametes elegans

Lara and co-workers (2003)

Monomethoxylate, dimethoxylated lignin derivatives

Phanerochaete chrysosporium, vertryl alcohol, radicals

Harvey and co-workers (1986)

Autoclaved lignin suspension

Mycelium of Phanerochaete chrysosporium

Johansson and co-workers (2000)

Softwood and hardwood lignin

Streptomyces viridosporus NRR 2414 white rot fungus, Coriolus versicolor IRRL 6 102, Phanerochaete chrysosporium IRRL, 6359 and Phanerochaete chrysosporium NRRL 6361

El-Gammal (1998)

Corn stover lignin

Irpex lacteus CD2

Yang and co-workers (2010)

Wheat lignin

Pleurotus sp.

Martinez and co-workers (2001)

Lignophenols

Rhus vernicifera laccase

Xia and co-workers (2003)

Lignin

Bacteria

Zimmermann (1990)

6.5 Chemical Modification of Lignin As we have understood from the above section, lignin is basically a cementing agent of cellulose ibres. Lignin in its pristine form is aromatic polyphenolic in nature.

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Lignin: Amorphous Natural Biopolymer-based Blends and Composites for Various Applications Being polyfunctional in nature, it offers researchers the opportunity to tailor the physical and chemical properties. Lignin needs to be chemically modiied to improve its performance properties as a biopolymer. This becomes highly important when it has to be used for a particular application. The modiication of lignin can be done chemically, thermally, genetically or by the irradiation method. The processing conditions for modiication greatly inluence the product and chemical properties of lignin. In this section, we will primarily focus on the various types of modiication of lignin reported. The one common observation was the cleavage of ether linkages and condensation of smaller units. In a recent report [22], chemically modiied lignin model compound was prepared with the help of arylboronate ester. The ester used to modify the lignin resulted in better solubility and thermal properties. Different types of lignin such as oil palm trunk ibre lignin, poplar lignin, maize stem lignin, barley, wheat and rye straw lignin were modiied with succinic anhydride. The authors observed that different types of lignin after undergoing succinolation produced different types of modiied lignin as revealed from Fourier transform spectroscopy. Also, all the modiied lignins were comparatively more stable than the original ones [23]. Another method to modify lignin and its derivatives is through heat treatment. Recently, two types of lignin i.e. Klason lignin and milled wood lignin along with holocellulose were thermally treated. The heat treatment depolymerised these natural biopolymers and this was followed by the recondensation reactions involving guaiacyl units as predicted by spectroscopic analysis. The degradation of the lignin occurred through the cleavage of β-aryl-ether linkages [24]. Barsberg [25] applied electron-abstracting oxidative system for the modiication of the lignin. For this, laccase-ABTS diammonium salt was used where the latter mediated for electron abstraction between the polymer and the enzyme. This type of modiication produced different temporal phases. The grafting of amino acids onto the lignin was done with the help of laccase enzyme. A number of amino acids such as glycine, phenylalanine, serine, arginine, histidine, alanine and aspartic acid were evaluated. This was done to increase the number of carboxylic acid functional groups. This type of modiication improved the tensile properties and tear resistance properties as well as the wet tensile strength [26]. Using sodium hydroxide and potassium hydroxide lignin was chemically activated by different reaction conditions to produce activated carbon. The optimal temperature reported here was 700 °C along with other physical parameters such as activating agent and time of activation to obtain highly porous product [27]. In a unique study, the modiication of lignocellulosic material was done using NdYAG pulsed laser beam used at 1064 nm. This tool of modiication was found to act speciically on the lignin part of the lignocellulose. The major change observed in this case was the reduction of polydispersity in modiied lignin [28]. The auto-hydrolysis process was used to modify aspen wood lignin. This was able to induce cleavage of

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas β-O-4 linkages of the lignin biopolymer through acidolysis and homolysis. A low degree of deligniication and poor lignin reactivity was reported with the probable reason of simultaneous condensation of lignin units. The use of sodium hydroxide and 2-naphthol played a role in improving the number of carboxylic acid groups for the former and the number of phenolics hydroxyl groups and the naphthalene rings for the latter [29].

6.6 Lignin-based Blends Polymer blends are developed to form a material that has superior and desirable properties that a pristine polymer cannot deliver. It is the technique that can be used to develop a new material with tailor-made properties. Polymer blends can be homogenous or heterogeneous based on the nature of the polymers mixed together. Lignin, a polyphenolic, amorphous polymer and the second most abundant polymer existing in nature, has been exploited for very many applications. In this section, lignin-based polymer blends that were investigated have been reported. Kraft lignin was mechanically mixed with polyethylene oxide followed by thermal extrusion in different ratios of between 5 and 10% wt./wt. It was found that a small amount of polyethylene oxide was able to disrupt the supramolecular structure of lignin and led to superior physical properties. It was interesting to note that all the blends were homogeneous in nature as predicted by differential scanning calorimetry. A negative value of the glass transition temperature suggested that there were weak intermolecular forces, which were very speciic between the two polymers. Also, there was string hydrogen bond formation between the aromatic hydroxyl proton of lignin and oxygen of ether in polyethylene oxide [30]. The otherwise brittle kraft lignin and its acylated and alkylated derivatives were plasticised using polymers with low glass transition temperatures. The polymers chosen in this case were miscible aliphatic polyester, polyethylene glycol and polyethylene glycol bis-phenol. The blends were prepared by the solvent casting method. It was observed that although the alkylated lignin and its adduct were able to plasticise signiicantly by polyethylene glycol, these were found to have poor mechanical properties when compared to their acylated counterparts [31]. In a unique study, researchers compared the nature of the lignin-based blends. Here, the lignin was mixed with apolar and highly polar polymer matrices and it was recommended that it is the phenolic character that plays a huge role in deining the ultimate properties of the blend rather than its polymer characteristics. Lignin was blended with unplasticised polyvinyl chloride and polystyrene [32]. p-Aminobenzoic acid/phthalic acid anhydride was used to produce a kraft lignin derivative, which was later blended with polyvinyl alcohol by casting. The ilms of blends were irradiated

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Lignin: Amorphous Natural Biopolymer-based Blends and Composites for Various Applications with a mercury vapour lamp. It was observed that the kraft lignin derivative signiicantly promoted the antioxidant and photoprotective properties in polyvinyl alcohol. The results also showed an increase in thermal stability due to the kraft lignin derivative. However, an increase in the amount of (≅25%) lignin was not able to form ilms [33]. Using the solvent evaporation technique, kraft lignin was incorporated into polyaniline matrix in emarlidine form [34]. Increased thermal stability was reported as the blend decomposed at 680 °C and a decreased tan value suggested a signiicant interaction between the two polymers. This was further conirmed when X-ray photoelectron spectroscopy (XPS) revealed an increase in protonated nitrogen of 3%. Dimethylformamide cast ilms with 4.2 and 23.2% lax soda lignin were prepared to study their applicative potentials. The polyurethane used in this case was derived from polyethylene adipate, ethylene glycol and 4,4 diphenylmethanamidiisocynate in 1 : 5 : 6 mole ratio. Strong interactions between the carbonyl groups of lignin and polyurethane were observed. It was recommended that 5% of lignin was able to provide the strength of a polyurethane elastomer and was found to be biodegradable [35]. In another study lignin was used up to 30% as iller using low-density polyethylene and polypropylene matrices to study its effect on melt low index, degradation behaviour and tensile properties. It was reported that lignin acted as a good processing stabiliser and light stabiliser for polypropylene whereas for polyethylene it had no effect on processing stability. It was also found to be an initiator for degradation for polypropylene at higher dosage. The choice of matrix to meet the degradation characteristics of the blend and stress conditions plays an important role where the lignin has to be used as iller [36]. Epoxy-modiied lignosulfonate in the binary polyolein mixture was reported to affect the mechanical properties only after 10 wt.% addition and biocompatibility showed signiicant improvement between 0 and 20 wt.% of the iller. Most of the blend properties were found to be dependent upon the lignin content. Soil burial tests done in the close vicinity of Vicia X hybrida hort plant resulted in the week negative inluence of the physiological process of the plants [37]. As a compatibilising agent, the 5 wt.% lignin along with 7 wt.% ethylene propylene rubber was eficiently used in poly(ethylene terephthalate)/low-density polyethylene blend. The lignin/ethylene propylene rubber at the above wt.% showed the optimal coupling process yielding a homogenous structure of the blend [38]. Polyfurfuryl alcohol/lignin thermosets were developed by introducing plasticised lignin into furfuryl alcohol during polymerisation. Oxidative thermal degradation was delayed with a lower rate of decomposition. The surface morphology revealed the monophasic system that indicated good penetration of lignin into the polyfurfuryl alcohol matrix [39]. The inal threshold limits between 68/32 and 54/46 for polyfurfuryl alcohol/plasticised lignin were recommended to avoid scariication of polyfurfuryl alcohol properties. Sugarcane bagasse lignin was blended with polyvinylpyrrolidone. In this study, lignin was extracted from sugar cane bagasse using formic acid. The blend was further

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas irradiated with ultraviolet light to study the changes in the thermal properties of this blend. It was observed that an overall decrease in thermal stability of pure polyvinylpyrrolidone when it is exposed to ultraviolet radiation. This decrease in thermal stability was minimised for the blend. On the other hand, the presence of lignin substantially improves the photostability [40]. The solutions of kraft lignin/ acetone soluble fraction of kraft lignin and polyvinyl alcohol were mixed using dimethylsulfoxide as solvent to obtain the ilms of the lignin/polyvinyl alcohol blend. The presence of lignin improved the thermal stability of the polymer by almost 50 °C. In a similar study, the researchers isolated kraft lignin from pulp and paper mill industry waste. The kraft lignin was then modiied by formic acid to be used as a photoinhibitor for polyamide. The modiied kraft lignin and UV irradiation inluenced the crystallinity ratio of polyamide ilms. The scanning electron micrographs of the blend show that the introduction of lignin into polyamide ilms yielded more spherulites onto the surface of the ilms [41]. 10%, 20% and 30% lignin was blended with polyethylene and polypropylene to study the effect on biodegradation by lignin degrading enzyme Phanerochaete chrysosporium. The presence of lignin peroxidase and Mn(II) peroxidase were observed in the cultivation medium. It was suggested that the biotransformation of lignin during the cultivation process enhanced the partial biodegradation of the polyethylene and polypropylene matrix [42]. A study of the effect of chemical modiication of lignin to prepare lignin/phenolics resin was done to investigate the glue bond performance. It was concluded that methylation of lignin was able to improve the reactivity of the phenol-formaldehyde resin prepared from it. The presence of lignin decreased the blowouts and had a better spread [43]. Lignin- and molasses-based polyurethane foams were prepared to analyse the changes in the thermal properties using differential scanning calorimetry and thermogravimetric analysis. The results show that both the glass transition temperature and the decomposition temperature were not much altered due to incorporation of lignin and molasses with the thermal conductivity of PU foam in the range 0.030 to 0.040 W mk–1. The researchers recommended its use for insulating material [44]. Cellulose acetate butyrate was blended with lignin to produce ibres to study the surface morphology and the mechanical properties. The blend was found to be liquid crystalline in nature with mixed phases. The lignin considerably affected the rheological properties of the blend; however, the mechanical properties were found to be better [45]. Straw lignin powder was mixed with various types of polyethylene i.e. linear low-density polyethylene, low-density polyethylene, high-density polyethylene and polystyrene. The lignin was able to protect the blend from UV radiation and also enhanced the modulus with reduced tensile strength. The authors concluded that lignin in polyethylene blends had poor adhesion and non-uniform distribution [46].

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Lignin: Amorphous Natural Biopolymer-based Blends and Composites for Various Applications Table 6.2 Lignin-based polyblends Lignin/lignin derivatives Kraft lignin Alkylated and acylated lignin

Polymer

Reference

Kraft lignin derivative

Poly(ethylene oxide) Polyester, polyethylene glycol and polyethylene glycol bisphenol Low-density polyethylene Polypropylene Polymethylene oxide Polyethylene oxide Polystyrene Polyacrylonitrile Plasticised PVC Polyvinyl chloride Polyvinyl acetate Poly(hydroxybutyrate-cohydroxyvalerate) Polyester amide (PEA) Poly(butylene succinateadipate) (bionolle) Starch Polyvinyl alcohol

Kraft lignin Flax soda lignin

Polyaniline Polyurethane

Fernandes and co-workers (2001) Rodrigues (2001) Ciobanu and co-workers (2004)

Lignin

Polyethylene and polypropylene

Alexy and co-workers (2000)

Epoxy modiied lignosulfonate

Binary polyolein mixture

Cazacu and co-workers (2004)

Esteriied lignin

Poly(ethylene terephthalate)/ low-density polyethylene

Aradoaei and co-workers (2010)

Lignin

Poly furfuryl alcohol

Guigo and co-workers (2010)

Sugarcane bagasse lignin Kraft lignin

Polyvinylpyrrolidone

Silva and co-workers (2005)

Polyvinyl alcohol

Bittencourt (2005)

Beech wood lignin

Polyethylene and polypropylene

Mikulášová (2001)

Eucalyptus wood lignin

Phenol-formaldehyde resin

Vazquez and co-workers (1997)

Sodium lignosulfonate lignin Organosolv lignin

Molasses-based polyurethane

Hatakeyama and co-workers (2008) Dave and co-workers (1997).

Kraft lignin

Straw lignin

Cellulose acetate butyrate

Soda lignin

Linear low-density polyethylene, low-density polyethylene and high-density polyethylene Poly(hydroxybutyrate)

Black liquor lignin

Polyvinyl chloride

Kadla and co-workers (2003) Li and co-workers (2005) Pouteau and co-workers (2004)

Pucciariello and co-workers (2004) Mousavioun and co-workers (2010) Mishra and co-workers (2007)

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas In another study, poly(hydroxybutyrate) was blended with the soda lignin to investigate the miscibility and thermal properties. A single peak of glass transition temperature suggested the miscibility of the two polymers with enhanced thermal stability. The interfacial interaction of the soda lignin with poly(hydroxybutyrate) was best when 40 wt.% of the former was mixed [47]. Lignin extracted from the black liquor was used to prepare polyblends with polyvinyl chloride using the solution blending technique. Tetrahydrofuran was the chosen solvent for the two polymers to prepare ilms. The result revealed an increase in surface relectance and rigid behaviour of the polyblends, probably due to the aromatic nature of the lignin polymer [48]. Table 6.2 is a summary of the blends discussed in this section.

6.7 Lignin-based Composites In this section we will focus on the work done in the ield of lignin- and lignocellulosicbased composites. Polylactide was incorporated with lignocellulosic materials such as corn stovers, wheat straws and soy stalks and hybrid ibres were developed using twin screw extrusion followed by injection moulding. The surface morphology revealed that there was poor adhesion of the ibres onto the polylactide matrix. This probably resulted in the poor tensile properties of the composites. Interestingly, the mixture of ibres when used as illers did not affect the physico-mechanical properties of the composites. However, the densities of these green composites were observed to be lower than those of glass composites [49]. Sequential extraction was carried out to obtain three different fractions of pine kraft lignin using various organic solvents. The fractions of extracted pine kraft lignin were then used to prepare lignin starch composite material. The important aspect that was observed here was that the three fractions had structurally different lignins that affected the plasticisation/compatibilisation phenomenon [50]. Polypropylene matrix was incorporated with Kenaf (Hibiscus Cannabinus) ibres, which possess some excellent speciic properties. The interaction of the ibres with the polymer was enhanced by using maleated polypropylene as compatibiliser. Based on the results obtained, Kenaf ibres were recommended as an alternative to inorganic/mineral-based ibres. The composites obtained showed low impact and high water absorption [51]. In a unique study, ionic liquid 1-allyl3-methylimidazolium chloride was used to prepare a composite of starch, cellulose and lignin. It was concluded that the cellulose and lignin affected the mechanical properties whereas the starch inluenced the lexibility of the composites. The composites showed high gas permeability for CO2 : O2 permeation ratio, which allows it to be used for food packaging applications [52]. Single screw extrusion was used to compound lignin with poly(ethylene terephthalate) to study the supramolecular structure and thermal properties. The introduction of lignin as a iller into the matrix of 136

Lignin: Amorphous Natural Biopolymer-based Blends and Composites for Various Applications poly(ethylene terephthalate) enhances the overall crystallinity and crystal dimensions. Also, lignin particles ranging from nanometre scale to micrometre scale have been reported [53]. Esteriied lignin was mixed with low-density polyethylene using maleic anhydride grafted low-density polyethylene as compatibiliser. The compatibiliser improved the adhesion between the polymer matrix and the modiied iller, which resulted in better mechanical properties. The scanning electron micrographs show a highly deformed fractured surface that suggested good adhesion and thermal studies resulted in lower weight loss [54]. Modiied montmorillonite and/or sepiolite were dispersed into combined matrices of lignin and natural ibres. The internal structure of a composite where modiied montmorillonite was used as reinforcement revealed mixed morphologies of intercalation and exfoliation, whereas, in the case of sepiolite, the iller was seen as needles dispersed into the matrices. A high lexural modulus, a high glass transition value and better thermal stability were found in the case of montmorillonite-based composites [55]. Lignin is amorphous and exhibits poor thermal behaviour. In this investigation, lignin was used as a carbon source with nano silica obtained using tetraethoxysilane as precursor material. To develop an organic-inorganic hybrid, the hybrid material was pyrolysed at a high temperature of 1400 °C in inert atmosphere, which was followed by oxidation. The heat treatment gave rise to silicon carbide nanorods found in the 50–200 nm range [56]. Sugarcane bagasse-based lignin was used to replace phenol in phenol formaldehyde resin to form a lignin-resin composite. There was a rise in the glass transition temperature of 25 °C and it was observed that the amount of lignin was one factor that improved the rate of cure and the heat of reaction. The composites were tested for coating cardboard, which resulted in good water-resistant behaviour with poor contact angle [57]. In a unique study, Langmuir–Blodgett metastable composite ilms of lignin and cadmium stearate at an air-water interface were developed. Cadmium stearate and lignin had independent phases in the composite and the latter was found to affect the stacking order of the former component. The ilm uniformity was evident from the surface potential measurements [58]. Mechanical properties, especially focusing on non-linear viscoelastic and viscoplastic for hemp/ lignin composites, were studied. Signiicant stiffness degradation was also observed with the rise in strain [59]. Butyrated kraft lignin was incorporated into a lax ibrereinforced thermoset composite as compatibiliser. The thermoset resin was composed of styrene and acrylated epoxidised soybean oil. Incorporation of compatibiliser improved the lexural strength of the composite. Scanning electron micrographs show an improvement in adhesion of the resin with the ibres in the presence of butyrated kraft lignin. However, the occurrence of dry patches in the composite was observed due to an increase in viscosity when 10 wt.% of lignin was solubilised in 150 wt.% of resin [60]. Suspension of multiwalled carbon nanotubes with kraft lignin was used to fabricate the ilms by the casting method. Raman spectroscopy indicated a strong interaction between the amorphous natural biopolymer and that of the carbon

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas nanotubes. The composites were successfully applied to the electrode by decreasing the overpotential for the electrochemical oxidation of dihydronicotinamide adenine dinucleotide [61]. The technology developed was found to open the doors for the use of lignin in sensor applications. Adipic acid-modiied starch microparticles were used as iller for glycerol-modiied corn starch matrix. The incorporation of lignin improved the water resistance and the thermal properties of the composite with reduced transparency. The composite showed high tensile strength and rigid behaviour with low elongation [62]. In another study, lignin was used as adhesion promoter for cotton ibre reinforced polylactic acid composite. A 9% rise in tensile strength and 19% rise in Young’s modulus were observed when lignin was used. There was a decrease in the impact strength by 17% that could be reduced by embrittlement of the composites as per the author’s recommendation [63]. Poly(butyleneadipate-co-terephthalate), a biodegradable copolymer matrix, was chosen to be incorporated with lignocellulosic illers obtained from the industrial fractionation process of wheat straw to produce different biocomposites. The fractionation process was able to modify the granulometry distribution that affected the overall mechanical properties of the composites [64]. In a unique study, lignin was used as iller in asphalt concrete using environmental temperature and water conditions. A higher lexural strength and ultimate lexural strain were observed where lignin was used as reinforcement. On the other hand, it was able to substantially reduce the water-freezing thaw effect [65]. Recently, Karade [66] wrote a review about the studies performed for cement-bonded lignocellulosic composites. He concluded that the lignocellulosic waste material has a great potential to be used for cement-based composites that can be successfully applied for building components, although these were found to be lighter in weight and with reduced tensile strength. The lignin-based composites reported above are presented in Table 6.3.

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Lignin: Amorphous Natural Biopolymer-based Blends and Composites for Various Applications Table 6.3 Lignin-based composites

Lignin/lignocellulosic

Polymer

Reference

Wheat straw, corn stover, soy stalks

Polylactide

Nyambo and co-workers [2010]

Pine kraft lignin

Starch

Baumberger and co-workers [1998]

Kenaf ibres

Polypropylene

Sanadi and co-workers [1995]

Lignin

Starch and cellulose

Wu and co-workers [2009]

Hydrolytic lignin

Poly(ethylene terephthalate)

Canneti and co-workers [2007]

Esteriied lignin

Polyethylene

Sailaja and co-workers [2010]

Lignin

Silicate clays and natural ibre

Guigo and co-workers [2009]

Kraft lignin

Polysiloxane

Mishra and co-workers [2009]

Sugarcane bagasse lignin

Phenol-formaldehyde resin

Park and co-workers [1998]

Lignin

Cadmium stearate

Constantino and co-workers [1998]

Lignin

Hemp

Marklund and co-workers [2008]

Butyrated kraft lignin

Acrylated epoxidised soybean oil and styrene

Theilemans [2004]

Kraft lignin

Carbon nanotubes

Milczarek [2010]

Lignin reinforced plasticised starch

Adipic acid modiied starch microparticles

Spiridion and co-workers [2010]

Lignin

Cotton ibre reinforced polylactic acid

Graupner [2008]

Lignocellulosic ibre

Poly(butyleneadipate-coterephthalate)

Digabel and co-workers [2006]

Lignin

Asphalt concrete

Xu and co-workers [2010]

Lignocellulosic waste

Cement

Karade [2010]

6.8 Other Applications of Lignin Lignin is a rich biopolymer that can undergo a variety of chemical reactions and degrades into a number of degradation products. It has been reported to be a renewable

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas material and should be considered as a replacement for at least part of the world’s inite store of petroleum. There are a number of applications of lignin, which are summarised in Figure 6.4. Some other applications are based on the properties of lignin i.e. colloidal, electrolyte, polarity, solubility and molecular weight distribution. These include its utility in the domain of suspending agents, emulsiiers and additives in plastic or substitutes for phenol and ion exchangers. Biochemical conversion of lignin could lead to its use as microbial proteins and organic fertiliser. Further ammonia-oxidised lignin could be replaced as humus in the soil.

Figure 6.4 Flowchart of lignin-based applications

Pyrolysis of lignin is an approach widely investigated to upgrade this material into higher value-added products. This is reported to be a potential technique to obtain products such as acetic acid, methanol, charcoal and phenolic compounds in large quantities. Combination of pyrolysis with other degradative procedures aims at better results of the utilisation of lignin. Another area of growing interest is biomethanation of black liquor by making use of aerobic and anaerobic treatment. This technique forms valuable gaseous hydrocarbons and biogases like carbon dioxide. Such a procedure solves two purposes simultaneously i.e., decolourising the efluent as well as utilisation of the lignin present in the black liquor into a value-added product to restrict the environmental pollution from further growth. Lignin obtained by precipitation could be used in forming polymer blends and complexes that could be considered as fertilisers, as adsorbent for heavy metal ions and for obtaining chemicals by depolymerising the lignin. In this section we will focus on the various other applications reported. The ilms of kraft lignin from softwood and milled wood lignin from soft- and hardwood

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Lignin: Amorphous Natural Biopolymer-based Blends and Composites for Various Applications were spin coated onto the oxidised silicon wafers. Evaluation of the surface energy of lignin was found to be comparable to that of cellulose. The water contact angle of lignin was found to be more than 90°, which renders the surface of the lignin to be wettable and therefore supports water transport in plants. The study on vapoursolid surface energy was done to investigate the possible applications in the ield of paper-making, ibre board and polymer blends and composites [67]. The solubility of lignin using the Flory–Huggins solubility theory along with the Hoy theory in non-polar solvent (styrene-based thermoset resin) was investigated. The kraft lignin obtained from both the hardwood and the softwood was esteriied with different types of anhydrides. It was concluded that the butyrated and methyacrylated/ butyrated lignin was able to solubilise in styrene and could be used for unsaturated thermoset resins [68]. The antioxidant activity of organosolv lignin as free radical scavenger was studied. Various parameters were found to inluence the antioxidant behaviour such as number of phenolics hydroxyl groups and aliphatic hydroxyl groups, molecular weight, polydispersity and the processing conditions where the regression model was used [69]. Lignin has prominently played a role of both the natural adsorbent and the activated adsorbent. Many researchers have investigated lignin as an adsorbent from all types of wood extracted from pulp and paper mill industry wastewater. Lignin either in natural form or in activated form (physical or chemical activation) has been successfully applied onto the uptake of heavy metal ions. In general, it was reported that natural lignin is a poor adsorbent when compared to the activated form [70]. Carbon ibres were drawn from hardwood lignin/polymer blend. The lignin was blended with poly(ethylene terephthalate) and polypropylene, respectively, and the carbon ibres were spun and thermally stabilised, followed by a carbonisation step. Lignin was reported to be miscible with the poly(ethylene terephthalate), which resulted in a smooth surface whereas immiscibility with the polypropylene matrix gave rise to a rough surface of the carbon ibres. Various other ibre properties were investigated to recommend the use of lignin in developing the ibres for various applications [71]. Starch acryl amide-based hydrogels with lignin or peat as reinforcement were developed for the removal of copper and nickel ions. The performances of lignin-based hydrogels were found to be superior to the peat-based ones, which made the system more rigid because of the presence of heterogeneity. The importance of lignin as a iller in hydrogels promoted adsorption due to the presence of different functional groups that acted as active sites for metal ion adsorption [72]. Commercially available kraft lignin and its blend with poly(ethylene oxide) were used to produce carbon ibres. The total yield was close to 45% after thermal treatment. Tensile strength of 400–500 GPa and tensile modulus of 30– 60 GPa were observed. The researcher recommended the lignin to be investigated for carbon ibre applications [73]. Jian and co-workers [74] reported the kinetic study of pyrolysis of alkali lignin, hydrolytic lignin, organosol lignin and Klason lignin using Kissinger’s method. He concluded the type of char and volatiles produced during

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas thermal degradation depends upon the separation method, which also inluences the order of reaction. Also the origin of lignin from a particular plant affects the activation energy to a large extent. In another study [75], lignin-based carbon ilms were fabricated using lignin/phenol formaldehyde resin. Nanopores were observed when 14% lignin was used as iller offering 22.7% of total porosity. By making use of enzymatic saccahriication and physical comminution, highly dispersed nanometric particles were obtained. These particles were found to be soluble in polar organic solvent and exhibited lame retardency. The structures of these particles were found to be core-shell type and were recommended for possible use in nanoscience and technological applications [76]. Suparno and co-workers [77] used kraft lignin and naphthol for leather tanning. Biomimetic degradation of kraft lignin yielded phenolics compounds, which were polymerised to obtain the products. These polymerised products were used as synthetic tanning agents for collagen. Allelochemicals are the organic prebiotic compounds that are released into the soil after decomposition of vegetal wastes by microorganisms. Popa and co-workers [78] in their review have extensively discussed the allelochemicals from lignin discharged into the soil by the physiological action of soil microbes. They had reported the successful biostimulating effect of lignin that was responsible for the formation of allelochemicals, which could possibly be used as herbicides, pesticides and growth stimulants. Lignin was phosphorylated in the presence of urea to be used as an adsorbent. With 16.6 wt.%, phosphorylated lignin showed the highest adsorption eficiency. Fourier transform spectroscopy revealed that the phosphorylation occurred at the phenolics hydroxyl and carbonyl groups of lignin [79]. Wood panel adhesives were prepared using low-molecular-mass lignin and tannin. The method developed to produce lignin-based adhesive was found to be advantageous for the fact that no ‘fortiication’ with synthetic resin was required. 94% of the formulation was of natural origin and non-toxic and 0.5% of non-natural material like glyoxal was ecofriendly and non-polluting in nature. No formaldehyde emissions were observed for tannin/hexamine binders [80]. Miretzky and co-workers [81] compiled the research done in the ield of trivalent and hexavalent chromium uptake by lignin and modiied lignin. They concluded that most of the researchers did work on the initial adsorption capacity of the lignin and modiied lignin and there was a need to develop a pilot plant to identify the challenges of adsorption at the industrial level. Due to the abundance, low cost and high adsorption capacity, lignocellulose and its modiied form became an easy choice for researchers for adsorption purposes. In a unique study, organosolv lignin was tested for rheological behaviour when used as a iller in inks, varnishes and paints. It was found that addition of organosolv lignin to ink, paint or varnish signiicantly enhanced the viscosity. A brown colour was observed in these products due to lignin but this had no negative effects [82]. Lignin, the natural amorphous and aromatic biopolymer, has been investigated for

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Lignin: Amorphous Natural Biopolymer-based Blends and Composites for Various Applications various applications. As given in the introduction, applications of lignin include some of the value-added products such as energy, polymers, resins, paints and fertilisers, etc. Alkali lignin could be used for preparing drilling mud additives, plugging water agents or proile control agents in water or stream looding. Black liquor was also used as an emulsifying agent for heavy crude oil with relatively low viscosity and high acid value. Another lab-scale preparation of vanillin from lignin has been described by alkali digestion of black liquor. This was followed by chloroform extraction [83]. Using homogeneous blends containing 85% (wt./wt.) underivatised industrial kraft lignin with polyvinyl acetate and two plasticisers, a series of thermoplastics has been fabricated [84]. Melt-low index measurements concluded that these polymeric materials were amenable to thermal processing by extrusion moulding. In a study of black liquor it was converted to anhydrous colloidal dispersion with the help of surfactants like DDBSA (dodecylbenzenesulfuric acid). This was followed by mixing with bitumen [85]. The black liquor with alkaline method and its fermented device were converted to fodder and fertiliser. The process includes a number of steps and the compound obtained was a combination of microbic and chemical fertiliser [86]. Lignin-based paint was recently studied, which included four major steps i.e. formation of lignin solution comprising sodium lignosulfonate and dye or pigment, mixing this solution with phenol oxidising enzyme and then incubating it for the desired viscosity [87]. Lignin was used as a substituent of phenol in phenol-formaldehyde resin. Lignin was found to replace up to 40% of phenol and resulted in a decrease in pollution caused by black liquor drainage [88]. The lignin was pretreated by different methods like acid hydrolysis, oxidation and thermal treatment. This pretreated lignin was used as a substituent of phenol in phenol-formaldehyde resin, which resulted in better properties [89]. Black liquor was clari-locculated with polyacrylamide and the sludge obtained was blended with various solid waste generated from paper plants and coal ines to briquettes that could be used as domestic as well as industrial fuel [90].

6.9 Developments in a Nutshell Lignin is an amorphous natural biopolymer that is the second most abundant polymer in nature and part of wood composite providing rigidity to plants for millions of years. Lignin in its native form is commonly known as protolignin and is aromatic polyphenolic with ether linkages. The lignin occurs in vascular plants and is absent in non-vascular plants. Lignin is usually classiied as hardwood, softwood and grass and is discharged into the environment in gallons across the world as lignin derivatives. These derivatives inhibit light penetration and affect the organic productivity causing threats to aquatic life.

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas The basic properties of lignin include its insolubility in most organic solvents, its brown colour and its amorphous nature. For real-time application where the performance properties of the polymeric product need to be upgraded to be best suited to the requirement, it becomes highly important to modify lignin chemically, photochemically, thermally or genetically. In general, chemically modiied lignin always has new functional groups introduced into the lignin polymer chains. The other forms of modiication result in degradation of lignin, especially the cleavage of ether linkages into low-molecular-weight lignin compounds, which either condense or polymerise. Lignin in its pristine form is not harmful to living species. However, when discharged into wastewater in modiied forms by pulp and paper mill wastewater, it poses a threat to aquatic life as it inhibits the penetration of light and thus affects organic productivity. Lignin is biodegradable and can be transformed into small molecules such as phenolics derivatives or aliphatic compounds. The biodegradation is usually carried out by various enzymes produced by fungi and bacteria. The most commonly investigated fungi are white rot fungi for the biodegradation. The multienzyme approach used in nature degrades cellulose and lignin simultaneously. The use of lignin in various modiied forms has been investigated to develop polymer blends with superior properties. Different methods were adopted to prepare lignin blends, among which the most commonly used were mechanical mixing, solution blending, solvent casting, thermal extrusion, polymerisation process, etc. The overall properties of the blend depend upon the reaction conditions, the solvent chosen and the type of lignin or lignin derivative used to prepare the blend. The presence of lignin in various studies indicates that it provides photostability, thermal stability, improved solubility, interfacial interactions and homogeneity. Lignin has also been extensively investigated for its use as iller or reinforcement in polymer matrices to develop polymer composites. Researchers across the world have studied the various composite properties that are substantially affected when lignin or its derivatives were used. Some of the important composite properties such as plasticisation, compatibilisation, crystallinity, thermal behaviour, tensile and rheological properties, water resistance and biodegradation were of special interests. The changes in these properties of the lignin composites thus deined applicability and limitations of the use of lignin composites. The properties of lignin-based polyblends and composites can be tailor made as lignin with polyfunctional molecular structure provides ample opportunity to researchers to develop the material’s unique and ultimate properties. Commercial application of lignin includes its use as a dispersant, emulsion stabilisers, antioxidants, resins, reinforcements and energy. It can be considered as a chemical reservoir that can

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Lignin: Amorphous Natural Biopolymer-based Blends and Composites for Various Applications produce small phenolics compounds upon fragmentation. Other applications include its use as radical scavengers, an eficient adsorbent for heavy metal uptake and a source for producing activated carbons. In some cases, lignin had been used to produce carbon ibres and tanning agents. It has also been considered a source of allele-chemicals and fertiliser.

6.10 Conclusion and Future Prospects Lignin in its native form or as protolignin will always remain an undisputed part of wood composite existing on Earth for billions of years. It is the strongest naturally occurring cementing agent of cellulose ibres that otherwise is discharged into pulp and paper mill industrial wastewater. Although lignin poses a severe threat to the environment, it is an extremely useful natural biopolymer and needs to be explored in different research areas. Lignin recovery from wastes and its possible use is still a subject of discussion. We must realise that the future of Earth depends upon waste minimisation and management. It is high time for researchers to make use of such waste that remains in abundance in wastewater. Thus, this is one huge challenge that will be directly proportional to the world-wide demand of paper. Also, an important issue that requires serious thought is the limitation of lignin for commercial applications due to lack of research at pilot scale. Lignin, being an aromatic and carbonaceous material, can also be explored for generation of energy and a source of gas using a biomethanation type of process. It can also be subjected to composite-based applications. In the age of nanotechnology, researchers can focus their objectives to discover the valuable nanoscale range of lignin and lignin derivatives for its possible use in nanotechnolgical-based applications.

Acknowledgements The author is thankful to Dr Ajay Kumar Mishra from the Department of Chemical Technology, University of Johannesburg, South Africa, for his valuable guidance and immense support.

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7

Biodegradable Polymers and Polymeric Composites

Ravi B. Srivastava and Tanushree Vishnoi

7.1 Introduction The use of polymers has gained momentum over the past several years because of their stability and durability over long periods of time. In the initial period, the non-degradable polymers were mainly used for various applications like packaging, aerospace, etc. but soon it was realised that the long-lasting nature of these polymers, even after their functional period was over, caused excessive damage to the environment. A need was then felt for a balance with nature and it was soon understood that it was time to return what was taken from the environment. Since then polymer degradation has aroused the interest of various researchers and extensive reports on rubber and gutta-percha degradation were published. Yet systematic and logical reports regarding polymer degradation started coming out only in the 1930s, with the advent of the plastic industry. Although the real thrust in the ield of degradation started between 1950 and 1955, until then most of the published work was based on theoretical knowledge about the polymers [1]. A polymer is a large macromolecule made up of either similar (homopolymer) or different repeating units (heteropolymer). Based on the source, polymers can be classiied into natural and synthetic/artiicial polymers. Natural polymers are obtained from renewable resources whereas synthetic polymers are from renewable/non-renewable petroleum resources [2]. Natural polymers can be protein (collagen, silk), polysaccharide (starch, cellulose) or lipid in nature as shown in Figure 7.1. Natural polymers, for example starch, cellulose, etc., are degradable in nature compared to most of the non-degradable synthetic polymers [3].

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas CLASSIFICATION OF POLYMER

NATURAL POLYMERS

PROTEIN

COLLAGEN GELATIN

POLYSACCHARIDE

SYNTHETIC POLYMERS

DEGRADABLE

NONDEGRADABLE

AGAROSE ALGINATE SILICONES

POLYESTER

Figure 7.1 Classiication of polymers

In general, physical forces are mainly responsible for the degradation of solid polymers whereas chemical and biological forces are the main cause of degradation for liquid polymers [4,5]. In order to achieve optimised mechanical properties and degradation rate of polymers, the mixing of two or more polymers is carried out by various methods resulting in enhanced intrinsic properties of the composite [6]. It has been shown that mixing of a non-biodegradable polymer with a biodegradable polymer, such as the blend of starch and poly(3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV), resulted in diminished mechanical properties and increased degradation [6].

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7.2 Polymer Composites Composites, as the term suggests, are composed of two or more types of constituents, in order to achieve enhanced properties. In composites matrix and reinforcement are the continuous and discontinuous phase, respectively. In the case of polymeric composites, the continuous phase is mostly a polymer and the illers used are resins, clay [7], etc. Biocomposite foams of polyvinyl alcohol, sodium alginate and bioactive glass as the iller were synthesised in order to provide effective physical and biological properties. Bioactive glass, apart from providing strength needed for bone regeneration, is also osteo-inductive in nature [8].

7.2.1 Polymer Nanocomposites Polymeric nanocomposites are a new class of material in which the polymer matrix is reinforced with nanoscale illers (tubes, spheres or platelets) that have at least one dimension in nanoscale [9]. The use of nanocomposites dates back to 1990 when clay/ nylon-6 nanocomposites were irst used by Toyota for belt covers [10,11]. Mostly the illers used are clay, silica, alkaline earth metal compounds, alumina and carbon nanotubes less than 5% by weight [12]. In order to produce a homogenous polymer/ iller nanocomposite, a certain modiier/surfactant like alkyl ammonium is added [13]. This helps in uniform dispersion of illers into the polymer. Three major types of known nanocomposites are [14]: • Phase-separated nanocomposites: incompatibility between the constituents leads to phase separation between the two constituents. The chemical connectivity controls the dimensions of phase separation to nanoscale. • Intercalated nanocomposites, in which the long polymeric chains interact with the illers resulting in alternate iller and polymer layers. • Exfoliated nanocomposites, in which the iller is uniformly distributed throughout the polymeric matrix. The various methods used for the synthesis of polymeric nanocomposites are the melting intercalation [15] method, the solution method [16] and the in situ/ interlamellar polymerisation method [17]. These nanocomposites have shown better mechanical properties compared to the neat polymer. Nanocomposites of PVA (polyvinyl alcohol) and bacterial cellulose ibres have been synthesised and their degradation was studied in different media. A1, A2 and A3 were the three types of synthesised nanocomposites based on increasing concentration of bacterial cellulose (biopolymer). The rate of degradation was found

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas to be highest for A2 compared to A1 and A3, which clearly showed degradation was solely based on the different concentration of BC in the composites as everything else was kept constant [18]. It has been shown that PLLA/PCL (poly-L-lactide acid/ polycaprolactone) nanocomposites with clay degraded much faster compared to neat PLLA/PCL [19]. This can be attributed to the fact that uniformly dispersed clay interferes more in the oxygen path compared to the neat PLLA/PCL, as a result of which oxygen has enough time for faster degradation of the composite. It has also been shown by Puglia and co-workers that carboxylated functionalised SWCNT (single-walled carbon nanotubes) lead to a faster degradation of the nanocomposite of SWCNT and poly(DL-lactide-co-glycolide) copolymer (50 : 50 PLGA) compared to the non-functionalised nanocomposite. This is because the carboxylated groups lead to higher hydrolytic degradation. Thus, the degradation rate of PLGA can be altered by incorporation of carbon nanotubes, and other functionalised groups [20]. The slow degradation of materials and their adverse effects on the environment will be discussed later in the chapter [21].

7.3 Degradation Mechanism The American Society for Testing and Materials (ASTM) and the International Organization for Standardization (ISO) deine degradation as ‘An irreversible process leading to a signiicant change of the structure of a material, typically characterised by a loss of properties (e.g. integrity, molecular weight, structure or mechanical strength) and/or fragmentation. Degradation is affected by environmental conditions and proceeds over a period of time comprising one or more steps’ [2]. Polymer degradation can be brought about naturally by either abiotic factors (light, temperature) or biotic factors (enzymes and exudates from microorganisms). Mostly degradation is a combined effect of both abiotic and biotic factors. This is due to the fact that the cell walls of microorganisms are impermeable to these macromolecules and the intake of these polymers is only possible if they are broken down into their monomers and oligomers. So the initial degradation occurs outside the microorganism either by the extracellular enzymes and exudates secreted by the microorganism or by the abiotic factors [22]. Thus degradation is an inevitable process that starts at the time of the polymer processing and continues until the end with signiicant decline in its properties.

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7.3.1 Abiotic Involvement

7.3.1.1 Mechanical Degradation Mechanical degradation can occur due to various factors such as stress, strain, ageing, load, water and air pressure, etc. Physical forces such as freezing/thawing, heating/cooling or drying/wetting can cause cracking of polymers as a result of the mechanical damage [5,23]. The damage due to mechanical degradation is rarely visible macroscopically as most of the changes occur at the molecular level. Vicryl, a commercially available suture material, was used to study the effect of strain on (mechanical) degradation [24]. It was found that unstrained, pre-strained and strained specimens fractured at 275 MPa, 180 MPa and 150 MPa, respectively, after exposure to water, suggesting the strain might have caused changes in their molecular orientation and crystalline structure, thus bringing about faster degradation of the strained and pre-strained material.

7.3.1.2 Photodegradation Light degradation is the most prominent factor in polymer degradation as most polymers absorb radiant energy, especially the UV spectrum of light. The photon molecules of this high-energy radiation cause the activation of the electrons from their ground state to the excited state, thus bringing about the oxidation and cleavage of the polymers leading to enhanced degradation. Energy transfer can occur through photoionisation, luorescence, thermal radiation and luminescence [25]. Poly(ether ether ketone) (PEEK) sheets [26] clearly evaluate the relation between light and degradation. It was shown that PEEK sheets after treatment with UV radiation became more brittle owing to a change in molecular chain orientation and cross-linking. Similarly, photodegradation of conjugated polymeric structures like poly(2,4-dimethyl-1,4-phenylene oxide) (PPO), poly(benzo[1,2-d:5,4-d] bisoxazole2,6-diyl-1,4-phenylene) (PBO) and poly(benzo[1,2-d:4,5-d] bisthiazole-2,6-diyl-1,4phenylene) (PBZT) have been shown to undergo photoinduced oxidation [27] as shown in Figure 7.2.

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas A + hv

A*

A + hv′

A* + hv

AA*

2A + hv′

ET

AA* – [A• +, A* ] + O2

ET

– [A• +, A* ]

BET

2A + hv′ A• + + 2A + O2• –

[A• +, A, O2• –]

BET N X

X N

+

X–

N

+

N

X N X

X H N

Figure 7.2 Schematic representation of PBX [PBO and PBZT] photooxidation by photoinduced electron transfer (PET). In this igure, the electron transfer mechanism of PBX during its photodegradation is shown. The photoinduced excited unit produces a radical cation/anion pair, after undergoing electron transfer, with another unit. In the presence of oxygen, the radical anion reacts with O2 to form superoxide, which further reacts with the radical cation to form an unstable compound. This is followed by other reactions, inally resulting in the degraded products, whereas in the absence of oxygen, the radical cation/anion pair undergoes back electron transfer. Reproduced with permission from Y.H. So, Polymer International, 2006, 55, 127. ©2006, Wiley

7.3.1.3 Thermal Degradation Thermal degradation of thermoplastic polymers can be brought about in various ways such as depolymerisation, chain scission, etc. Thermal degradation of polymers occurs when the solid polymer state is changed to its liquid state and is thus related to their melting temperature. Moreover, thermal degradation is also inluenced by the structural framework of these polymers. Above the glass transition temperature (Tg), polymers have enhanced segmental motion and a more distorted arrangement of the molecular chains, leading to increased accessibility to abiotic and biotic factors, whereas under Tg formation of cracks and brittleness occurs [28]. Polyamino acids are naturally occurring polyionic molecules. They are biocompatible and biodegradable in nature. Experiments have shown that polylysine, one of the examples of polyamino

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Biodegradable Polymers and Polymeric Composites acids, can be degraded to its lysine monomers by a membrane-bound enzyme of a bacterium Streptomyces albulus, whose optimum temperature is 50 °C, which is relatively high [29]. Thermal degradation usually brings about physical and optical changes including molecular weight changes, colour changes, reduced ductility, chalking and embrittlement. Pyrolysis and combustion are the two ways by which polymers can be made to undergo thermal degradation [30,31].

7.3.1.4 Chemical Degradation The two main ways reactions of chemical degradation are oxidation and hydrolysis. O2 is the most potent chemical for the oxidation of polymers. Oxygen either in its native form i.e. atmospheric oxygen or in the form of ozone (O3) brings about the cleavage of the covalent bonds in the polymers and in the process releases free radicals like peroxy radicals, which further leads to cross-linking/chain scission. The mechanism is illustrated in Figure 7.3. It has been shown that polymers containing unsaturated bonds, branched polymers, etc, are more prone to oxidation than the others [32].

Polymer (P.H) poly alkyl radical (P* + H*) P* (radical) + O2 P.O.O* (polymer peroxy radical) P.O.O* + P.H P.O.OH + P* P.O.OH P.O.O* + OH* P* + P* P* + P.O.O* P.O.O* + P.O.O*

Inert Product

INITIATION PROPAGATION

PROPAGATION

(a)

Figure 7.3 General mechanism of degradation. Reproduced with permission from E.L. Dane, S.B. King and T.M. Swager, Journal of the American Chemical Society, 2010, 22, 7758. ©2010, ACS

7.3.1.5 Hydrolysis Reaction Moreover, polymers containing hydrolysable bonds like ester, anhydride, ether and so forth undergo hydrolytic degradation as shown in Table 7.1 [33]. For example degradation of PLA occurs due to its ester bonds [34]. The degradation rate is also inluenced by the pH of the environment and it has been shown that degradation of poly(ether ester amide) (PEEA) copolymer increased in the alkaline medium [35]. A few examples of hydrolysable bonds are given in Table 7.1.

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Table 7.1 Examples of hydrolysable bonds

Reproduced with permission from G. Scott, Degradable Polymers: Principles and Applications, 2nd edition. ©2002, Kluwer Academic Publishers

7.3.1.6 Ultrasonic Degradation Ultrasonic waves are high-frequency waves (more than 20 kHz) that have been shown to cause degradation of polymers. The literature shows that use of accepted values of ultrasound causes degradation of polyanhydrides [36,37]. This polymer degradation upon irradiation with ultrasonic waves can be explained by the Cavitation theory [38]. Thus, the above examples clearly reveal that abiotic parameters weaken the polymeric framework and structure making the polymers amenable to biodegradation.

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Biodegradable Polymers and Polymeric Composites Biodegradation can be deined as a ‘degradation process, which is caused by biological activity, especially by the enzymatic action of microorganisms’ [2]. Biodegradation mainly consists of three different phenomena: biodeterioration, biofragmentation and bioassimilation [25].

7.3.1.7 Biodeterioration Biodeterioration is the supericial degradation of polymers resulting in alteration in their properties due to the physical, chemical and biological activity of microorganisms [39]. Microorganisms responsible for biodeterioration are bacteria, fungi, protozoa and algae.

7.3.1.8 Physical Activity The protruding mycelia of the ilamentous fungi penetrate the polymeric materials, which leads to the formation of cracks and also brings about increments in the pore sizes, thus making them more amenable to degradation [40]. Polymeric composites, of glass and reinforced carbon ibre, when examined by scanning electron microscopy (SEM) showed bacterial and fungal colonisation leading to penetration and bond breakage between the ibres and resins, which ultimately cause physical disintegration of the composites [41].

7.3.1.9 Chemical Activity Chemolithic [42] and Chemoorganotrophic [43] bacteria release inorganic compounds like H2S, NH3 and organic acids like fumaric acid, glycoxalic, etc. respectively which react with the polymeric material and bring about deterioration of these polymers.

7.3.1.10 Enzymatic Activity Enzymes such as esterases, lipases, protease, urease, etc. synthesised by microorganisms are involved in the breaking down of large polymeric molecules and thus help in biodeterioration. Mainly it is either bulk or surface erosion. Muller has recently shown the degradation of aromatic polyester by lipases synthesised by Thermobifida Fusca leading to new avenues in the ield of polyester recycling [44].

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7.3.1.11 Biofragmentation It is necessary for microorganisms to disintegrate the polymers, into monomers and oligomers, which can then be used by them as a source of energy. Enzymes with their catalytic nature and high speciicity bring about the degradation of polymers into their simpler units by the process of depolymerisation. It is mostly carried out by oxidoreductases and hydrolases such as cellulases, amylases, etc. In the absence of speciic enzymes for the degradation of polymers, certain oxidising enzymes like oxygenases, peroxidases and oxidases bring about the chain scission. The extracellular polymerases of these microorganisms bring about the biotransformation reactions, thus bringing about the lysis of the long polymer chains into short fragments. It has been reported that lignolytic microorganisms synthesise enzymes such as lignin peroxidase, which breaks down the three-dimensional polymeric network of lignin [45].

7.3.1.12 Assimilation In this process the fragmented polymeric materials transported inside the microbial cell are used as a source of energy. Some monomers are brought directly into the cells with the help of receptors and speciic carriers without the need of biofragmentation. These molecules are then catabolised to produce ATP, which is used for their cellular activity, reproduction and elements of cell structure. Depending on the environment in which the microorganisms grow, they can follow three major catabolic pathways: • Aerobic respiration, in which oxygen is the ultimate electron acceptor. In these microorganisms, the products are assimilated by various pathways such as glycolysis, -oxidation, oxidative phosphorylation, etc. to produce H2O and CO2 as their inal product [46]. • Anaerobic respiration, in which the ultimate electron acceptor is other than oxygen such as CO2, SO42–, Fe3+, etc. It inally leads to the formation of CH4, H2O and CO2 [47]. • Fermentation, where an organic molecule is the electron acceptor. It leads to incomplete oxidation of the fragments into lactate, acetate, ethanol and CO2 [47].

7.3.1.13 Mineralisation Simple and complex molecules such as CH4, CO2, H2O and organic acids, aldehydes, antibiotics, respectively, may be released into their extracellular environment after assimilation. The release of such simple molecules into the environment is referred

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Biodegradable Polymers and Polymeric Composites to as mineralisation [48]. Figure 7.4 clearly summarises the steps involved in biodegradation.

THERMAL DEGRADATION

ULTRASONIC DEGRADATION

CHEMICAL DEGRADATION

PHOTODEGRADATION

MECHANICAL DEGRADATION

ABIOTIC DEGRADATION

POLYMER

BIODETERIORATION BIOFRAGMENTATION

ASSIMILATION

AEROBIC DIGESTION CO2 + H2O + Biomass + Energy

ANAEROBIC DIGESTION CO2 + CH4 + Biomass + Energy

Figure 7.4 Summary of polymer degradation

7.4 Factors Affecting Degradation The rate of degradation depends on the environmental conditions in which it occurs such as temperature, pH, availability of nutrients apart from the chemical composition and structural framework of the polymer. It also depends on the microbial consortium that is bringing about the biodegradation. In order to understand the importance of the above parameters, a few examples are cited.

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7.4.1 Temperature of Medium As explained in thermal degradation, the glass transition temperature and melting point of polymers inluence the degradation rate to a great extent by affecting the mobility and the orientation of the molecular chains. In short, the polymeric molecular structure and the arrangement of their chains inluence the degradation by temperature [49].

7.4.2 pH of Medium The acidic or basic environmental conditions can bring about different degradation mechanisms and products. Some polymers such as polyesters and PPC (polyester powder coated) are degraded in strong and mild alkaline conditions, respectively, whereas PCL is degraded in strong basic conditions more easily. These features are attributed to the differences in their nucleophilicities and electrophilicities to the carbonyl carbon atom in acidic and basic conditions [50].

7.4.3 Availability of Nutrients Fungi predominantly bring about polymer degradation when there are fewer available nutrients compared to bacteria. Moreover, fungi can cause degradation even in limiting conditions such as acidic microenvironment [51,5].

7.4.4 Chemical Composition Polymers in the presence of hydrolysable bonds such as ester, ether and carboxyl are easily degraded by enzymes [33]. Polymer degradation also depends on coniguration, structure and the presence of C-C bonds. Cellulose acetate (CA) is a chemically modiied natural polymer that shows enhanced mechanical properties and processability. The rate of degradation of CA depends on the degree of its acetylation. CA with a substitution value less than 2.5 is degradable and its degradation decreases as the substitution value increases [39]. Examples include aliphatic polyesters, polyamides and polyanhydrides. Viera and co-workers have in their review discussed the effect of hydrolysable bonds such as esters, and their hydrolytic reaction is illustrated in Figure 7.5 [52].

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Figure 7.5 Hydrolysis of ester bond to release aldehyde and carboxylate end group. Reproduced with permission from A.C. Vieira, R.M. Guedes and A.T. Marques, Journal of Biomechanics, 2009, 42, 2421. ©2009, Elsevier

7.4.5 Crystallinity/Amorphous The rate of enzymatic degradation depends on the amount of crystalline and amorphous components of a polymer. It has been postulated that degradation is inversely related to the crystallinity of a polymer. This is because the organised structure in the crystalline component of the polymer is less accessible to the enzymes compared to that in the amorphous component. Moreover the formation of spherulites during the crystallisation also affects their degradation as they make the polymer more brittle [53]. Polymorphism in crystal also plays a role in the rate of their degradation. Although their chemical composition is the same, the different forms have different abilities to present themselves to the highly speciic enzymes resulting in different degradation rates [54].

7.4.6 Microbial Consortium A varied range of microorganisms such as fungi, algae and bacteria can bring about the biodegradation of polymers. The rate of degradation depends on the type of microorganism as fungi prefer degradation in acidic conditions whereas bacteria 165

Biotechnology in Biopolymers Developments, Applications & Challenging Areas degrade equally in both acidic and alkaline conditions. The microbes found to be associated with the degrading polythene and plastics were identiied as Streptococcus, Staphylococcus, Micrococcus (Gram +ve), Moraxella and Pseudomonas (Gram –ve) and two species of fungi (Aspergillus glaucus and A. niger) [55]. Bioilm technology has been proposed for faster degradation of the polymeric materials as bioilms are believed to be a combination of a large number of highly complex microorganisms and their extracellular polysaccharides. Due to this close association, optimal conditions for the microorganisms are present. The bioilm can also provide a different microenvironment altogether for the degradation of the polymeric sample, for example pH. Moreover, the bioilm can be a source of a large amount of extracellular polymer degrading enzymes, which generally diffuse away in a liquid environment [56]. Bioilm formation on polyurethane ilm has been reported by Srivastava and co-workers and their study showed the formation of pits on the surface where these fungal colonies were formed [57]. Polyethylene is hydrophobic in nature, as a result of which adhesion of the hydrophilic surfaces of bacteria is unlikely, whereas adhesion of fungi to a hydrophobic surface is not a limiting factor. Recently in Science correspondence, inoculum of fungal-bacterial (Penicillium-Bacillus) bioilm has been proposed to enhance the biodegradation of the above hydrophobic polyethylene as Penicillium (fungi) would effectively help in colonisation and transportation of Bacillus (bacteria) [58]. Recalcitrant polymers like polyimide have high strength and durability and are resistant to biodegradation. However, these chemically synthesised resistant polymers have been shown to biodegrade through bioilm formation [59].

7.5 In Vivo Degradation of Implantable Devices In previous years, non-functional/injured tissue was mainly replaced by non-degradable implants such as metal implants. Either a secondary surgery had to be done for these implants to be removed or the implant remained in the patient for life, which led to immunity problems. However, in recent years there has been a paradigm shift from replacement to regeneration in tissue engineering. The non-degradable implants have been replaced by degradable implants made from natural and synthetic polymers. These polymers are biocompatible as well as biodegradable, as a result of which no secondary surgery or problem of immunity would arise [60]. These can be synthesised by various techniques such as solvent casting [61], cryogelation [62], etc. The rate of their degradation is such that it is in proportion to the rate of regeneration of the lost/ injured tissue such that the degradable scaffold would provide support and guidance to the regenerating cells until the native/host tissue is regenerated. The degradation of the implanted scaffold is followed by a chain of reactions. Primarily after implantation in the extracellular luid there is diffusion of aqueous

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Biodegradable Polymers and Polymeric Composites solution into the implant, leading to hydrolytic degradation by both enzymes and water. Polymers that are hydrophilic in nature such as polyamides, polyanhydrides and polyesters undergo hydrolytic degradation compared to hydrophobic polymers such as polyurethanes and polycarbamates, which are degraded by oxidation. Hydrolysis results in smaller fragments in contrast to surface cracking and pitting caused by oxidation. Moreover, hydrolysis causes faster degradation compared to oxidation [1,63]. Hydrolysis can result in surface erosion or bulk erosion of the implanted polymer. Water molecules being smaller in size can diffuse into the implant and bring about bulk erosion of the polymer, leading to homogenous degradation of the entire polymeric matrix. Conversely, enzymes are large molecules and cannot penetrate deeper into the polymer, thus enzymes are mainly responsible for surface erosion [64]. Enzymatic degradation also depends on the nature of the implant, types of bonds, molecular structure and dimensions, temperature, pH, etc. Amorphous domains of the polymer are more susceptible to enzymatic attack compared to the crystalline domains [65]. Moreover, hydrolysis causes the formation of more carboxylic ends, leading to autocatalytic hydrolysis as the -COOH groups are highly subjected to hydrolysis [66]. Oligomers resulting from hydrolysis can either leach into the aqueous medium from the surface of the polymer or be trapped in the core of the polymeric matrix resulting in an autocatalytic effect. Upon implantation, monocytes adhere to the scaffold and, over a period of several weeks, differentiate into macrophages. These macrophages secrete hydrolytic enzymes and reactive oxygen species. This secretion is further enhanced when these macrophages fuse to form foreign body giant cells [67]. The reactive oxygen species leads to oxidation and chemical degradation of the implanted biomaterial. Chapanian and co-workers have shown in vivo enzymatic and oxidative degradation of trimethylene carbonate-based elastomers upon implantation. The SEM image shown in Figure 7.6 clearly shows the deformation of the implant primarily after oxidative degradation due to reactive oxygen species secreted by adherent phagocytes [68].

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SEM image (100x) of trimethylene carbonate based elastomers

SEM image (100x) of trimethylene carbonate based elastomers

Figure 7.6 SEM image (100×) of trimethylene carbonate-based elastomers. Reproduced with permission from C. Rai, M. Yattse, S.C. Pang and B.G. Amsden, Biomaterials, 2009, 30, 295. ©2009, Elsevier

7.6 Mechanisms Responsible for Alteration in the Rate of Polymer Degradation Degradation of polymers is a requisite step for the recycling of compounds and maintenance of geochemical cycles such as the carbon cycle, the nitrogen cycle, the hydrogen cycle, etc. Polymers have varied physical, mechanical and chemical properties and applications owing to their different hydrocarbon bonds (C-C, C-R and C-H bonds), conigurations, stereochemistry and substituent groups [69]. These properties also inluence the degradation behaviour of these polymers and categorise them into either biodegradable or non-biodegradable. Polymers follow the ‘Freundlich’ isotherm during the early part of their degradation [70]. In order to achieve degradation in non-biodegradable polymers, we can modify the above properties by either incorporating certain peptides or mixing them with degradable polymers.

7.6.1 Nanocomposites/Blends Recently nanocomposites have gained a lot of interest as it has been proven that the incorporation of illers in these composites help in faster degradation than their

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Biodegradable Polymers and Polymeric Composites individual constituents. Formation of these blends and of nanocomposites also affects their intrinsic properties. Biodegradation of montmorillonite (MMT), LDPE and biodegradable cassava starch nanocomposite was enhanced with increased amounts of cassava starch. The polymers of PCL, poly(styrene-co-acrylonitrile) and their blends were compared and it was shown that the nanocomposite degraded faster than their individual constituents [71]. Incorporation of 50% starch with PHBV altered certain properties of PHBV. Its mechanical properties and lexibility decreased whereas its Young’s modulus increased. Moreover, the degradation rate of these blends was faster compared to only PHBV degradation [72]. Kriegel and co-workers electrospun blended nanoibres of chitosan-polyethylene oxide with surfactant, resulting in largediameter, smoothed or beaded ibres against the bead formation that was obtained with chitosan only [73].

7.6.2 Copolymerisation/Cross-linking Two methods that are commonly used in order to achieve a controlled degradation rate are copolymerisation and cross-linking. Copolymers are made up of two or more different constituents, for example graft copolymers, block polymers, etc. [74]. It has been reported that copolymers of starch-g-acrylamide/itaconic acid with N,N′-methylenebisacrylamide (N-MBA) as the cross-linking agent resulted in biodegradable scaffolds [75]. Cross-linking of two polymers can be brought about by either irradiation or chemicals. Varying the cross-linking concentration can help in modulating the degradation rate of the molecule [76]. Multifunctional cross-linking molecules have been shown to enhance the mechanical strength and degradation time of hydrogels [77], thus cross-linking altered the degradation rate of the molecule.

7.6.3 Incorporation of Additives Additives enable the degradation and lysis of non-biodegradable polymers. A few techniques to mention are Scott–Gilead technology and Totally Degradable Plastic Additives (TDPA) technology. In the former technology iron dithiocarbamate functional groups are introduced into the polymer. This molecule initially acts as an antioxidant and maintains the mechanical properties but later acts as a photoinitiator and brings about polymer degradation. The latter technique was introduced by EPI (Environmental Plastics Inc.) to obtain controlled degradation and shelf-life of polymers, by adding TDPA formulations [78]. Organic materials such as starch, cellulose or resins like ethylene vinyl alcohol (EVOH) are also added to the polymer for degradation. It has been shown that a small portion of additive like starch added to plastic degrades to carbon dioxide and water, while the remaining 95% of the plastic fragments enabling further degradation [79]. 169

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7.6.4 Photoinitiators Polymers can be made to degrade photochemically either by introduction of a chromophore such as a carbonyl group into the polymeric chain [80], which absorbs UV light and undergoes degradation by Norish type I and II reactions [81], or by incorporating a radical initiator/pro-oxidant (for example, TiO2, ZnO, FeCl3, quinines, peroxides, etc.) into the polymer, leading to auto-oxidation cycles and thus degradation of the polymer. These radical initiators also require ultraviolet light for their activation and hence degradation [80]. Although recently methods have been employed to degrade the polymers in visible light by the incorporation of metalmetal bonded organo-metallic dimers, these polymers have not been very successful in commercial use [80]. Recently the widely used packaging material polyethylene has been made biodegradable by adding nanoclay as the iller. Apart from providing higher mechanical strength, the nanoclay brings about degradation by photo-oxidation and thus enhances its degradation rate [82].

7.6.5 Thermal Initiators Polymers can similarly be thermally degraded by introducing certain peptides or moieties into the polymeric backbone. These polymers when exposed to heat undergo degradation, resulting in chain scission. Chain scission results in smaller fragments, which are easily degraded by microorganisms. For example, polyurethanes have been reported to degrade thermally after the introduction of a di-functional azo compound into their polymeric chains [83].

7.6.5.1 Development of Genetically Modified Microorganisms Synthetic polymers do not occur naturally. Moreover, the coniguration of synthetic polymers is different from natural polymers, as a result of which microorganisms are unable to recognise these naturally as their substrates. Therefore, microorganisms capable of using a speciic carbon source as a substrate have been synthesised by either plasmid transformation or de novo enzyme synthesis so that they are capable of degrading the desired polymers. Degradation depends mainly on the type of enzymes and transporters present in the membrane of the organism as they would be responsible for breaking the polymers into smaller compounds and enabling them to cross the membrane and be further metabolised. Introduction of such enzymes in genetically modiied organisms also helps polymer degradation [84,85].

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7.6.5.2 Synthesis of Artificial Biopolymers Artiicial biopolymers are made to support controlled biodegradation. In this, bior multifunctional metabolites of the biochemical cycles such as amino acids and hydroxyl acids are used to make polymers with degradable backbones. Therefore these polymers release only metabolites upon degradation leading to recycling of the polymer [86].

7.7 Measurement of Degradation In order to have a controlled degradation rate and a limited shelf-life it is essential to study the degradation pathway of the particular polymer and thus deduce methods to alter/modify it according to its applications. For example, polymers that have a limited function to perform should be degraded after they have performed their function or else their deposition would disturb our ecosystem. In the early years when proper methodologies and equipment were not available, preliminary experiments on polymer degradation was performed whereby the loss in molecular weight and the rate of volatile production were studied. Viscometry and osmometry were used to study the loss in molecular weight [87]. These methods, along with analysis of the volatile compounds, enabled the researchers to study a simple degradation processes. With the advent of new tools and techniques, the ield of polymer degradation has been reformed and revolutionised to achieve better understanding and knowledge of the topic. It is important to emphasise that, based on the ability of a polymer to degrade (biodegradability), polymers can be classiied into three categories [88]: • Ready biodegradability: these materials degrade rapidly in the most common/ natural environment. The tests performed to determine such materials are the closed bottle and die away test. • Inherent biodegradability: these materials degrade in the most favourable conditions/environment and their degradation is a slow process. The tests performed to determine such materials are the Zahn–Wellens test and semicontinuous activated-sludge (SCAS). • Ultimate biodegradability: these materials degrade in a particular chosen environment. The test performed to determine such materials is CO2 evolution.

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas Mainly degradation tests can be classiied into three types [88]: • Field tests: these tests are performed in the natural environment where there is no control of pH, temperature, humidity, etc. It is mainly performed by burying the polymers, such as plastic, into soil, lakes or rivers. Therefore, the methods used for analysis are limited and elementary tests can be performed such as visible change in colour, change in molecular weight. • Simulation tests: to overcome the above issues, a simulation test is done where the material is degraded in the laboratory reactors where environmental conditions and parameters like soil, composting, temperature and humidity are provided in a controlled manner. Analytical tests such as O2 consumption and CO2 production rate, residual analysis, controlled composting test [89], aquarium test [90], etc. are performed. • Laboratory tests: this is the most reproducible test among all the tests. In this method synthetic media are inoculated with a mixed microbial consortium to carry out degradation. The conditions are optimum for microbial growth and survival, as a result of which the amount of degradation achieved in this test is high compared to that achieved naturally.

7.8 Techniques Used to Assess Degradation Visual changes can be observed using the SEM or atomic force microscopy (AFM) [91] techniques, apart from detecting changes in colour, surface roughness, hole formation, etc. of the polymer. The SEM image of Figure 7.7 clearly shows the degraded HEMA/ gelatin scaffolds with closed pores and deformed shape after incubating the scaffold for eight weeks in PBS at 37 °C [92]. Alterations in the length of the polymer chain can be monitored by measuring the change in their mechanical properties such as tensile strength as the polymeric chain length is associated with the strength of the material. Techniques such as size exclusion/gel permeation chromatography have been used to monitor loss in molecular weight of polymers apart from using conventional techniques like osmometry, gravimetry, light scattering, etc. For example, in reports [52] by Vieira and co-workers the degree of degradation was assessed by measuring the loss in molecular weight by size exclusion chromatography (SEC) or gel permeation chromatography (GPC).

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SEM image of HEMA-gelatin scaffold

SEM image of degraded HEMA-gelatin after eight weeks

Figure 7.7 SEM images of gelatins. Reproduced with permission from D. Singh, A. Tripathi, V. Nayak and A. Kumar, Journal of Biomaterial Science Polymer Edition, 2010, 10, 23. ©2010, Brill

The percentage weight loss was calculated using the following formula:

Wl % = 100(W0 – Wr) / W0

(7.1)

where W0 is the initial weight and Wr is the residual weight of dried/partially degraded sample. With modiication in temperature during initial degradation the Tg of the polymer changes, which modiies the crystalline and amorphous component in the polymer. The degradation of blends of PHB-PHV/PCL matrix and changes in its crystal structure were monitored through various techniques like XRD by Yasin and coworkers [93]. Surface energy measurements were performed using electrochemical impedance spectroscopy (EIS). EIS is considered to be the least destructive in nature and provides information about the electrochemical analysis. This was used to measure the interfacial phenomena between painting coatings and fungi by Gu and co-workers. The fungi penetrates deep into the polymer, as a result of which the polymer becomes accessible to a large amount of ions and water leading to its degradation [94]. The respirometric test for O2 consumption and the Sturm test/BOD/DOC tests [95–97] for CO2 production are commonly used to monitor polymer degradation. Recently infrared and paramagnetic O2 detectors have been used for the detection of these gases [98]. These tests are restricted to aerobic microorganisms. Instead anaerobic 173

Biotechnology in Biopolymers Developments, Applications & Challenging Areas microorganism releases CO2 and CH4 as their end products, which are measured using either a manometer or the Buswell equation [98]. The problem associated with measuring the CO2 concentration is the additional amount of CO2 released from the biowaste/matrix (where polymer degradation is carried out). So in order to overcome this problem the polymers to be monitored for degradation are labelled with 13C. An interesting and simple technique referred as the ‘clear zone formation’ technique is used to study whether the organism is capable of causing depolymerisation of the chain. It is a semiquantitative technique, in which the polymer is distributed in synthetic agar as very minute particles. Then the microbial inoculum is added to it. If the microorganisms can degrade the dispersed polymer, a halo/clear zone is formed around the colony, which is shown in Figure 7.8 [99].

Figure 7.8 Photograph showing the clear zone test for PHB-co-HV agar and strain 4A-2 after 96 h. Reproduced with permission from J. Augusta, R.J. Müller and H. Widdecke, Applied Microbial Biotechnology, 1993, 39, 673. ©1993, Springer

Nowadays, novel non-destructive methods are being employed to study polymer degradation. Wrobel and co-workers deduced methods such as thermography and ultrasonic diagnosis to study polymer degradation. These methods are based on the hypothesis that an explicit relationship exists between the strength of the polymer

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Biodegradable Polymers and Polymeric Composites and its thermal properties (active or passive thermography) and for the ultrasonic technique it is hypothesised that acoustic properties and strength are related [100]. In reality it is not a single factor that causes a polymer to degrade. Many factors such as temperature and humidity are responsible. Therefore, novel equipment has been designed that has the ability to measure the synergistic effect of mechanical stress, humidity, UV exposure and temperature on the polymer, as all of these would affect the physical properties and so the degradation of the polymer. This was determined by observing the creep behaviour of the sample in diverse environmental conditions [101]

7.9 Applications

7.9.1 Agriculture Biodegradable pots are used for plantation as these pots break down to carbon dioxide and water, eliminating double handling and recycling of conventional plastic containers [102]. Degradable polymers are also used for controlled release of fertilisers [103].

7.9.2 Plasticulture India is an agricultural country and its economy is dependent on agriculture for the success and prosperity of the country. Therefore, there is always a constant struggle for innovation and improvisation in this ield, as a result of which plastics are being innovatively used in the ield of agriculture, to enhance the quality and quantity of the products. The American Society of Plasticulture has deined plasticulture as ‘The use of plastics in agriculture for both plant and animal production including; plastic mulch, drip irrigation, row covers, low tunnels, high tunnels, greenhouses, silage bags, hay bales and in food packaging and nursery pots and containers for growing transplants’ [104]. In order to build economical greenhouses (polyhouses), drip or trickle irrigation (which supply water and nutrients to crops, especially in places where soil has a very low water holding capacity) [105], mulching (where the area is covered with plastics around plants to prevent loss of moisture, and for weed and temperature control) [106] and plastics in the form of LDPE (low-density polyethylene) are used. Thus, plasticulture can be considered as an eficient management tool that enables producers to use resources more eficiently. However, the replacement and disposal costs of these plastics is very high. Therefore, biodegradable polymers have been shown to have a great potential as these polymers are degradable in nature

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas and there is no requirement of disposing of them, which makes them cost effective. Photodegradation and biodegradation are the two major ways by which degradation of these polymeric ilms occurs. Polyester ilms, modiied Ecolex® or Poly(butylene adipate-co-terephthalate) (PBAT) (white and black ilms) and LDPE have been used as mulch ilms for tomato cultivation and it has been shown that these polymers degraded over the period of time without any extra effort for its removal, as shown in Figure 7.9 [104].

Figure 7.9 Pictures showing the degradation of white ilms after the growing season. Reproduced with permission from K. Thitisilp, A. Rafael, R. Maria, N. Mathieu and T.F. Rodney, Chemosphere, 2008, 5, 942. ©2008, Elsevier

7.9.3 Medicines Biodegradable polymers have paved their way gradually in the ield of medicine and are now being used in the closure of wounds as sutures and staples [105] and in orthopaedics as ixation devices such as pins, rods, screws, tacks and ligaments [106]. Moreover, degradable polymeric implants are being seen as a potential candidate to replace metal implants as they do not need second surgery for the removal of the implant because these polymers degrade over the period of time. Importantly their rate of degradation is in proportion to the rate of tissue regeneration in that area [107]. Moreover, unlike metal implants, these do not result in leaching of toxic ions

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Biodegradable Polymers and Polymeric Composites from the implant nor do they cause any distortion to techniques like MRI used for radiological imaging [108].

7.9.4 Polymers for Drug Delivery Biodegradable nanoparticles are used as drug delivery vehicles because they are less immunogenic in nature as most natural polymers resemble the glycosaminoglycans of the extracellular matrix. Most importantly, encapsulated polymers have a controlled release rate of the drugs [109]. The polymeric vehicles used mostly undergo degradation via surface erosion, leading to regulated and controlled release of drugs [110]. Moreover, after the drug has been released the polymeric nanoparticle undergoes degradation.

7.9.5 Construction Industry Biodegradable polymers used in the construction industry lead to greening of the area and thus keep the environment clean. These polymers are environmentally friendly [108]. Recently bioadmixtures have been used to regulate material properties, and are biodegradable in nature e.g. lignosulfonate, pine root extract, sodium gluconate, guar gum, protein hydrolysates, tartaric acid, Xanthan and casein [108].

7.9.6 Packaging Bioplastics: Recycling of Plastics There has been a tremendous increase in the use of plastics in recent years for various applications such as packaging, transportation, automobiles, etc., but their nondegradable nature has always been a matter of great concern. Plastic degradation in landills is the common and it has been shown that the decomposition of these dumped plastics takes many years. Therefore, recycling is considered a promising solution to these illed landills. The Environmental Protection Agency (EPA) has reported that instead of 80% of plastic being dumped into landill now only 20% has been reported after the effect of recycling processes. The method of plastic recycling is shown in Figure 7.10 [111].

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Figure 7.10 Schematic representation of plastic recycling. Plastic collected from various sources as commercial, agricultural and municipal wastes is cleaned and sorted, after which it is subjected to size reduction by cutting (with scissors, saws, etc.), shredding (this is done for smaller pieces using a rotor blade; it results in the formation of irregular plastic lakes) and agglomeration (heating, then rapid cooling of soft plastic to solidify the material, which is then cut into smaller coarse and irregular pieces called crumbs). These pieces are again sorted and decontaminated for further manufacturing techniques as mentioned above. Reproduced with permission from Recycling plastics (practical action – technology challenging poverty): http://practicalaction.org/docs/technical_ information_service/recycling_plastic.pdf One of the most serious problems with plastic waste is the presence of additives such as metals, which lead to the release of toxic compounds into the environment and also make degradation a problem. Plastics are organic polymers and are generally of two kinds [111]: • Thermoset plastics: cannot be remoulded on heating. • Thermoplastics: can be remoulded on heating. In order to provide a uniform coding for the available plastics for all manufacturers and consumers, the Society of the Plastics Industry (SPI) in 1988 designed codes from 1 to 7 for differentiating types of plastics. The codes and their explanations

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Biodegradable Polymers and Polymeric Composites are summarised in Table 7.2 [112]. Plastic recycling is a necessity in order to avoid detrimental effects on the environment. The different steps of plastic recycling are differentiated into four types [112], which are summarised in Table 7.3. Considering the problems associated with plastics, bioplastics are thought to be a promising solution. Bioplastics can be either biodegradable (from fossil resources) or biobased plastics (from biomass or renewable resources) in nature. These are gaining importance as they are environmentally friendly; polylactic acid (PLA) and polyhodroxyalkanoates (PHA) are two examples. Biodegradable plastics have many advantages such as reduced greenhouse gas emissions, increased fertility, recyclable to useful metabolites. PHA are a group of biopolymers that are gaining importance in the ield of bioplastics [113,114]. Microorganisms synthesise both organic and inorganic intracellular or spherical inclusion bodies. Examples of inorganic and organic inclusions are magnetosomes and high-molecular-weight biopolyesters (PHA). These polyesters are formed by eubacteria and archaea in the presence of limiting nutrients and an excess of carbon source. In the starvation conditions the microbes use these water insoluble inclusion bodies as a source of energy [115]. There are mainly two kinds of polyhydroxyalkanoates: short chain length (scl-) PHA, which have four or ive carbon atom monomers; and medium-chain-length- (mcl-) PHA with six or more carbon atoms [116]. Around 150 hydroxyalkanoates have been known, of which only PHB and copolymers of 3HB and 3-hydroxyvaleric acid (3HV) by the name of Biopol are being used in the market [117]. The two kinds of enzymes used for the degradation of PHA are intracellular and extracellular PHA depolymerises, respectively, for intracellular and extracellular PHA (PHA releases after the lysis and death of the bacteria). In order to consider the degradation of these, it is important to emphasise that native/intracellular PHA are amorphous in nature in contrast to the partial crystalline nature of the extracellular PHA. Most importantly the enzymes degrade the polymers to release carbon dioxide and water in aerobic conditions and methanol and carbon dioxide in anaerobic conditions [118]. A few examples of PHA-degrading microorganisms from different environments are (Pseudomonas lemoignei) from soil, (Almligenes faecalia) from activated sludge, (Comamonas testosterone) from sea water, (Pseudomonas pickettii) from laboratory atmosphere and (Pseudomonas stutzeri) from lake water [119]. Fortunately PHA have been shown to resemble plastic in many of their physical properties like moulding, lexibility to acquire various shapes, etc., which opens new avenues for PHA as bioplastics. Genetically engineered plants and bacteria with PHA-synthesising enzymes are being used to increase the production of PHA. Transgenic alfalfa plant was used for the production of the biodegradable plastic poly-β-hydroxybutyrate (PHB) after the expression of three bacterial enzymes by Agrobacterium-mediated transformation [120]. Cheap carbon sources like molasses, methanol, etc. are also being exploited to be used for PHA production otherwise the cost of their production is 5–10 times higher than production of normal plastic [121].

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas Copolymers of PHA are mostly used in order to achieve better properties. Biopol [copolymer of polyhydroxybutyrate (PHB) and polyhydroxyvalerate (PHV)] was the irst biodegradable polyester to be available in the market [116].

Table 7.2 Coding for different types of plastic Type

Polyethylene Terephthalate (PET)

Properties

Examples

Single-use, inexpensive, lightweight, easy to recycle, low risk of recycling rate is around 20%

Life jacket, winter coat

Versatile plastic, carries low risk of leaching and is readily recyclable into many goods

Oil bottles, fencing

PVC is tough, releases toxins

Decks, looring , cables

Not often recycled through curbside programmes

Compost bins, loor tiles

Can be recycled through some curbside programmes.

Auto battery cases, ice scraper

Can be recycled through some curbside programmes

Insulation, egg cartons

Cannot be recycled, although some curbside programmes now take them

Polycarbonate, plastic resin

High Density Polyethylene(HDPE)

Vinyl/Polyvinyl Chloride (PVC)

LDPELow-Density Polyethylene

PP Polypropylene

PS Polystyrene

Reproduced with permission from L. Ahmad, Plastic Recycling [online] available at: http://www.loti.net/recycle/plastic.html [Accessed 04 April 2011]

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Biodegradable Polymers and Polymeric Composites Plastic recycling is mainly differentiated into the following four types [112]:

Table 7.3 Types of plastic recycling Type of recycling

Type of product

Uses

Primary recycling

Product has features similar to the original product

Semi-clean industrial plastics It is rarely used

Secondary recycling

Moulded into new, recyclable, non-food packaging products (less mechanical strength )

Fenceposts, substitute of wood, metal

Tertiary recycling

Adapt to higher levels of contamination, recyclable product

Chemicals and fuels

Quaternary recycling

Uses energy from plastic by burning

Incinerators (materials left from this process are placed in landills)

Reproduced with permission from L. Ahmad, Plastic Recycling [online] available at: http://www.loti.net/recycle/plastic.html [Accessed 04 April 2011]

7.10 Wastewater Treatment – Secondary Effluent Considering the shortage of water resources due to environmental pollution, efforts are being made to reuse wastewater after treatment, especially for agricultural purposes. The stepwise protocol for the treatment of wastewater is given in Figure 7.11 [122].

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas

Figure 7.11 Flowchart for the treatment of wastewater. Wastewater treatment is divided into preliminary treatment (coarse screening is done to remove large materials like grit, sand), primary treatment (removal of settable and loating materials, respectively, by sedimentation and skimming in sedimentation tank; 20–50% BOD, total suspended solids, organic N2, P, etc. are removed resulting in primary efluent) and secondary treatment (removal of remaining organic materials (EPS/SMP) and solids is done by the aerobic digestion of organisms). Activated sludge process or trickling ilters are used. The secondary sludge obtained by secondary sedimentation is combined with primary sludge for further processing. Tertiary treatment removes speciic substances still present after secondary treatment. Reproduced with permission from G. Tchobanoglous, Wastewater Engineering: Treatment, Disposal and Reuse, 3rd edition, Eds., G. Tchobanoglous and F. Burton, McGraw-Hill, New York, 1991, p.1848. ©1991, McGraw Hill

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Biodegradable Polymers and Polymeric Composites It has been reported that the secondary efluent has extracellular polymer substances (EPS)/soluble microbiological products (SMP) as its major foulant, leading to a higher organic (polymer) contamination [123]. Membrane/ultrailtration has been considered as a promising technique for the removal of undesirable products from the secondary efluents so that it can be reused [124]. Due to the organic foulants as contamination, dynamic membrane polyacrylic acid is used prior to membrane iltration as these membranes can be removed and replaced [125]. Slow sand iltration is another method employed as a pretreatment for the removal of the polymers. The biopolymer content is degraded by the microorganisms in the upper layer of the sand. Temperature, pH and secondary efluent concentration have a high inluence on the removal of the biopolymers [126].

7.11 Standardisation and Certification In the past few years there has been rapid progress in the ield of polymer science with the development of a large number of degradable polymers. It is essential to keep a check on their degradability so that when disposed in the environment they do not cause any harm. So in order to be qualiied as ‘biodegradable’ various standardisation bodies have set certain benchmarks. The International Standard Organization (ISO), an international standardisation body, the American Society for Testing and Materials (ASTM) and the Committee for European Standardization (CEN), regional standardisation bodies, and the Austrian Standard Institute (ÖNORM), the Italian Ente Nazionale Italiano di Uniicazione (UNI), the Association Francaise de Normalization (AFNOR) and the Biodegradable Plastics Society (BPS) of Japan are the various national standardisation bodies [2]. Plastic biodegradability standards come under TC61/SC5/WG22. For testing the biodegradation of plastics nine standards exist. One standard deals with speciicity and one for preparation of the sample for the biodegradation tests. All the standards for the biodegradation of plastic can be categorised based on the test environment like compost, aqueous solution, etc. [127]. These bodies therefore keep a check on the degraded polymers to be released in the market so that they do not pose any threat to the environment.

7.12 Conclusion and Future Prospects All the various sectors such as medicine, agriculture, automobiles, etc. must try and switch to the use of environmentally friendly polymers. The use of non-degradable commodities needs to be regulated and alternatives like bioplastics, natural polymers, etc. have to be promoted in order to regulate this incessant and uncontrolled practice. Intensive efforts have to be made to develop a niche market for these kinds of polymers.

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas Moreover, the mechanism for biodegradation is well established and if these polymers can be altered by genetic, recombinant technology as well as polymer chemistry then the abiotic and biotic factors can be used for the biodegradation of these polymers, which would enhance the use of these polymers worldwide.

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Biodegradable Polymers and Polymeric Composites 109. M.L. Hans and A.M. Lowman, Current Opinion in Solid State and Materials Science, 2002, 6, 319. 110. J.A. Tamada and R. Langer, Proceedings of National Academy of Science USA, 1993, 90, 552. 111. Recycling plastics (practical action – technology challenging poverty), available at: http://practicalaction.org/docs/technical_information_service/recycling_ plastic.pdf. 112. L. Ahmad, Plastic Recycling available at: http://www.loti.net/recycle/plastic.html [accessed 04 April 2011] 113. Y. Tokiwa and B.P. Calabia, Journal of Polymers and the Environment, 2007, 15, 259-267. 114. Y. Tokiwa, B.P. Calabia, C.U. Ugwu and S. Aiba, International Journal of Molecular Science, 2009, 10, 3722. 115. B.H.A Rhem, Current Issues in Molecular Biology, 2006, 9, 41. 116. D. Jendrossek and R. Handrick, Annual Review Microbiology, 2002, 56, 403. 117. D. Jendrossek, Extracellular Polyhydroxyalkanoate Depolymerases: The Key Enzymes of PHA Degradation, Biotechnology Online, Wiley-VCH Verlag GmbH & Co. KGaA, 2005. 118. K. Mukai and Y. Doi, Riken Focused on Microbial Diversity, 1993, 3, 21. 119. S.D. Marco, MMG 445 Basic Biotechnology eJournal, 2005, 1, 1. 120. P. Saruul, F. Srienc, D.A. Somers and D.A. Samac, Crop Science, 2002, 42, 919. 121. C. Nawrath, Y. Poirier and C. Somerville, Molecular Breeding, 1995, 1, 105. 122. G. Tchobanoglous, Wastewater Engineering: Treatment, Disposal and Reuse, 3rd edition, Metcalf and Eddy, Inc., New York, McGraw-Hill, 1991, p.1334. 123. C. Jarusutthirak and G. Amy, Environmental Science and Technology, 2006, 40, 969. 124. I.S. Chang and S.N. Kim, Process Biochemistry, 2005, 40, 1307.

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8

Smart Chitosan Matrices for Application to Cholesterol Biosensors

Ashutosh Tiwari

8.1 Introduction Chitosan (CHIT), a polycationic biopolymer, is usually obtained from alkaline deacetylation of chitin, which is the main component of the exoskeleton of crustaceans such as shrimp, crab and lobster [1]. It is α (1→4) 2-amino 2-deoxy β-D-glucan, having structural characteristics similar to glycosaminoglycans (Figure 8.1). It is the characteristic polysaccharide of several important phyla, like arthropoda, annelida, mollusca, coelenterata and of many fungi, e.g. euascomycetes, zygomycetes, basidiomycetes and deuteromycetes. CHIT displays interesting characteristics including biodegradability, biocompatibility, chemical inertness, high mechanical strength, good ilm-forming properties and low cost [2,3]. The physicochemical and biological properties of CHIT led to recognition for a number of its applications such as fabrication of artiicial muscles [4,5], wastewater treatment [6], functional membranes [7], food packaging [8–10], drug delivery systems [11,12] and biosensors [13–15].

Figure 8.1 Chemical structure of CHIT

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas Moreover, CHIT has three reactive groups, i.e. primary (C-6) and secondary (C-3) hydroxyl groups on each repeat unit and the amino (C-2) group on each deacetylated unit. These reactive groups can be chemically modiied for altering its physical and chemical properties. Typical reactions involving the hydroxyl groups are etheriication and esteriication. Selective O-substitution can be achieved by protecting the -NH2 groups during the reaction. As N-protected CHIT derivatives, several schiff bases of CHIT and N-phthaloyl CHIT have been reported [16]. CHIT can also be modiied by either cross-linking [17] or graft copolymerisation [18–22]. In many cases, the chemically modiied CHIT shows greater activity than the original polymer. In general, the smart properties of the natural CHIT are altered to a remarkable degree by the introduction of very small amounts of substituent groups of either neutral or ionic types. In recent years, CHIT has drawn attention in the ield of smart materials, which offer a reversible and yet discontinuous molecular phase change in response to various external physico-chemical factors not only because of their unique properties but also because of their potential for signiicant technological applications [1]. Chemical signals, such as pH, metabolites and ionic factors, will alter the molecular interactions between polymer chains or between polymer chains and ions present in the material system. Physical stimuli, such as temperature or electrical potential, may provide various energy sources for altering molecular interactions. These interactions will change the properties of polymer materials such as solubility, swelling behaviour, conigurations of conformational change, redox (i.e. reduction-oxidation) state and crystalline or amorphous transition [23–27]. In general, smart materials are the class of materials having one or more properties that can be signiicantly changed in a controlled manner by changing the external stimuli, for example stress, temperature, moisture, pH, electric and magnetic ields. There are numerous types of smart materials; however, we can categorise them on the basis of their responsive behaviours during the stimuli. Based on the properties, we can categorise smart materials in the following classes: • Piezoelectric materials: The ceramics or polymers are characterised by a swift, linear shape change in response to an electric ield. The electricity makes the material expand or instantly contract. This class of materials has potential applications in actuators as control chatter in precision machine tools, improving robotic parts for faster and accurate movement, microelectronic circuits in machines e.g., computers, photolithography printers, etc. and also in healthmonitoring ibres for bridges, buildings and wood utility poles [28]. • Electrostrictive and magnetostrictive materials: This class of materials refers to the change in size of materials with response to either an electric or a magnetic

194

Smart Chitosan Matrices for Application to Cholesterol Biosensors ield and, conversely, producing a voltage when stretched. These materials show promising applications for the manufacturing of pumps and valves, aerospace wind tunnels, shock tube instrumentation, landing gear hydraulics and further biomechanics force measurement of orthopaedic gait and posturography, sports, ergonomics, neurology, cardiology and rehabilitation [29]. • Rheological materials: Smart materials cover not only solids but also luids like electro-rheological and magneto-rheological luids that can change state instantly using an electric or magnetic ield [30]. These types of luids used in dampers for vehicle seats, shock absorbers, exercise equipment and optical inishing. • Thermo-responsive materials: The shape memory alloys are obtained from this class of materials. Such alloys demonstrate a change in shape in response to heat and/or cold. Nitinol or nickel and titanium are usually combined to form such intelligent materials. The rarely used materials of this class are gold/cadmium, silver/cadmium, copper/aluminium/nickel, copper/tin, copper/zinc and copper/ zinc/aluminium [31]. They are frequently used in couplers, thermostats and automobile, plane and helicopter parts. The pH-sensitive materials are used as indicators and can change colours as a function of pH. This type of smart material shows promise in paints that change colour when the metal beneath begins to corrode [32]. • Electrochromic materials: The phenomenon is deined as the ability of a material to change its optical properties when a voltage is applied across it. These materials are used as antistatic layers, electrochrome layers in liquid crystal displays (LCDs) and cathodes in lithium batteries. • Smart gels: These gels are engineered to shrink or swell by a factor of 1000, and can be programmed to absorb or release luids with response to chemical or physical stimulus. They are used for agriculture, food, drug delivery, prostheses, cosmetics and chemical processing applications [32]. Overall, smart polymers undergo fast and reversible changes in micro-structure from a hydrophilic to a hydrophobic state that are triggered by small stimuli in the environment. The changes are apparent at the macroscopic level as order of magnitude changes in hydrogel size [33]. It is a reversible process, the system returning to the initial stage with removal of the trigger including neutralisation of charged groups either by a pH change or by adding an oppositely charged polymer, changing the eficiency of hydrogen bonding with increase in temperature or ionic strength and by breaking up hydrogels. Nowadays electric, magnetic and radiation-induced reversible phase transitions are also known and have found versatile biological applications [34]. Further, smart materials respond to environmental stimuli with particular changes in some variables and for this reason this category of materials is often also called 195

Biotechnology in Biopolymers Developments, Applications & Challenging Areas responsive materials. Depending on changes in some external conditions, ‘smart’ materials change their properties e.g. mechanical, electrical, appearance and/or structure and/or composition and/or functions. Mostly, smart materials are embedded in systems whose inherent properties can be favourably changed to meet the desirable performance. It is well known that one or more properties of polymeric materials can be signiicantly changed in a controlled fashion under external stimuli including stress, temperature, moisture, pH, electric or magnetic ields. These materials could be piezoelectric materials, shape memory alloys and polymers, magnetic shape memory alloys, pH- and temperature-responsive polymers, halochromic and chromogenic materials and non-Newtonian luids [35]. Recently, great attention has been paid to the development of stimuli-responsive CHIT-based redox materials with unique properties such as biocompatibility, biodegradability and biological functions, especially for biosensor applications. They may be prepared by combining pH-responsive redox polymers with natural-based polymeric components to form graft copolymers. A number of graft copolymers have been considered to be incorporated into pH-responsive materials in this respect, among them polyaniline (PANI) has the greatest potential. The aim of this chapter is to focus on the different methods of preparation of CHIT-based smart materials such as CHIT grafted/blended with PNIPAAm (poly(N-isopropylacrylamide)), PANI, PPy (polypyrrole), etc. designed as stimuli-responsive polymeric materials for biomedical device applications. In this review, various types of strategies involved in the preparation of CHIT-based stimuli-responsive materials as well as nanocomposites and properties such as phase transition temperature, swelling behaviour, redox behaviour, mechanical strength, morphology, conductivity, etc. are discussed in detail.

8.2 Biosensors: Biorecognition Devices A biorecognition device, or so-called ‘biosensor’, is an analytical device that converts a biological response into an electrical signal. Biosensors are used to determine the concentration of substances and other parameters of biological interest. A successful biosensor must possess a few properties such as it should be speciic stable under normal storage conditions accurate, precise, reproducible and it should produce a linear response over the useful analytical range, it should also be economical, portable and capable of being used by semiskilled persons. The biocatalyst (a) converts the substrate to product. This reaction is determined by the transducer (b), which converts it to an electrical signal. The output from the transducer is ampliied (c), processed (d) and displayed (e) as shown in Figure 8.2.

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Smart Chitosan Matrices for Application to Cholesterol Biosensors

Figure 8.2 Schematic diagram of a biosensor

The transducer has a key role in a biosensor, which makes use of a physical change accompanying the reaction. This may be the heat output (or absorbed) by the reaction (calorimetric biosensors) [36], changes in the distribution of charges causing an electrical potential to be produced (potentiometric biosensors), movement of electrons produced in a redox reaction (amperometric biosensors), light output during the reaction or a light absorbance difference between the reactants and products (optical biosensors) or effects due to the mass of the reactants or products (piezo-electric biosensors). There are three so-called generations of biosensors; irst-generation biosensors where the normal product of the reaction diffuses to the transducer and causes the electrical response, second-generation biosensors, which involve speciic ‘mediators’ between the reaction and the transducer in order to generate improved response, and thirdgeneration biosensors where the reaction itself causes the response and no product or mediator diffusion is directly involved. The electrical signal from the transducer is often low and superimposed upon a relatively high and noisy (i.e. containing a high-frequency signal component of an apparently random nature, due to electrical interference or generated within the electronic components of the transducer) baseline. The signal processing normally involves subtracting a ‘reference’ baseline signal, derived from a similar transducer without any biocatalytic membrane, from the sample signal, amplifying the resultant.

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas The analogue signal produced at this stage may be output directly but is usually converted to a digital signal and passed to a microprocessor stage where the data is processed, converted to concentration units and output to a display device. Many enzyme-catalysed reactions are exothermic i.e. evolving heat [37], cf. Table 8.1, which may be used as a basis for measuring the rate of reaction and, hence, the concentration of analytes. This represents calorimetric biosensors. Thermistors are usually used to measure the temperature changes incorporated at the entrance and exit of small packed bed columns containing immobilised enzymes within a constant temperature environment. Under such closely controlled conditions, up to 80% of the heat generated in the reaction may be registered as a temperature change in the sample stream. This may be simply calculated from the enthalpy change and the amount reacted. If a 1 mM reactant is completely converted to product in a reaction generating 100 kJ mole–1, then each ml of solution generates 0.1 J of heat. At 80% eficiency, this will cause a change in temperature of the solution amounting to approximately 0.02 °C. This is about the temperature change commonly encountered and necessitates a temperature resolution of 0.0001 °C for the biosensor to be generally useful.

Table 8.1 Molar heat enthalpies of enzyme-catalysed reactions Reactant

Enzyme

Molar heat enthalpies –∆H (kJ mole–1)

Cholesterol

Cholesterol oxidase

53

Esters

Chymotrypsin

Glucose

Glucose oxidase

80

Hydrogen peroxide

Catalase

100

Penicillin G

Penicillinase

67

Peptides

Trypsin

10–30

Starch

Amylase

8

Sucrose

Invertase

20

Urea

Urease

61

Uric acid

Uricase

49

4–16

The sensitivity (10–4 M) and range (10–4 to 10–2 M) both are quite low for the majority of applications with biosensors using thermistors. The sensitivity can be increased

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Smart Chitosan Matrices for Application to Cholesterol Biosensors by using the more exothermic reactions (e.g. catalase). The low sensitivity of the system can be increased substantially by increasing the heat output by the reaction. In the simplest case this can be achieved by linking together several reactions in a reaction pathway, all of which contribute to the heat output. Thus the sensitivity of the glucose analysis using glucose oxidase (GOD) can be more than doubled by the co-immobilisation of catalase within the column reactor in order to disproportionate the hydrogen peroxide produced [38]. Potentiometric biosensors use ion-selective electrodes to transduce the biological reaction into an electrical signal. It consists of an immobilised enzyme membrane surrounding the probe from a pH meter [39], where the catalysed reaction generates or absorbs hydrogen ions. There are three types of ion-selective electrodes used in biosensors: • Glass electrodes for cations (e.g. normal pH electrodes) in which the sensing element is a very thin hydrated glass membrane, which generates a transverse electrical potential due to the concentration-dependent competition between the cations for speciic binding sites. The selectivity of this membrane is determined by the composition of the glass. The sensitivity to H+ is greater than that achievable for NH4+. • Glass pH electrodes coated with a gas-permeable membrane selective for CO2, NH3 or H2S. The diffusion of the gas through this membrane causes a change in pH of a sensing solution between the membrane and the electrode, which is then determined. • Solid-state electrodes where the glass membrane is replaced by a thin membrane of a speciic ion conductor made from a mixture of silver sulide and a silver halide. The iodide electrode is useful for the determination of I– in the peroxidase reaction and also responds to cyanide ions [40]. Reactions involving the release or absorption of ions may be used in potentiometric biosensors: (a) H+ Cation glucose oxidase H2O D-glucose

+ O2 → D-glucono-1,5-lactone + H2O2 → D-gluconate + H+

penicillinase penicillin → penicilloic acid + H+ urease (pH 6.0) H2NCONH2 + H2O + 2H+ → 2NH4+ + CO2 199

Biotechnology in Biopolymers Developments, Applications & Challenging Areas urease (pH 9.5) H2NCONH2 + 2H2O → 2NH3 + HCO3- + H+ lipase neutral lipids + H2O → glycerol + fatty acids + H+ (b) NH4+ Cation L-amino

acid oxidase L-amino

acid + O2 + H2O →keto acid + NH4+ + H2O2

asparaginase L-asparagine

+ H2O → L-aspartate + NH4+

urease (pH 7.5) H2NCONH2 + 2H2O → H+ + 2NH4+ + HCO3– (c) I– Anion peroxidase H2O2 + 2H+ + 2I– → I2 + 2H2O (d) CN–Anion glucosidase amygdalin + 2H2O → 2 glucose + benzaldehyde + H+ + CN– Amperometric biosensors function by the generation of a current when a potential is applied between two electrodes [41]. They generally have response times, dynamic ranges and sensitivities similar to the potentiometric biosensors. The simplest amperometric biosensors in common usage involve the Clark oxygen electrode. It consists of a platinum cathode at which oxygen is reduced and a silver/silver chloride reference electrode. When a potential of –0.6 V, relative to the Ag/AgCl electrode, is applied to the platinum cathode, a current proportional to the oxygen concentration is produced. Normally both electrodes are bathed in a solution of saturated potassium chloride and separated from the bulk solution by an oxygen-permeable plastic membrane (e.g. Telon, polytetraluoroethylene). The following reactions occur: Ag Anode: 4Ag + 4Cl– → 4AgCl + 4e–

200

Smart Chitosan Matrices for Application to Cholesterol Biosensors Pt Cathode: O2 + 4H+ + 4e– → 2H2O The eficient reduction of oxygen at the surface of the cathode causes the oxygen concentration there to be effectively zero. The rate of this electrochemical reduction therefore depends on the rate of diffusion of the oxygen from the bulk solution, which is dependent on the concentration gradient and hence the bulk oxygen concentration. Thus, a small, but signiicant, proportion of the oxygen present in the bulk is consumed by this process; the oxygen electrode measuring the rate of a process that is far from equilibrium, whereas ion-selective electrodes are used close to equilibrium conditions. This causes the oxygen electrode to be much more sensitive to changes in the temperature than potentiometric sensors. A typical application for this simple type of biosensor is the determination of glucose concentrations by the use of an immobilised glucose oxidase membrane. The reaction results in a reduction of the oxygen concentration as it diffuses through the biocatalytic membrane to the cathode, this being detected by a reduction in the current between the electrodes [42]. Other oxidases may be used in a similar manner for the analysis of their substrates (e.g., alcohol oxidase, D- and L-amino acid oxidases, cholesterol oxidase, galactose oxidase and urate oxidase). A potential is applied between the central platinum cathode and the annular silver anode. This generates a current (I), which is carried between the electrodes by means of a saturated solution of KCl. The electrode compartment is separated from the biocatalyst (such as glucose oxidase (GOD)) by a thin plastic membrane, which is permeable only to oxygen. The analyte solution is separated from the biocatalyst by another membrane, permeable to the substrate(s) and product(s). There are two main areas of development in optical biosensors. These involve determining changes in light absorption between the reactants and products of a reaction, or measuring the light output by a luminescent process [43]. The former usually involve the widely established, if rather low technology, use of colorimetric test strips. These are disposable single-use cellulose pads impregnated with enzyme and reagents. The most common use of this technology is for whole-blood monitoring in diabetes control. In this case, the strips include glucose oxidase, horseradish peroxidase (HRP) (EC 1.11.1.7) and a chromogen (e.g., o-toluidine or 3,3′,5,5′-tetramethylbenzidine). The hydrogen peroxide is produced by the aerobic oxidation of glucose that oxidises the weakly coloured chromogen to a highly coloured dye. D-glucose

+ oxygen → D-glucono-1,5-lactone + hydrogen peroxide

201

Biotechnology in Biopolymers Developments, Applications & Challenging Areas peroxidase chromogen (2H) + H2O2 → dye + 2H2O The evaluation of the dyed strips is best achieved by the use of portable relectance meters, although direct visual comparison with a coloured chart is often used. A wide variety of test strips involving other enzymes are commercially available at the present time. A most promising biosensor involving luminescence uses irely luciferase (Photinus-luciferin 4-monooxygenase, ATP-hydrolysing, EC 1.13.12.7) to detect the presence of bacteria in food or clinical samples. Bacteria are speciically lysed and the ATP released (roughly proportional to the number of bacteria present) reacted with D-luciferin and oxygen in a reaction that produces yellow light in high quantum yield. luciferase ATP + D-luciferin + O2 → oxyluciferin + AMP + pyrophosphate + CO2 + light (562 nm) The light produced may be detected photometrically by use of high-voltage, and expensive, photomultiplier tubes or low-voltage, cheap photodiode systems. In general, biosensors are easy to operate, analyse over a wide range of useful analyte concentrations and give reproducible results. The diffusion limitation of substrate(s) may be an asset to be encouraged in biosensor design due to the consequent reduction in the effects of analyte pH, temperature and inhibitors on biosensor response. The nanolevel matrices design may have potential to provide a new era of advanced biosensors.

8.3 Matrices Fabrication Methodology

8.3.1 Chemical Oxidative Method A polymer comprises molecules with one or more species of block connected to the main chain as side-chains, these side chains having constitutional or conigurational features that differ from those in the main chain. In a graft copolymer, the distinguishing feature of the side chains is constitutional, i.e. the side chains comprise units derived from at least one species of monomer different from those that supply the units of the main chain. The graft copolymerisation of PANI onto CHIT was reported using ammonium persulfate, (NH4)2S2O8/HCl as redox initiator as shown in Scheme 8.1 [44]. It was observed that (NH4)2S2O8/HCl redox system can be eficiently used in the graft copolymerisation of PANI onto CHIT. Silva and co-workers synthesised

202

Smart Chitosan Matrices for Application to Cholesterol Biosensors pH-responsive PANI colloids by using CHIT as a steric stabiliser. The process was enzymatic polymerisation using toluenesulfonic or camphorsulfonic acid as a doping agent [45,46].

Scheme 8.1 Synthesis of HCl doped CHIT-co-PANI using (NH4)2S2O8 as oxidising agent in acidic medium

8.3.2 Electrochemical Method Electrochemical synthesis in organic chemistry is the synthesis of chemical compounds in an electrochemical cell [47]. The main advantage of electrochemical synthesis over an ordinary redox reaction is avoidance of the potential wasteful other halfreaction and the ability to precisely tune the required potential. Tiwari and co-

203

Biotechnology in Biopolymers Developments, Applications & Challenging Areas workers have reported electrochemical synthesis of CHIT-co-PANI in an acidic medium [48]. In a typical procedure, aniline and CHIT solution was mixed with HCl in an electrochemical cell and the CHIT-co-PANI was chronoamperometrically synthesised onto an ITO-coated glass surface using a three-electrode assembly with ITO glass as working, platinum as counter and Ag/AgCl as reference electrodes. The resulting CHIT-co-PANI/ITO electrode was washed with deionised water followed by a phosphate buffer saline solution of pH 7.0 in order to neutralise the electrode surface (Figure 8.3).

Figure 8.3 Electrochemical synthesis of CHIT-co-PANI onto indium tin oxide (ITO) coated glass plate. Reproduced with permission from A. Tiwari and S. Gong, Electroanalysis, 2008, 20, 1775. ©2008, Wiley

204

Smart Chitosan Matrices for Application to Cholesterol Biosensors In the electrochemical copolymerisation of CHIT-co-PANI, the aniline monomer initially became protonate with acid (i.e. H+ of HCl) and propagated to form an intermediate called PANI radical cation (Scheme 8.2 [a]).

(a)

(b)

Scheme 8.2 Formation of (a) PANI radical cation and (b) CHIT macro radical

PANI radical cation simultaneously generated CHIT macro radicals (Scheme 8.2 [b]) by the abstraction of hydrogen from the -OH and -NH2 groups of the CHIT macromolecules. The PANI cation radicals and CHIT macro radicals then copolymerised and yielded CHIT-co-PANI (Scheme 8.3).

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas

Scheme 8.3 Synthesis of CHIT-co-PANI by graft copolymerisation of PANI radical cation (a) onto the CHIT macro radical (b)

8.4 Physico-chemical Blending The use of nanomaterials such as CNTs to fabricate matrices for biosensors is one of the most exciting approaches because nanomaterials have a unique structure and high surface-to-volume ratio [49]. The surfaces of nanomaterials can also be tailored in the molecular scale in order to achieve various desirable properties [50]. The diverse properties of nanocomposites materials such as unique structure and good chemical stability enable them to provide a wide range of applications in sensor technology [51]. Further, nanocomposites do not suffer from the drawback of sensing complications, synthesis complexities, they have a long shelf life and eficiency. In addition, the fundamental electronic characteristics of CNTs could also be used to facilitate the uniform current within the anocomposites biosensing electrodes. There are many

206

Smart Chitosan Matrices for Application to Cholesterol Biosensors reports on integration of CNTs with sol-gel derived SiO2-CHIT to fabricate biosensors to gain synergistic action using organic-inorganic bionanocomposites. The sol-gel SiO2-CHIT is prepared by mixing alcoholic silica precursor such as tetraethoxysilane (TEOS) and CHIT solution under magnetic stirring at room temperature. To this mixture, homogeneously dispersed CNTs in ethanol are added. The mixture initially comprising two phases is made uniform by stirring vigorously until -SiO2 is distributed evenly in the aqueous solution while the hydrolysis reaction occurs. After a certain time period, the opaque and black sol is formed as shown in Scheme 8.4 [50].

Scheme 8.4 Reaction scheme for the preparation of SiO2-CHIT/CNTs bionanocomposites. Reproduced with permission from A. Tiwari and S. Gong, Electroanalysis, 2008, 20, 2119. ©2008, Wiley

In a control process, tetraethoxysilane underwent hydrolysis and formed tetrahydroxy silane (silanol) at acidic pH [52]. The resulting silanol then reacted with CHIT via a condensation reaction between the -OH groups and led to the formation of a CHITSiO2 composite network, in which CNTs are homogeneously dispersed. Both CNTs and SiO2 improve the mechanical properties of the CHIT-SiO2-CNT bionanocomposites, primarily CNTs enhance the electrical conductivity of the biocomposite. In other works, Thanpitcha and co-workers prepared PANI/CHIT blend ilms by using a solution casting method and mechanical and electrical properties of blends were investigated in terms of blend composition and the doping conditions: acid 207

Biotechnology in Biopolymers Developments, Applications & Challenging Areas type, acid concentration and doping time [53]. Smooth, lexible and mechanically robust blend ilms were obtained at PANI content lower than 50 wt.%. To become electrically conductive, they doped PANI with HCl. The electrical conductivity of the doped ilms increases with increasing PANI content. However, high concentrations of HCl (2–6 M) and long doping times (15–24 h) led to a decrease in electrical conductivity. This result indicated the over-protonation of PANI chains in the blend ilms. Moreover, increasing strength and using smaller anions for acid dopant induced higher electrical conductivity values of the blend ilms. The mechanical properties of the blend ilms were strongly affected by the doping treatment with HCl. The inferior mechanical properties of the blend ilms after doping were presumed to be due to hydrolysis of CHIT. The comparatively poor mechanical strength of conducting polymer-based actuators has led researchers to investigate the possibility of obtaining composite materials and interpenetrating networks with other polymers, carbon nanotubes (CNT), etc. [54]. Combination of a conducting polymer and CHIT (hydrogel) is expected to lead to improved properties that can be used for designing actuator devices. A hydrogel can produce a large swelling strain, even though the strain offered by a conducting polymer is relatively low. However, the conducting polymer improves the property by providing a low operational voltage, a short diffusion path and a fast response time. PANI/ CHIT semi-IPN networks and dual-mode actuation anocomp in CHIT/PANI/CNT composite ibres was reported previously [55]. Ismail and co-workers have fabricated a new type of electroactuating biopolymer hydrogel/PANI microibre by wet spinning a CHIT solution, followed by the in situ chemical polymerisation of aniline [56]. This novel biomaterial showed an enhanced chemical and electrochemical actuation in response to pH and an electrical stimulus. The ibres showed a reasonable electrical conductivity of 2.856 × 10−2 S/cm at room temperature. This novel semiconducting biomaterial showed enhanced electrochemical actuation, resulting in a strain of about 0.39%, and a chemical actuation corresponding to a strain of 6.73% upon switching the pH between pH = 0 and 1. Yavuz and co-workers are the first group who reported substituted PANI/ CHIT composites [57]. Composites were synthesised chemically and ammonium peroxydisulfate was used as oxidant and composites were characterised by measurements of conductivity, FT-IR, UV-vis, SEM and TGA techniques. FTIR spectra of the composites revealed that there is a strong interaction between substituted PANIs and CHIT. Among the substituted PANI/CHIT composites synthesised, poly(Nethylaniline)/CHIT (PNEANI/CHIT) has the highest conductivity with a value of 1.68 × 10–4 S/cm. The P2EANI/Ch composite exhibited higher thermal stability than the other composites. SEM images of the composites showed an agglomerated granular morphology of substituted PANI particles coated on the surface of CHIT (Figure 8.4).

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Smart Chitosan Matrices for Application to Cholesterol Biosensors

Figure 8.4 Scanning electron micrographs of the composites; (a) CHIT, (b) PANI/CHIT, (c) P2EANI/CHIT, (d) PNEANI/CHIT and (e) PNMANI/CHIT. Reproduced with permission from A.G. Yavuz, A. Uygun and V.R. Bhethanabotla, Carbohydrate Polymers, 2009, 75, 448. ©2009, Elsevier

Electrically conductive polymer biomaterials have been proven to be another promising alternative for developing new biodegradable conduits used for restoring the function of injured peripheral nerves or the generation of a nerve gap since the early 1990s by using electrical stimulation in situ [58–60]. Apart from being effectively used for nerve regeneration [61,62] these conductive biomaterials have also shown a capacity to facilitate the growth of other types of cells, such as endothelial cells [63], bone cells [64] and chromafin cells [65]. In most cases, these conductive biomaterials are obtained in the form of blends or composites by using biodegradable polymers as matrices and an intrinsically conductive polymer, PPy, as a conductive component because PPy has an acceptable biocompatibility with mammalian cells [66]. Wan and co-workers worked on the fabrication of novel conductive poly(DL-lactide)/ CHIT/polypyrrole (PPy) complex membranes [67]. Using poly(DL-lactide)/CHIT blends as matrices and PPy as a conductive component, several kinds of membranes with various compositions are prepared. A percolation threshold of PPy as low as 1.8 wt.% is achieved for some membranes by controlling the CHIT proportion

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas between 40 and 50 wt.%. SEM images exhibit that the membranes with a low percolation threshold show a two-phase structure, which consists of poly(DL-lactide) and CHIT phases. Dielectric measurements indicate that there is limited miscibility between the poly(DL-lactide) and CHIT but PPy is nearly immiscible with the other two components. Based on the structural characteristics of the membranes, the PPy particles are suggested to be localised at the interface between two phases. Yang and Lu [68] have demonstrated the one-step synthesis of AgCl/PPy core/shell nanostructures with controllable shell thickness in the presence of CHIT and thus hollow PPy nanoparticles were reported. AgCl, as a template, was formed during the initial polymerisation by the interaction between metal cations and oxidising anions and adsorbed with CHIT whereupon the coating of PPy layers was induced (Figure 8.5). The method shows the merits of easier preparation compared with the conventional stepwise process and controlled shell thickness could be obtained by adjusting the quantity of pyrrole added.

Figure 8.5 The possible mechanism for the formation of core/shell and hollow particles. Reproduced with permission from X. Yang and Y. Lu, Polymer, 2005, 46, 5324. ©2005, Elsevier

Another study conducted by Abdi and co-workers [69] prepared PPy-CHIT composite ilm and investigated the electrical and optical properties of PPy-CHIT composite. Their work focused on the effects of CHIT content on the electrical conductivity, photoacoustic effect [70], EMI-SE [71,72] and dielectric constant of the resulting ilms. The refractive indexes of the samples were successfully measured by SPR technique (Figure 8.6). Since the SPR angle shifted clearly to the right and produced a sharp peak of resonance angle, this technique is suitable for using in sensitive optical sensors. It seems that the EMI shielding effectiveness of 33.9 dB for the PPy-CHIT composite ilms is very

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Smart Chitosan Matrices for Application to Cholesterol Biosensors attractive in any electromagnetic interference (EMI) shielding applications where a minimum shielding effectiveness of 30 dB is required (Table 8.2).

Figure 8.6 Relectivity versus surface anocom resonance angle curves for Au, Au-PPy and Au-PPy-CHIT thin ilms. Reproduced with permission from M.M. Abdi, A. Kassim, H.N.M.E. Mahmud, W.M.M. Yunus, Z.A. Talib and A.R. Sadrolhosseini, Journal of Material Science, 2009, 44, 3682. ©2009, Springer

Table 8.2 EMI shielding effectiveness of PPy-CHIT composition films with various concentrations of CHIT and PPy film without CHIT CHIT conc. (%w/v)

C (S cm–1)

Tr (%)

Ab (%)

Re (%)

Total Atten.

SE (dB)

0.3

39.3

0.75

16.05

83.2

99.25

21.2

0.5

49.4

0.20

8.09

91.7

99.79

26.9

0.7

69.1

0.04

7.56

92.4

99.96

33.9

0.9

42.1

0.29

11.91

87.8

99.71

25.3

1.1

33.5

1.50

18.12

80.4

98.52

18.3

PPy

35.2

1.01

16.21

82.8

99.01

19.8

C = Conductivity; Tr = Transmittance; Ab = Absorbance; Re = Relection; SE = Shielding effectiveness

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8.5 Cholesterol Bioelectrodes Rapid and accurate detection of biomolecules is essential in medical diagnosis as it permits fast treatment. Small size and greater sensitivity mean less-invasive sampling and detection of molecules such as neurotransmitters or hormones at biologically relevant levels [73,74]. Greater speciicity allows assays to be performed in complex luids such as blood or urine without false negative or false positive results. Nanotechnology promises to improve biosensing on all of these fronts [75]. Nanofabricated biomaterials can bind directly to biomolecules and/or act as transducers to extremely small and sensitive detectors. The unique electrical, chemical, thermal and catalytic properties of namomaterials offer excellent prospects in the development of electrochemical biosensors [76]. The high sensitivity of such devices, coupled to their compatibility with modern microfabrication technologies, portability, low cost (disposability), minimal power requirements and independence of sample turbidity or optical pathway make them excellent candidates for clinical diagnostics. The SiO2-CHIT/CNTs sol thin ilm is fabricated by spreading it uniformly onto a substrate such as ITO glass plate using the spin-coating technique and it is subsequently dried at room temperature. SiO2-CHIT/CNTs/substrate electrode is washed with deionised water followed by phosphate buffer saline of pH 7.0 in order to maintain pH over the electrode surface. SiO2-CHIT/CNTs electrode is treated with aqueous glutaraldehyde as a cross-linker. The freshly prepared enzyme solution is uniformly spread onto glutaraldehyde treated SiO2-CHIT/CNTs electrode and is kept in a humid chamber for 12 h at 4 °C, Figure 8.7.

Figure 8.7 Schematic diagram of the fabrication of CHIT-SiO2-CNTs/ITO and ChOx/CHIT-SiO2-MWNT/ITO electrodes. Reproduced with permission from A. Tiwari and S. Gong, Electroanalysis, 2008, 20, 2119. ©2008, Wiley

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Smart Chitosan Matrices for Application to Cholesterol Biosensors The enzyme-SiO2-CHIT/CNTs bioelectrode is immersed in phosphate buffer solution of pH 7.0 in order to wash out unbound enzyme from the electrode surface. A stable enzyme-substrate coupling is achieved with glutaraldehyde as a cross-linking agent [77]. At one end glutaraldehyde is attached to the -NH2 side of the CHIT-SiO2-CNTs/ ITO electrode through a reaction between the -CHO end group of glutaraldehyde, while other end of the glutaraldehyde is attached to enzyme through a reaction between the -CHO group of glutaraldehyde and the terminal -NH2 group of enzyme, which resulted in an enzyme/CHIT-SiO2-CNTs/ITO bioelectrode as shown in Figure 8.8.

Figure 8.8 Covalent immobilisation of an enzyme on SiO2-CHIT/CNTs bionanocomposites matrices using glutaraldehyde as a linker

8.6 Characterisations

8.6.1 Redox Behaviour PANI is a typical redox material that can easily create an electronic moment by producing electrons. In fact, the reversibility of PANI is very well-behaved and can be achieved by a cyclic voltammetry (CV) study [48]. The acid-doped analogues of PANI are an especially powerful oxidising as well as reducing agent, due to inductive receiving/donation of electrons. Usually, the electroactivity of emereldine salt is higher than that of emereldine base due to charge mobility appearing after doping with HCl. Figure 8.9 shows the anodic as well as cathodic current peaks of CHITco-PANI that inluence the voltage scan rate. This is because of reversible electron

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas transfer in between the protonated PANI and CHIT. With the increase in the scan rate, peak current was increased due to the reversible electron transfer reaction on the surface of CHIT-co-PANI.

Figure 8.9 Scan rate and rate constant dependence of the CV curves. CV of the CHIT-co-PANI ilm using Ag/AgCl as a reference electrode in 0.5 M HCl at different scan rates: (i) at 10 mVs–1; (ii) at 20 mVs–1; (iii) at 30 mVs–1; (iv) at 40 mVs–1; and (v) at 50 mVs–1. The oxidation peaks are shown at 250 and 710 mV and the reduction peak is shown at 405 mV. Reproduced with permission from A. Tiwari and S. Gong, Electroanalysis, 2008, 20, 1775. ©2008, Wiley

Likewise, the bare and enzyme immobilised SiO2-CHIT/CNTs bionanocomposite can be characterised with Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM) and cyclic voltammetry (CV). In Figure 8.10, the infrared peaks of CHIT (curve A) in SiO2-CHIT (curve B) become wider and sharper due to overlap of functional groups of CHIT and SiO2, i.e. stretching vibration bands of Si-O-Si, Si-O-C and C-O bond. Also, two new peaks appear at 1300 and 785 cm–1 pertaining to stretching vibration of C-Si and bending vibration of C-H corresponding to the CH3-Si group [50].

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Figure 8.10 FT-IR spectra of the (A) CHIT-SiO2/CNTs nanocomposite and (B) CAH/CHIT-SiO2/CNTs. Reproduced with permission from A. Tiwari and S. Gong, Electroanalysis, 2008, 20, 2119. ©2008, Wiley

On incorporation of CNTs in CH-SiO2 hybrid, the infrared band corresponding to SiO2 becomes broader and a new infrared band appears at 890 cm–1 revealing the presence of CNTs, which affects the vibration mode of CHIT and SiO2 resulting in the formation of SiO2-CHIT/CNTs bionanocomposite. After immobilisation of enzyme, FT-IR bands corresponding to -NH/-OH group in the bionanocomposite become broader suggesting interaction between the amino and hydroxyl groups of CHIT. However, the presence of a peak at 1672 cm–1 (corresponding to amide bands) indicates immobilisation of enzymes [78,79].

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas The surface morphology of SiO2-CHIT/CNTs (Figure 8.11, image A) reveals the mono-dispersed rope-like structure of CNTs surrounded with the globular appearance of SiO2 particles into the CHIT matrix indicating that CNTs and SiO2 are uniformly dispersed into the backbone of CHIT. We speculate that CNTs are entrapped with CHIT and SiO2 via electrostatic interactions. The surface morphology of SiO2-CHIT/ CNTs bionanocomposite further changes after the immobilisation of enzyme revealing attachment of enzymes over the electrode surface (image B).

Figure 8.11 SEM images of the (A) CHIT-SiO2/CNTs and (B) enzyme/CHIT-SiO2/ CNTs. Reproduced with permission from A. Tiwari and S. Gong, Electroanalysis, 2008, 20, 2119. ©2008, Wiley

It may be noted that the appearance of CNTs is less predominant due to immobilisation of enzymes onto bionanocomposites via electrostatic interactions and covalent binding. A multifunctional matrix presumably provides a mesoporous surface resulting in enhanced enzyme loading at the enzyme-loaded SiO2-CHIT/CNTs bioelectrode. Figure 8.12 shows a probable mechanism of enzyme immobilisation onto the bionanocomposite using glutaraldehyde as linker. It appears that available -NH2 groups of CHIT are covalently attached with the aldehyde group of glutaraldehyde at one end and another aldehyde group is linked with available -NH2 groups of enzymes via covalent bonding and electrostatic interactions between bionanocomposite and enzymes.

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Figure 8.12 Proposed mechanism for preparation of ChEt-ChOx/CNTs/SiO2CHIT/ITO bioelectrode. Reproduced with permission from P.R. Solanki, A. Kaushik, A.A. Ansari, A. Tiwari and B.D. Malhotra, Sensors and Actuators B, 2009, 137, 727. ©2009, Elsevier

8.6.2 Electrocatalytic Properties Electrocatalytic properties of novel nanomaterials display a fundamental role in the organisation of electrochemical biosensing devices. A variety of nanostructures have been investigated to determine their properties and propose their possible applications in the development of electrochemical biosensing devices [80]. These nanomaterials include metal nanoparticles, oxide nanoparticles, semiconductor nanoparticles, polymeric nanomaterials, carbon nanotubes and even nanocomposite materials have been widely used in the construction of electrochemical biosensors because of their unique electrocatalytic properties. Such metal-nanoparticles (e.g. platinum) catalyse the redox process of some biomolecules with analytical interest,

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas which can be monitorised using electroanalytical techniques. Nanomaterials have high surface-to-volume ratio, electrocatalytic activity as well as good biocompatibility and novel electron transport properties make them highly attractive materials for ultra-sensitive detection of biological macromolecules via bioelectronic devices [74]. Some nanoscale materials exhibited remarkable electron transport properties, which are strongly dependent on their nanocrystalline structure. Morphologicalbased nanomaterials show new capabilities that are generated by combination of novel nanobuilding units and strategies for assembling them. These extraordinary electrocatalytic characteristics of the nanomaterials are exploiting the fabrication of eficient electrochemical biorecognition devices. The surface features of nanoscale materials such as shape, size, diameter, surface condition, crystal structure and its quality, chemical composition, crystallographic orientation along the axis, etc., are very important parameters, all of which inluence the electron transport mechanism of the nanomaterials [76].

8.6.3 Electrochemical Response Electrochemical study measures the movement and separation of charge in matter, i.e. it is the study of the transfer of electrons. Most of the chemical reactions involve charge transfer, and matter may hold charge, either positive or negative. The charges could be discrete and measurable or partial and diffuse; charged matter can maintain separations leading to interesting effects. Figure 8.13 shows the CVs of the electrochemical cells using either CHIT-SiO2-CNTs/ITO or enzyme/CHIT-SiO2-CNTs/ ITO electrode at a constant 20 mVs–1 scan rate in 50 mM phosphate buffer solution (pH 7.0, 0.9% NaCl) containing 5 mM Fe(CN)63−/4−. The current of the electrochemical cell using the electrode CHIT-SiO2-MWCNTs/ITO (2.5 × 10–3 A) was about five times that using the enzyme/CHIT-SiO2-CNTs/ITO bioelectrode (0.5 × 10–3 A). Thus, immobilising enzyme onto the bare electrode reduced the current. A decrease in current after the immobilisation of enzyme may be attributed to a slower redox behaviour compared with the bare CHIT-SiO2-CNTs/ITO electrode. The covalent binding of enzyme on the CHIT-SiO2-CNTs/ITO electrode controls the moment of the supporting electrolytes [81]. Also, the non-conducting nature of the enzyme molecules might have contributed to the decrease in current when using the enzyme/ CHIT-SiO2-CNTs/ITO electrode. The study indicates a high affinity of enzyme to the CHIT-SiO2-CNTs nanocomposite matrix over the electrode surface, which may be attributed to: (1) the advantageous nanoporous surface of the CHIT-SiO2-CNTs matrix for the enzyme immobilisation that can favour conformational changes of the enzyme, and (2) the high surface-to-volume ratio, which can help to effectively immobilise enzyme onto the CHIT-SiO2-CNTs nanocomposite [82].

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Smart Chitosan Matrices for Application to Cholesterol Biosensors

Figure 8.13 Cyclic voltammograms of the (A) CHIT-SiO2-CNTs/ITO and (B) enzyme/CHIT-SiO2-CNTs/ITO electrodes in PBS (50 mM, pH 7.0, 0.9% NaCl, 5 mM Fe(CN)63-/4-) at a 20 mVs–1 scan rate. Reproduced with permission from A. Tiwari, Journal of Inorganic and Organometallic Polymers, 2009, 19, 361. ©2008, Elsevier

The electron transport properties of the nanomaterials can also be altered by introducing some doping materials into the matrix, which enhance the surface properties and electrical conductance of the nanomaterials. The small change in the surface properties of the nanomaterials can cause remarkable changes in the transport behaviour. It has been reported that the change in electrical conductivity of the bioelectrode is inluenced by minor surface perturbations such as binding of biomacromolecular species on a long conduction channel. The semiconductor electronic nanomaterials, in particular, have active surfaces that can easily be modiied for immobilisation of numerous biomolecules [77]. Additionally, the sizes of biological macromolecules, such as proteins and nucleic acids, are comparable to nanoscale building blocks. Therefore, any interaction between such molecules should induce signiicant changes in the electrical properties of nanostructures. Due to the extreme smallness of these nanomaterials, it is possible to pack a large number of biomacromolecule-functionalised nanomaterials onto a remarkably small footprint of an array device. These nanometre-scale electronic transducers reduce the pathway for direct electron communication from the redox biomolecule to the electrode for sensitive and speedy detection of analyte without any hindrance [76].

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas This article illustrates the usefulness of nanoscale biocomposite materials for the designing of eficient electrochemical biosensing devices and also highlights the potential analytical applications in terms of nanostructured-based electrochemical biosensors and bioreactors. All these properties of nanoscale composite materials strongly depend on the synthesis procedures used to grow them. Therefore, extensive efforts have been made to synthesise novel morphological based nanosize biocomposite materials such as nanotubes, nanoglobules, nanoparticles, etc., because these morphological nanosize biocomposite materials-based electrochemical biosensing devices show higher performance such as sensitivity, selectivity and real-time detection limit compared to those fabricated from other forms of the nanomaterials [74]. These novel nanomaterials with controlled size, shape and structure can be tuned by altering the physical, chemical and biological routes. CV studies of CHIT/ITO, SiO2-CHIT/ITO, CHIT-CNTs/ITO, SiO2-CHIT/CNTs/ ITO, ChEt-ChOx (cholesterol esterase-cholesterol oxidase)/SiO2-CHIT/CNTs/ITO electrodes have been conducted to understand the synergy between the various components in phosphate buffer saline containing [Fe(CN)6]3−/4− at a scan rate of 50 mV/s, Figure 8.14A (a–c). The CV of pure CHIT (curve a) shows well-defined reversible redox behaviour attributed to highly positively charged species on the matrix indicating that electrons originate from the negatively charged medium. After addition of -SiO2- in CHIT, the magnitude of the current decreases (curve b) due to formation of a complex between SiO2 and the -OH/-NH2 group of CHIT. In the case of CHIT and CNTs (curve c), the magnitude of current decreases and the potential is shifted towards the lower side at 0.213 V compared to that of CHIT (0.301 V) (curve a). It appears that CHIT-CNT biocomposites have increased the number of electrons compared to that of CHIT since ∆Ep is inversely proportional to the number of transferred electrons (∆Ep µ 1/n) [83], while curve (d) exhibits the highest voltammetric response on the SiO2-CHIT/CNTs/ITO electrode because SiO2/ CNTs provides an electroactive surface area that enhances the electron conduction pathway and promotes electron transfer between the enzymes and electrode. The redox potential of ChEt-ChOx/SiO2-CHIT/CNTs/ITO bioelectrode (curve e) is much less compared to the other electrode due to a slow redox process during the biochemical reaction. The inset in Figure 8.14A shows oxidation (Ipa) and reduction (Ipc) peak current against ν1/2 (ν is the scan rate) for SiO2-CHIT/CNTs/ITO and ChEt-ChOx/ SiO2-CHIT/CNTs/ITO bioelectrodes. The diffusion control process yields a linear response obtained by plotting peak current versus the square root of the scan rate and it depends on the value of the diffusion coefficient. The values of slope of the peak oxidation current obtained using d(I)/d(ν1/2) (square root of the scan rate is proportional to D1/2 for electrode and nanobiocomposite) have been found to be 94.6 and 99.3 µA (mV/s)1/2, respectively. The high value of slope of the electrode compared to the other bioelectrode is probably due to the binding of ChEt and ChOx onto the electrode and possibly controls transport of the ions. Figure 8.14B exhibits CV

220

Smart Chitosan Matrices for Application to Cholesterol Biosensors of SiO2-CHIT/CNTs/ITO bioelectrode as a function of scan rate varying from 10 to 100 mV/s. The magnitudes of the cathodic peak and anodic peak currents increase with increasing scan rate. The increased peak-to-peak separation reveals electron transfer between ChEt-ChOx/SiO2-CHIT/CNTs/ITO bioelectrode and the medium. The variation of potential difference (∆Ep) between cathodic (Epc) and anodic (Epa) peaks for SiO2-CHIT/CNTs/ITO bionanocomposite matrix and ChEt-ChOx/SiO2CHIT/CNTs/ITO bioelectrode is shown in Figure 8.14C. The decrease in the ∆Ep value for the bioelectrode indicates faster kinetics of electron transfer on the surface [84]. The surface concentrations of CHIT/ITO, SiO2-CHIT/ CNTs/ITO; ChEt-ChOx/SiO2-CHIT/CNTs/ITO bioelectrode have been estimated from the plot of current versus potential (CV) using the equation: Ip = n2F2I*AV/4RT (Brown–Anson model) where n is the number of electrons transferred, which is 1 in the case of CHIT/CNTs/ITO electrode, F is the Faraday constant (96,584 C/mol), I* is the surface concentration (mol/cm2) obtained for the SiO2-CHIT/CNTs/ITO bionanocomposite matrix, A is the surface area of the electrode (0.25 cm2), V is the scan rate (50 mV/s), R is the gas constant (8.314 J/mol K) and T is the absolute temperature (298 K). The values of surface concentrations for the CHIT/ITO electrode and SiO2-CHIT/CNTs/ITO bionanocomposite matrix have been found to be 11.8 × 10–8 and 13.0 × 10–8 mol/cm2, respectively [85]. These results indicate that SiO2-CHIT/CNTs/ITO matrix provides an increased electroactive surface area for loading of enzymes (ChEt and ChOx). The ability to mass produce biosensors in an eficient and cost-effective fashion has been a major by-product of this research. Hence, CHIT-based SiO2-CHIT/CNTs bionanocomposite matrices can potentially be used for the production of enzyme electrodes, immunodiagnostic arrays or as a luidic handling system in more elaborate high-throughput analytical systems. The crucial role of diffusion membranes in both the kinetics and selectivity of amperometric biosensors must not be underestimated if devices are to be accurately reproduced.

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas

Figure 8.14 (A) Cyclic voltammogram of (a) CHIT/ITO electrode; (b) SiO2CHIT/ITO electrode; (c) CHIT/CNTs/ITO electrode; (d) SiO2-CHIT/CNTs/ITO electrode; (e) ChEt-ChOx/SiO2-CHIT/CNTs/ITO bioelectrode at 50 mV/s in PBS containing 5 mM Fe[(CN)6]3−/4−. The inset shows oxidation and reduction peak current with square root of scan rate for SiO2-CHIT/CNTs/ ITO electrode. (B) CVs of ChEt-ChOx/SiO2-CHIT/CNTs/ITO bioelectrode as a function of scan rate (10–100 mV/s). (C) Square root of scan rate for (a) SiO2-CHIT/CNTs/ITO electrode; (b) ChEt-ChOx/SiO2-CHIT/CNTs/ITO bioelectrode and difference between the cathodic and anodic peak shifts. (D) Nyquist plot of (a) CHIT/ITO electrode, (b) SiO2-CHIT/CNTs/ITO electrode and (c) ChEt-ChOx/SiO2-CHIT/ CNTs/ITO bioelectrode. (E) DPV of (a) CHIT/ITO electrode, (b) SiO2-CHIT/ CNTs/ITO electrode and (c) ChEt-ChOx/SiO2-CHIT/CNTs/ITO bioelectrode in PBS containing 5 mM Fe[(CN)6]3−/4− at potential height 0.4995 V, potential period 0.07 ms and interval period 0.14 ms. Reproduced with permission from P.R. Solanki, A. Kaushik, A.A. Ansari, A. Tiwari and B.D. Malhotra, Sensors and Actuators B, 2009, 137, 727. ©2009, Elsevier

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Smart Chitosan Matrices for Application to Cholesterol Biosensors Figure 8.14D demonstrates the Faradaic impedance spectra, presented as Nyquist plots, obtained from real (Z′) and imaginary (–Z′) in the frequency range 0.01–105 Hz for the CHIT/ITO electrode, SiO2-CHIT/CNTs/ITO bionanocomposite and ChEtChOx/SiO2-CHIT/CNTs/ITO in phosphate buffer saline (pH 7.0, 0.9% NaCl) containing 5 mM [Fe(CN)6]3−/4−, which yields information about electrical properties at desired interfaces. The values of electron-transfer resistance (RCT) derived from the diameter of the semicircle of impedance spectra are obtained as 4.59 × 103 Ω for CHIT/ITO electrode (curve a), 3.35 × 103 Ω (curve b) for SiO2-CHIT/CNTs/ ITO electrode and 7.15 × 103 Ω for ChEt-ChOx/SiO2-CHIT/CNTs bioelectrode, respectively. The semicircle of CHIT/ITO (curve a) exhibits charge transfer phenomena between the electrode and the medium. Compared to the CHIT/ITO electrode, the charge-transfer resistance (RCT) value obtained for the SiO2-CHIT/CNTs/ITO electrode (curve b) decreases resulting in enhanced electron transfer or conductive pathway towards electrode. This suggests that the presence of both SiO2 and CNTs enhance ionic transport in CHIT, resulting in the formation of complex and improved charge transfer. It can be seen that RCT increases after immobilisation of ChEt and ChOx onto SiO2-CHIT/CNTs/ITO bionanocomposite (curve C) due to the hindrance provided by macromolecular coniguration of ChEt and ChOx to electron transport between electrode and redox mediator indicating immobilisation of ChEt and ChOx onto the SiO2-CHIT/CNTs/ITO surface. The value of RCT is dependent on the electrochemical reaction time constant, τ (τ = ½πfmax = Rp·Cdl, where fmax is the frequency at which maximum Z″ is obtained, Rp is the polarisation resistance and Cdl is the double layer capacitance). DPV studies have been carried out on SiO2-CHIT/CNTs/ITO electrode (Figure 8.14E). DPV experiments have been conducted in phosphate buffer saline (50 mM, pH 7.0) containing 5 mM [Fe(CN)6]3−/4− in the range –0.1 to 0.6 V. The DPV measurements have been carried out at 0.49 V, potential period 0.07 ms and interval period 0.14 ms. The value of maximum response current obtained as 7.58 × 10–5 A for CHIT (curve a) increases to 8.19 × 10–5 A on incorporation of SiO2 particles and CNTs into CHIT. This suggests that the conducting nature of CNTs results in increased ionic transport in CHIT enhancing electron transfer towards the electrode. The magnitude of current decreases to 5.30 × 10–5 A for ChEt-ChOx/SiO2-CHIT/ CNTs/ITO bioelectrode indicating a slow redox process at the bionanocomposite due to insulating characteristics of ChEt and ChOx revealing immobilisation of ChEt-ChOx/SiO2-CHIT/CNTs/ITO bionanocomposite. The electroactive molecule H2O2 results from the enzymatic reaction between the cholesterol oxidase and the cholesterol (Figure 8.15).

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas

Figure 8.15 Liberation of electrically active H2O2 as a sensing element resulting from the reaction of cholesterol and ChOx. Reproduced with permission from A. Tiwari and S. Gong, Electroanalysis, 2008, 20, 2119. ©2008, Wiley

In the biochemical reaction, the positive charge on the SiO2-CHIT/CNTs/ITO bionanocomposite accepts electrons generated during re-oxidation of ChEt and ChOx prior to the evolution of oxygen resulting in enhanced current response of the SiO2-CHIT/CNTs/ITO bionanocomposite matrices. The enhancement in peak current suggests that this bionanocomposite provides a favourable microenvironment to the enzyme wherein CNTs provide enhanced electron transfer to the electrode. Direct acceptance of electrons by the matrix is attributed to enhanced charge transport in the SiO2-CHIT/CNTs/ITO ilm due to electrons hopping via conductive CNTs that mediate electron transfer via the bionanocomposite in the presence of [Fe(CN)6]3−/4− as mediator. The value of the enzyme-substrate kinetics parameter (Michaelis–Menten constant, Km) estimated using the Lineweaver–Burk plot reveals the affinity of the enzyme for the desired analyte. It is noted that Km is dependent both on the matrix and on the method of immobilisation of enzymes that often results in conformational changes resulting in different values of Km. Besides this; the value of Km for the bound enzyme can be lower or higher than that of purified enzyme. In this study the value of Km is obtained for the ChEt-ChOx/SiO2-CHIT/CNTs/ITO bioelectrode as 3.4 mg/dL (0.052 mM), which is smaller than the reported value [84]. The lower Km value indicates a high afinity for cholesterol oleate attributed to the immobilisation of ChOx and ChEt onto SiO2-CHIT/CNTs/ITO bionanocomposite for faster biochemical reaction. The value of the sensitivity of ChEt-ChOx/SiO2-CHIT/CNTs/ITO bioelectrode estimated

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Smart Chitosan Matrices for Application to Cholesterol Biosensors from the slope of the curve has been found to be 3.8 μA/mM. The values of standard deviation and correlation coeficient obtained from the linear regression analysis for the bioelectrode have been found to be 1.23 μA and 0.994, respectively. Hence, the CHIT-SiO2-CNTs bionanocomposite matrices provided a longer shelf life, higher selectivity and shorter response time to immobilised enzymes.

8.6.4 Photometric Response The rate of enzyme reactions is usually measured with enzyme assays because enzymes do not catalyse reactions without their consumption. The enzyme assays principally track the changes in the concentration of either substrates or products to measure the rate of reaction. There are many methods of measurement, among them spectrophotometric assays observe changes in the absorbance of light between products and reactants. A photometric study was performed to calculate the apparent enzyme activity of the enzymes. The apparent enzyme activity (enzaapp) was calculated using the equation enzaapp = AV/ εts, where A is the difference in absorbance before and after incubation, V is the total volume of the solution, ε is the millimolar extinction coeficient, t is the reaction time and s is the surface area of the electrode. The cholesterol oleate concentration can be measured by measuring the change in the intensity of colour based on the following reaction (Scheme 8.5):

Scheme 8.5 Reaction steps for the photometric detection of cholesterol

The value of the apparent Michaelis–Menten constant (Km) has been estimated using a Lineweaver–Burk plot, i.e. a graph of the inverse of absorption and the inverse of cholesterol oleate, and has been found to be 0.14 mM, revealing high afinity of ChEt-ChOx with cholesterol oleate in the presence of HRP. However, a higher Km value in comparison to that obtained using electrochemical response (i.e. 0.052 mM) may be due to the presence of HRP [77].

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8.7 Conclusion and Future Perspectives The combination of functional properties like thermal, electric, conducting, optical and biological with properties of CHIT has led to the development of a wide range of biofunctional materials that could produce an overabundance of biological compatible strategies for the development of eco-friendly technology. Engineering of CHIT as stimuli-responsive advanced biomaterials as biomimetic materials, hybridtype composite materials, molecular device materials, functionalised polymers, supermolecular systems, information- and energy-transfer materials, environmentally friendly materials, etc. at various levels has established its outstanding applications in a wider mode. Smart materials based on CHIT are prepared by graft copolymerisation, oxidative copolymerisation, electrochemical and blending with polymer and/or metals, and would be capable of giving fascinating direction such as cholesterol for modern machinery.

References 1.

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9

Biopolymeric Scaffolds for Tissue Engineering

Ashok Kumar and Anuj Tripathi

9.1 Introduction A paradigm shift is taking place in regenerative medicine from using synthetic grafts and tissue transplants to a tissue engineering approach that uses porous polymeric degradable scaffolds integrated with active cells and biological molecules to restore tissue functions and regenerate damaged tissues or organs. Tissue engineering is one of the most important branches of biomedical engineering, which is an advanced area of chemical engineering as suggested by Peppas and Langer [1]. This concept was accepted by many researchers after realising that living cells are able to exchange signals with their neighbouring cells when placed close to each other. Eventually, they form analogous structures that resemble the native structures formed by the same cells in a living body [2,3]. Various disciplines of biomedical science such as cell and molecular biology, materials science, bioreactor technology and clinical operations are required for achieving success in tissue regeneration [4]. It requires a balanced growth of cells in culture with bioactive scaffold to support, enhance and direct the cell growth. This new prototype uses culturing of functional tissue cells on biomimetic scaffolds that temporarily stabilise mass transport functions with substantial mechanical integrity during application to aid cell functions and biological delivery followed by tissue regeneration [5]. Tissue regeneration requires a well-organised integration of various cellular and molecular events such as cell adherence, proliferation, migration and differentiation, which eventually lead to a re-forging of functional tissue in a three-dimensional (3-D) architecture. To fabricate a complex information-coded biomimetic scaffold that can enhance cell behaviours and properties is still a central dilemma. In earlier times, less information was available about the use of tissue engineering porous materials, which were fabricated using synthetic and natural substances but not with precise porous architecture, mechanical properties and bioactivity [5,6]. Being an important component in tissue engineering, polymeric matrix interaction and its consequences on cell behaviour are interesting but still not well established. The design of optimum and well-characterised scaffolding materials for speciic tissue engineering applications is currently a major challenge

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas in material science. In contrast, polymeric scaffolds are a key component in tissue engineering applications and their modulation in functions holds great therapeutic promise [7,8]. Since the emergence of tissue engineering research, polymeric scaffolds have been considered as an important component that provides support material for cell growth and comprises an essential part in tissue regeneration. Recent advances in the fabrication of scaffolds have made it possible to create biomimetic scaffolds using biopolymers that provide well-deined cell interactive properties with controlled architecture [5]. In this chapter, we review the use of biopolymers to construct tissue engineering scaffolds and also describe their speciic properties to achieve artiicial tissue regeneration. In vitro and in vivo studies on the use of biopolymers and their biological responses such as immune reactions is also discussed.

9.2 Biopolymers One of the most exciting and rewarding areas of advanced materials science research is the application of biomaterials to healthcare and especially to reconstructive surgery. At a time when technological research should ideally contribute both to wealth creation and to improvements to the quality of life, it is obvious that the developments that lead to the applications of advanced materials and devices either inside or outside the body in order to treat disease and injury have a very high priority. With the advent of new sciences and technologies, there has been a need to develop new polymeric materials and techniques to put the novel knowledge into application. The variety of natural chemical structures, together with the precise control of their molecular architecture and morphology, rationalise the numerous uses of biopolymers (natural polymers) as biomaterials in high technological and biological applications. Biopolymers are the material evolution in reparative medicine. ‘Biopolymers are the degradable class of polymers which are the part of or produced by living organisms.’ Polysaccharides like cellulose, starch and chitin, proteins like collagen and its hydrolysed form gelatin and nucleotides like deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are all examples of biopolymers and the building blocks of these three major classes are sugars, amino acids and nucleotides, respectively. These polymers are attaining importance with time and with increasing environmental awareness. Primarily, biopolymers that are safe and obtained by processing of monomers or polymers found in nature according to good manufacturing practices (GMP) and applicable regulations are used for various technical applications such as in food, pharmaceuticals, cosmetics, animal feed and other industrial uses [9]. There is great demand for biopolymers to assist or replace organ functions and to improve patients’ quality of life. Many researchers have suggested bottom-up design of biomimetic assemblies and pointed out that such design requires development

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Biopolymeric Scaffolds for Tissue Engineering of functional molecular building blocks that arrange to form biologically active components [5]. To achieve this goal one must be able to fabricate molecular pieces in a controlled manner and anticipate their ability to self-assemble so as to form macromolecular aggregates of the required form. Researchers are aiming to create a tool box of biomimetic molecules, which will self-assemble with biological precision. Because of their inherited properties, biopolymers are of great interest as a biomimetic component for tissue engineering application but there are also some concerns, for example biopolymers are active components that may provoke some immune response.

9.3 Types of Biopolymers

9.3.1 Polysaccharides Complex carbohydrate moieties of mono- or disaccharide units, repeatedly linked by glycosidic bonds, are called polysaccharides. The formation of polysaccharides is a result of intermolecular dehydration between ring structures of disaccharides. Commonly, these polysaccharides are heterogeneous in nature depending upon the presence of types of monosaccharide in the repeated structure of polysaccharide macromolecules, which result in distinct properties of polysaccharides. Polysaccharides are basically of two types i.e. homo-polysaccharides (the presence of similar monosaccharides in structure) and hetero-polysaccharides (the presence of two or more types of monosaccharides in a structure). Homo-polysaccharides like starch, cellulose and glycogen are made up of glucose units. Starch and glycogen are storage polysaccharides, which differ in structure by the arrangement of glucose units. Glucose is converted into starch by plants and into glycogen by animals. These storage polysaccharides can be hydrolysed into glucose inside the body when an energy source is required. Starch is not generally stable when constructed as a scaffold and is thus commonly mixed with thermoplastic polymers such as cellulose acetate or ethylene vinyl alcohol, which provide easy processing, make them less brittle and have better resistance for thermo-mechanical degradation [10]. However, cellulose, chitin, glycosaminoglycan, alginate and agarose are structural polysaccharides, which provide structural integrity to the living organism. The orientation of the sugar units (β-D-glucose) in cellulose is little different whereas other sugar molecules are ‘upside-down’. Cellulose is the primary component of all plants and provides structural strength rather than serving as a glucose storage component. For example, cotton and paper are processed forms of plant component that is almost pure cellulose. Cellulose is a non-reducing sugar and contains β-1,4 linkages. Humans and some other animals do not have an enzyme to break down these beta linkages, 235

Biotechnology in Biopolymers Developments, Applications & Challenging Areas but it can be hydrolysed to form glucose. The controlled degradation of this polymer is possible due to the unavailability of a speciic enzyme required for digestion inside the living organism. These features make it a good scaffolding material. On the other hand, chitin, which is derived from sea crustaceans, mainly shrimps and crab shell, is one of the most abundant polymers found naturally. It is a long-chain polymer composed of N-acetylglucosamine units (glucose derivative), which are covalently cross-linked by β-1,4 linkages. It is a well-known biodegradable polymer that can be degraded by chitinase enzyme produced in some plants and mainly secreted by microorganisms like bacteria and fungi. It mainly serves as an exoskeleton in arthropods, which gives an idea for its use as a structural support for tissue-engineering applications. Chitosan, a deacetylated form of chitin, is a soluble form of the polymer that has shown signiicant commercial utility. Chitosan is composed of randomly arranged β-1,4-linked glucosamine and N-acetylglucosamine units (Figure 9.1).

Figure 9.1 Chemical structure of chitosan

Glycosaminoglycan (GAG) is an unbranched polysaccharide that can structurally resemble a bottle-brush-like morphology. It is made up of repeated disaccharide units of hexose (six carbon sugar) and hexosamine (six carbon sugar containing nitrogen). These polysaccharides create highly hydrophilic regions (density of sugar molecules and the net negative charges attract cations such as Na+ resulting in attraction of water molecules) that aid luid absorption and inluence the mechanical properties of connective tissue. It is hypothesised that in cartilage, the GAGs interact with collagen (proteoglycans) to bind water molecules. This reduces tissue permeability and increases resistance to pressure i.e. stiffness (water is nearly incompressible), which protects the solid state of cartilage. Examples of GAGs include hyaluronic acid, chondroitin sulfate and heparin, which are commonly used for biopolymeric scaffold preparations.

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Biopolymeric Scaffolds for Tissue Engineering Alginate is a linear heteropolysaccharide obtained from the cell walls of brown algae and is composed of D-mannuronic acid and L-guluronic acid. Chemically, it is a linear copolymer with homopolymeric blocks of (1-4)-linked β-D-mannuronate (M) and its C-5 epimer α-L-guluronate (G) residues, respectively (Figure 9.2). These units can covalently link together in different sequences or blocks. The polysaccharide contains block copolymer regions rich in mannuronic or guluronic acid as well as random copolymer regions of the two sugars. The average molecular weights of alginate chains range from 200 to 500 kDa [11]. Due to the presence of the carboxyl groups along the polymer chain, alginates can form gels in the presence of divalent ions such as calcium ions. The soft structure and high water absorbency of alginate gels combined with the haemostatic potential make them an attractive candidate in a broad range of biotechnology and biomedical ields. They have also shown good candidature for wound dressing [12] and several alginate-based wound dressings are commercially available under trade names such as AlgiDERM, Algisite, Hyperion and Kaltostat.

Figure 9.2 Structural coniguration of alginate polymer

Agarose is a modiied form of the agar used for bacterial cell culture and is isolated and puriied from certain Asian seaweeds. Typically, it is a long-chain polysaccharide; 1-4 and 1-3 alternate repeated unit of 1-4-linked 3,6-anhydro-α-L-galactopyranose and 1,3-linked β-D-galactopyranose (Figure 9.3). This arrangement allows two chains to join together and adopt a left-handed (LH) double helix structure. The two chains wrap together so tightly that gaps are closed and water is trapped inside the helix. The two chains have four ends and these remain as random chains that are able to link up with random coil ends of other helices so giving extensive cross-linking and form water containing complex 3-D structure.

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas

Figure 9.3 Chemical structure of agarose

9.3.2 Polypeptides More than 10 amino acids linked by peptide bonds but not translated into their secondary structure are referred to as polypeptides, and one or more chains of polypeptides combines to form protein. Amino acids are the basic building blocks of several macromolecules such as proteins, enzymes, hormones and body tissues. Folding of proteins (the most complicated macromolecules in the human body) in a particular fashion gives speciic functions, for example they can act as enzymes, serve to provide structural stability to connective tissues and regulate metabolism. Protein macromolecules are broken down into small peptides and amino acids by proteases, enzymes present in the body. Deiciency of dietary protein may prevent the production of an adequate amount of peptide hormones as well as structural proteins to maintain normal body functions. In various physiological processes, individual amino acids serve as neurotransmitters, while several proteins regulate gene expression, catalyse various chemical reactions, regulate immunological reactions and also form the major constituents of muscle as well as structural elements of cells in the body. Insuficient quality and quantity of proteins in the body apparently cause symptoms such as diabetes, fatigue, obesity, blood pressure problems, infection susceptibility, sexual dysfunction and loss of bone mass leading to osteoporosis. Many body parts and structures are made of different types of proteins such as hair and nails, which are made of keratins. Protein is also a major component of animal claws, feathers, scales, horns and hooves. One of the most common proteins in the body is collagen, which constitutes approximately 20–30% of all body proteins. It serves structural and mechanical functions in the body and greatly contributes to the tissue responsible for the above functions such as tendons, ligaments, tooth enamel and bones. Gelatin (Figure 9.4) is a denatured form of collagen produced by boiling collagen for a long time until it becomes water soluble and gummy. Primarily, collagen, gelatin, silk and ibrin are attractive protein members for biomimetic scaffold development.

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Biopolymeric Scaffolds for Tissue Engineering

Figure 9.4 Chemical structure of gelatin

9.3.3 Polynucleotides Polynucleotides are linear biopolymers that consist of a large number of nucleotide monomers (more than 13) covalently cross-linked by phosphodiester bonds between the C3′ atom of one nucleotide and the C5′ atom of the other nucleotide such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). The structure of DNA consists of two spiral chains of polynucleotides while RNA is a single chain structure. Polynucleotides are naturally present in all living organisms and show a variety of functions. DNA shows potential to be structurally engineered for the fabrication of nanoscale devices [13]. The double helical structure of DNA usually suited for threedimensional design could make it possible to fabricate 3-D scaffolds. In a recent study, a structurally tunable scaffold has been fabricated using DNA and further examined for good cell growth and migration [14]. These indings attract the use of polynucleotides as 3-D structures and it will expectedly lead to new insights about their functions as bioactive scaffolds for tissue regeneration. Polynucleotides have been suggested as the next frontier for processing of informative scaffolds that may lie at the interface of nanotechnology and biotechnology. Self-assembly of molecules is a natural process in which one molecule attracts another and forms small structures. The attraction of DNA molecules can be programmed to assemble in a desired format within a deined system. It is possible to design different sizes of DNA molecules in the laboratory that can be programmed by a deined code for speciic functions. The approach of highly selective attachment of nanoparticles on DNA scaffolds offers the design of electronic circuits at the nanoscale level that could be programmable [15]. Such a type of electrically active structure can resemble the electrically active tissue part of the brain such as neural cells. These polynucleotide properties potentially open new ways to design informational as well as electrically active scaffolds.

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9.3.4 Polyhydroxyalkanoates Polyhydroxyalkanoates (PHA) are the true biodegradable polyesters, which are produced by fermentation of sugars or lipids in various microorganisms. The intracellular accumulation of PHA occurs under unfavourable growth conditions such as limitations of oxygen, nitrogen, phosphorus or magnesium in the abundant supply of carbon source [16–18], which is used by microbes as a carbon/energy storage [19]. PHA and its homopolymers (Figure 9.5) can be either thermoplastic or elastomeric with melting points ranging from 40 to 180 °C, and are known as bioplastics. Containing a number of carbon atoms in monomer units, bacterial PHA can be classiied into two main groups: short-chain-length (SCL) PHA, comprising 3 to 5 carbon atoms, and medium-chain-length (MCL) PHA, comprising 6 to 14 carbon atoms [20]. MCL PHA has been suggested to be used as a biodegradable rubber and has potential applications for medical and pharmaceutical purposes. Poly(3hydroxybutyrate) (PH3B) homopolymer is the most broadly studied biopolyester in the class of PHA. The properties that propelled PHA applications in biomedical science are biodegradability, non-toxicity, thermo-processibility and adaptability to suit the requirements of many applications [21]. PHA has been widely employed in biomedical devices such as adhesion barriers, surgical mesh, tendon repair devices, articular cartilage repair devices, cardiovascular patches, skin substitute sutures, wound dressings, meniscus repair devices, rivets, bone plates and bone plating systems, orthopaedic pins, bone marrow scaffolds, tacks, staples, screws, slings, stents, bone graft substitutes, bone dowels, nerve guide devices, atrial septal defect repair devices, pericardial patches, vein valves, meniscus regeneration devices, ligament and tendon grafts, ocular cell implants, etc. [22].

Figure 9.5 Structure of polyhydroxyalkanoate (PHA) homopolymers: poly(3-hydroxybutyrate) (PH3B), polyhydroxyvalerate (PHV) and poly(3hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) 240

Biopolymeric Scaffolds for Tissue Engineering

9.4 Chemistry of Biopolymers Natural macromolecules like proteins and carbohydrates are a very diverse and complex class of polymeric materials. These materials have evolved to execute very speciic structural, biochemical and mechanical functions. Capturing such properties in the development of synthetic polymers has been well established. However, we are still far from imitating the natural biomaterials that possess the advantages inherently. For example, within the protein and polypeptides structure, amino acid sequences are specially arranged and have multiple functional groups with predisposed 3-D structures and appropriate molecular weights. Moreover, carbohydrates are comparatively simple in structure and also present various functional groups and show high bioactivities with intricate physico-chemical properties. Both corporate and academic researchers have recognised the advantage of using natural extracellular matrix (ECM) as biomaterials for device development. In general, manufacturing of a typical biomaterial for biomedical application is synthesised using a puriied form of a group of proteins such as collagen, elastin and even may also contain a polysaccharide. The body is able to integrate the demandable components required for growth, maintenance and repair of tissue more effectively and eficiently compared to the attempts that are made in a laboratory. So, the biomedical materials designed from natural materials must be designed around the known physico-chemical information and observed results of the material.

9.5 Polymeric Scaffolds Engineered biologically active polymeric scaffolds are gaining importance for use in tissue regeneration in a variety of clinical applications [23]. The interest in such materials continues to grow because they display signiicant versatility with a wide range of physical properties including biodegradability, compared to other biomaterials like ceramics, metals, etc. [24]. As mentioned earlier, in future it could be possible that the polymeric scaffolds from natural materials may be able to provide an alternative to the standard metallic implant technologies. The advantage of incorporating scaffold by this approach is that the scaffold matrix mimics the natural extracellular matrix (ECM) and provides support for cell adhesion, migration and proliferation. They also allow for differentiated function, new tissue generation and its 3-D organisation. Scaffolds potentially provide initial 3-D structural support for cells to retain in the damaged site and then its degraded components are replaced by the cell secreted natural matrix i.e. ECM. The scaffold matrix can act as a carrier for bioactive agents. Ideally, a porous scaffold favours the permeation and migration of seeded cells or iniltration of native cells from the host tissue. The surface properties of a scaffold should be biocompatible, promote cell adhesion and have substantial mechanical 241

Biotechnology in Biopolymers Developments, Applications & Challenging Areas strength to support cell in-growth. Of course, scaffolds need to be biodegradable and the degradation products should have no cytotoxicity or other undesirable effects. Various biopolymers do show good potential as biomaterials but interface suitability between the body tissue and polymeric scaffolds is still a major problem. The non-speciic protein adsorption followed by non-speciic cell adhesion and ibrous encapsulation occurs very often in polymer surfaces because of their high wetting functionality [24,25]. Successful strategies to fabricate polymeric scaffold devices are involved in providing the desired cell growth, improved device biointegration and most importantly provide biocompatiblity. The surface of currently used metallic implant materials can be modiied with cell attractive peptide Arg-Gly-Asp (RGD), which can promote signiicant cell adhesion and growth [26]. However, signiicant results have not yet been attained using polymeric modiied devices, as the surface treatments using biopolymers do not give suficient material surface coverage [24], although improved bioactivity of polymeric scaffold materials has been achieved by copolymerisation [27–30], blending [31–34] or physical treatment [35,36]. These methods can alter the bulk properties of the polymer [36] and concede only low peptide surface coverage [37,38]. One important feature of surface chemistry in vivo is that it is highly dynamic. Thus, the subsequent adsorption and availability of serum proteins on the surface, recruitment and adhesion of cells, and deposition and remodelling of ECM must all be considered in the design of a scaffold for tissue engineering. This is to the extent that initial conditions and surface chemistry of a biomaterial at all length scales are important in successful scaffold design. Most of the biological processes that can inluence the performance of the implant take place on a relatively rapid time scale just after implantation. Such a dramatically inluenced tissue regeneration process by early response of material surface chemistry may increase the rate of success of implantation. In order to tailor the biofunctional properties of a biomaterial, one can (a) add functional surface moieties to a polymeric scaffold that will encourage protein or cell adhesion, (b) add functional surface moieties to a polymeric scaffolds that will restrict non-speciic protein and cell adhesion or (c) use the balanced combination of both factors as per the desired application. The topography of the scaffold surface is also important at all three principal length scales i.e. subcellular, cellular and supracellular [39]. Many studies have shown that subcellular features can modulate cell growth and migration. The signal transmission mechanism of cell to surface is not yet well understood. In particular, the surface topographic features may affect the integration of tissue engineered construct to their surrounding tissue. Such a concept has been successfully used in biomedical metallic implantation such as the use of roughened titanium for bone implants. In order to modulate the topography of scaffolds one can (a) use a mould to direct the surface

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Biopolymeric Scaffolds for Tissue Engineering topography of biomaterial, (b) add molecules, (c) remove molecules after development or (d) use a balanced combination of all. The scaffold tailored physico-chemically by these approaches can be modular. For example, one can begin with a natural biomaterial and engineer it to design for multifunctional needs [40]. Over the years, the basic tissue engineering concept has provided an inert biocompatible material to allow revascularisation and cell invasion in engineered scaffolds followed by developed functional tissue implantation. It is therefore essential for cells to cultivate in a favourable environment, wherein the native-like environment can promote cell attachment, proliferation and differentiation and can help to conserve the differentiated phenotype. Such a type of materialistic native-like environment could promote the deposition of a new extracellular matrix throughout the entire scaffold. By virtue of scaffold strength and pore structure, it provides specialised 3-D volume in which effective cell proliferation and differentiation can occur. However, in biomaterial science, the selection of suitable polymers and designing them in a desired format is mainly a trial-and-error approach [41]. So, the selection of a polymer and design to create an ideal scaffold is a most promising step towards successful tissue regeneration. The scaffold network is formed by cross-linking of polymer chains via covalent, ionic or hydrogen bonding or through physical entanglement. The cross-linked polymer chains provide the network structure and physical integrity in the form of a 3-D matrix. The chemical structure and attribution of functional groups in the hydrogels directly affect their swelling ratios, where the gel that contains hydrophilic groups swells to a higher degree than those containing hydrophobic groups. Scaffold characteristics such as surface roughness, stiffness and porosity (curvature of pores, interconnectivity, micro- and macro porosity) inluence the cellular responses, while these properties also collectively regulate the penetration of cells, the degree of nutrient supply and metabolic waste removal. The cell-seeded scaffolds can convey the mechanical signals when subjected to a strain environment, which can increase our understanding of cell response under mechanical stimulation. In the case of in vitro bioreactor application for culturing the cells, the engineered scaffold must provide an appropriate environment, while its properties must also be suitable for in vivo environment when implanted. Factors such as pressure gradients, nutrient concentration gradients and luid velocities are different in both environments. For example, the transport of nutrients in the in vivo environment occurs by diffusion mechanism while in the in vitro it happens principally by low of luid. In order to consider a scaffold to be successfully fabricated, it is necessary that it offers a suficient nutrient low, which can enrich the cell requirement even in the core of scaffold and minimise cell necrosis. The interconnected channels and pore

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas connectivity within the scaffolds also inluence vascular invasion, delivery of cell nutrients and waste removal [42]. Several conventional techniques provide possibilities to fabricate the 3-D scaffold with random pores, which are not able to provide structural homogeneity for cell growth and tissue construction. The non-uniform microenvironments in the scaffold cause some void regions for non-uniform cell adhesion as well as insuficient nutrient concentrations, which can inhibit cellular activity and prevent the formation of homogeneous new tissue.

9.5.1 Conventional Polymeric Scaffolds In general, hydrogels are cross-linked polymeric materials that can absorb a large quantity of water without dissolving or losing physical identity [43]. The presence of hydrophilic functional groups in the polymeric backbone provides the property to absorb a high amount of water while the cross-linking between their chains makes hydrogels stable [44,45]. If such a conventional hydrogel dries by heating, no void volume (pores) will remain in its structure. However, after reswelling of the dried hydrogel in water it shows relatively higher porosity between the polymeric networks known as molecular porosity. Thus, the molecular porosity is dependent on the degree of swelling in the case of a conventional polymeric hydrogel where polymer regions are located in the range of a few nanometres. Importantly, the presence of a number of hydrophilic groups is also responsible for the degree of lexibility and strength of hydrogel. Large numbers of hydrophilic groups are able to retain higher water content and thus provide higher lexibility while the less hydrophilic properties may tend to generate a stiffer hydrogel. Hydrogels have attracted great interest and considerable progress has been made in manipulation, fabrication and their use for various bioengineering applications. Hydrogels have been widely used in wound dressing, medical and biological sensors, breast implants, drug and protein delivery system, water absorbent pads, hygiene products, enzyme and cell immobilisation, tissue engineering and cell culture applications, etc. [46]. However, early studies suggested that hydrogels are a potential candidate in many biomedical and bioengineering applications but sometimes they do not meet the required standard for more specialised applications. Moreover, most of the hydrogels show poor luid low, poor mechanical strength [47] and slow swelling kinetics due to small pore architecture [48]. These problems have driven scientists in a new direction to employ new strategies for the design of polymeric scaffolds with relatively high porous architecture and increasing characteristics for different biotechnological applications.

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Biopolymeric Scaffolds for Tissue Engineering

9.5.2 Supermacroporous Polymeric Scaffolds Supermacroporous gels refer to porous materials having a stable porous structure that remains unchanged even in the dry state [49]. Supermacroporous gels contain macroor micron-size pores or channels separated by cross-linked polymer regions, which provide adequate 3-D mechanical integrity. According to the IUPAC nomenclature, pores of width 200 kDa

[3]

Antheraea mylitta

By solubilisation with 8 M urea along with 2% SDS and β mercaptoethanol

Fractions of 70 kDa, 200 kDa and higher than 200 kDa

[17]

By solubilisation with NaOH and LiSCN

A single polypeptide of 66 kDa and is glycoprotein in nature

[25]

Antheraea mylitta

Antheraea mylitta Silk cocoon ibroin

Comments

Philosamia ricini

Philosamia ricini Antheraea assama

12.1.4.2 Spider-silk Fibroin The molecular weight of the polypeptide chain of spider silk ranges from 70 to 700 kDa depending upon the source. The most extensively studied spider silk is

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas dragline silk of Nephila clavipes. The silk proteins of Araneus diadematus and N. clavipes appear to be block copolymers, comprising large hydrophobic blocks with a highly conserved repetitive sequence of glycine and alanine. The intervening small hydrophilic blocks are very complex with bulkier side chains and charged amino acids [26,27]. The presence and arrangement of ordered hydrophobic regions and less ordered hydrophilic regions in spider silk are responsible for the impressive tensile strength of silk ibres [28]. Thus, after a clear understanding of the silk structure and its biophysics, silk-based studies in the last decade have mainly focused on the modiication of silk proteins by genetic engineering, which allows displays of new characteristics alongside the native properties.

12.2 Challenges of Natural Silks An enormous quantity of industrial silk is obtained from silkworm cocoons that protect the pupal stage of the insect metamorphose into the adult imago state, the moth [20]. This is in stark contrast to spider silk’s yield and function. The characteristic crystalline properties of silks from different arachnid species are somewhat alike. However, spider dragline silk is considerably stronger than silkworm silk and considered for its unique properties like high-performance, toughness, strength, biodegradability and lightness [1,29,30]. Silk changes its properties depending on different factors like temperature, the spinning processes and silk chemistry [31,32]. Silk is produced by specialised glands in a very speciic way but the mechanism of the attainment of metastable states inside the spinning organism’s glands is still unclear [31,33]. Silk is available in a simple solution state in nature; this technology is still lacking in the case of synthetic materials [6]. Major challenges that need to be approached for spider-silk applications are the control of silk quality and scale-up. Spider silk can not be harvested easily like silkworms’ cocoons. It is very dificult to breed and domesticate spiders in large factories as it may ultimately lead to them killing one other. On the other hand, the present method of extracting silk from silkworms is laborious and time-intensive. It involves separating the cocoons from silkmoth larva, boiling and removing the contaminating the glycoproteins and further processing to produce an implantable and cytocompatible end product [34]. In synthetic methods of silk production, the puriication processes can be avoided, and quality control and yield can effectively be maintained. To produce highperformance protein ibres of spider silk at an industrial scale, genetic engineering of silkworms seems to be a promising technique [35–38] to obtain super-strong sutures for biomedical applications and tissue engineering applications [4,39,40], as well as for the construction of bullet-proof vests stronger than Kevlar. The silk produced by

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Biotechnology of the Silk Proteins: Challenges, Approaches and Applications this process is a hybrid protein, which is a combination of silkworm silk and spider silk, produced by the transgenic silkworm engineered with spider-silk gene fused with natural silk gene. This is stronger and iner than silkworm silk, but not quite as strong as spider silk [41]. Hence, various improved properties can be obtained in the form of the hybrid protein. This type of protein produced on an industrial scale can be by genetic engineering technologies that are not present in the natural production system of the protein [12]. To increase the yield of spider silk, spider-silk genes are expressed in bacterium Escherichia coli, Salmonella, fungi, mammalian cells, insect cells and even designed to be secreted in the milk of goats [30,42]. One of the advantages of using genetic engineering technology for designing recombinant protein is that it eliminates the immune response generated by the glycoprotein sericin present in the cocoons of natural silk of silkworms [34]. The wax-coated sutures that are used to avoid immune responses are non-biodegradable. So these recombinant silks are biodegradable and can be safely implanted/deliver drugs into the body. Based on recent silk research, designs can be made for the various expected approaches in biomedical applications. Silk can be created synthetically to produce and improve the yield in economical and practical ways. To meet this expectation, the production of genetically engineered silk proteins in genetically altered organisms is anticipated.

12.3 Biotechnological Approaches for Modification for Native Silk Biopolymer Silk, the versatile biopolymer, is used not only in the textile industry but also in tissue engineering and regenerative medicine. The versatility and potential applications of silk as a biomaterial were recently rediscovered and are being investigated. To further diversify its applications, attempts are being made to improve its properties, especially biomaterial aspects. Silk proteins can be tailored to contain additional features for targeted applications. This can be achieved to a large extent by genetic engineering. A variety of recombinant silk, each tailor made for a particular application, is engineered. Synthetic gene technology can be used to control the size of protein that can allow studying the new compositions with structure–function relationships [43].

12.3.1 Recombinant Silkworm Silk The most well-known and common example of silk is silkworm silk from Bombyx mori. This silk is mostly used in the textile industries. Apart from this mulberry type of silk, there are several non-mulberry silks, which can be used just like mulberry silk

379

Biotechnology in Biopolymers Developments, Applications & Challenging Areas in textiles, medicine and bioengineering. Most of them are used in textiles, and they are not commonly used in the ield of biomaterials. Even research groups developing recombinant silkworm silk mainly use mulberry silk and the non-mulberry silks are yet to be thoroughly exploited. Mulberry silk lacks RGD sequences, which help to improve cell adhesion. RGD controls not only cell adhesion but also growth and proliferation. To overcome the lack of this RGD researchers have designed cell-adhesive recombinant silk with better cell-adhesive properties. In the ibroin light chain (L-chain) genetically tandem repeating of the Arg-Gly-Asp-Ser (RGDS) sequences are interfused to give the sequence (L-RGDS × 2 ibroin) for creating silk proteins with enhanced cell-adhesive properties [44]. To obtain recombinant silks composed of a partial collagen or ibronectin sequence, that is, [GERGDLGPQGIAGQRGVV(GER)3GAS]8GPPGPCCGGG or [TGRGDSPAS]8, respectively, the transgenic silkworms were constructed by inserting the modiied ibroin light chain genes. Transgenic silkworms are created by inserting the modiied light-chain genes of ibroin protein [45]. Recombinant human-like collagen (RHLC) is also added to ibroin to construct a hybrid biomaterial for skin tissue engineering having improved cell adhesion and proliferation properties [46]. Two silk-like proteins, as potential biomaterials with improved properties, were designed containing the sequences [TGRGDSPAGG (GAGAGS) 3AS]5 (FS5) and [TGRGDSPA-(GVPGV)2GG(GAGAGS)3AS]8 (FES8). Crystalline domain sequence (GAGAGS)n from B. mori silk ibroin is present in the irst protein and TGRGDSPA cell-adhesive sequence from ibronectin-containing RGD triplet. The other protein contains the sequence (GVPGV)n from elastin [47]. Researchers have also designed recombinant silk with improved physical and mechanical properties. To produce dragline silk in E. coli regenerated silk ibroin ibres are created containing silk-like proteins with characteristic sequences from a spider, Naphila clavipes. A small increase in the tensile strength was obtained by adding this silk-like protein to the silk ibroin [48]. By combining a polyalanine encoding region (Ala)18 (similar to the protein found in Samia cynthia ricini) and the VGAGYGAGAGYGVGAGYGAGVGYGAGAGY sequence, from silkworm B. mori, a synthetic gene encoding chimeric silk-like protein has been constructed. The chimeric protein in comparison to S. c. ricini silk demonstrated improved solubility [49]. Silk-elastin-like polymers (SELPs) are a family of genetically engineered protein block copolymers with structures comprising tandem repeat units of silk-like (GAGAGS) and elastin-like (GVGVP) peptide blocks [50,51]. It amalgamates the excellent physical and mechanical features of silkworm silk, and the chemical properties of elastin-like polypeptides [52] to form a new, enhanced biomaterial. The accuracy with

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Biotechnology of the Silk Proteins: Challenges, Approaches and Applications which SELPs can be synthesised helps the precise addition of motifs responsible for self-assembly, stimuli-sensitivity, biorecognition and biodegradation [53]. For these reasons the application of these biomaterials are used in controlled drug and gene delivery [50–57].

12.3.2 Recombinant Spider Silk The cause of superior strength and elasticity of spider silks is mainly the modular nature of their repetitive proteins, and this property can be effectively exploited to produce recombinant spider silk. The recombinant technology can be used to design and create a library of synthetic silk-like genes using a speciic protein sequence. This may produce the desired mechanical properties in the resulting ibres. Recombinant proteins comprise characteristic sequences from Anaphe silk ibroin and cell-adhesive regions from ibronectin are prepared to create biomaterials with high cell-adhesive ability [58]. Genetic constructs based on the sequence of Argiope aurantia are engineered to increase the number of GPGXX repeats. This increase leads to greater extensibility of the ibres [59]. Recombinant spider-silk proteins composed of four poly-Ala/Gly-rich cosegments and the C-terminal domain, 4RepCT, have been designed. They form macroscopic ibres resembling native spider silk [60,61]. They are the irst synthetic spider ibres, which are formed in vitro in a physiological buffer without the use of toxic additives and their application as a biomaterial has been greatly enhanced [62]. Some recombinant spidroins ibres are unable to show the extraordinary mechanical properties of the native material. Further, in order to improve the mechanical properties of 4RepCT ibres attempts are made selectively to introduce AACC mutations and allow ibre formation under physiologically redox conditions. The insertion of AACC mutations in the irst poly-Ala region enhances the stiffness and tensile strength in the miniature spidroin with no major changes in ibre formation or ibre morphology [63]. Studies have been carried out to ascertain the solubility and structure of genetically engineered spider silks [1]. Spider dragline silk has the tendency of self-assembling into microibrils, leading to precipitation and loss of the protein during the puriication process [43]. Genetically engineered silk protein derivatives containing molecular triggers depending on chemical or biochemical reactions are designed to control their assembly and solubility [64–66]. The repetitive gene structure of silk proteins is exploited to create recombinant spider-silk proteins using these consensus repeats or their variants [43]. Maintaining the highly repetitive gene sequences is dificult and often results in genetic deletions or premature termination errors during the process of protein synthesis [43]. In recent years, partial cDNA constructs of dragline silk protein have been cloned and expressed in prokaryotic E. coli [35], mammalian cell lines [30], insect-cell lines [67] and transgenic silkworm larvae [68]. Designer synthetic

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas genes based on N. clavipes spider dragline and lagelliform protein sequences have also been created and expressed in E. coli [69–75], Pichia pastoris [76] and plants [77–79].

12.4 Applications of Recombinant Silk Silkworm silks are used for various biomedical applications whereas spider silks are not very commercialised, due to the spiders’ predatory nature and the small quantities of silk available [6]. Nowadays, silk production is carried out by recombinant DNA technology methods, which provide substantial production of protein for use in various applications. The ability to introduce critical motifs leading to various profound and speciically designed effects on cell growth, differentiation and migration is one of the main advantages of using recombinant silk. This in turn permits various customised matrices for varied biomedical applications to be made [40]. This section introduces various matrices made of recombinant silk either alone or combined with other polymers as biomaterial for various biomedical and therapeutic applications.

12.4.1 Films Films have applications in skin tissue engineering, topical drug delivery and immobilisation of other bioactive molecules. Micro- and nanopatterned ilms can provide us with insights on the effects of surface characteristics on attachment, growth, proliferation and differentiation of cells. Spider-silk protein ilms have a number of potential biomedical applications related to cell growth, differentiation and proliferation. Various structural motifs such as β-sheet, β -turn, α-helix and spacers of spider-silk protein play an important role in the overall physical properties of the protein ilm [12]. Huemmerich and co-workers investigated the conformational changes in the ilms of recombinant spider silk after methanol treatment. The ilms made by spin-coating on silicon wafers were studied by circular dichroism, grazingincidence X-ray diffraction and small-angle X-ray scattering. The study reveals the occurrence of the transition of α-helix into β-sheet with a high degree of crystallinity [80]. Led by Conrad, another research group developed an elastin motif (elastin-like polypeptide) in combination with the MaSp1-derived SO1 protein fusion protein. In transgenic tobacco and potato this fusion protein was expressed and extracted by heat treatment and salt precipitation from leaves. The protein obtained was found to be biocompatible and supports the growth of human chondrocytes when cultured cell culture plates are coated with the same protein. The spherical morphology of chondrocyte is also preserved, suggesting that the spider-silk-elastin protein promotes chondrocytes attachment and viability. It prevents the dedifferentiation of chondrocytes [81]. The integrin-binding motif RGD (Arg- Gly-Asp) combined

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Biotechnology of the Silk Proteins: Challenges, Approaches and Applications with sequences derived from spider silk is processed into ilms and supported the growth and differentiation of human mesenchymal stem cells in the presence of an osteogenic medium [82]. Similar results are observed with ilms of RGD-containing blends of regenerated B. mori silk and recombinant RGD-spidroin [83]. The growth and differentiation of a murine undifferentiated osteoblastic cell line on the ilms in speciic media with calciication was observed. Recombinant silk proteins have also been used for hepatic tissue engineering. A novel biocompatible ilm prepared from recombinant human-like collagen blended with silk ibroin is used and shows enhanced liver cell growth and proliferations when they are compared to the tissue culture plates [84].

12.4.2 Scaffolds Tissue engineering uses biodegradable porous material scaffolds integrated with biological cells or molecules to regenerate and model tissue. They can be used in space illing, bioactive molecule delivery and as cell/tissue delivery agents. A lot of work has been carried out on silk scaffolds in biotechnological applications. A biocompatible 3-D scaffold using a combination of RGD-containing recombinant spider silk and polyvinyl alcohol polymers was fabricated and was found to support the growth of mouse embryonic ibroblast origin cell line [85]. In another study, the lyophilised recombinant spidroin 1 is used for the preparation of scaffolds by the salt leaching method. The 3-D scaffolds of pore size of 50–100 µm and 200–400 µm support the growth and proliferation of ibroblast 3T3 for long-term cell culture [86]. Recombinant spider-silk protein is also developed from pNSR-16/BL21 (DE3) pLysS strains by fermentation. The recombinant protein scaffolds blended with polyvinyl alcohol (1:1 w/v) were fabricated by the salt leaching method. These recombinant scaffolds are observed to be of non-toxic nature and allow good attachment and growth of NIH-3T3 cells [87].

12.4.3 Hydrogels A recombinant silk-elastin-like protein polymer, SELP-47 K hydrogel, for the delivery of therapeutics was prepared by a research group led by Ghandehar [88–92]. Human mesenchymal stem cells (hMSCs) in SELP-47 K were encapsulated and cultured for 4 weeks in chondrogenic medium with growth factor TGF β3. Histological, immunohistological, RNA and biochemical analyses and reverse transcriptase (RT)PCR show chondrogenic differentiation and up-regulation in chondrogenic genes such as aggrecan, type II and type X collagen and SOX 9 in the presence of TGF-β3. This study suggests the suitability of this recombinant protein SELP-47 K hydrogel for cell encapsulation and chondrogenesis of hMSCs for soft tissue engineering [88]. A 383

Biotechnology in Biopolymers Developments, Applications & Challenging Areas hydrogel of silk-elastin-like protein polymer (SELP) was fabricated for DNA release. The further inluence of hydrogel geometry, DNA molecular weight and conformation was analysed in addition for DNA release from recombinant silk hydrogel. The concentration of hydrogel and the release of DNA are correlated, indicating the potential application for controlled gene delivery [89]. SELP-47K hydrogels are also characterised in terms of thermal properties and are evaluated both in vitro and in vivo for their potential in controlled adenoviral gene delivery to head and neck tumours [90–92].

12.4.4 Fibres Recently, a method of production of recombinant spider-silk ibres, 4RepCT, was developed that is structurally similar to those of native dragline silks [60]. The tensile strength and elastic modulus of 4RepCT are 0.2 GPa and 7 GPa, respectively, and are comparable to mammalian bone and tendons [93,94]. Recombinant spider silk is used as ibres with modelling of silk assembly and electrospinning. The generated nanoibrils are characterised with FTIR, SEM, TEM, AFM, mechanical testing and molecular dynamic studies. The ibres exhibit a relative tensile strength of 0.1–0.15 GPa and this suggests their suitability for tissue engineering applications [95]. Recently, artiicial spider silk was produced with mechanical and structural characteristics similar to those of native spider silk from recombinant mini spidroins. It is coated with the calcium phosphate with supersaturated simulated body luid in order to improve bone repair. These recombinant ibres supported the growth and proliferation of bone-marrow-derived hMSCs [96]. In vivo studies with recombinant silk ibres were carried out in rats. The recombinant spider-silk protein (4RepCT) macroscopic ibres were implanted subcutaneously in rats for 1 week. There were not any negative systemic or local reactions, even after 1 week of implantation, suggesting excellent biocompatible properties. The 4RepCT-ibres with newly formed capillaries and ibroblasts in growth are observed after 1 week of implantation and support the formation of vascularised tissue [97].

12.4.5 Microcapsules and Microspheres Recombinant silk microcapsules and microspheres have been fabricated for the delivery of bioactive molecules [98]. Self-assembly recombinant silk microcapsules from spider Araneus diadematus are used for drug delivery. At an emulsion interface these microcapsules were prepared by self-assembly of the proteins. These capsules are also used to encapsulate small active ingredients [99]. Microspheres of the same recombinant spider silks are fabricated by methods including dialysis and micromixing. The spider-silk microspheres, due to their material strength, biocompatibility and the 384

Biotechnology of the Silk Proteins: Challenges, Approaches and Applications possibility of modiication with recombinant protein techniques, have the potential to be used for targeted drug delivery systems [100].

12.5 Limitations The commercial applications of recombinant spider silks are limited mainly because of the inability of recombinant silk to produce suficient quantities of silk proteins with an accurate molecular weight at a reasonable cost [1]. Other limitations are that the recombinant spider-silk protein (spidroin) consists of glycine and alanine. Production of these amino acids is dependent upon the metabolism of fast-growing organisms such as yeast and bacteria [81]. Bacterial production of these amino acids creates genetic instability due to recombination. This leads to the formation of highly repetitive gene sequences encoding the repetitively composed spidroin protein. To overcome these problems, production of the recombinant proteins in transgenic plants might prove a feasible option. The production of recombinant spider silk is complicated by several factors, such as the highly repetitive nature of the genes, the length of the construct, speciic codon usage of the spider and high GC (guanine and cytidine) content [12]. These factors may cause the aforementioned secondary structure constraints and may lead to gene recombination and translation pause. This creates the requirement of a tRNA pool specially designed for the glycine alanine rich encoding mRNA transcripts [79,30]. Several research groups are able to develop successful production of silk proteins (ranging from 60 to 140 kDa) in mammalian cell lines. Although the recombinant ibre exhibits the same toughness and modulus as native silk, it shows lower tenacity than that of native silk. Moreover, production of recombinant silks from animals is expensive and limited in the amount produced. Recombinant protein obtained from E. coli is heterogeneous in size due to the premature termination of translation. Expression of a similar gene construct in Pischia pastoris results in a higher level of accumulation of the protein without any evidence of truncated protein synthesis [101]. In order to overcome all these problems and limitations, plants are chosen over any other means for the production of recombinant spider silk.

12.6 Conclusion and Future Prospects Considering the needs of different silks for various applications the inluence of genetic variation, and thus protein chemistry, on the material properties are to be further investigated. A proper understanding of the material is important and to get functional materials from silks the eficient use of synthetic genes and incorporating 385

Biotechnology in Biopolymers Developments, Applications & Challenging Areas the desired sequences in the various silks for their assembly are key factors. Achieving this goal will bring new light to the ield of recombinant silk protein systems in novel applications. Silk-protein-based biomaterials are already applied in several tissue-engineering studies. The biomaterial can be further advanced by recombinant technology. Using this technology the natural properties of silk can be enhanced and speciically modiied for each different application. Thus, recombinant silk, after achieving anticipated scientiic and engineering progress, has the potential to have an impact not only in materials science and engineering but also in chemistry and biomedicine ields.

Acknowledgements The authors wish to acknowledge inancial support from Indo-Australia Biotechnology Fund, Department of Biotechnology, Indo-Russia Biotechnology Programme, Department of Science and Technology, Government of India and Indo-US Science and Technology Forum, New Delhi.

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13

Plasticulture for Agriculture and Food Security

Ravi B. Srivastava and AshishYadav

13.1 Introduction The vast majority of polymer and polymeric composite products that are presently used in agricultural applications are made from petroleum-based synthetic polymers that do not degrade in a landill or in a compost-like environment. Therefore, the disposal of these products poses a serious environmental problem. Synthetic polymers i.e. polyethylene (Marlex), polytetraluroethylene (Telon), polyvinyl chloride (PVC), polycarbonate (Lexan) and phenol formaldehyde resin (Bakelite) are macromolecules and are non-biodegradable and so when they are dumped on soil and water bodies they remain there for many years and thus cause pollution to terrestrial and aquatic lora and fauna. They also pose a threat to irrigation channels and drainage systems. In many cases, cattle swallow polythene bags or other polymeric material, blocking their digestive tracts causing untimely deaths. When they are disposed of in agricultural ields, the fertility/physical texture of the soil deteriorates, disturbing soil aeration and root penetration and also disturbing the optimum nutrient intake from soil. An environmentally conscious alternative is to design/synthesise polymers that are biodegradable. Material use and the inal mode of biodegradation are dependent on the composition and processing method employed [1]. Renewable resource feedstocks include microbially grown polymers and those extracted from starch. It is possible to reinforce such materials with natural ibres, from plants such as lax, jute, hemp and other cellulose sources [2]. Fully biodegradable synthetic polymers, such as polylactic acid (PLA), polycaprolactone (PCL) and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), have been commercially available since 1990. However, these synthetic polymers are usually more expensive than petroleum-based polymers and also have slow degradability. Blending starch with these degradable synthetic polymers has recently become a focus of researchers. Advanced research results obtained by many scientists have established that the blending of starch with polyvinyl alcohol and ethylene vinyl alcohol can be used for production of degradable ilms, and that biodegradable polymer substitutes

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas can be produced by the blending of starch with degradable PHBV. The preparation of new degradable polymers by the blending of starch with degradable polycaprolactone (PCL) was the base for commercial trials. Unfortunately the mechanical strength properties of these blends were very limited. Fomin [3] reported that the end of the twentieth century saw the worldwide production of synthetic polymers reaching 130 million tonnes/year, while the demand for biodegradable polymers is reported to be growing by 30% each year [4].

13.2 Biodegradability of Polymers Biodegradable polymers disposed of in bioactive environments degrade by the enzymatic action of microorganisms such as bacteria, fungi and algae. The polymer chains may also be broken down by non-enzymatic processes such as chemical hydrolysis. The American Society for Testing of Materials (ASTM) and the International Standards Organization (ISO) deine degradable polymers as those that undergo a signiicant change in chemical structure under speciic environmental conditions. These changes result in a loss of physical and mechanical properties, as measured by standard methods. Polymers may also be designated as photodegradable, oxidatively degradable, hydrolytically degradable or those that may be composted. The option of composting based on the biodegradable nature of polymers is beginning to receive some attention.

13.3 Biotechnological Interventions The biodegradation of water-insoluble polymers is a heterogeneous reaction. The size, shape, surface area and surface texture greatly affect the rate of degradation. Polymer with varied morphological properties in terms of crystallinity and orientation can be expected to degrade at different rates under the same environmental conditions [5]. Most biodegradable synthetic polymers and biopolymers contain hydrolysable groups along with the main chains. For a polymer to be degradable, a main-chain hydrolysable group must be present and accessible. The presence of a hydrolysable or oxidisable linkage in the polymer main chain, suitable substituents, the correct stereoconiguration, the balance of hydrophobicity and hydrophilicity and conformation lexibility contribute to the biodegradation of hydrolysable polymers, which proceed in a diffuse manner with the amorphous regions degrading prior to the degradation of the crystalline and cross-linked regions. Cross-linked polymers containing one or more hydrolysable functional groups (amides, enamine, enolketons, esters, urea and urethane) have been synthesised and found to biodegrade at various rates. Possibilities have been explored for triggering degradation through

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Plasticulture for Agriculture and Food Security biological cycling using biological agents like microorganisms or released enzymes [6]. However, only a few studies have examined the capability of microorganisms to degrade polymers and polymeric composite materials [7,8]. Polymeric composites, although they are considered advanced engineering materials, they are also observed to be susceptible to environmental and microbiologically inluenced degradation [7].

Figure 13.1 SEM microphotograph of carbon ibre reinforced composite showing microbially assisted degradation in the presence of microbial cultures

Fibres in composites may promote fungal colonisation by serving as capillaries for transporting nutrients from susceptible regions or external surfaces, stimulating extensive microbiological invasion. Composites may contain a range of chemicals such as plasticisers, lame retardants, catalysts, colourants and organics in resins. These chemicals can be used as carbon and energy sources by microorganisms. In their studies on biosusceptibility on carbon ibre composites Patrisin and co-workers [9] reported differential colonisation of resin ibre interface in composites comprising epoxy and carbon ibre as constituents. Srivastava and co-workers [10] carried out scanning electron microscopic visualisation to provide evidence of active bioilm development on the material surface (Figure 13.1). Profuse growth of microbial cultures was observed on the ibre and resin matrix as well as debonding between the ibre and the resin matrix. Under the inluence of Aureobasidium pullulans, a fungal culture, rough surface areas were observed along with debonding between the ibre and the resin matrix. In the presence of sulfate reducing bacteria (SRB), bacterial colonisation on the ibre surface was noted and some deep hidden areas due to high bacterial growth were also observed. These observations suggested possible use of chemical components of composites by microorganisms as energy and carbon

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas sources favouring profuse microbial growth. A considerable amount of extracellular polymer production was also noted in the case of A. pullulans, supporting extensive colonisation. Mercedes and co-workers [11] have also observed through their scanning electron microscopic studies that bacteria were interconnected by similar extracellular polymers and were attached to the polymeric materials. In an earlier reported work, Srivastava and co-workers [7] obtained a signiicant decrease in the mechanical properties of glass ibre reinforcements in vinyl ester resin compared to epoxy resin exposed to SRB. Pankhurst and Davies [12] also observed a decrease in the tensile strength of adhesive PVS tapes exposed to two bacterial and one fungal culture. It is considered that there may be a synergistic effect amongst the bacteria and fungi present in combination cultures, which might have enhanced the ability of SRB and A. pullulans in particular to biodegrade carbon ibre composites. Studies conducted by Srivastava and co-workers [10] revealed also the susceptibility of polymeric coatings to microbial attack. Bioilm deposition was observed more on polyurethane coupons than on alkyl-coated coupons exposed to sterile mineral salt medium. They further observed that when lacquer extract was inoculated with fungal consortium, a higher fungal biomass as well as ATP content, carbohydrate content and protein content were estimated in the fungal mass growing on polyurethane lacquer extract compared to that of alkyd lacquer extract. This can be regarded as a direct indication of the nutritional relationship between lacquer and the growth of fungi. Polyurethane has been well regarded as a nutritive material to a variety of microorganisms. It was observed that polyester of polyurethane liquor might have been used as a carbon and energy source by microorganisms. Scanning electron microscopic observations of test coupons coated with alkyd and polyurethane lacquer exposed to fungal consortium suggest the possible use of coating materials as nutritive substances to microorganisms. It was also shown that loss of adhesion and blistering of the protective polymeric coatings was also due to fungal growth. Microorganisms as fungi play an important role in the biodegradation of polymeric materials by secreting enzymes that help in breaking the bonds of the polymers into monomers. Upreti and Srivastava [8] observed the potential of Aspergillus foetidus for polymer degradation under laboratory conditions using polyurethane as a test material. Polyurethanes are a class of polymeric materials that are widely used as a raw material in various industries developing products for agricultural applications. The fungal culture, A. foetidus, was isolated from a polymeric sheet under degradation due to fungal colonisation. The sheet was used as a greenhouse covering for the protected cultivation of plants. The fungus obtained from degraded polymeric sheet was cultured, and after puriication it was identiied as A. foetidus (Figure 13.2).

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Figure 13.2 SEM micrographs showing fungal degradation of greenhouse polymeric sheet (a) abiotic control (b) growth of A. foetidus (c) degraded polymeric sheet

Little and co-workers [13] reported that microorganisms grow on material surfaces and produce a viscoelastic layer or bioilm. The environment at the bioilm–material interface, in terms of pH, dissolved oxygen, organic and inorganic species, is readily different from the bulk medium to which the material is exposed. Srivastava and coworkers [10] observed a signiicant biogrowth of A. foetidus on the material surface during visual examination, covering 25–50% of the area of test samples. On biogrowth characterisation, 32.88 × 104 colonies/cm2 were recorded on the material surface. ATP (17.5 × 10–9 g/micron) and carbohydrates (1802.00 μg/ml) were estimated in the bioilm formed on the material surface. The 0.22% loss in weight of test coupons exposed to A. foetidus was also recorded compared to the abiotic control. Scanning electron micrograph of the abiotic control sample showed smooth surface morphology, although some darkened areas and white dots were seen. The polyurethane samples exposed to A. foetidus showed profuse biogrowth. Fungal mycelium and spores were also present. After washing the biogrowth, whitened areas with ridges and furrows were seen (Figure 13.3). The rough surface of the coupons showed its degradation due to A. foetidus, which induced signiicant reduction in the mechanical properties such as tensile strength, tensile modulus and elongation at break percentage of test polyurethane coupon. Losses of 11.15% in tensile strength, 1.87% in tensile modulus and 9.15% in elongation at break were noted compared to the abiotic control (Table 13.1), whereas Aspergillus flavus produced a 43% reduction in polyester polyurethane substrate and 40% in the case of polythene ilm materials [14].

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Figure 13.3 SEM micrographs of polyurethane coupons exposed to A. foetidus, (a) control, (b) showing biogrowth, (c) after washing surface biogrowth

Table 13.1 Alterations in mechanical properties of polyurethane coupons exposed to A. foetidus (Mean ± SE) Condition

Tensile strength (kg/cm2)

Tensile modulus (kg/cm2)

Elongation at break %

Positive control

284.10 ± 4.32a

32.35 ± 0.45a

922.60 ± 13.33a

Abiotic control

281.30 ± 4.32a

31.55 ± 0.67a

889.40 ± 9.05a

Aspregillus foetidus

249.94 ± 6.07b

30.96 ± 0.75a

808.00 ± 2.28b

* Mean values with different superscripts in a column differ signiicantly at (P < 0.05)

In recent years, the development of some of the polymers and polymeric blends has shown remarkably high susceptibility to microbial attack [15]. Looking at the biodegradability of such blends, Srivastava and co-workers [16] have prepared a polyethylene–glucose blend (10%) to study the biodegradability of this blend by two common fungal agents i.e., Aspergillus niger (Figure 13.4) and Paecilomyces varioti with a view to selecting potential biodegrading fungal species and acclimatising on a polymer augmented broth. They observed through a scanning electron microscope that the surface of the polymer/glucose blend, which had been incubated in a sterile medium for three weeks (control), was rather smooth, whereas the surface of the test blend exposed to test media inoculated with fungus was very rough. Different parts of the ilm were whitened and severely degraded by both the fungal species. Besides, a number of holes on the surface of the ilm incubated with P. varioti were observed. Microbial degradation of polymeric blends by different bacterial and fungal strains

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Plasticulture for Agriculture and Food Security has been reported from all over the world. Foust and co-workers [17] reported that Trichoderma virde showed 100% surface area growth on LDPE blended with 8% (wt./ wt.) cellulose in 90 days. Biodegradation of maleated linear LDPE and starch blends by mixed fungal cultures (including A. niger) after 28 days have also been reported by Chandra and Rustugi [18]. Grandils and co-workers [19] described the kinetics of the in vitro biodegradation of poly(DL-lactide). The studies carried out by Srivastava and co-workers [7] provided evidence that both the fungal isolates A. niger and P. varioti possess potential for biodegradation of the test polymeric blend indicating that they are capable of changing their metabolic reactions to suit the nutritional amendments and such studies will go a long way towards helping in developing useful microbial consortiums assisting in inducing biodegradability to the polymeric formulation in use for many packaging, food and agricultural-related applications.

Figure 13.4 SEM micrographs showing growth of Aspergillus niger on LDPE/ glucose blend

13.4 Biodegradable Polymers of Bio-origin Microbial biopolymer feedstocks produce biological polymers through microbial fermentation. The products are naturally degradable, environmentally friendly substitutes for synthetic polymers [20]. A number of bacteria accumulate

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas polyhydroxyalkanoates (PHA) as intracellular carbon reserves in the cytoplasm as discrete granule inclusions when nutrient deiciencies occur. The biopolymers, which are microbially produced polyesters, have the same thermoplastic and waterresistant qualities as synthetic polymers [1]. The PHA granules thus produced are usually spherical in shape, varying in size and molecular weight (average generally between 105 and 106 Da) with different microorganisms. Many microorganisms produced large quantities of PHA varying from 30 to 80% of their cellular dry weight, which appears as a refractile material when viewed under a microscope. Although more than 300 different microorganisms are involved, generally all prokaryotes are known to synthesise PHA in nature. However, only a few bacteria including Ralstonia eutropha, Alcaligenes latus, Azotobacter vinelandii, Chromobacterium violaceum, methylotrophs and pseudomonads can produce PHA to an extent that meets commercial interest [21]. PHA can be biodegraded to water and CO2 (or methane in an anaerobic environment) by a large variety of ubiquitous microorganisms present in many ecosystems like soil and compost. Their fairly easy biodegradability is interesting bearing in mind their inertness, water-insolubility and partial crystalline characteristics [22]. The degradation of PHA in soil is particularly suitable for its application in agriculture, as plastic sheets for seedling protection, as degradable seedling containers and most fascinatingly in the controlled release of insecticides. As PHA also degrades well in rumen of cattle, it may be used as a biodegradable matrix for drug release in veterinary medicine. The production of poly-β-hydroxybutyrate (pHB) is another eco-friendly alternative that has been reported as an intracellular reserve polymeric compound in many bacteria and is also of industrial interest along with other poly(βhydroxyalkanoates) because of its possible use as biodegradable polymers [23]. A copolymer of PHB, β-hydroxybutyrate and β-hydroxyvalerate have been produced by ICI Ltd, USA, under the trade name ‘Biopol’ using eubacterium A. eutrophus [24]. Bacillus megaterum BS1 was found to accumulate PHB of 10% of its cell dry weight, which increased slightly on UV-irradiation. It was suggested that chemical mutagenesis could be an alternative method to increase the PHB yield through the enrichment of RNA and amino acid syntheses in wild-type bacterial cells [23]. The biodegradability of blends of bacterial PHB/α-PHB has also been studied and it was attributed to the presence of a crystalline phase provided by bacterial components. In the study, mass spectrometric techniques used were considered as powerful tools in investigating the mechanism of enzymatic hydrolysis of PHA through the identiication of degradation products [25]. It is now fairly well established that PHA are biopolyesters accumulated as storage compounds by a large number of different prokaryotes. More than 100 different hydroxyl-alkanoic acids have been detected as constituents of these bacterial

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Plasticulture for Agriculture and Food Security polyesters. They are biodegradable thermoplastics and elastomers, which have attracted considerable attention from academia and the industry for their various technical applications in industry, agriculture, medicine, pharmacology and other areas [26]. PHA have been produced on an industrial scale by Zeneca Bio Products using environmental friendly methods for harvesting and drying PHA granules from bacterial cells. Subsequently, the US company Monsanto embarked on a signiicant research programme to develop PHA producing recombinant crops, particularly corn. Thereafter, US company Metabolix Inc. initiated work on direct production of PHA in plants, which may yield better economics compared to the existing large number of petrochemical polymers. It is expected that with many research programmes taken up on PHA the world over, on its cost-effective production, through biotechnological intervention, efforts will be successful to make it commercially viable for various promising applications particularly in the agricultural sector.

13.5 Applications in the Agriculture and Food Sectors By 1936, American, British and German companies were producing poly(methyl methacrylate) (PMMA), better known as acrylic. Although acrylics are now well known for their use in paints and synthetic ibres, such as fake furs, in their bulk form they are actually very hard and more transparent than glass, and are sold as glass replacements under trade names such as Plexiglas and Lucite. Plexiglas was used to build aircraft canopies during the Second World War, and it is now used as a marble replacement for countertops. Another important polymer, polyethylene (polythene, PE), was discovered in 1933 by Reginald Gibson and Eric Fawcett at the British industrial giant Imperial Chemical Industries (ICI). This material evolved into two forms, low-density polyethylene (LDPE) and high-density polyethylene (HDPE). Polyethylene is cheap, lexible, durable and chemically resistant. LDPE is used to make ilms and packaging materials, including plastic bags, while HDPE is used more often to make containers, plumbing and automotive ittings. While PE has low resistance to chemical attack, it was found later that a PE container could be made much more robust by exposing it to luorine gas, which modiied the surface layer of the container into the much tougher polyluoroethylene. Polymers and composites continue to be improved. General Electric introduced Lexan, a high-impact polycarbonate, in the 1970s. Du Pont developed Kevlar, an extremely strong synthetic ibre best known for its use in bullet-proof vests and combat helmets. Polymers and polymeric composites are gaining importance in all spheres of life including agriculture due to the useful properties such as high strength to weight ratio, lexibility to moulding to any shape and size with varying strength, colour

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas and transparency, corrosion resistance, etc. It can be created with any strength and resistance to any type of deterioration by the addition of some additives. This makes it suitable for almost all types of uses; however, economics may be one of the constraints [27]. Plasticulture has developed management systems that allow growers to achieve higher quality produce, superior yields and extended production cycles. Growers using plasticulture can produce vegetables, fruits and lowers for markets during winters, early spring and late autumn that would be otherwise impossible to cultivate. Polymers and polymeric composites in agriculture are used in such forms as ibres, ilms, sheets, pipes and tubes, woven fabrics, nettings, moulding, bags, etc. for a variety of uses. While the polymer industry continues to make rapid progress by way of making available newer and better materials, it is for the professionals to make plans for its use in the agriculture system (from seed to seed/market) based on the research experiences conducted under varying farming conditions, which change from location to location. Good efforts have been made to generate information on applicability of polymers in surface covered cultivation, pressurised irrigation systems, lining of ponds and conveyance systems, mulching and soil solarisation, ish hatching devices, ish culture mechanisms and related aspects. However, testing of technologies in farmers’ ield conditions, reining them to suit the real location-speciic farming conditions under varying socio-economic environments and development of a package of practices to achieve eficient use of the available resources are some of the challenges [27]. Biodegradable polymers are now gaining importance day by day and are being successfully used in agriculture and the packaging industry.

13.5.1 Protected Cultivation Protected cultivation in the form of greenhouses (Figure 13.5), net houses, high tunnels, low tunnels, trench cultivation (Figure 13.6), etc. offers several advantages to growing crops of high quality and yields, thus using land and other resources more eficiently. In India, only 600–700 ha could be brought under greenhouse cultivation until now with varying levels of climate control [27]. The greenhouses are usually covered with a material (i.e. glass or polymeric material) that has the ability to transmit light that provides essential energy for plant growth and production. Many factors affecting the physical and mechanical properties of polyethylene ilms used in greenhouse coverings can be grouped into three categories; product manufacturing and process speciications, climate and external conditions and microclimate conditions [28]. Important processes in the manufacturing of polymers and polymeric composites include temperature of the melt, die parameters, blow-up ratio, drawn ratio and frost-line height and cooling conditions that produce different mechanical properties of the ilm [29]. Molecular orientation during ilm blowing

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Plasticulture for Agriculture and Food Security inluences the tensile properties; higher in the direction of the covalently bonded carbon–carbon chain than in the transverse direction dominated by weaker van der Waals bonds [30,31]. Climatic conditions such as solar irradiation, temperature, humidity, rain and wind loads and environmental pollution inluence ageing and mechanical properties of low-density polyethylene (LDPE) greenhouse covers [28].

Figure 13.5 Strawberry cultivation in a greenhouse

Figure 13.6 Winter vegetable cultivation in trenches

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas Light intensity, duration and spectral distribution all affect plant response. Photosynthesis proceeds only with visible light or photosynthetically active radiation (PAR) in the 400–700 nm wavebands. Lozano and co workers [32] emphasised the importance of good solar radiation transmission in increasing greenhouse crop production. In addition, climatic factors such as air temperature and relative humidity are indirectly affected by the cover material. The major crop species grown under greenhouses are vegetables (>90%) and some lowers and fruit crops. Domestic animals were also reported to have been raised in solar greenhouses [33,34]. In recent years, new solar greenhouse technology has been developed in China. These greenhouses play a vital role in China’s vegetable production in winter. There are several different types of solar greenhouses, including the single-slope solar greenhouse. These are built facing south. The support and insulation walls are on the north, east and west sides. A short roof is installed on the top of the north wall. The south side is supported by metal or bamboo frames or a mixture of both, and is covered with plastic ilm and an insulating blanket. These greenhouses are used to produce warm season crops such as tomato and cucumber during the winter (Figure 13.7) without using other forms of heat, in locations at latitude 32°N to 43°N [35].

Figure 13.7 Greenhouses for winter vegetable production

High tunnels, although resembling the traditional plastic-covered greenhouses, are a completely different technology. In their purest form, high tunnels have a pipe or other framework covered by a single layer of greenhouse grade 4-6 mm plastic and

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Plasticulture for Agriculture and Food Security they have no electrical service, automated ventilation or heating system [36,37]. In temperate parts of the world, high tunnels are used to extend the growing season by creating a warmer environment for crop growth [38,39], while in the tropical regions of the world; high tunnels also extend the growing season by permitting crop production during the rainy seasons [40]. Cloches are generally employed to provide protection to young transplants in fruit orchards and forests. A cloche is a protective enclosure, consisting of a structural frame and a transparent/translucent glazing material for one plant. Once the young seedlings are well established and/or the harsh season is over, the cloches are removed. Cloches can also be used to provide protection to potted plants. Low-density polyethylene and polyvinyl chloride ilms are common glazing materials. The plasticulture system is based on integrated crop management (ICM) practices that avoid and/or reduce pest pressure. The system decreases dependency on chemical pesticides by maintaining a microclimate that is not conducive to pest development, and by physically excluding pests from the susceptible plant material.

13.5.2 Biocontainers Agricultural applications for biopolymers are not limited to ilm covers. Containers such as biodegradable plant pots (Figure 13.8) and disposable composting containers and bags are areas of interest [41]. The pots are seeded directly into the soil and break down as the plant begins to grow. Polycaprolactone planting containers have been used in automated machine planting for tree seedlings. It was found that the polycaprolactone had undergone substantial degradation after being in soil for six months, having lost 48% of its original weight, with 95% weight loss within a year [42]. Fertiliser and chemical storage bags, which are biodegradable, are also useful applications. From an agricultural standpoint, biopolymers that are compostable are important, as they may supplement the current nutrient cycle in the soils where the remnants are added [1]. Biocontainers are generally deined as containers that are not produced from petroleum and that degrade rapidly when planted into a ield or when placed in a composting operation. Biocontainers can be further described as being plantable or compostable. Plantable biocontainers are those that are designed to be left intact on the root ball and planted into the ield. They are designed to allow roots to grow through the container walls and to decompose after being planted into the ield. Compostable biocontainers are designed to be removed before inal planting, broken apart and composted [43].

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Figure 13.8 Strawberry cultivation in pots

Numerous types of biocontainers have been developed [44–47], and they have typically been composed of peat, paper, coconut ibre, rice hulls, poultry feather ibre, rice straw, dairy manure or organic components. One of the most common plantable biocontainers is the peat container, typically made from a combination of peat and paper. Wood-pulp containers, often marketed as DOT containers (Bethel Organics, Arcadia, FL) or Fertil containers (Fertil International, Boulogne Billancourt, France), are made from 80% cedar wood ibre, 20% peat and lime. Paper pulp containers are produced from paper pulp with a binder. CowPots (CowPots Co. Brodheadsville, PA) is the commercial name for a container made from composted dairy manure with a binder. Strawpots (Ivey Acres Baiting Hollow, NY) are made from 80% rice straw, 20% coconut ibre and a binder, while coconut ibre containers are made from medium and long coconut husk ibres and a binder. One type of compostable biocontainer is the rice hull container. These containers are made from ground rice hulls and a binder and are marketed in various shapes and sizes. Another compostable biocontainer is the OP47 container (Summit Plastic Co., Tallmadge, OH) fabricated from a biopolymer [43].

13.5.3 Mulching Organic mulches such as hay, straw, sawdust, etc. provide some of the beneits of polymer mulch but may be more expensive, harder to handle and do not provide the soil-warming effects encountered with polymers. Polymer mulches provide many positive advantages for the user, such as increased yields, earlier maturing crops,

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Plasticulture for Agriculture and Food Security crops of higher quality, reduced soil evaporation, solarisation, soil temperature control, enhanced insect management and weed control. Recently, biodegradable mulches have been developed for agricultural uses. Biodegradable ilms are often thinner than traditional polyethylene, but otherwise are quite similar. Biodegradable ilms are available in clear, black (Figure 13.9) and a variety of colours. They may be made from renewable resources such as starch, cellulose or degradable polymers. Although a variety of vegetables can be grown successfully using polymer mulches, muskmelons, tomatoes, peppers, cucumbers, squash, eggplant, watermelons and okra have shown the most signiicant responses. The production of strawberries and cut lowers is greatly improved by the use of plasticulture. The selection of which mulch type to use will depend on factors such as the crop to be grown, the season of the year, whether double or triple cropping is contemplated and if insect management is desired. Young plants, which are particularly susceptible to frost, may be covered with a thin Ecolex ilm. At the end of the growing season, the ilm can be worked back into the soil, where it can be broken down by the appropriate microorganisms [1]. Li and co-workers [48] concluded that the use of a clear polymer mulch cover immediately following seeding increases the yield of spring wheat if it is used for fewer than 40 days. Therefore, polymer ilms that begin to degrade in average soil conditions after approximately 1 month are ideal candidates to be used as crop mulches.

Figure 13.9 Black polyethylene mulching

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas The best qualities of both clear and black plastic are available with infrared transmitting (IRT) mulch. IRT mulches include wavelength selective or photoselective mulches, which selectively transmit radiation in some regions of the electromagnetic spectrum but not in the photosynthetic wavelength [49]. These mulches absorb photosynthetically active radiation (PAR) and transmit solar infrared radiation, providing a compromise between black and clear mulches. These IRT mulches give the weed control beneits of black mulch, but are intermediate between black and clear mulch in terms of increasing the soil temperature. IRT mulches are typically translucent green or brown. These mulches warm the soil in the same way as clear mulch, but without the accompanying weed problems. Additional colours that are currently being investigated are red, blue, yellow, grey and orange. All of these have distinct optical characteristics and thus relect different radiation patterns into the canopy of a crop, thereby affecting plant growth and development [50,51]. Plastic mulches directly affect the microclimate around the plant by modifying the radiation budget (absorbitivity versus relectivity) of the surface and decreasing the soil water loss [52,53]. The colour of mulch largely determines its energy-radiating behaviour and its inluence on the microclimate around a vegetable plant. Colour affects the surface temperature of the mulch and the underlying soil temperature. Ham and Kluitenberg [54] found that the degree of contact between the mulch and soil, often quantiied as a thermal contact resistance, greatly affects the performance of mulch. If an air space is created between the plastic mulch and the soil by a rough surface, soil warming can be less effective than expected.

13.5.4 Irrigation System Water is the most important input for agricultural production. As fresh water resources are increasingly strained by agricultural and industrial usage, water conservation has gained importance. Practices that conserve water and maximise its use are increasingly important in agriculture and these include recycling water, reducing waste and helping plants to make the most of the water available to them. The irrigation sector is the largest consumer of polymers in agriculture. It has vast potential and opportunity to increase the area under micro-irrigation considering the limited water availability for crop production and the ever-shrinking available water resources around the world. In developing countries, the agricultural production is mostly rainfed or based on some diverted spring water, which contribute an almost negligible amount to the overall production. However, a very great opportunity for rainwater harvesting exists in upland humid areas, which can provide good amounts of water for irrigation, although there is limited availability based on the catchmentcommand area ratio, and opportunity for water harvesting through the construction of ponds [27]. Lining of such ponds is very important to retain the water for longer

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Plasticulture for Agriculture and Food Security periods. Polythene lining is a cheap, easy-to-construct and simple technology for the purpose. However, designs and needs for safety mechanisms are very important for satisfactory performance of the ponds for longer periods. Use of an LDPE-lined tank (Figure 13.10) to supplement the water for vegetable production in hilly terraces resulted in enhanced productivity per unit of area. A 14.7% increase in productivity was observed for French bean, 27.3% for tomato, 21.1% for garden pea, 27.3% for cabbage, 21.4% for potato and 16.7% for capsicum in Uttarakhand, India [27].

Figure 13.10 Polyethylene-lined, low-cost water tank for drip irrigation

Drip irrigation (Figure 13.11) is helpful in water conservation by reducing evaporation and deep drainage compared to other types of irrigation such as lood or overhead sprinklers, since water can be more precisely applied to the plant roots through a network of valves, pipes, tubing and emitters. Each dripper/emitter oriice supplies a measured, precisely controlled uniform application of water directly into the root zone of the plant. It saves up to 70% water compared to lood irrigation. Fertilisers and chemicals can be added with the irrigation water low for fertigation and chemigation and thus its use eficiency increases by 30%. This is a result of improved water and fertility management and reduced disease and weed pressure. When drip irrigation is used with polyethylene mulch, yield increases can be even greater. These beneits are only possible when a drip irrigation system is properly designed, managed and maintained.

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Figure 13.11 Drip irrigation systems in a pear orchard

The drip irrigation method offers an opportunity for precise application of watersoluble and other nutrients to the soil at the appropriate time and with the desired concentration. In addition, drip irrigation can eliminate many diseases that are spread through water contact with foliage. With drip irrigation, root development of temperate fruit crops is extensive in a restricted volume of soil, and fertigation can effectively place the plant nutrients in the plant root zone of highest root concentration. Fertigation also provides lexibility of fertiliser applications in various fruit crops with speciic nutritional requirements to be met at different stages of its growth and it further increases nutrient-use eficiency by the plants throughout the reproductive cycle. Water and nutrients enter the soil from the emitters, moving into the root zone of the plants through the combined forces of gravity and capillary. In this way, the plant’s withdrawal of moisture and nutrients are replenished almost immediately, ensuring that the plant never suffers from water stress, thus enhancing fruit and vegetable quality, promoting optimum growth and also increasing the yield.

13.5.5 Controlled-release Fertilisers Nutrient-use eficiency, particularly for nitrogen fertiliser, is still low despite signiicant improvements in crop production over the last few decades. Development of coated or controlled-release fertilisers has been shown to increase nutrient-use eficiencies and reduce the environmental impact of agricultural production [55]. The agricultural sector consumes only about 10% of the total slow-release fertiliser used, but demand has been increasing at an annual rate of 10% [56]. The coating limits or controls the

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Plasticulture for Agriculture and Food Security rate of water penetration to the soluble fertiliser core and, in some products, controls the release rate of the solubilised fertiliser from within the granule to the soil. The three categories of coated fertilisers are based on the coating material and include sulfur, polymer, and both sulfur and polymer coatings [57]. Developed in the late 1970s, polymer-coated fertilisers represent the most technically advanced state-of-the-art in terms of controlling product longevity and increasing nutrient-use eficiency. The granular urea is covered with resin, and nitrogen release occurs as water moves in to the coated prill, with nitrogen release as the urea solution diffuses out into the soil [58]. Reliable information regarding release characteristics of nutrients from a polymer-coated controlled-release fertiliser (CRF) is essential for beneicial agronomic and environmental results. Most polymer-coated products release by diffusion through a semi-impermeable membrane; the rate of release can be altered by the composition of the coating and the coating thickness. Coated substrate can consist of urea alone or a complete fertiliser containing nitrogen, phosphorous and potassium. Water vapour penetrates the resin coating and dissolves the water-soluble fertiliser core. The dissolved nutrients then diffuse through the coating into the soil. The release patterns are much more linear than sulfur-coated urea technology. Soil temperature inluences the rate of diffusion, therefore having the greatest inluence on fertiliser release rate. The release durations of these materials vary greatly depending on the coating material and thickness, and range from 3 to 16 months [59]. A reactive layer coating (RLC) is a technology where a mixture of diisocyanate and polyol are reacted, producing a coating that bonds to a urea prill [60]. Signiicant knowledge regarding nitrogen release from polymer-coated urea was gained while the information regarding the release of the different nutrients contained in polymer-coated compound N–P–K CRF remains limited. Du and co-workers [61] conducted an experiment in which major factors affecting the differential release of nutrients from two coated compound CRF was performed in free water, water saturated sand and sand at ield capacity. In general, nitrate release was the fastest, followed by ammonium and potassium whereas phosphate was signiicantly slower, with a rate of linear release in free water 45–70% slower than that of nitrate. Few differences were obtained for the lag periods of nitrate, ammonium and potassium release (2–10 days) under the experimental conditions, whereas for P they were one order of magnitude larger. The main factor slowing the release was assumed to be the lower solubility of ions, with P being the least soluble. Release into free water was, expectedly, somewhat faster than that into saturated sand and signiicantly faster compared to sand at ield capacity, and particularly so for P. Raising the temperature from 20 °C to 40 °C increased the rate of linear release of the different nutrients. Shaviv and co-workers [62] developed a mathematical model based on vapour and nutrient diffusion equations. The model predicts the release stages in terms of measurable geometrical and chemophysical parameters such as the following: the

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas product of granule radius and coating thickness, water and solute permeability, saturation concentration of the fertiliser and its density. The model successfully predicts the complex and ‘sigmoidal’ pattern of release that is essential for matching plant temporal demand to ensure high agronomic and environmental effectiveness. It also lends itself to more complex statistical formulations, which account for the large variability within large populations of coated CRFs and can serve for further improving CRF production and performance.

13.5.6 Packaging Biopolymers that are employed in packaging continue to receive more attention than those designated for any other applications. It is estimated that 41% of polymers are used in packaging (Figure 13.12), and that almost half of that volume is used to package food products [1]. Polymers have gained a unique position in packaging technology because of their physical properties, notable strength and barrier properties against water-borne microorganisms, which cause spoilage of perishable commodities like foodstuffs. Among the promising packaging materials are long-chain esters of starch. Blends of starch with biodegradable polymers are now commercially available as packaging materials [63]. When blended with starch, the microbial consumption of the starch component in fact leads to increased porosity, void formation and loss of integrity in polymeric materials. Research is continuing on further development of biodegradable polymers based upon polyester and starch [3]. Ecolex is a fully biodegradable polymeric material that was introduced to consumers in 2001. The material is resistant to water and grease, making it appropriate for use as a hygienic disposable wrapping, it to decompose in normal composting systems. Consequently, Ecolex has found a number of applications as a packaging wrap [1]. Edible ilms and coatings have received considerable attention in recent years because of their advantages over synthetic ilms. The main advantage of edible ilms over traditional synthetics is that they can be consumed with the packaged products. Biodegradable polymer ilms derived from natural polymers or polymers derived from natural monomers provide excellent opportunities as edible ilms since their biodegradability and environmental compatibility can be assured. Since there is no package left to dispose, even if the ilms are not consumed, they could still contribute to the reduction of environmental pollution. Rice bran-illed biodegradable low-density polyethylene ilms have been developed for food packaging applications [64]. Biodegradable polymer ilms, non-edible in nature, include certain cellulose-based products (e.g. cellophane), microbial polyesters (e.g. polyhydroxybutyrate/valerate copolymers produced by bacteria), biodegradable synthetic polymers (e.g., polylactic acid produced from fermentation lactic acid) and combinations of starch with biodegradable synthetic polymers (e.g., polyvinyl alcohol) for food packaging applications [65]. The ilms are produced exclusively from renewable, edible ingredients and therefore are anticipated 414

Plasticulture for Agriculture and Food Security to degrade more readily than polymeric materials. The ilms can enhance the organoleptic properties of packaged foods provided they contain various components i.e. lavourings, colourings, sweeteners, etc. [66]. Production of edible ilms causes less waste and pollution; however, their permeability and mechanical properties are generally poorer than those of synthetic ilms [67].

Figure 13.12 Packaging of apricot pulp for long-term storage

13.6 Climate Change and Food Security Climate is an important factor of agricultural productivity. Because of the fundamental role of agriculture in human welfare, concern has been expressed by many organisations and others regarding the potential effects of climate change on agricultural productivity. Interest in this matter has motivated a substantial body of research on climate change and agriculture over the past decade. Climate change is expected to inluence agricultural and livestock production, hydrologic balances, input supplies and other components of agricultural systems. Population growth will mean more land must be used for rice cultivation and other crop production and an increase in the number of farm animals without signiicant improvements in yield rates in the future. These factors will lead to an increase of methane and other greenhouse gases released to the atmosphere. Adjustments will be necessary in order to a counterbalance any negative impacts of a changing climate. Looking into the increasing role of plasticulture in agriculture, adapting more and more polymeric

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas materials of biodegradable nature, augmenting the use of such materials in agriculture and food security applications may contribute to a large extent in combating the adverse affects of climate change on agricultural productivity. Farmers must have the ability to adjust to changes by adapting such farming practices. Adaptation, such as changes in crops and crop varieties, improved water management and irrigation systems and changes in planting schedules and tillage practices, along with use of biodegradable polymeric materials, will be important in limiting the negative effects and taking advantage of the beneicial effects of changes in climate. More eficient use of mineral fertilisers and other adjustments in agricultural practices could also help to counteract the effects of climate change [68]. The massive increase of world population and people’s need for healthy food during the last decades have put a tremendous pressure on limited natural resources around the globe. The scarcity of water and land has been intensiied by increases in soil salinity and climate change, seriously affecting poorer, rural people, whose livelihoods depend on conventional agriculture. Plasticulture and its associated production techniques can signiicantly improve productivity per unit of area and land for the production of high-value cash crops. Plasticulture can provide rural people with nutritious vegetable crops to help address food security and alleviate poverty. The increased consumption of vegetables, together with the additional food that rural families could purchase through the increased income generated by protected agriculture, will contribute to improved food security and nutrition of the most vulnerable, such as children, pregnant women and the elderly. Plasticulture reduces seasonality, enabling farmers to supply local markets with high-quality food out of season and creating more permanent jobs. Plasticulture is labour intensive, therefore it could provide rural society with a number of job opportunities for the disadvantaged, particularly women. In addition, supporting the production facilities requires a number of off-farm technicians, roles that can be occupied by local youths. Furthermore, it also offers potential for the development of a private service sector in the construction and supply of protected agriculture equipment. High-yielding quality produce from protected agriculture could be expanded to serve the export market and generate a valuable source of foreign revenue [69].

13.7 Biodegradable Polymers and Eco-management The development of biodegradable polymers and polymeric composites for agricultural and food-related application is certain to increase in importance due to the emerging problems of environmental contamination and waste disposal problems associated with polymers. Substantial quantities of plastic have accumulated in the natural environment and in landills. Around 10% by weight of the municipal waste stream is plastic [70]. The use of polymers based on renewable feedstocks that are biodegraded 416

Plasticulture for Agriculture and Food Security is a more sensible choice than recycling conventional polymers, as the end products are organic matter, and toxic emissions are avoided. The growth of polymers that are compostable or easily degraded must be encouraged [1] for agricultural applications. Compostable polymers undergo biological degradation during composting to yield carbon dioxide, water, inorganic compounds and biomass at a rate consistent with other known compostable materials, and leave no visually distinguishable or toxic residues [71]. Most polymeric packaging materials are also non-biodegradable and may take hundreds of years to photodegrade if exposed to sunlight. The accumulation of these wastes will also increase at a rate that will lead to a waste management crisis in the near future. A recycle approach here again will offer only a partial solution to this problem, while the use of degradable polymers in packaging will certainly provide a better option to solve the problem. This and several other reasons have successfully led to the development of ‘biodegradable’ polymers to a certain extent during recent years. Such development includes consideration of ecological aspects and requirements such as ever-expanding landill sites, political or social trends involving discussion on renewable versus fossil resources. Other developments considered the biotechnological potentials observed in certain soil resident microbes or otherwise as possible interventions to achieve biodegradability in the polymers and polymeric composite materials used for agricultural, food packaging and several other applications. Development of polymers and polymeric blends that exhibit high susceptibility to microbial attack, on the other hand, is a visible approach of great importance for relieving environmental concerns caused by the current use of non-biodegradable polymers [15]. Polyethylene was discovered in 1933 and has found ubiquitous usage, as it can be easily produced to give inished products with different properties. Blends of polyethylene and starch have gained increasing scientiic attention in the past two decades [72]. When a biodegradable additive is employed, microorganisms can use the additive. The porosity of the material is thereby increased and a mechanically weakened ilm is obtained. The surface area is increased and the ilm becomes more susceptible than the original ilm to all degradation factors including biodegradation. The biotechnological approach is also being increasingly recognised as a key to developing better biodegradable, low-cost, polymeric formulations. Some of the new and improved catalysts (both synthetic and natural), as well as use of engineered bacteria directly producing polymers based on proteins, are being looked as assisting in expanding the capabilities of polymers such as polyethylene into the range of engineering polymeric formulations useful for applications in the packaging and agricultural sectors.

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13.8 Conclusion and Future Prospects The production of plastics has increased substantially over the last 60 years from around 0.5 million tonnes in 1950 to over 260 million tonnes today [73]. In Europe alone the plastics industry has a turnover in excess of 300 million euros and employs 1.6 million people [74]. Almost all aspects of daily life involve plastics, in agriculture, transport, telecommunications, clothing, footwear and as packaging materials that facilitate the transport of a wide range of food, drink and other goods. There is considerable potential for new applications of plastics that will bring beneits in the future, for example as novel medical applications, in the generation of renewable energy and by reducing energy used in transport [75]. Barnes and co-workers [70] show that plastic wastes, including packaging, electrical equipment and plastics from end-of-life vehicles, are major components of both household and industrial wastes; our capacity for disposal of waste to landill is inite and in some locations landills are at, or are rapidly approaching, capacity [76]. So from several perspectives it would seem that our current use and disposal of plastics is a cause for concern [70,77]. There are accounts of inadvertent contamination of soils with small plastic fragments as a consequence of spreading sewage sludge [78], of fragments of plastic and glass contaminating compost prepared from municipal solid waste [79] and of plastic being carried into streams, rivers and ultimately the sea with rain water and lood events [80]. Around 4% of world oil production is used as a feedstock to make plastics and a similar amount is used as energy in the process. Yet over one-third of current production is used to make items of packaging, which are then rapidly discarded. Given our declining reserves of fossil fuels, and the inite capacity for disposal of waste to landill, this linear use of hydrocarbons, via packaging and other short-lived applications of plastic, is simply not sustainable [73]. There are solutions, including material reduction, design for end-of-life recyclability, increased recycling capacity, development of biobased feedstocks, strategies to reduce littering, the application of green chemistry life-cycle analyses and revised risk assessment approaches. Such measures will be most effective through the combined actions of the public, industry, scientists and policymakers [73]. Therefore, there is a clear need for more research on the quantities and effects of plastic debris in natural terrestrial habitats, on agricultural land and in freshwaters. It is increasingly being realised that the use of long-lasting polymers for short-lived applications is not entirely justiied and that bioplastics or biodegradable polymers need to be developed from renewable sources for future applications, particularly agriculture and food-related areas. There is a need to increase the use of food grade and biodegradable polymers taking into consideration the effect on human health

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Plasticulture for Agriculture and Food Security and the environment. The challenge related to the use of biodegradable polymers in terms of edible ilms or food-packaging-related products is to achieve a controlled lifetime. Such products must remain stable during useful functional applications. According to Jerry Conklin, who works with cellulose ilms at Dow Chemical Co., ‘Successful growth in the area of edible ilms will require a fusion of technologies of food and polymer science’ [65]. Plasticulture applications are inevitable for increasing production and productivity of horticultural crops to meet the food and nutritional needs of a population but use of biodegradable polymers in loose-ill packaging, mulching, compost bags, greenhouses and other agricultural applications has yet to gain momentum. Greater efforts are also needed towards developing biodegradable polymer matrices for the controlled release of plant growth factors (nutrients and fertilisers) or carriers for long-term dosage of germicides, fungicides and insecticides. Current landilling regulations, recycling fees and development of composting infrastructure are increasing the demands for biodegradable polymers. At present, however, biodegradable polymers appear to be much more costly than the commodity materials they are seeking to displace, resulting in signiicant hurdles in the way of their market acceptance. It is a welcome approach that a number of biodegradability tests have been developed to establish biodegradability and many producers of biodegradable polymers are making their biodegradation studies available with a view to demonstrating the environmentally friendly nature of their products.

References 1.

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10. R.B. Srivastava, T.C. Shami, M.C. Upreti and S.K. Pandey in Recent Advances in Polymers and Composites, Eds., G.N. Mathur, L.D. Khandpal and S.N. Sen, Allied Publishers India, 2001, p.675. 11. M. Mercedes, P. Ana, R. Santiago, O.M. Lusia and L. Fernando, International Biodeterioration & Biodegradation, 1995, 35, 269. 12. E.H. Pankhurst and M.J. Davies in Biodeterioration of Materials, Eds., A.H. Walters and J.J. Elphick, Elsevier Science Publishers, New York, USA, 1968, p.303. 13. B. Little, R. Ray, P. Wagner, Z. Lewandovski, W.C. Lee, W.G. Characklis and F. Mansfeld, Biofouling, 1991, 3, 1, 45. 14. S. Lal, M. Awasthi and R.B. Srivastava, Proceedings of the International Symposium on Biodegradable Polymers and Packaging Materials, Hyderabad, India, November 2001. 15. P. Dave, R.A. Gross, C. Brucato, S. Wong and S.P. McCarthy, Biotechnology and Polymers, Ed., C.G. Gebelenin, Plenum Press, New York, USA, 1991. 16. R.B. Srivastava, S.K. Pandey, M.C. Upreti, S.K. Pradhan, K.N. Pandey and G.N. Mathur, in Proceedings of Nat. Conf. on Electron Microscopy, Eds., G.N. Mathur and C.D. Dwivedi, 1999, p.180. 17. C.J. Foust, R.R. Mills, T. Haas, C. Castevens and R.M. Ottenbrite, Macromolecular Symposia, 123, 1. 18. R. Chandra and R. Rustugi, Polymer Degradation and Stability, 1991, 56, 185. 19. C. Grandils, P. Flandroy and R. Jeroma, Controlled Release, 1996, 38, 109.

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Plasticulture for Agriculture and Food Security 20. H. Chau and P. Yu, Water Science and Technology, 1999, 39, 10/11, 273. 21. S.Y. Lee and J. Choi, Advances in Biochemistry and Biotechnology, 2001, 71, 183. 22. D. Jendrossek, Advances in Biochemical Engineering and Biotechnology, 2001, 71, 293. 23. S. Sabat, M.K. Deshpande and P.V. Khandwekar, Journal of Scientific & Industrial Research, 1998, 57, 654. 24. Y. Doi, Microbial Polyesters, VCH Publishers, New York, USA, 1990. 25. M. Scandola, M.L. Focarete, G. Adamus, W. Sikorska, I. Baranowsks, G. Swierczek, M. Kowalczuk and Z. Jedlinski, Macromolecules, 1997, 30, 2568. 26. V.C. Kalia, N. Raizada and V. Sonakya, Journal of Scientific & Industrial Research, Bioplastics, 2000, 59, 433. 27. P.R. Bhatnagar, in Advances in Agriculture Environment & Health, Eds., S.B. Singh, O.P. Chaurasia, A. Yadav, A.M. Rimando and T.H. Terrill, Satish Serial Publishing House, Delhi, India, 2009, 159. 28. A.M. Alhamdan and I.M. Al-Helal, Journal of Material Processing Technology, 2009, 209, 63. 29. P.A. Dilara and D. Briassoulis, Polymer Testing, 1998, 17, 549. 30. G. Gruenwald, Plastics: How Structure Determines Properties, Hanser Publishers, Munich, Germany, 1992. 31. S. Vishu, Handbook of Plastics Testing Technology, Wiley, New York, USA, 1995. 32. G.M. Lozano, D. Gonzalez and E. Santos, Plasticulture, 1996, 110, 15. 33. M.S. Chen, S.P. Ma, P.G. Zhou and J.W. Li, China Vegetables, 2007, 1. 34. T.L. Li and J. Shenyang, Agricultural University, 2005, 36, 131. 35. L.H. Gao, M. Qu, H.Z. Ren, X.L. Sui, Q.Y. Chen and Z.X. Zhang, Horticulture Technology, 2010, 20, 3, 626. 36. W.J. Lamont, M. McGann, M. Orzolek, N. Mbugua, B. Dye and D. Reese, Horticulture Technology, 2002, 12, 447.

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas 37. O.S. Well, Horticulture Technology, 1996, 6, 172. 38. W.J. Jiang, D. Qu, D. Mu and L. Wang, Horticulture Review, 2004, 30, 115. 39. O.S. Well, Proceedings of Vegetable Production Using Plasticulture Seminar, American Society of Horticultural Science and American Society of Plasticulture, Charlotte, NC, 1998, p.49. 40. M. Jensen and A.J. Malter, Protected Agriculture: A Global Review, World Bank Tech. World Bank, Washington DC, USA, 1995, Paper No. 253. 41. J.C. Huang, A.S. Shetty and M.S. Wang, Advances in Polymer Technology, 1990, 10, 1, 23. 42. J.E. Potts, R.A. Cleudinning, W.B. Ackart and W.D. Niegich, Polymer and Ecological Problems, Plenum Press, New York, 1973. 43. M.R. Evans, M. Taylor and J. Kuehny, Horticulture Technology, 2010, 20, 3, 549. 44. M.R. Evans and D. Hensley, Horticulture Science, 2004, 39, 1012. 45. S.K. Gayed, Canadian Plant Discovery Survey, 1971, 51, 4, 142. 46. E. Lahde and K. Kinnonen, Folia Forestalia, 1974, 197, 1. 47. F. Mrazek, Beitrage fur-die Forstwirtschaft, 1986, 20, 3, 128. 48. F.M. Li, A.H. Guo and H. Wei, Field Crops Research, 1999, 63, 79. 49. B. Loy, J. Lindstrom, S. Gordon, D. Rudd and O. Wells, Proceedings 21st National Agricultural Plastics Congress, 1989, p.193. 50. D.R. Decoteau, M.J. Kasperbauer and P.G. Hunt, Journal of American Society of Horticulture Science, 1989, 114, 216. 51. M.D. Orzolek and J.H. Murphy, Proceedings 24th National Agricultural Plastics Congress, Kansas City, USA, 1993, 157. 52. A. Liakatas, J.A. Clark and J.L. Monteith, Agricultural for Meteorology, 1986, 36, 227. 53. C.B. Tanner, HortScience, 1974, 9, 555. 54. J.M. Hamand Kluitenberg, Agriculture for Meteorology, 1994, 71, 403.

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Plasticulture for Agriculture and Food Security 55. J.B. Sartain, W.L. Hall, R.C. Littell and E.W. Hopwood, in Soil and Crop Science Society of Florida Proceedings, 2004, 63, 72. 56. M.E. Trenkel, Controlled-release and Stabilized Fertilizers in Agriculture, International Fertilizer Industry Association, Paris, France, 1997. 57. E.H. Simmone and C.M. Hutchinson, Horticulture Technology, 2005,15, 36. 58. N.W. Hummel Jr., Agronomy Journal, 1989, 81, 290. 59. K.T. Morgan, K.E. Cushman and S. Sato, Horticulture Technology, 2009, 19, 10. 60. C.H. Peacock and J.M. DiPaola, Agronomy Journal, 1992, 84, 946. 61. C. Du, J. Zhou and A. Shaviv, Journal of Polymers and the Environment, 2010, 14, 3, 223. 62. A. Shaviv, S. Raban and E. Zaidel, Environmental Science and Technology, 2003, 37, 10, 2251. 63. C. Bastioli, L. Bellotti, D. Giudice and G. Gilli, Journal of Environmental Polymer Degradation, 1993, 1, 3,181. 64. J. George, R. Kumar, C. Jayaprakash, A. Ramkrishna, S.N. Sabapathy and A.S. Bawa, Journal of Applied Polymer Science, 2006, 102, 4514. 65. M. Druchta and C.D. Johnston, An Update on Edible Films, CSA Library Series, 1997, Article #24, Lifeline, XV, 2, 1. 66. T. Bourtoom, International Food Research Journal, 2008, 15, 3, 1. 67. J.J. Kester and O.R. Fennema, Food Technology, 1986, 40, 12, 47. 68. C. Aydinalp and M.S. Cresser, American-Eurasian Journal of Agricultural and Environmental Science, 2008, 3, 5, 672. 69. M. El-Solh, A.T. Moustafa and A.A. Hassan, in Proceedings of the International Conference on Horticulture, 9–12 November 2009, Bangalore, India, pp.535–546. 70. D.K.A. Barnes, F. Galgani, R.C. Thompson and M. Barlaz, Philosophical Transactions of Royal Society B, 2009, 364, 1985.

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas 71. ASTM Standards, D883-96: Standard Terminology Relating To Plastics, New York, NY, ASTM, 1998, 08, 01. 72. J.D. Gu, C. Lu, R. Mitchell, K. Thorp and A.J. Corsto, International Microbial Biotechnology, 1997, 18, 364. 73. R.C. Thompson, C.J. Moore, F.S. Saal and S.H. Swan, Philosophical Transactions of Royal Society B, 2009, 364, 2153. 74. Plastics Europe, The Compelling Facts about Plastics 2007. An Analysis of Plastics Production, Demand and Recovery in Europe, Brussels, Austria, 2008, p.24. 75. A.L. Andrady and M.A. Neal, Philosophical Transactions of Royal Society, 2009, 364, 1977. 76. E. Defra, S. Wilson and M. Hannan, Review of England’s Waste Strategy, Environmental report under the ‘SEA’ directive, London, UK, DEFRA, 2006, p.96. 77. J. Hopewell, R. Dvorak and E. Kosior, Philosophical Transactions of Royal Society B, 2009, 364, 2115. 78. K.A.V. Zubris and B.K. Richards, Environmental Pollutants, 2005, 138, 201. 79. W.F. Brinton, Compost Science and Utilization, 2005, 13, 274. 80. R. Thompson, C. Moore, A. Andrady, M. Gregory, H. Takada and S. Weisberg, Science, 2005, 310, 1117.

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14

Adoption of Biopolymers: An Analysis Based on American, Japanese and European Patent Data

Avrath Chadha

14.1 Introduction With a worldwide plastics production of over 250 million tonnes the plastics industry is one of the most important sectors within the chemical industry [1]. Over the years petro-based polymers have dominated the market over other types of plastics [2]. As a result, the plastics industry tends to endorse incremental process-oriented innovations [3]. Compared to that, the adoption of radical innovations is rare as it requires major shifts in competencies and is more risky [4–6]. However, in the coming years radical change in the plastics industry might be necessary [7]. As most plastics are produced from fossil-fuel raw material resources, their depletion is a widely discussed issue in the industry [8,9]. Some studies expect oil reserves to run dry in approximately 30–50 years [10–12]. However, the economic impact of oil depletion could hit the plastics industry much earlier, for example if the cost increase due to rising oil prices cannot be passed on to customers [13–15]. To gain independence from crude oil the plastics industry has started to focus on the development and adoption of technologies that substitute for petro-based plastics. In line with a ‘green chemistry’ orientation and an emphasis on renewable resources, considerable research and development efforts for so-called biopolymers or bioplastics are currently taking place [9]. They are made from renewable feedstock like starch and the end products may or may not be biodegradable [16–18]. Although market reports suggest that with technological progress, economies of scale in production and thus lower costs the potential to substitute petro-based polymers by biopolymers will increase in the years ahead [14,19], the extent of research and development (R&D) investments in biopolymer technology and its industry-wide adoption are not well understood [20–22]. This is mainly the result of the fact that comprehensive studies on biopolymers are still limited. Yet, since technical progress in biopolymer technology is the direct output of R&D investments, it can be mapped via patent analysis.

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas However, to my knowledge, only limited information is publicly available on biopolymer-related patent analysis and research has not yet used patent information in assessing the future potential of biopolymer technology. Therefore, this study is a irst step towards quantifying the extent of progress in biopolymer research activities. In doing so, this chapter particularly explores the following research questions: • How much biopolymer technology has been iled for patent application in the USA, Europe and Japan and in which technological development stage is biopolymer technology? • In which industrial sectors are biopolymer technology patents being applied for? • Which irms and organisations have a high biopolymer patenting activity? This research provides an overall picture and new insights on biopolymer technology development by viewing the latter from an international perspective using information provided by biopolymer-related patent applications in the world’s largest patent database WPINDEX. (The Derwent World Patents Index is run by Thomson Reuters and has over 16 million patent application records.) Hereby, I use the number of biopolymer patent publications as a metric of the extent of investment and activity in R&D and technologies by different industry segments. To answer the research questions, I begin by briely presenting the characteristics of biopolymer technology. I then give a brief overview on patent analysis, discuss the research methods, present the results and conclude with an outlook.

14.2 Biopolymers

14.2.1 Definition and Classification Biopolymers cannot be understood as a single class of polymers but rather as a class of materials that can differ considerably from each other [23]. Two different classes of biopolymers can be distinguished [7,8,13]: (1) Polymers based on renewable resources and (2) biodegradable polymers. Products from the irst class do not necessarily have to be biodegradable and products from the second class do not necessarily have to be based on renewable resources, as even numerous synthetic polymers are certiied as biodegradable [24,25]. (Synthetic biodegradable polymers are not part of this analysis since I deine biopolymers for the purpose of this study as polymers that have the potential to substitute petro-based polymers and are thus based on renewable resources. In addition, so-called functional polymers – e.g. additives for industrial

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Adoption of Biopolymers: An Analysis Based on American, Japanese and European Patent Data paints, colours and adhesives – and DNA-biopolymers, which are applicable for genomic sequencing and clinical diagnosis, are excluded from the study.) Currently there are three different kinds of biopolymers which have reached the commercialisation stage and are available on the market: (1) starch materials, (2) polylactic acid and (3) cellulose materials. A promising fourth class of biopolymers are so-called polyhydroxyalkanoates, which are produced using fermentation processes [26]. However, currently polyhydroxyalkanoates are commercially available only in very limited quantities [14,27]. Compared to petro-based polymers, biopolymers possess a number of new and speciic material characteristics such as antistatic properties, good printability and high degree of water permeability, which make them suitable for a new range of applications in agriculture, medicine and packaging [28]. Initial applications such as loose foils, bottles, trays, crockery, etc. have already become successfully established in important markets [7,29,30]. Alongside these short-lived applications there are also durable applications, e.g. injection-moulded parts from polylactic acid, which can be found in products in the automotive and electronics industries [14].

14.2.2 Production The production chain for biopolymers consists in general of three steps: (1) agricultural crop production, harvest and processing, (2) production of basic materials and (3) inal processing of end products [14,21,27]. Whereas in the value chain of petro-based polymers the petrochemical industry is the supplier for raw materials, in the value chain of biopolymers this role is taken by agricultural producers and the biotechnology sector [31]. These irms derive starch, sugar and cellulose from renewable raw materials and convert them to chemical basic materials with the help of thermocatalytic or biotechnological processes (step 1). In a next step these materials are processed to biopolymers through polymerisation or polycondensation (step 2). Depending on the desired properties of the biopolymers, additives from fossil or renewable resources are added [32]. Finally, the biopolymers are delivered to converters in the form of pellets or ibres and are manufactured with injection moulding of extrusion processes to inished products (step 3). Conventional production machinery is used, which after slight modiication can be charged with biopolymers [19]. Thus, the processing of biopolymers is comparable to petro-based polymers. A fundamental difference between the two polymer types lies in the recycling possibilities of biopolymers in the end-of-life-phase. Similar to their petro-based counterparts, biopolymers can be materially and thermally recycled [16,33].

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas Additionally, most biopolymers such as polylactic acid can also be microbiologically degraded in compost facilities [2].

14.2.3 Drivers In the past, biopolymers have been in general signiicantly more expensive and less eficient than petro-based polymers due to their processing properties. However, increasing oil prices have made biopolymers more cost competitive and have spurred interest in research in molecular science and genetic engineering to improve the technology and technical performance of biopolymers [15,34]. While interest in biopolymers at the irm-level is a combination of new market opportunities, more sustainable solutions for established markets and increasing independence from fossil resources, regional interests served by biopolymers differ substantially: in the USA resource security and resource utilisation have priority; in Japan, a strong drive towards products with a green image can be observed; in Europe, the focus is on resource utilisation, reduction of greenhouse gas emissions and compostability [2,7]. Expectations are that in 2012 there will be a much greater alignment of regional interests steering biopolymer development at the global scale since environmental beneits and a stronger focus on renewable feedstock are coming to the fore [31]. In summary, while depleting oil reserves, biodegradability and the use of legislative instruments are signiicant driving forces for the use of biopolymers, the suitability of material properties for converters, the technical feasibility of processing options, the versatility of applications and, ultimately, commercial viability of production and processing are the key factors that are going to decide the actual use of biopolymers in the coming years [9,35].

14.3 Patent Analysis The disclosure of a patent is public information and patent data exist for many countries and in long time series. For legal reasons, applications to obtain a patent (‘patent applications’) and (granted) patents are systematically registered by government bodies. A patent speciication contains and describes technical knowhow and it provides information on technological development [36] and industrial innovation [37]. For the purpose of research enquiries and monitoring, patent applications are categorised by technical ields. Furthermore, they give an indication of the technical applicability, which in turn is an indicator for the commercialisation potential of a technology.

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Adoption of Biopolymers: An Analysis Based on American, Japanese and European Patent Data Next to being used as an instrument for analysing the relationship between technological development and economic growth [38,39] or assessing the research and innovation process in a national or international context [40,41], patent data analysis is also an important method for the assessment of numerous aspects of technological change [42]. Thus, the increasing supply of patent information has particularly facilitated the possibilities of applying patent analysis for technology forecasting. For example, Ernst [36] distinguishes three different development stages in the analysis of patenting activity over the technological life cycle (Figure 14.1).

Figure 14.1 Theoretical development of patenting activity over the technological life cycle. Reproduced with permission from H. Ernst, Small Business Economics, 1997, 9, 361. ©1997, Springer

Stage 1 is labelled ‘emerging phase’ of a new technology. This stage is characterised by stable patenting activity, which is interrupted by a considerable rise in the number of patent applications. The overall quantity of patent assignees is in this phase rather low. The irst peak of patenting activity highlights the end of the emerging phase as irst products based on the new technology are introduced into the market. In the second stage, the so-called ‘consolidation phase’, patenting activity decreases and the number of patent applications is lower than during the peak. This is typically

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas the result of re-focusing of R&D resources based on irst market experiences with the new technology. The third stage is characterised by a strong increase in overall patenting activity. Next to incumbent irms, new irms apply for patents in particular technological ields. In this so-called market penetration phase, the number of patent applications reaches a new peak, which is substantially higher than the irst peak in the emerging phase [36,43]. According to Basberg [44] this is an indicator for the inal commercial breakthrough of the new technology.

14.3.1 Methods For my research, biopolymer patent families were extracted from the WPINDEX database, which contains information on over 16 million patent applications and patents from different countries that have been published since 1962 (I focused in the analysis entirely on patent families, except in Figure 14.2 due to search constraints in the WPINDEX database). The database provides information on patent publications from the 41 most important issuing authorities in the world, including the United States Patent and Trademark Ofice (USPTO), the Japan Patent Ofice (JPO) and the European Patent Ofice (EPO). Each patent family in the WPINDEX database consists of a irst patent application, the so-called priority application and information about patent activity based on the priority application in other countries. (In the following, for simplicity I use the term ‘patent application’ to refer to patent documents [‘publications’] more broadly. In the USA, publications before 2001 refer to granted patents. From 2001 onwards, publications refer to patent applications and granted patents. In Europe and Japan, publications always refer to applications. If I refer to patent applications exclusively, i.e. if I exclude granted patents, I say so explicitly.) Among other data, the database contains patent classiication, title, inventor data, abstract and patent assignee data. Since no unique patent classiications are assigned in the WPINDEX database for biopolymer technology, I identiied biopolymer patents for the priority years 1976 to 2006 based on title and abstract using a list of biopolymer-related keywords. (Since the irst patent applications are iled only in 1979, all graphs and igures have been adjusted accordingly. At the JPO and EPO, and – since 2001 – also at the USPTO, there is a maximum 18-month period between the iling of a patent application and its publication. Since the patent analysis was conducted in April 2009 not all biopolymer patent applications iled between November 2007 and April 2009 have yet been published and therefore could not be retrieved in our analysis.) The list was then extended and veriied by industry experts. Keywords for the positive list included different biopolymers based on renewable resources such as polyhydroxyalkanoates or polylactic acid. Variations were used to encompass alternative terms (e.g. polylactic acid and poly(lactic acid)). In addition, I also searched for commercially available

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Adoption of Biopolymers: An Analysis Based on American, Japanese and European Patent Data products and their producers in order to have as complete a keyword list as possible. I used this list as a search ilter for two reasons: (1) only patent documents regarding biopolymers based on renewable resources and not petro-based biodegradable biopolymers were extracted from the database and (2) the search ilter allowed the separation of patent documents regarding DNA-biopolymer. (However, the search possibilities in the WPINDEX database did not allow an exclusion of biopolymer patent documents related to medical science in the results list. The main driver for these materials is not resource substitution but biocompatibility.) A full description of the patent search ilter is given in the annex.

14.4 Discussion

14.4.1 Biopolymer Patent Activity The research process resulted in 3291 publications in the analysed regions USA, Japan and Europe (Figure 14.2). (In the USA, publications before 2001 refer to granted patents. From 2001 onwards, publications refer to patent applications and granted patents. In Europe and Japan, publications always refer to applications.) The USA has with 1272 the largest number of publications, followed by Japan with 1148 and Europe with 871 publications. All three regions show a similar trend, yet to a different degree. The number of publications in all three regions between 1979 and 1984 was very marginal. Between 1985 and 1992 the number of publications per year averaged 12 in the USA, 15 in Japan and 10 in Europe. In all three regions a signiicant increase publications for the years 1993–2006 can be observed. The results show that up until 2002 all three regions had similar dimensions in terms of the quantity of publications. From the year 2002 until its peak in the year 2006, I observed a sharp increase in publications in all regions, which was, however, more pronounced in the USA compared to Japan and Europe. The main reason for the strong increase in the USA was a revision of the US patent law, which introduced the publication of patent applications whereas before only granted patents had been published. Furthermore, the increase in patent activity can also be an indication that the US market for biopolymers is seen as the most important one by the USA as well as by foreign irms.

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Figure 14.2 Biopolymer technology publications in USA, Japan and Europe during the years 1979 and 2006

In order to understand which development stage biopolymer technology is currently in, I analysed whether biopolymer patenting activity follows the pattern as discussed in the above section on patent analysis. Following Ernst [36], biopolymer technology is currently still in the emerging phase where the basics of the technology are developed. In this phase, the signiicant increase of worldwide biopolymer patenting activity beginning from the year 2002, which corresponds to the sharp bend in the in the emerging phase in Figure 14.1, could indicate the end of the technological development stage and the introduction of biopolymer technology in irst pioneer niche applications such as catering products. Basic technological obstacles were solved in the emerging phase such as the use of slightly modiied standard machinery for the conversion into inished products [19] or the polymerisation of lactide for the production of polylactic acid [45]. However, following Anderson and Tushman [46] no ‘dominant design’ has yet evolved in biopolymer technology. In the beginning of the emerging phase there was only a small number of pioneer irms as only a few are willing to bear the high R&D risk. Therefore, the concentration ratio, deined as published patent applications per assignee, is relatively high in the beginning and decreases to lower numbers later (Figure 14.3).

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Adoption of Biopolymers: An Analysis Based on American, Japanese and European Patent Data

Figure 14.3 Publications per assignee (concentration ratio)*

*Worldwide assignees who have iled patent applications in USPTO, JPO and EPO WPINDEX database allows the yearly tracking of patent applications for a speciic assignee only from the starting year 1983 The analysis of the assignee countries of publications in the USPTO, JPO and EPO databases shows that the focus of biopolymer R&D and patenting activity has shifted over the years from Japan to the United States. Figure 14.4 shows the historical evolution of biopolymer patenting activity by US, Japanese and European assignees in USPTO, JPO and EPO databases over the years 1979 to 2006. Until 1998 Japanese irms iled the majority of biopolymer patent applications and patents. Since then, however, the focus has shifted to the USA, where the emphasis on biopolymer technology has grown steadily. For the year 2006, US organisations hold 60% of all biopolymer patent applications and patents, Japanese organisations 35% and, surprisingly, European organisations a mere 5%.

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas

Figure 14.4 Number of patent families from different assignee regions in the USPTO, JPO and EPO databases

14.4.2 Biopolymers by Industrial Sector In my research I used the third-level International Patent Classiication (IPC) label of the World Intellectual Property Organization to analyse speciic patent technological ields. The IPC is a hierarchical system and is divided into eight sections, several technology classes and subclasses and almost 70,000 groups. The analysis of the IPC sections shows that overall the largest industrial sector with regard to biopolymer patent applications is the chemical sector, accounting for 39% of all biopolymer patent applications worldwide, exceeding the number of patent applications by the next sector by over 60% (Table 14.1). A more in-depth analysis of the IPC sections (Table 14.2) reveals that the top IPC technology classes are mainly in organic chemistry and medical sciences. Worldwide, the technology class ‘C08: Organic macromolecular compounds’ had the highest number of biopolymer patent families published, almost 30% more than the second largest technology class: ‘A61: Medical science and hygiene’. The irst IPC technology class refers mainly to processes using enzymes or microorganisms. These processes are applied during fermentation to breakdown various organic substances, which are the feedstock for biopolymers. The technology class ‘A61: Medical science and hygiene’ summarises patent applications on medical applications such as in medical devices, tissue engineering and drug delivery applications, for which biopolymers are increasingly used.

434

Adoption of Biopolymers: An Analysis Based on American, Japanese and European Patent Data Table 14.1 Distribution of biopolymer technology patent applications by sector and region for the priority years 1979–2006* Sector**

Description

USA***

Japan

Europe

Others

Worldwide

Chemistry

Inorganic chemistry, organic chemistry, manufacture of fertilisers, natural resins, adhesives

937

916

673

695

3221

Performing operations

Physical and chemical processes, working of plastics, micro-structural technology, nanotechnology

537

581

426

415

1959

Human necessities

Agriculture, forestry, hygiene

592

389

457

408

1846

Physics

Optics, computing, measuring, photography

373

380

211

204

1168

Textiles, paper

Yarns, weaving, non-woven fabrics, paper-making, production of cellulose

168

181

135

134

618

Electricity

Basic electric elements, electric communication technique

118

78

62

69

327

Fixed constructions

Building, drilling, mining

133

25

57

77

292

Mechanical Engineering

Machines in general, engines for liquids, luidpressure actuators, heat exchange in general

18

17

20

20

75

Total

2876

2567

2041

2022

9506

* Multiple entries possible ** According to the International Patent Classiication (IPC) of the World Intellectual Property Organization (WIPO) *** Granted patents until 2000, and patent applications and granted patents from 2001 onwards

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas

Table 14.2 Top ten technology fields in US, Japan and Europe for the priority years 1979–2006* Rank

US** IPC Class

Number of patents applications

Japan IPC Class

Number of patents applications

Europe IPC Class

Number of patents applications

1

Medical science; hygiene

449

Organic macromolecular compounds

576

Organic macromolecular compounds

337

2

Organic macromolecular compounds

399

Medical science; hygiene

254

Medical science; hygiene

322

3

Natural resins and adhesives

262

Measuring and testing

212

Natural resins and adhesives

150

4

Measuring and testing

244

Layered products

140

Measuring and testing

146

5

Physical or chemical processes

178

Physical or chemical processes

123

Physical or chemical processes

108

6

Drilling and mining

121

Natural resins and adhesives

107

Layered products

101

7

Layered products

113

Working of plastics

100

Working of plastics

67

8

Organic chemistry

106

Conveying and packaging

85

Food treatment

62

9

Basic electric 76 elements

Organic chemistry

76

Organic chemistry

57

10

Working of plastics

Natural or artiicial threads of ibres

67

Conveying and packaging

51

73

* Multiple entries possible ** Granted patents until 2000, and patent applications and granted patents from 2001 onwards

The three repositories have numerous top technology ields in common. For example, eight technology ields appeared in all three repositories. In spite of the fact that the top ten technology ields are very similar in the three regions, their rankings and number of publications differ signiicantly. For instance, the technological ield ‘C09:

436

Adoption of Biopolymers: An Analysis Based on American, Japanese and European Patent Data Natural resins and adhesives’ is ranked at third position in the USA and Europe but is only at sixth position in Japan. Traditionally, the US resin producers play a major role and have also been the pioneers for these new materials. This phenomenon is an indicator that, although worldwide biopolymer research has to some extent common focus, different regions have their speciic strengths in certain technology ields.

14.4.3 Assignee Analysis For the analysis I used patent assignee codes provided by the Derwent Patents Index database to identify irms, universities and government institutions. The research did not ind a mentionable number of patent documents that were held by individual inventors. Table 14.3 lists the top 50 irms ranked by biopolymer patent application count, Table 14.4 the top 25 universities and government institutions.

Table 14.3 Firms ranked by total number of biopolymer patent applications for the priority years 1979–2006* Rank

Firm

Sector

Country

Size

Total patent families

1

Halliburton Energy Services

Energy

USA

106,000

101

2

Unilever

Consumer goods

Great Britain

174,000

63

3

Procter & Gamble

Consumer goods

USA

138,000

52

4

Kimberly Clark

Paper

USA

55,000

40

5

Agilent Technologies

Electronics

USA

19,000

38

6

Boston Scientiic Corp.

Medical

USA

25,000

37

7

Advanced Cardiovascular Systems

Medical

USA

n/a

36

8

Fujitsu

Computers

Japan

161,000

31

9

Shimadzu

Electronics

Japan

9,300

30

10

Mitsui Chemical

Chemicals

Japan

12,500

28

11

Monsanto

Agriculture

USA

16,500

27

12

Fuji Photo Film

Photographic equipment

Japan

78,000

25

437

Biotechnology in Biopolymers Developments, Applications & Challenging Areas 13

Eastman Chemical

Chemicals

USA

11,000

23

14

Scimend Life Systems

Medical

USA

n/a

22

15

BioTec Biologische Packaging/ Naturverpackungen bioscience

Germany

< 100

21

15

Mitsubishi Plastics

Chemicals

Japan

4,200

21

17

Metabolix

Bioscience

USA

60

20

17

Sony

Electronics

Japan

180,500

20

17

Hitachi Engineering Electronics

Japan

2,600

20

20

Unitika

Chemicals

Japan

1,300

19

20

Toppan Printing

Packaging

Japan

11,100

19

20

Sumitomo Industries

Chemicals

Japan

2,800

19

23

Kaneka

Chemicals

Japan

3,200

18

24

Daicel Chemical Industrial

Chemicals

Japan

2,180

17

24

Ethicon

Medical

USA

n/a

17

24

Toray Industrial

Chemicals

Japan

38,560

17

24

Ciba Speciality Chemicals

Chemicals

Switzerland

13,300

17

28

Mobil Oil

Oil

USA

106,100

16

29

SynX Pharma Inc.

Pharmaceutical

USA

n/a

15

29

Zeneca

Pharmaceutical

Great Britain

65,000

15

29

Texas United Chemical

Chemicals

USA

n/a

15

29

Johnson & Johnson Consumer goods

USA

122,000

15

33

Terumo

Medical

Japan

7,500

14

33

Tosoh

Chemicals

Japan

11,000

14

35

Novamont

Chemicals / bioscience

Italy

110

12

36

Mitsubishi Gas Chemicals

Chemicals

Japan

4,680

11

36

Medtronic Vascular

Medical

USA

38,000

11

38

Bruker Daltonik GmbH

Life science

USA

n/a

10

38

Bayer AG

Chemicals

Germany

106,200

10

38

Idemitsu Kosan

Chemicals

Japan

7,500

10

41

Playtex

Consumer goods

USA

n/a

9

438

Adoption of Biopolymers: An Analysis Based on American, Japanese and European Patent Data 42

Toyota Motor Company

Automotive

Japan

316,000

8

43

Cadbury Adams

Consumer goods

Great Britain

55,800

7

43

BASF Corporation

Chemicals

Germany

95,100

7

43

3M

Multitechnology

USA

76,200

7

46

Cargill

Agriculture

USA

158,000

6

46

Dow Chemical

Chemicals

USA

62,500

6

48

DuPont de Nemours

Chemicals

USA

60,000

4

48

Archer Daniels Midland

Agriculture

USA

27,000

4

48

Applied Biosystems

Bioscience

USA

n/a

4

* In the US, granted patents until 2000, and patent applications and granted patents from 2001 onwards

Overall, the private sector possesses the majority of biopolymer patent applications (>90%). The inding is analogue to other studies and research of patent activity, which found that corporations own more than 80% of patent applications [47–49]. Of the top 50 biopolymer patenting irms in 2006, US irms account for 24, Japanese irms for 18, while only 8 European irms were included in the list. Regarding the industrial backgrounds of the top 50 patent assignees, the majority are based in the chemical sector. There are 10 Japanese chemical irms in the list, owning in total 174 biopolymer patent applications compared to 4 US chemical irms, which have 48 patent applications. The results indicate that in biopolymers the Japanese chemical industry currently has a competitive advantage with regard to material technology, especially against the USA. Interestingly, in the agricultural sector, which provides feedstock for biopolymers; the USA dominates with 3 irms and 37 patent applications in the list. This might be an indication that the biopolymer value chain is connected worldwide and no region has a competitive advantage in all technological ields.

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas

Table 14.4 Universities and government institutions ranked by total number of biopolymer patent applications for the priority years 1979–2006* Rank

University/Institution

Country

Total patent families

1

Agency of Industry, Science and Technology

Japan

16

2

Michigan State University

USA

10

3

Korea Institute of Science and Technology

Korea

9

4

University of California

USA

8

5

Industrial Technology Research Institute

Taiwan

6

5

Massachusetts Institute of Technology

USA

6

5

University of Texas

USA

6

8

Battelle Memorial Institute

USA

5

8

University of Minnesota

USA

5

8

U.S. Department of Agriculture

USA

5

11

Kinki University

Japan

4

11

Northeastern University

USA

4

11

National University of Singapore

Singapore

4

14

California Institute of Technology

USA

3

14

Cornell Research Foundation

USA

3

14

Japan Atomic Research Institute

Japan

3

14

National Institute of Advanced Industrial Science and Technology

Japan

3

14

National Institute for Materials Science

Japan

3

14

Max-Planck Gesellschaft zur Förderung der Wissenschaften

Germany

3

14

Columbia University

USA

3

14

University of Connecticut

USA

3

14

University of Massachusetts

USA

3

14

Rice University

USA

3

24

Agrotechnological Research Institute

Netherlands

2

24

National Research Council Canada

Canada

2

* In the US, granted patents until 2000, and patent applications and granted patents from 2001 onwards

440

Adoption of Biopolymers: An Analysis Based on American, Japanese and European Patent Data Only very few patent assignees have a high number of biopolymer patent applications since the technology is very new and irms are confronted with high R&D costs. Halliburton Energy Services own 101 biopolymer patent applications, which correspond to about 10% of all biopolymer patent applications held by the top 50 irms. At the bottom of the list is Applied Biosystems, with 4 biopolymer patent applications, which correspond to about 0.4% of all biopolymer patent applications held by irms in the top 50 list. Such a large spectrum in the list in terms of number of biopolymer patent applications indicates that the industry is still in its infancy and only a few irms dominate the growing market for biopolymers. The relatively low percentage of biopolymer patent applications owned by universities and government institutions (around 10%) suggests that they play a rather minor role in the development of biopolymer technology compared to the private sector. Among this group, most of the patent applications are iled by universities or research laboratories that are located in the United States. This may be the result of US laws (Bayh-Dole-Act) of the 1980s and related legislation that dealt with intellectual property arising from federal government-funded research. It allows US government grants recipients to retain the title to inventions resulting from government-sponsored R&D and generally encourages universities to conduct patenting [47]. Interestingly, Japanese government institutions, such as the Agency of Industry, Science and Technology or the National Institute of Advanced Industrial Science and Technology, focus on biopolymer technology, whereas European or US government institutions do so only a little or not at all. This might indicate that research in biopolymer technology in Japan is steered by public authorities whereas in the United States and Europe it is conducted by universities.

14.5 Conclusion and Future Prospects This paper is a irst exploratory study on the development efforts and diffusion of biopolymer technology. The proliferation of biopolymer technology is a worldwide phenomenon, which has increased rapidly since the early 1980s and with regard to its technological development stage is about to leave the emerging phase in the near future. I focused in my regional assessment of biopolymer patent activity on the United States, Japan and Europe. In general, the identiied dissimilar trends of innovation suggest that speciic regions may have different incentives for pursuing biopolymer technology trends. The study shows that US irms have a competitive advantage in biopolymer technology since they hold the majority of biopolymer patent applications. This fact is not surprising since US irms are often larger in size compared to other regions and irms based inside and outside the United States have different patent strategies. Furthermore, the US government already published a technology roadmap a decade ago deining the future use of plant-based renewable resources in the year 2020 441

Biotechnology in Biopolymers Developments, Applications & Challenging Areas for fulilling a vision to enhance US economic security by gaining independence from imported crude oil [50]. The National Research Council further speciied priorities for research on and commercialisation of biopolymeric products [51]. Biopolymer research activity in Japan and especially in Europe is quite segmented due to different country-speciic environmental laws such as the German Packaging Directive. Therefore, investigation of biopolymer patent activity within Europe could enhance understanding of regional competencies in this area of technology. Biopolymer technology is emerging rapidly, especially in the chemical sector. However, other industrial sectors such as the converter industry are not moving at the same speed towards biopolymer-based processes and manufacturing strategies. Compared to industry, academia and the government sector place much less emphasis on biopolymer research activities. This relects the important role of industry in developing and commercialising this new technology. Although I believe that the biopolymer patent search ilter was well designed, I acknowledge some limitations. Due to the lack of common quality among the searched patent databases and evaluation standards on patent technology, the patent analysis can only represent the general results of each database under its unique patent laws. In addition, the scale of biopolymer innovation activity may not be fully disclosed by patent application counts since different industrial sectors and countries have different intellectual property protection strategies. While this study has provided insights into the overall trends of biopolymer patenting activity, it also points to avenues of future research. For example, there is a high degree of uncertainty on which type of biopolymer is going to dominate the market. This prevailing uncertainty makes irms hesitant to allocate large R&D budgets to a speciic biopolymer since they do not know which biopolymer will be accepted by the market and inally emerge as a ‘dominant design’ [46]. Therefore, researchers as well as managers would beneit from further work exploring the development trends of speciic biopolymers such as polylactic acid via patent analysis. Future research could also focus on the collaboration pattern of assignees, e.g. BASF and Metabolix, to analyse the relationship between collaboration and biopolymer patent activity. At a time when environmental laws are being strengthened worldwide, corporate environmental consciousness is increasing, and public pressure for environmental technologies and fossil fuel scarcity are high, the strong increase in biopolymer technology patenting indicates the important role of these factors in helping this new technology gain acceptance in various industrial sectors. To use the advantages of biopolymers further research and development is required in order to optimise production by increasing the eficiencies of the different chemical and biotechnical processes involved. Substantial scope for improvement is expected in terms of

442

Adoption of Biopolymers: An Analysis Based on American, Japanese and European Patent Data economics of scale and the fact that all biopolymers are in still in their infancy while the productions of petro-based polymers has been optimised for decades.

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Biotechnology in Biopolymers Developments, Applications & Challenging Areas 15. H. Langeveld, M. Meeusen and J. Sanders in The Bio-based Economy: Biofuels, Materials and Chemicals in the Post-oil Era, Earthscan, London, UK, 2010. 16. A.K. Mohanty, M. Misra and G. Hinrichsen, Macromolecular Materials Engineering, 2000, 267, 1. 17. European Bioplastics, 2008, http://www.european-bioplastics.org/. Accessed on 10.10.2008. 18. L. Shen and M.K. Patel, Journal of Polymer and Environment, 2008, 16, 154. 19. M. Crank and M. Patel, Techno-economic Feasibility of Large-scale Production of Bio-based Polymers in Europe, European Commission – Institute for Prospective Technological Studies, Sevilla, 2005. 20. D. Michael, Bioplastics Supply Chains – Implications and Opportunities for Agriculture, Rural Industries Research and Development Corporation, Barton, 2004, p.244. 21. M. Paster, J.L. Pellegrino and T.M. Carole, Industrial Bio-products: Today and Tomorrow, Energetics, Columbia, 2003, p.199. 22. W. Runge, Innovation, Research and Technology Intelligence in the Chemical Industry, Fraunhofer IRB Verlag, Stuttgart, 2006, p.1063. 23. R.P. Wool and X.S. Sun, Bio-based Polymers and Composites, Elsevier, Amsterdam, The Netherlands, 2004, p.640. 24. S. Mojo, Journal of Polymer and Environment, 2007, 15, 289. 25. I. Kyrikou and D. Briassoulis, Journal of Polymer and Environment, 2007, 15, 125. 26. J. Yu, X.L. Lilian and L. Chen, Environmental Science and Technology, 2008, 42, 6961. 27. A.K. Mohanty, M. Misra and L.T. Drzal, Natural Fibres, Biopolymers, and Biocomposites, CRC Press, USA, 2005, p.896. 28. E.T.H. Vink, D.A. Glassner, J.J. Kolstad, R.J. Wooley and R.P. O’Connor, Industrial Biotechnology, 2007, 3, 58. 29. T. Kijchavengkul and R. Auras, Polymer International, 2008, 57, 793.

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Adoption of Biopolymers: An Analysis Based on American, Japanese and European Patent Data 30. D.D. Cornell, Journal of Polymer and Environment, 2007, 15, 295. 31. S. Beucker and F. Marscheider-Weidemann, Zukunftsmarkt Biokunststoffe, Umweltbundesamt, Berlin, Germany, 2007, p.45. 32. C. Bastioli, Handbook of Biodegradable Polymers, 2005, Rapra Technology, Shawbury, UK. 33. R. Gross and B. Kalra, Science, 2002, 297, 803. 34. V. Dornburg, B.G. Hermann and M.K. Patel, Environmental Science and Technology, 2008, 42, 2261. 35. B.G. Hermann, K. Blok and M.K. Patel, Environmental Science and Technology, 2007, 41, 7915. 36. H. Ernst, Small Business Economics, 1997, 9, 361. 37. D. Archibugi and M. Pianta, Technovation, 1996, 16, 451. 38. E. Penrose, The Economics of the International Patent System, Johns Hopkins University Press, Baltimore, USA, 1951, p.247. 39. C.T. Tayler and Z.A. Silbertson, The Economic Impact of the Patent System: A Study of the British Patent System, Cambridge University Press, Cambridge, MA, USA, 1973, p.416. 40. D. Schiffel and C. Kitti, Research Policy, 1978, 7, 324. 41. R. Paci and A. Sassu, Technovation, 1997, 17, 25. 42. K. Debackere, A. Verbeek, M. Luwel and E. Zimmermann, International Journal of Management Reviews, 2002, 4, 213. 43. H. Ernst, Patent information für die strategische Planung von Forschung und Entwicklung, Deutscher Universitäts-Verlag, Wiesbaden, 1996. 44. B.L. Basberg, Research Policy, 1987, 16, 132. 45. E.T.H. Vink, K.R. Rábago, D.A. Glassner, B. Springs, R.P. O’Connor, J. Kolstad and P.R. Gruber, Macromolecular Bioscience, 2004, 4, 551. 46. P. Anderson and L. Tushman, Administration Science Quarterly, 1990, 35, 604.

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446

A

nnexure

Search Filter (biopolymer technology search terms) Keywords for biopolymers based on renewable resources* Polymer/material class

Abbreviation

Polyhydroxyalkanoates

PHA

Polyhydroxybutyrate

PHB

Poly(3-hydroxybutyrate)

P(3HB)

Poly(3-hydroxybutyrate-co-3-hydroxyvalerate)

P(3HB-co-3HV)

Poly(3-hydroxybutyrate-co-4-hydroxybutyrate)

P(3HB-co-4HB)

Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate)

PHBHHx

Polylactic acid and 1. Polybutylene succinate 2. Polybutylene succinate-co-adipate 3. Poly(butylene adipate-co-terephthalate) 4. Polycaprolacton

PLA PBS PBSA PBAT PCL

Input material

Polyesters from biobased intermediates Polytrimethylene terephthalate

PTT

Biobased 1,3-propanediol (bio-PDO)

Polybutylene terephthalate

PBT

Biobased 1,4-butandiol

Polybutylene succinate

PBS

Biobased succinic acid

Starch polymer and 1. Polycaprolactone 2. Poly(butylene adipate-co-terephthalate)

PCL PBAT

Polysaccharide

* Polymers in bold represent a class of biopolymers

447

Biotechnology in Biopolymers Developments, Applications & Challenging Areas Keywords for commercially available biopolymers and their producers Commercial name

Producer

Mater-BI™, Origo-Bi™

Novamont

Solanyl™

Rodenburg Biopolymers

Biopar™

BIOP Biopolymer Technologies AG

Cornpol™

Japan Corn Starch

Placorn™

Nihon Shokuhin Kako

Ecovio™

BASF

Bioplast™

Biotec

Cereplast Hybrid Resins™

Cereplast

Bio-PDOTM (propandiol), Sorona™

DuPont

Bioflex™

FKuR Kunststoff GmbH

Biograde™

FKuR Kunststoff GmbH

Nodax™

Procter & Gamble/Kaneka

Mirel™

Metabolix

Biopol™

Metabolix

NatureWorks Polymer™

Natureworks

Plantic™

Plantic Technologies

Purac™

Purac

Bioplast™

Stanelco

Starpol 2000™

Stanelco

Earthshell™

Earthshell

AgroResin™

Grenidea Technologies

Okopack™

NNZ BV

Hycail™

Hycail

Lacea™

Mitsui Chemicals

Biomer™

Biomer

Eco-Foam™

National Starch

Biogreen™

Mitsubishi Gas

448

A

bbreviations

2-D

Two-dimensional

3-D

Three-dimensional

ABB

Anorganic bovine bone

ABTS

2,2′-Azinobis (3-ethylbenzothiazoline-6-sulfonate)

AFM

Atomic force microscopy

AFNOR

Association Francaise de Normalization

ASAIO

American Society for Artiicial Internal Organs

ASTM

American Society for Testing and Materials

ATP

Adenosine-5′-triphosphate

bBFGF

Basic ibroblast growth factors

BMP

Bone morphogenetic proteins

BPS

Biodegradable Plastics Society

CA

Cellulose acetate

CAC

4,4′-(Adipoyldioxy) dicinnamic acid

CEN

Committee for European Standardization

CF

Cotton ibre

ChEt

Cholesterol esterase

CHIT

Chitosan

451

Biotechnology in Biopolymers Developments, Applications & Challenging Areas ChOx

Cholesterol oxidase

CIP

Cleaning-in-place

CNS

Central nervous system

CNT

Carbon nanotube

CRF

Controlled-release fertiliser

CV

Cyclic voltammetry

DI

Deionised water

DMA

Dynamic mechanical analysis

DMTA

Dynamic mechanical thermal analysis

DNA

Deoxyribonucleic acid

DSC

Differential scanning calorimetry

DTA

Differential thermal analysis

ECM

Extracellular matrix

EIS

Electrochemical impedance spectroscopy

EPA

Environmental Protection Agency

EPI

Environmental Plastics Inc.

EPO

European Patent Ofice

EPS

Extracellular polymer substances

EVOH

Ethylene vinyl alcohol

FAO

Food and Agriculture Organization

FF

Flax ibre

FTIR

Fourier transform infrared

GAG

Glycosaminoglycan

452

Abbreviations GMP

Good manufacturing practices

GOD

Glucose oxidase

GPC

Gel permeation chromatography

HA

Hydroxyapatite

HCl

Hydrochloric acid

HDPE

High-density polyethylene

HEMA

2-hydroxyethyl methacrylate

HIPS

High-impact polystyrene

HPC

Hydroxypropyl cellulose

HRP

Horseradish peroxidase

HTV

High-temperature vulcanizing

HUCB

Human umbilical cord blood

ICI

Imperial Chemical Industries

ICM

Integrated crop management

IDT

Initial decomposition temperature

IL

Ionic liquid

IPC

International Patent Classiication

IPCC

Ionic polymeric conductor composites

IPMC

Ionic polymer metal composites

IPN

Interpenetrating polymer network

IRT

Infrared transmitting

ISO

International Organization for Standardization

ITO

Indium tin oxide 453

Biotechnology in Biopolymers Developments, Applications & Challenging Areas JPO

Japan Patent Ofice

LbL

Layer-by-Layer

LCD

Liquid crystal displays

LDPE

Low-density polyethylene

LED

Light emitting diode

LH

Left-handed

LSR

Liquid silicone rubbers

MAH-PP

Maleated polypropylene

MAPP

Maleated polypropylene

MAS

Magic-angle spinning

MCL

Medium-chain-length

MCP

Metacarpophalangeal

MEMS

Micro-electro-mechanical systems

MMT

Montmorillonite

MPC

2-methacryloyloxyethyl phosphorylcholine

MPEG

Monomethoxypoly(ethylene glycol)

MRDT

Maximum rate of decomposition temperature

MS

Mass spectroscopy

NBCS

Natural bone collagen scaffold

N-MBA

N,N′-methylenebisacrylamide

NMR

Nuclear magnetic resonance

NPCS

N-palmitoyl chitosan

NSC

Neural stem cell

454

Abbreviations ÖNORM

Austrian Standards Institute

OP

Osteogenic protein

ORMOSIL

Organically modiied silicates

PAA

Polyacrylic acid

PAAM

Polyacrylic-acid-bis-acrylamide

PAMPS

Poly(2-acrylamido-2-methyl-1-propane sulfonic) acid

PAN

Polyacrylonitrile

PANI

Polyaniline

PAR

Photosynthetically active radiation

PB

Polymeric biomaterials

PBAT

Poly(butylene adipate-co-terephthalate)

PBO

Poly(benzo[1,2-d:5,4-d] bisoxazole-2,6-diyl-1,4-phenylene)

PBZT

Poly(benzo[1,2-d:4,5-d] bisthiazole-2,6-diyl-1,4-phenylene)

PCL

Polycaprolactone

PDL

Poly-D-lysin

PDMS

Polydimethylsiloxane

PE

Polyethylene

PEA

Polyester amide

PEEA

Poly(ether ester amide)

PEEK

Poly(ether ether ketone)

PEG

Polyethylene glycol

PEI

Polyethylene imine

PEO

Polyethylene oxide 455

Biotechnology in Biopolymers Developments, Applications & Challenging Areas PET

Photo-induced electron transfer

PGA

Polyglycolic acid

PHA

Polyhydroxy acid

PHA

Polyhydroxyalkanoate

PH3B

Poly(3-hydroxybutyrate)

PHB

Polyhydroxybutyrate

PHB-HA

Poly(3-hydroxybutyrate)-hydroxyapatite

PHB/nHA

Poly(3-hydroxybutyrate)/nanohydroxyapatite

PHBV

Poly (3-hydroxybutyrate-co-3-hydroxyvalerate)

PHV

Polyhydroxyvalerate

PLA

Polylactic acid

PLGA

Polylactic-co-glycolic acid

PLLA

Poly-L-lactide acid

PMMA

Poly(methyl methacrylate)

PNEANI

Poly(N-ethylaniline)

PNIPAAm

Poly(N-isopropylacrylamide)

PNS

Peripheral nervous system

POEMA

Poverty and Environment in Amazon

PP

Polypropylene

PPC

Polyester powder coated

PPO

Poly(2,4-dimethyl-1,4-phenylene oxide)

PPy

Polypyrrole

PRP

Platelet rich plasma

456

Abbreviations PSA

Pressure sensitive adhesives

PSS

Polystyrene sulfonate

PTFE

Polytetraluoroethylene

PU

Polyurethane

PVA

Polyvinyl alcohol

PVC

Polyvinyl chloride

PVMQ

Phenyl vinyl methyl silicone

PVP

Polyvinylpyrrolidone

R&D

Research and development

REDOX

Reduction-oxidation

RGD

Arg-Gly-Asp

RH

Relative humidity

RHDPE

Recycled high-density polyethylene

RLC

Reactive layer coating

RNA

Ribonucleic acid

RTV

Room-temperature vulcanizing

SBF

Simulated body luid

SCAS

Semicontinuous activated-sludge

SCL

Short-chain-length

SEC

Size exclusion chromatography

SELP

Silk-elastin-like polymers

SEM

Scanning electron microscopy

SIC

Strain-induced crystallisation 457

Biotechnology in Biopolymers Developments, Applications & Challenging Areas SIP

Sterilisation-in-place

SIS

Small intestinal submucosa

SMP

Soluble microbiological products

SPI

Society of the Plastics Industry

SWCNT

Single-walled carbon nanotube

TDDS

Transdermal drug delivery systems

TDPA

Totally degradable plastic additives

TEM

Transmission electron microscopy

TEOS

Tetraethoxysilane

TESPT

(Triethoxysilylpropyl)tetrasulide

Tg

Glass transition temperature

TGA

Thermogravimetric analysis

UDT

Ultimate decomposition temperature

UNI

Ente Nazionale Italiano di Uniicazione

UP

Unsaturated polyester

USPTO

United States Patent and Trademark Ofice

VMQ

Vinyl methyl siloxane

WPINDEX

Derwent World Patents Index

XPS

X-ray photoelectron spectroscopy

XRD

X-ray diffraction

458

I

ndex

A Abrasion 254, 342 Absorb 55, 126, 157, 195, 197, 211, 225, 244, 338, 410 Absorption 18-19, 21-22, 78, 93, 110, 136, 199, 201, 225, 236, 335, 338, 340, 346 Acceptor 162 Accident 249, 326 Account 302, 414, 439 Acid 12, 16-17, 23-25, 42-43, 46, 48, 59, 77, 128-129, 131-134, 138-140, 143, 156, 161, 164-166, 169, 177, 179, 183, 198-201, 203-205, 207-208, 213, 234, 236-237, 239, 241, 247, 251, 253-254, 256-257, 259, 263, 287-289, 293, 317, 331-333, 335, 338, 372, 395, 402, 414, 427-428, 430, 432, 442, 447, 449-451, 453-455 Acrylic 89, 331-333, 403 Acrylonitrile 169 Activation 42-43, 48, 69, 131, 141-142, 157, 170, 297, 322, 337, 339, 352 energy 42-43, 142 Addition reaction 320 Additive 8, 51, 65, 121, 140, 143, 169, 178, 188-189, 381, 404, 417, 426-427, 456 Adduct 132 Adhesion 5-6, 93-94, 99, 114, 134, 136-138, 166, 240-242, 244, 255, 263, 328, 332, 335, 344, 380, 398 Adhesive 78, 142, 255, 265, 319, 322, 352, 355, 368, 380-381, 398 Adipose tissue 77 Adsorption 141-142, 242, 331, 335, 338, 344, 352-353 Afinity 5, 100, 114, 117, 218, 224-225, 338-339, 364 Ageing 48, 60, 70-73, 157, 262, 349, 405 Agency 104, 177, 440-441, 443, 450 Agent 19, 70, 78, 94, 99, 105-106, 110-111, 124, 127, 130-131, 133, 143, 145, 169, 203, 213, 288, 295-296, 320, 355

459

Biotechnology in Biopolymers Developments, Applications & Challenging Areas Agglomeration 93, 178 Aggregation 329, 339 Agriculture 7, 13-14, 81, 83, 89, 91, 93, 103, 112, 175, 183, 195, 395, 397, 399, 401-405, 407, 409-411, 413, 415-419, 421-423, 427, 435, 437, 439-440, 444, 446, 450 Alignment 58, 69, 428 Aliphatic 77, 124, 129, 132, 141, 144, 164 Alkali 90, 99, 102, 141, 143, 155, 159, 164, 166, 193 Allergy 184 Aluminium 23, 195, 336 Amorphous 1, 6, 23-24, 28-30, 45, 49, 51, 56, 58, 72-74, 123-127, 129, 131-133, 135, 137, 139, 141-145, 147, 149, 151, 165, 167, 173, 179, 194, 375, 396 polymers 58 Anaerobic 129, 140, 162-163, 173, 179, 402 Analogue 74, 198, 263, 265, 439 Analysis 1-3, 15, 17-25, 27, 29, 31, 33, 35, 37, 39, 41-43, 45-49, 51-54, 121, 131, 134, 148, 171-173, 186-187, 199, 201, 225, 255, 263, 295, 333, 339, 350, 354, 424-435, 437, 439, 441-443, 445, 450, 456 Analyte 201-202, 219, 224 Anhydrous 143 Anion 43, 158, 200, 291, 288, 305 Anode 200-201, 296-297 Anti-inlammatory 184 Antibacterial 59, 263, 265 Antimicrobial activity 128, 265 Antioxidant 3, 6, 56, 124, 133, 141, 144, 169 Antistatic properties 427 Apolar 132 Appearance 45, 70, 96, 126, 196, 216 Application 6, 8, 20, 70, 72, 100, 103, 131, 144, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225, 227, 229, 231, 233235, 241-243, 246, 250-251, 260, 262, 265, 269, 309, 323, 334, 338, 340-341, 355-356, 379, 381, 384, 386, 402, 411-412, 416, 418, 426, 430, 437, 442 Applied stress 64 Aqueous 3-4, 18, 55, 59, 74-75, 166-167, 183, 207, 212, 293, 340, 351 solution 4, 18, 183, 207, 293 Aromatic 46, 124, 128-130, 132, 136, 142-143, 145, 161 Array 219, 330 Arthritis 341, 366

460

Index Aspect ratio 98 Assay 128 Assembly 39, 204, 239, 247-248, 266, 353, 381, 384, 386 Assessment 86, 90, 115, 418, 429, 441 Association 46, 166, 183, 288, 336, 348, 365, 368, 376, 423, 449 Asymmetric 21, 46 Atmosphere 42-43, 45, 104, 137, 179, 331, 415 Atom transfer radical polymerisation 43 Atomic force microscopy 172, 295, 449 Attachment 10, 216, 239, 243, 333, 351, 382-383 Autocatalytic 167 Autoclave 340, 347

B Backbone 12, 42, 44, 51, 59, 68, 170, 216, 244, 290, 309-313, 319, 322, 343 Bacteria 12, 14, 59, 128, 130, 144, 161, 164-166, 179, 202, 236, 263, 345, 385, 396-398, 401-402, 414, 417 Balance 1, 39-40, 78, 110, 153, 349, 396 Ball 339-340, 343, 407 Band 20-21, 215 Bar 4, 258, 267-268, 315 Bark 97, 102, 124 Barrier 46, 262, 349, 351, 357, 414 Base 16, 116, 213, 247-248, 319, 328, 357, 396 Bead 169, 292 Beam 27, 131, 327, 329 Bearing 91, 290, 341, 357, 402 Bending 19-21, 70, 108, 214, 296-298, 301-302, 432 Binary 56, 133, 135 Binding 105, 199, 216, 218-220, 253, 372, 382 Bioactivity 233, 242, 335, 354 Biobased 3, 8, 17, 37, 76, 91, 179, 418, 446-447 Biocompatible 12, 23, 43, 56, 158, 166, 241, 243, 248, 251, 323, 328, 333, 336337, 364, 371, 382-384 Biodegradable 1, 3, 5-7, 12, 14, 16-17, 24, 37, 42, 46, 55, 58-59, 76-77, 91, 93, 98, 108-109, 123, 114, 133, 138, 144, 153-155, 157-159, 161, 163, 165-171, 173, 175-177, 179-181, 183-185, 187-191, 193, 196, 209, 236, 240-242, 251, 253-254, 257, 267, 344, 371, 378-379, 383, 395-396, 400-404, 407, 409, 414, 416-420, 425-426,128, 431, 443, 445, 449

461

Biotechnology in Biopolymers Developments, Applications & Challenging Areas Biological 7-8, 18, 32, 37, 49, 66, 70, 103, 126-128, 154-155, 161, 193, 195-196, 199, 218-220, 226, 231, 233-235, 242, 244, 247-249, 251, 253, 263, 268-269, 271, 274, 276, 323, 326-327, 336, 338-339, 350, 354, 358, 362-363, 371, 383, 387, 390, 392, 397, 401, 417 activity 161, 251 properties 7, 155, 193, 263, 268, 338, 358 Biomass 5, 103, 112, 114, 117, 148, 163, 179, 398, 417 Biomaterial 8, 12, 17, 29, 31, 80, 167, 173, 190, 208, 241-243, 247, 250-251, 253, 259, 265, 323-324, 326-327, 337-339, 344, 359-361, 363, 379-382, 386 Biomimetic 8, 87, 142, 226, 233-235, 238, 252, 287-288 Blend 7-8, 16, 31, 37, 45, 49, 56, 105, 132-134, 136, 141, 144, 154, 206-208, 226, 242, 292, 327, 355, 358, 395-396, 400-401 Blistering 398 Block 43, 45, 77, 169, 202, 237, 278, 378, 380, 395 copolymer 43, 45, 237 Blood pressure 238 Board 141, 323 Boiling 238, 292-293, 378 Bond 21, 25, 60, 66, 68-69, 128, 132, 134, 138, 161, 165, 170, 214, 310-311, 322, 330, 338, 377, 405 Bonding 1, 19, 23, 42, 51, 69, 195, 216, 243, 247, 337, 354 Bone 59, 77-78, 155, 190, 209, 238, 240, 242, 252-254, 272, 274-275, 279, 325, 337, 341-342, 354, 365, 384, 449, 452 Bottle 171, 236 Box 235, 248 Branch 3, 51, 55, 58, 159, 317 Breakage 68, 161, 249 Breaking 4, 60, 161, 170, 195, 398 Bridge 6, 94, 367 Brittle 57, 70-72, 111, 132, 157-158, 165, 235 Buffer 204, 212-213, 218, 220, 223, 381 solution 213, 218 Building 55, 91, 100, 104, 110, 138, 150, 219, 234-235, 238, 248, 435 Bulk 1, 55, 60, 69-70, 161, 167, 200-201, 242, 288, 326-328, 332, 337, 345, 355, 357, 399, 403 By-product 100, 221

C Calorimetry 2, 44, 52-54, 132, 134, 148, 187, 450

462

Index Can 2, 4-6, 8-13, 16, 18-19, 24, 28, 39, 42, 49, 58-59, 61, 64-66, 70, 72, 75-78, 89-90, 92-97, 100, 105-106, 112, 114, 117, 124, 130-132, 138-139, 144-145, 153, 156-162, 164-172, 174-175, 178-180, 183-184, 194-196, 198-199, 202, 206, 208, 212-214, 218-221, 223-225, 233, 235-245, 247-248, 250-251, 253254, 259-260, 263, 265-267, 269, 287-292, 294-297, 301-305, 317-318, 320, 323, 325-335, 337-341, 344-346, 348, 351, 357-358, 373, 376, 378-379, 381383, 386, 395-398, 402, 404, 407, 409-417, 425-428, 431, 442 Capacity 59, 73, 126, 129, 142, 175, 209, 262, 346, 413, 418 Capillary 351, 412 electrophoresis 351 Carbohydrate polymer 80, 150, 226-227, 229, 231 Carbon nanotubes 85, 137, 139, 155-156, 208, 217, 287, 289, 306 Carboxylic group 21 Carrageenan 20, 45 Carrier 241, 267, 285, 355, 357 Case 5, 12, 19, 23, 27-28, 45, 48, 72, 75, 90, 93-94, 96, 131-133, 137, 155, 199, 201, 220-221, 243-244, 249, 263, 327, 378, 398-399 Casting 132-133, 137, 144, 166, 207, 245, 341 Catalysis 187, 359 Catalyst 310, 317, 319-320 Cathodic 213, 221-222 Cation 158, 199-200, 205-206, 288, 291, 303-305 Cationic 28, 255, 262, 288 Cavity 68, 344, 346 Cell 9-10, 64, 78, 81, 103, 124, 126, 156, 162, 203-204, 218, 233-234, 237, 239244, 251, 253-255, 257-262, 265-270, 275, 278, 332-333, 335-339, 350-353, 368, 372, 380-383, 385, 387, 402, 452 growth 233-234, 239, 242, 244, 254, 351, 382-383 proliferation 78, 243, 255 viability 333 wall 126 Cellophane 414 Cellular 162, 233, 242-244, 249, 251, 263, 301, 337, 351-352, 388, 402 Cellulosic 106 Centrifugation 92 Ceramic 185, 368 Chain 1, 38, 46, 50-51, 55, 58, 60, 66-69, 75, 126, 129, 157-159, 162, 166, 170, 172, 174, 179, 202, 236-237, 239-240, 248, 309-312, 316-319, 322, 331, 335, 352, 376-377, 380, 396, 405, 414, 427, 439, 452, 455

463

Biotechnology in Biopolymers Developments, Applications & Challenging Areas Chain end 67 extension 46 length 50, 172, 179 segment 69 Chamber 212, 266, 330 Channel 219 Characterisation 18, 22, 24, 27, 99, 102, 119, 399 Charge transfer 218, 223 Chemical 3, 5-6, 9-10, 12, 15, 18, 24-27, 32, 34-35, 42, 45, 50-51, 54, 56-58, 60, 65-66, 69-70, 75-76, 79, 83-84, 90, 94, 96, 105, 107, 109, 117, 121, 123-127, 130-131, 134, 139-141, 143-145, 154-155, 159, 161, 163-165, 167-169, 181, 186, 188-189, 193-195, 202-203, 206, 208, 212, 218, 220, 227-229, 231, 233234, 236, 238-239, 241, 243, 245, 254, 269-271, 273, 275, 289, 292-294, 311, 322, 326-328, 330, 335, 340, 350-351, 355, 358-359, 363, 367, 380-381, 391392, 396-397, 402-403, 407, 411, 419, 425, 427, 434-439, 442, 444, 448, 451 attack 12, 403 bond 25, 69 composition 50, 76, 163-165, 218 environment 328 industry 15, 425, 439, 444 modiication 6, 27, 94, 107, 130, 134, 330 properties 6, 56, 75, 131, 168, 194, 241, 254, 322, 328, 380 reaction 330 shift 26 stability 206, 289, 326 structure 51, 60, 105, 124-125, 188, 193, 236, 238-239, 243, 311, 340, 396 Chemistry 6, 25, 31, 33-34, 53-54, 69, 79-81, 83-85, 87, 123-124, 146, 149-150, 184, 187-190, 203, 227-231, 241-242, 273, 276, 280, 306-307, 309-310, 312, 328, 330, 358-364, 367-368, 378, 385-386, 390, 418-419, 425, 434-436 Chip 351, 367 Chitosan 8-11, 21, 23, 28-29, 42, 45, 48, 59, 66, 74-75, 169, 193, 195, 197, 199, 201, 203, 205, 207, 209, 211, 213, 215, 217, 219, 221, 223, 225-227, 229, 231, 236, 251, 253-255, 257, 263, 266, 287, 290-292, 296-297, 449, 452 Chlorinated 128 Chromatography 172, 451, 455 Chromogen 201-202 Chromophore 170 Circular 266, 382 Classiication 57, 96, 154, 426, 430, 434-435, 451

464

Index Clay 7, 19, 155-156 Clean 104, 124, 177, 181, 249 Clear 25, 28, 70, 174, 269, 298, 357, 378, 409-410, 418 Cleavage 66, 68, 129, 131, 144, 157, 159 CNT 207-208, 220, 450 Coagulation 326, 344 Coated 141, 164, 199, 204, 208, 259, 266, 333, 338, 344, 352-353, 355, 357, 379, 382, 384, 398, 412-414, 454 ilm 355 Coating 9, 48, 50, 55, 78, 114, 137, 210, 212, 327-328, 332, 338, 344-345, 351, 355-358, 364, 382, 398, 412-414, 455 Coding 178, 180 Coeficient 51, 220, 225, 297-301, 329 Coil 21, 29, 45-46, 67-68, 72, 237, 291 Colloid 34, 188, 361 Colour 8, 14, 70, 126, 142, 144, 159, 172, 195, 201-202, 225, 354, 375, 403, 410 Column 199, 264, 400 Combustion 159 Compatibiliser 136-137 Compatible 3, 6, 8, 31, 49, 55-56, 93-94, 105, 114, 212, 251, 226, 326, 334-335, 337, 343, 351, 357-358, 391, 414 Complex 23, 27, 43, 45-46, 55, 58, 66, 77, 162, 166, 209, 212, 220, 223, 233, 235, 237, 241, 245, 247, 262-263, 265-266, 290, 293, 315, 349, 376, 378, 414 Compliance 2, 65, 355 Component 28, 48, 62, 78, 91, 137, 165, 173, 193, 197, 209, 233-235, 238, 247, 253-254, 260, 265, 316, 319, 338, 355, 414 Composite 5-8, 10, 48, 59, 78, 90, 93-94, 99, 105, 109-110, 113, 115, 118-120, 122-123, 127-128, 136-138, 143-145, 149, 154, 156, 207-208, 210, 220, 226, 253, 287, 289, 291, 293, 295, 297, 299, 301, 303, 305, 307, 351, 376, 395, 397, 417 Composition 18, 23, 50, 72, 76, 116, 163-165, 196, 199, 207, 211, 218, 252, 315, 340, 395, 413 Compound 3, 27, 39, 56, 121, 127-128, 131, 136, 143, 158, 170, 250, 317, 319, 402, 413 Compression 5, 92-93, 105, 112-113, 322 moulding 92, 105 set 322 stress 253

465

Biotechnology in Biopolymers Developments, Applications & Challenging Areas Computer 333, 349 Concentration 28, 46, 69, 76, 105, 124, 155-156, 169, 174, 183, 196, 198-201, 208, 221, 225, 243, 263, 300-305, 341, 352, 357, 384, 412, 414, 432-433 Condensation 40, 131-132, 207, 319 reaction 207 Conduction 219-220 Conductivity 134, 196, 207-208, 210-211, 219, 288-289 Conformational 8, 20, 24, 27, 29, 43, 67-69, 194, 218, 224, 382 Construction 91, 100, 104-105, 110, 150, 177, 184, 217, 244, 344, 355, 373, 378, 410, 416 Consumer 5, 114, 117, 410, 437-439 Consumption 37, 76, 91, 112, 172-173, 225, 414, 416 Contact angle 137, 141 Container 403, 407-408 Contamination 51, 110, 181, 183, 346, 355, 416, 418 Continuous 7, 55, 155, 246, 322, 331 Contract 194, 368 Contraction 289, 301-302 Contrast 24, 64, 74, 167, 179, 234, 253-254, 289, 340, 352, 378 Conversion 57-58, 140, 432 Cooling 45, 157, 178, 404 rate 45 Copolymerisation 8, 169, 194, 202, 205-206, 226, 242, 322, 333, 355 Copper 141, 195, 315 Core 112, 142, 167, 210, 243, 268, 355, 375, 413 Core-shell 142 Correlation 188, 225 Corrosion resistance 8, 14, 404 Cosmetics 195, 234 Cost 1, 5, 14-15, 17, 89, 98, 114, 117, 142, 175-176, 179, 193, 212, 221, 323, 325, 347, 351, 354-355, 385, 403, 411, 417, 425, 428, 441 Cotton 43, 89, 92, 96-97, 107-108, 116, 121, 138-139, 235, 449 Coupling 6, 18, 25, 66, 94-95, 99, 105-106, 110-111, 124, 133, 213, 302, 321, 335, 350 Cover 59, 195, 263, 323, 351, 375, 398-399, 406, 409 Crack 69-71, 74, 90, 157, 167 Crazing 69-70 Creep 61, 64, 175 Crosslinking 60, 70, 320

466

Index agent 70, 320 Crude oil 143, 425, 442 Crushing 104 Crystal 27, 70, 74, 77, 137, 165, 173, 195, 218, 230, 330, 452 Crystalline 1, 6, 24, 27, 30, 44, 51, 56, 59, 72, 74, 123, 134, 148, 157, 165, 167, 173, 179, 194, 259, 368, 378, 380, 396, 402 phase 402 polymers 51 structure 30, 44, 157 Crystallinity 24, 27, 29, 45, 48, 58-59, 70, 72, 134, 137, 144, 165, 382, 396 Crystallisation 27, 44-47, 72, 74, 165, 322, 455 Crystallite 27, 72, 74 Culture 233, 237, 244, 251, 255, 258, 263, 265, 268, 325, 336, 352, 382-383, 386, 397-398, 404 Curable 317, 320 Curing 2, 41, 44, 48, 92, 95, 137, 317-321, 323, 348 reaction 318, 320 Current 10, 64, 81, 85, 187, 191, 200-201, 206, 213-214, 218, 220-224, 228-229, 250, 254, 257, 271, 281, 285, 297, 301-302, 305, 371, 393, 407, 417-420 Curvature 243, 248, 298 Cutting 110, 178 Cyclic 43, 48, 213-214, 219, 222, 315-316, 339, 341, 450 Cylindrical 63 Cytotoxicity 77, 242, 343

D Damage 5, 66, 70, 117, 153, 157, 317, 324, 337, 346 Damping 2, 48-49, 102 Data 20-21, 39, 63, 70, 198, 255, 299, 339, 425, 427-431, 433, 435, 437, 439, 441, 443, 445 Debonding 397 Decay 46, 94 Decomposition 2, 39-43, 45, 51, 112, 133-134, 142, 177, 340, 451-452, 456 Defect 77, 240 Deformation 2, 48, 58, 60-61, 63, 65-66, 69-72, 74, 76, 167, 298, 339 Degradation 1, 3, 5, 7, 12, 16, 32, 38-39, 41-44, 50-53, 56, 58-59, 66, 80, 90, 92-93, 95, 112, 128-131, 133, 137, 139, 142, 144, 146-148, 153-179, 183-188, 191, 235-236, 242, 253-254, 269, 340, 347, 395-400, 402, 407, 417, 420, 423 Degree of crystallinity 27, 58, 382 Degree of swelling 244

467

Biotechnology in Biopolymers Developments, Applications & Challenging Areas Dehydration 48, 235, 357 Dehydrogenation 124, 127 Deionised 74, 204, 212, 450 Demand 37, 91, 123, 145, 234, 396, 412, 414, 424 Density 5, 10, 13, 51, 58, 70, 74, 91-92, 96-98, 105, 112, 117, 133-135, 137, 175, 180, 236, 255, 266, 288, 292, 297, 302-303, 305, 323, 352, 403, 405, 407, 414, 451-452, 455 Depolymerisation 112, 129, 158, 162, 174, 317, 340 Deposition 171, 242-243, 252, 336, 340, 344, 353-354, 398 Depth 1, 434 Derivative 39-40, 59, 126, 132-133, 135, 144, 236 Design 1, 5, 12, 57, 90-91, 114, 149, 202, 233-234, 239, 242-244, 249, 251, 265, 309, 323, 325, 328, 342, 346, 381, 395, 418, 432, 442 Deterioration 8, 38, 161, 340, 404 Determination 2, 18, 24, 34, 48, 199, 201, 299 Development 1-2, 8, 15, 17, 23, 30, 58, 76, 90, 108, 112, 170, 183, 186-187, 196, 201, 212, 217, 226, 234, 238, 241, 243, 245, 247, 249-253, 255, 257, 263, 265, 279, 309, 324, 326, 328, 344, 348, 369, 371, 375, 397, 400, 404, 407, 410, 412, 414, 416-419, 425-426, 428-429, 432, 441-442, 444, 455 Diameter 4, 96, 218, 223, 248, 256 Die 121, 171, 404, 422, 445 Dielectric 86, 210, 290, 309, 322 Differential scanning calorimetry 2, 44, 53, 132, 134, 450 thermal analysis 46-47, 450 Differentiation 233, 243, 251, 253, 255, 257-258, 266, 351-352, 382-383 Diffraction 1, 27-29, 35, 337, 382, 456 Diffusion 41, 166, 197, 199, 201-202, 208, 220-221, 243, 295, 299-302, 304, 351, 355, 357, 413, 441 Difunctional 317 Dimension 8, 155, 302 Diode 77, 452 Directive 15, 424, 442 Discontinuous 8, 155, 194 Disease 112, 127, 234, 250, 256, 259, 262, 345, 348, 411 Dispersion 55, 143, 155, 295-296, 321, 351, 356 Displacement 24, 61 Display 51, 55, 60-61, 198, 217, 241, 336 Dissipation 322

468

Index Dissociation 38, 42 Dissolution 356 Dissolving 244 Distortion 177 Distribution 47, 93, 134, 138, 140, 197, 256, 293, 301, 351, 406, 435 Disubstituted 312-313 DMA 2, 48, 450 Domain 140, 380-381 Doping 77, 203, 207-208, 213, 219 Dosage 133, 356, 419 Double bond 25 Drawn 8, 39-40, 112, 141, 194, 330, 404 Dried 71, 114, 173, 212, 244, 250 Drug 2, 12, 17-18, 31, 59, 66, 78, 177, 193, 195, 228, 244, 250, 324-326, 354357, 369, 371, 381-382, 384-385, 389, 392, 402, 434, 456 Drying 56, 70-71, 109, 157, 245-246, 261, 403 DSC 2, 43-46, 51, 72, 450 DTA 46-48, 450 Ductility 57, 70, 159 Dumbbell 67-68 DuPont 291, 439, 448 Durability 90, 153, 166, 260, 339-340, 343 Dye 143, 201-202, 421 Dynamic 2, 18, 24, 43, 48-49, 54, 67, 75, 94, 183, 200, 242, 290, 302, 304-305, 322, 339-340, 384, 450 mechanical analysis 2, 48, 54, 450 mechanical thermal analysis 43, 48-49, 450

E Edible 116, 414-415, 419, 423 Eficiency 91, 93, 104, 142, 195, 198, 206, 255, 263, 309, 315, 411-413 Elastic 3, 27, 48-50, 56, 58, 60-61, 63-65, 72, 75-76, 115, 254, 260, 298, 326, 355, 384 modulus 3, 48, 50, 56, 58, 115, 384 Elasticity 50, 64-65, 72, 108, 245, 265, 372, 375, 381 Elastomer 58, 167-168, 311, 317, 320, 322-323, 326, 133, 336-341, 343-344, 346-348, 355, 357-358, 403 Electric 8, 10-11, 43, 57, 66, 76, 194-197, 199, 207-210, 212, 219, 223, 226, 288-289, 296-303, 305-306, 329, 341, 350, 403, 407, 418, 435-436 Electrically conducting polymers 288 469

Biotechnology in Biopolymers Developments, Applications & Challenging Areas Electrode 9, 77, 138, 199-201, 204, 212-214, 216, 218-225, 292-293, 295, 351 Electrolyte 140 Electron 27, 77, 131, 134, 137, 158, 161-162, 209, 213-214, 218-221, 223-224, 267, 320, 327, 337, 359, 397-400, 420, 454-456 Electronic 24, 77, 86, 93, 197, 206, 213, 219, 239 Electrostatic 216, 289, 353 Elongation 68, 72, 95-96, 100, 105, 138, 399 at break 96, 105, 399 Embedded 196, 257 Emission 329 Emulsion 144, 384 Enamel 238 Encapsulate 177, 242, 249, 253, 259, 265, 319, 349, 383 Encoding 12, 373, 380, 385 End group 165, 213 End point 39 Endothermic 44-46, 72 Energy 4-6, 8, 22, 27, 37, 42-43, 48, 57, 76, 90-91, 93, 98, 124, 141-145, 157, 162-163, 173, 179, 181, 194, 226, 235, 240, 287, 289, 311, 320, 322, 327-328, 332, 375, 397-398, 404, 410, 418, 437, 441, 443, 446 consumption 37 source 235, 398 Engineering 5, 8-9, 12, 15, 17, 31, 54, 73, 77-81, 83, 85, 87-88, 90, 92, 95, 103, 106, 115, 118, 120-122, 149-150, 166, 182, 189-191, 226, 228, 233-237, 239, 241-257, 259-267, 269-273, 275-277, 279-285, 309-311, 313, 315, 317, 319, 321, 323-331, 333, 335, 337, 339, 341, 343, 345, 347, 349, 351, 353, 355, 357-361, 363, 365, 367, 369, 373, 378-380, 382-384, 386, 392, 397, 417, 421, 428, 434-435, 438, 444 Enhancement 224, 348 Enthalpy 46-47, 70-73, 198 Environmental 1, 3, 5-7, 15, 17, 37-38, 54, 59, 75-76, 90, 104, 111, 116-117, 138, 140, 146, 156, 163-164, 169, 172, 175, 177, 181, 185, 187-188, 191, 195, 234, 267, 340, 350, 387, 395-397, 403, 405, 412-414, 416-417, 423-424, 428, 442, 444-445, 450 protection 37, 104, 146, 177, 450 Enzyme 124, 128-129, 131, 134, 143, 147, 159, 170, 188, 198-199, 201, 212216, 218-219, 221, 224-225, 235-236, 244, 248, 351 Epithelial cell 333 Epoxy resin 93, 398

470

Index Equation 38, 61-63, 65, 94, 174, 221, 225, 297-298, 300-306, 321 Equilibrium 61-64, 67, 72, 201, 293, 300, 302 Equipment 5, 47-48, 91, 117, 123, 171, 175, 195, 317, 320, 416, 418, 437 Esteriication 95, 99, 135, 137, 139, 141, 194 Etching 327, 330, 336 Ether ester 159, 453 Etheriication 95, 194 Ethylene 16, 133, 135-137, 139, 141, 169, 235, 395, 450, 452 Evaluation 141, 202, 337, 340, 364, 442 Evaporation 22, 40, 43, 133, 409, 411 EVOH 169, 450 Exclusion 172, 431, 455 Exothermic 44, 48, 198-199, 315 Expansion 51, 301-302 Export 98, 106, 416 Exposure 157, 175, 247 Expression 12-13, 179, 238, 254, 266, 385, 388 Extension 46, 259 Extraction 100, 103, 111, 136, 143, 250, 377 Extrusion 5, 38, 92-93, 132, 136, 143-144, 320, 344, 427 Exudate 372

F Fabrication 9, 22, 29, 77, 93, 193, 202, 209, 212, 218, 234, 239, 244, 247, 249250, 261, 266, 287, 290, 323, 335, 344, 350, 371 Facing 77, 406 Failure 38, 65, 70-71, 77, 259, 281, 340 Fan 274, 276, 389 Fatigue resistance 5, 90, 341 Feed 58, 146, 234, 372 Feedstock 37, 418, 425, 428, 434, 439 Fertiliser 140, 143, 145, 407, 412-414, 450 Fibre 3-6, 12, 17, 29, 43, 76, 89, 91-117, 131, 138-139, 141, 161, 245, 381, 385, 397-398, 403, 408, 449-450 Fibroblast 10, 262-263, 265, 338, 343, 383, 449 Fibrous 23, 103, 242, 254, 375 Filament 4, 92, 371 Filled 18, 177, 348, 414 Filler 6-7, 124, 133, 136-138, 141-142, 144, 155, 170, 321

471

Biotechnology in Biopolymers Developments, Applications & Challenging Areas Film 13-14, 16, 27-28, 43, 45, 50, 55, 60, 70, 108-109, 132-134, 136-137, 140, 142, 149, 166, 176, 193, 207-208, 210-212, 214, 224, 257, 323, 325, 334-336, 343, 351, 353-356, 382-383, 395, 399-400, 403-404, 406-407, 409, 414-415, 417, 419, 423, 437 thickness 353 Filter 10, 325, 431, 442, 447 Filtration 183 Fish 28, 404 Flax 89, 92, 96-97, 108, 116, 133, 135, 137, 395, 450 Flexibility 1, 8, 14, 98, 136, 169, 179, 244, 248, 309, 311, 325, 341, 346, 357, 372, 396, 403, 412 Flexural modulus 98, 137 Flow 40, 44, 49, 60, 64-68, 72, 96, 133, 143, 243-244, 302, 342, 345, 351, 411 Fluid 3, 63, 66, 166, 236, 243-244, 262, 326, 329, 337, 346, 354-355, 384, 455 Fluorescence 157, 248, 351 Flux 10, 297, 301 Foam 7, 134, 448 Focus 8, 12, 16, 131, 136, 140, 145, 196, 262, 395, 425, 428, 433, 437, 441-442 Fold 67, 238, 250 Food 3, 7, 12-14, 33, 53, 55, 59, 61-62, 66, 76, 78-83, 85, 93, 98, 111-112, 116, 136, 149-150, 175, 181, 193, 195, 202, 234, 350, 371, 375, 395, 397, 399, 401, 403, 405, 407, 409, 411, 413-419, 421, 423, 436, 450 industry 78, 112 packaging 14, 136, 175, 181, 193, 414, 417 Force 4, 61, 66-70, 76, 172, 195, 295, 301, 353, 449 Foreign 167, 267, 416, 431 Forestry 435 Formaldehyde resin 134-135, 137, 139, 142-143, 395 Formation 25, 28, 42, 45-46, 49, 56, 69-70, 75, 132, 142-143, 146, 158, 161162, 165-167, 169, 172, 174, 178, 205, 207, 210, 215, 220, 223, 235, 244, 246, 258-260, 288, 293, 310, 323-324, 343, 349, 351, 354-355, 372, 381, 384385, 414 Forming 6, 126, 140, 193, 250, 354, 372, 375 Formula 173, 311 Formulation 50, 142, 296, 298, 401 Fossil fuel 58, 442 Foundation 275, 347-348, 440 Fourier transform 18, 131, 142, 214, 450 Fraction 134, 289

472

Index Fractionation 138 Fracture 71, 76, 90, 341-342 Fragmentation 129, 145, 156 Frame 372, 407 Framework 158, 160, 163, 372, 406 Free energy 332 radical 23, 43, 127, 141, 331 radical polymerisation 23, 43, 331 volume 46, 355 Frequency 2, 22, 48-49, 58, 75, 160, 197, 223, 299 Friction 329, 341, 344 Frozen 72, 245, 262 Fruit 91, 97, 100, 111-112, 124, 406-407, 412 FTIR 1-2, 18-23, 30, 40, 42-43, 208, 384, 450 spectra 2, 19, 208 spectroscopy 20, 22, 43 Fuel 58, 91, 104, 120, 143, 425, 442 Functional group 58, 331 polymer 227 Functionalisation 8, 156, 219, 226, 330, 338, 355 Functionality 6, 94, 242, 247, 259-260, 262, 265, 317, 319, 376 Fungi 28, 124, 128-129, 144, 161, 164-166, 173, 193, 236, 379, 396, 398 Furnace 39-40, 46 Fusion 13, 44-45, 47, 382, 419

G Gap 15, 209, 269, 309 Gas 2, 37, 39-40, 55, 79, 90, 95, 136, 145, 173, 179, 199, 221, 245, 301, 327, 329, 344, 355, 357, 403, 415, 428, 438, 448 Gaseous 40-42, 140, 246, 335 Gel 3, 45, 56, 58, 61-64, 68, 172, 195, 207, 237, 243, 245, 247, 255, 260, 266, 287, 290, 292, 296, 348, 368, 451 permeation chromatography 172, 451 Gelation 20, 25, 46, 245 Gene 12-13, 238, 379-381, 384-385 Geometry 66, 322, 384

473

Biotechnology in Biopolymers Developments, Applications & Challenging Areas Glass 7, 39, 44-45, 47-49, 56, 60, 72-73, 91, 96-97, 101, 106, 110, 114-115, 132, 134, 136-137, 155, 158, 161, 164, 199, 204, 212, 249, 288, 292, 322, 326, 351, 398, 403-404, 418, 456 ibre 91, 110, 114, 398 transition temperature 39, 44, 49, 56, 60, 72, 132, 134, 136-137, 158, 164, 322, 456 Glassy 49, 60, 72-73, 79 Glazing 407 Glue 134 Gold 195, 291, 293 Government 15, 86, 386, 428, 437, 440-442 GPC 172, 451 Grade 14, 406, 418 Gradient 10, 67-68, 201, 255, 297-298, 300-301, 329, 357 Graft 8, 17, 78, 95, 131, 169, 194, 196, 202, 206, 226, 240, 249, 253-254, 259, 262-263, 266, 327, 330-335 copolymer 202 copolymerisation 8, 194, 202, 206, 226, 333 Grain 27 Graph 225 Green chemistry 150, 418, 425 Growth 37, 51, 59, 74, 77, 90-91, 105, 123, 126, 140, 142, 172, 209, 233-234, 239-242, 244, 250-251, 253-254, 257-259, 262-263, 265-266, 317, 323, 333, 344, 351, 354, 380, 382-384, 397-399, 401, 404, 407, 410, 412, 415, 417, 419, 429, 449 Gum 21, 59, 177, 317

H Handling 175, 221, 246, 321, 334, 344, 352, 408 HDPE 13-14, 105, 180, 403, 451 Head 35, 249, 384 Healing 59, 66, 250, 253, 262, 265, 309, 319, 324, 336 Health 8-9, 14, 77, 90, 93, 98, 112, 184, 309, 323, 418, 421 Heart 26, 259-261, 281-282, 324-325, 339-344, 346, 366 Heat 12, 38-39, 44-46, 48, 73, 96, 112, 131, 137, 170, 195, 197-199, 317, 320, 340-341, 382, 406, 435 low 44 resistance 96 Heating 39-43, 45, 47, 51, 99, 157, 178, 244, 247, 315, 407 rate 39-43, 51

474

Index Height 222, 256, 404 Hemicellulose 103-104, 109, 112, 116, 123 Heterogeneous 132, 235, 245, 372-373, 385, 396 High density 180, 255 density polyethylene 13, 105, 134-135, 403, 451, 455 impact polystyrene 112, 451 molecular weight 128, 316 resolution 24, 27, 290 temperature 46, 72, 95, 102, 137, 309-310, 314, 317, 340, 451 Hips 112-113, 451 History 24, 89, 249, 274, 324, 326 Homogeneous 67, 93, 105, 132, 143-144, 244-245 Homopolymer 45, 153, 240 Housing 91, 252 Human immunodeiciency virus (HIV) 349 Humidity 51, 60, 70, 172, 175, 405-406, 455 Hybrid 8-9, 43, 45, 111, 136-137, 215, 255, 269, 292, 296-297, 306, 357, 379380, 448 Hydrated 18, 50, 55, 199, 289-290 Hydration 298, 357 Hydrocarbon 50, 168, 309 Hydrogel 49, 59, 75, 195, 208, 244, 265-266, 357, 383-384 Hydrogen bond 19, 132, 195, 243, 247 Hydrolysable 1, 159-160, 164, 396 Hydrolysis 45, 131, 143, 159, 165, 167, 207-208, 310, 315-316, 322, 330, 396, 402 Hydrophilic 1, 3, 5-6, 56, 58, 93, 114, 117, 166-167, 195, 236, 243-244, 290, 335, 338, 355, 357-358, 375, 378, 396 Hydrophobic 1, 3, 5-6, 56, 59, 75, 93, 114, 117, 166-167, 195, 243, 290, 326, 329, 333, 338-340, 355, 357-358, 375, 378, 396 Hydroxyl group 21 Hydroxylated 364 Hygroscopic 60, 94 Hysteresis 72

I Ice 180, 245-246 Identiication 351, 402 Imaging 24, 177

475

Biotechnology in Biopolymers Developments, Applications & Challenging Areas Immiscible 141, 210 Immobilisation 199, 213, 215-216, 218-219, 223-224, 244, 250, 254, 382 Impact 5, 90, 94, 99, 102, 105, 108-110, 112-115, 117, 136, 138, 338, 386, 403, 412, 425, 445, 451 resistance 112-113 strength 94, 105, 108-109, 114-115, 138 Impedance 173, 223, 450 Impermeable 156, 297, 413 Impregnate 111, 201 Impurities 18 In situ 92, 155, 208-209 In vitro 29, 77, 234, 243, 250-251, 253-255, 257-258, 260-261, 263, 265, 335, 337-340, 354, 381, 384, 401 In vivo 29, 34, 77, 166-167, 234, 242-243, 253-255, 257-258, 260-261, 265-266, 268, 337-339, 384 Inclusion 23, 179 Incompatible 64, 93, 155 Incubation 225 Indicator 15, 428, 430, 437 Induction 8, 253 Industry 3, 12, 14-15, 57, 66, 78, 112, 123, 127, 134, 141, 153, 177-178, 372, 379, 403-404, 418, 423, 425-427, 430, 439-442, 444, 456 Inert 12, 43, 46, 128, 137, 159, 243, 249, 254, 309, 323, 337, 347 Infection 238, 253, 263, 266, 347 Iniltration 241, 258, 263 Inlammation 254, 257-258, 267 Infrared 18, 20, 22, 173, 208, 214-215, 231, 410, 450-451 spectroscopy 18, 20, 214-215, 231 Infrastructure 419 Inhibitor 339 Initiation 70, 159, 262 Initiator 133, 170, 202 Injection 92, 96, 110, 114, 136, 257, 320, 427 moulding 96, 114, 136, 320, 427 Inks 142 Innovation 15, 175, 428-429, 441-444 Inorganic 23, 27, 51, 97, 136-137, 161, 179, 207, 219, 231, 253, 273, 309, 399, 417, 435 Insect 376, 378-379, 381, 387, 409

476

Index Insoluble 126, 179, 289, 376, 396 Instability 385 Institute 183, 189, 270, 275, 307, 440-441, 444, 453 Institution 440 Instrument 2, 39, 44, 429 Instrumentation 195 Insulation 100, 180, 340, 406 Integration 24, 207, 233, 242, 251, 253, 258, 260-261 Intensity 23, 27-28, 225, 329, 406 Interaction 3, 22, 56, 75, 90, 100, 133, 136-137, 208, 210, 215, 219, 233, 251, 267, 337, 358 Intercalation 10, 23, 137, 155 Interconnected 243, 245-246, 398 Interface 6, 18, 34, 93-95, 99, 101, 110, 137, 210, 239, 242, 251, 279-280, 283, 337, 351, 361, 384, 397, 399 Intermediate 49, 55, 70, 95, 124, 205, 410 Interpenetrating polymer network 50, 355, 451 Interphase 323 Intrinsic 7, 154, 169, 339 Investment 355, 426 Ion 66, 140-141, 199, 201, 266, 287-290, 292-293, 297, 300-304, 327 exchange 288-289, 292 Ionic 8, 10-11, 23, 27, 59, 136, 194-195, 223, 243, 287-301, 303-307, 327, 451 liquid 27, 136, 451 Ionisation 95, 114, 329 IPN 50, 208, 355, 451 Irradiation 6, 45, 131-132, 134, 160, 169, 320, 331, 402, 405 Irreversible 10, 156, 296, 298 Isolate 129, 251, 377 Isothermal 39, 42-43, 168 Isotropic 256 Izod 99

J Joint 3, 66, 190, 252, 272, 274-275, 279, 325-326, 339-342, 365

K Key 1, 9, 14, 18, 37, 55, 58, 191, 197, 234, 248, 250, 252-253, 265, 386, 417, 428 Kinetic 3, 43, 46, 48, 56, 59, 141, 221, 224, 244, 301, 304, 306, 401 477

Biotechnology in Biopolymers Developments, Applications & Challenging Areas

L Lacquer 129, 398 Lamella 124 Laser 22, 131, 327, 329-332, 344 Last 17, 59, 63, 76, 129, 378, 412, 416, 418 Lateral 256, 374 Latex 325, 344, 356 Law 302, 366, 431 Layer 77, 94, 183, 223, 263, 265-266, 327, 331, 351-354, 357, 399, 403, 406, 413, 452, 455 LDPE 13, 105, 169, 175-176, 401, 403, 405, 411, 452 Leaching 176, 180, 245, 351, 383 LED 4, 8, 77, 99, 132, 166, 193, 207-208, 226, 269, 324, 345, 382-383, 417, 452 Legislation 7, 90, 441 Length 50, 100, 116, 172, 179, 240, 242, 290, 298, 311, 385, 452, 455 Lenses 325-326, 357 Library 381, 423 Light 22, 38, 77, 123, 126, 128, 133-134, 143-144, 156-157, 170, 172, 197, 201202, 225, 329, 337, 376-377, 380, 386, 404, 406, 452 scattering 172 Lignin 6, 43, 47, 97, 101, 103-104, 109, 111-112, 116, 123-145, 147, 149, 151, 162 Linear 10, 49, 51, 58, 61, 63-64, 72, 76, 134-135, 137, 194, 196, 220, 225, 237, 239, 288, 291, 296, 298, 305, 312, 315-316, 401, 413, 418 low-density polyethylene 134 Lining 14, 404, 410-411 Linkage 1, 67, 396 Lipid 2, 153, 340 Liquid 18, 27, 48-49, 65, 72, 134, 136, 154, 158, 166, 195, 230, 245, 291, 320, 451-452 crystalline 134 Load 57, 76, 91, 105, 107, 157, 216, 221, 253-254, 337, 341, 356 Long chain 331 Long term 251, 255, 257, 259-260, 269, 326, 336, 339-340, 349, 352, 383, 415, 419 Loss 39-42, 48-49, 72, 77, 137, 156, 171-173, 175, 238, 253, 256, 259, 262, 381, 396, 398-399, 407, 410, 414 modulus 48-49

478

Index Low cost 1, 14, 351, 411, 417 density 13, 105, 133-135, 137, 175, 403, 405, 407, 414, 452 density polyethylene 13, 105, 133-135, 137, 175, 403, 405, 407, 414, 452 molecular weight 355 temperature 309, 322, 357 toxicity 17, 257 Lubrication 3, 66 Luminescence 157, 202

M Machinery 194, 226, 407, 427, 432 Macromolecular 3, 25, 34, 56, 79, 83-85, 120, 153, 188, 223, 227, 229-230, 235, 247, 272-273, 293, 295, 301, 312, 359, 388, 420, 434, 444-445 Macroporous 10, 245-246, 255, 263 Macroscopic 57, 65, 69, 195, 251, 302, 381, 384 Magnetic 24, 31, 33-34, 105, 194-196, 207, 452 Magniication 102, 256 Main chain 1, 202, 309, 312, 317-318, 322, 396 Maintenance 168, 241, 357 Maleated 105-106, 109-110, 136, 401, 452 Management 13-14, 91, 104, 145, 175, 341, 355, 357, 404, 407, 409, 411, 416417, 420, 443, 445-446, 451 Manipulation 244, 248, 269 Manufacturing 10, 15, 90, 98, 100, 102, 104-105, 109, 114, 117, 122, 178, 190, 195, 234, 241, 291-292, 294-295, 326, 328, 338, 352, 357, 368, 404, 435, 442, 446, 451 Market 15, 51, 96, 100, 179-180, 183, 250, 262, 276, 404, 416, 419, 425, 427431, 441-442 Mass spectroscopy 452 Material 1, 8-9, 12, 17-18, 23, 26-27, 30, 39-51, 60-61, 63, 65, 69-70, 72, 74-75, 77-78, 87, 90-91, 93-95, 100, 103-106, 109-110, 112, 123, 126, 131-132, 134, 136-138, 140, 142, 144-145, 149-150, 155-157, 161, 170, 172, 177-178, 184185, 190, 194-195, 211, 213, 234, 236, 241-243, 247-251, 253, 259, 265-267, 269, 271-273, 275-276, 282, 288, 291-292, 297, 300, 303, 305, 323, 325-326, 333, 336-337, 339-340, 344-347, 349, 354, 357-359, 362, 371, 381, 383-385, 395, 397-399, 402-404, 406-407, 413-414, 417-418, 420-421, 425, 427-428, 439, 447

479

Biotechnology in Biopolymers Developments, Applications & Challenging Areas Matrix 3, 5-6, 8-9, 29, 55-56, 63, 78, 90, 92-95, 99-101, 105, 108-111, 114, 117, 126, 133-134, 136-138, 141, 155, 167, 173-174, 177, 216, 218-221, 224, 233, 241, 243, 246, 250-251, 254-255, 260, 262-263, 266-267, 288, 324, 337-338, 355, 397, 402, 450 Measurement 18, 64, 72, 171, 189, 195, 225, 351 Mechanical properties 3, 5, 7, 12-13, 30, 37, 49, 55-59, 61, 63-65, 67, 69, 71-73, 75-77, 79, 81, 83, 85, 87, 93-96, 99, 101-102, 105, 107, 109, 111, 114, 117, 132-134, 136-138, 154-155, 164, 169, 172, 207-208, 233, 236, 253-254, 259-260, 338-340, 372, 380-381, 396, 398-400, 404-405, 415 property measurements 105 resistance 60 strength 16, 30, 72, 77, 156, 169-170, 181, 193, 196, 208, 244, 289, 291, 323, 396 testing 384 Mechanism 1, 12, 38, 42-43, 45, 68, 70-71, 156, 158-159, 184, 210, 216-218, 242-243, 257, 269, 301, 318, 354, 378, 402 Mediator 129, 197, 223-224 Melt 2, 44, 105, 133, 143, 404 low index 133 Melting 44-47, 51, 92, 155, 158, 164, 240, 322 point 45, 51, 164 temperature 46, 158, 322 Membrane 66, 159, 170, 183, 197, 199-201, 226, 263, 266-267, 290, 293, 296, 300-302, 304-305, 351, 355, 413 Mesh 240, 259, 325 Metabolism 238, 262, 385 Metallic 170, 241-242, 292-293, 310, 314-315, 357 Metastable 137, 378 Methodology 202 Micelle 70 Microbe 129 Microcrystalline 45 Microelectronic 194 Microorganism 156, 165, 174 Microscope 247, 400, 402 Microscopic 10, 69, 71, 254, 290, 302, 397-398 Microscopy 161, 172, 214, 267, 295, 337, 420, 449, 455-456 Microstructure 3, 5, 56, 90, 344

480

Index Migration 3, 56, 78, 233, 239, 241-242, 253, 255, 257-258, 300-301, 337, 339, 352, 382 Mill 6, 104, 123, 129, 134, 141, 144-145 Miscibility 31, 45, 49, 56, 132, 136, 141, 210 Mixture 46, 105, 133, 135-136, 199, 207, 245, 261, 315, 357, 406, 413 Mobility 24, 55, 60, 164, 213, 288, 352 Model 10, 61-65, 68, 76, 131, 141, 221, 257, 261, 298, 302, 338, 340, 383, 413414 compound 131 Modelling 24, 289, 296, 299-301, 384 Modiication 6, 9, 27, 42, 56, 93-95, 101-102, 105, 107, 128-131, 134, 144, 173, 248, 269, 326-328, 330-332, 335, 337-338, 345, 355, 357, 371, 378-379, 385, 427 Modiied 6, 12, 25, 27, 42, 49, 58, 101, 105, 131, 133-135, 137-139, 142, 144, 164, 170, 176, 194, 219, 237, 242, 251, 253, 259-260, 265, 269, 321, 327, 329, 331-332, 335, 338, 345, 348, 354, 359, 380, 386, 403, 432, 453 Modiier 155, 327 Modulation 234 Modulus 2-3, 48-50, 56, 58, 61-63, 72, 76, 91-92, 96, 98, 100, 105, 108, 111, 115, 134, 137-138, 141, 169, 253, 257, 298, 317, 322, 384-385, 399-400 Moiety 25 Moisture 5, 55, 93, 106, 110, 114, 117, 175, 194, 196, 263, 321, 412 Molecular mass 58 structure 33, 43, 52, 72, 144, 164, 167, 291-292 weight 40-41, 47-48, 50, 60, 66, 126, 128, 140-141, 156, 159, 171-172, 316317, 319, 355, 377, 384-385, 402 weight distribution 47, 140 Molten 95 Moment 213, 218, 298 Momentum 14, 153, 419 Monitor 1, 23, 30, 50, 76, 127, 172-173, 201, 309, 344, 350, 428 Monochromatic 22 Monolayer 267 Monomer 202, 205, 240, 331, 335 Morphology 56, 133-134, 136, 196, 208, 216, 234, 236, 251, 255, 295, 331, 381382, 399 Motion 158, 282, 342 Motor 4, 439 Mould 8, 242, 249, 319, 325 481

Biotechnology in Biopolymers Developments, Applications & Challenging Areas Moulded 110, 112, 181, 325, 427 Moulding 13-14, 38, 92, 96, 105, 114, 136, 143, 179, 245, 249, 320, 403-404, 427 MS 4, 40, 42, 222-223, 452 Multifunctional 169, 171, 216, 243, 251, 289, 306

N Nanocomposite 155-156, 169, 215, 217-218, 287, 289-292, 294, 298 Nanometre 137, 219, 290 Nanoparticle 177, 271, 293 Nanoporous 218 Nanostructure 253, 358 Nanotechnology 31, 145, 212, 239, 274, 358, 435 Natural polymer 29, 77, 164 resource 110 Net 180-181, 191, 236, 404 Network 3, 46, 49-50, 55-57, 60, 63-65, 70, 126-127, 162, 207, 243, 289, 293, 295, 300-301, 351, 355, 411, 451 formation 49 structure 243 Neutral 194, 200, 289, 300, 329 Neutralisation 195, 317 Nickel 141, 195 Noise 19, 27, 91 Non-homogeneous 93 Non-ionic 59 Non-linear 137 Non-polar 5, 93, 141, 288, 290, 311 Non-toxic 55, 142, 248, 257, 323, 337, 347, 383 Novel 1, 24, 30, 81, 91, 124, 174-175, 208-209, 217-218, 220, 234, 250, 253, 257, 266, 293, 383, 386, 418 Nuclear magnetic resonance (NMR) 18, 20, 24-27, 31, 33-34, 105, 290, 452 spectra 24-26, 290 spectroscopy 18, 24-27, 31, 33-34 spectrum 24-25 Nucleation 74, 338 Nucleophilic 95 Nylon 72, 89, 155

482

Index

O Oligomeric 26 One-step 210, 245 Opaque 207, 245 Optical 8, 77, 86, 159, 195, 197, 201, 210, 212, 226, 266, 329-330, 335-336, 350, 410 properties 195, 210 Optimisation 5, 328 Organic 23, 34, 127-128, 136-137, 140, 142-144, 161-162, 169, 178-179, 182183, 203, 207, 309-311, 399, 408, 417, 434-436 Organosol 141 Orientation 23, 27, 70, 100, 157, 164, 218, 235, 396, 404, 425 Oriice 340, 411 Oscilloscope 330 Output 196-199, 201, 302, 425 Oxidant 170, 208 Oxidation 44, 129-130, 137-138, 143, 157, 159, 162, 167, 170, 194, 201, 214, 220, 222, 224, 287, 327, 330, 335, 338, 340, 455 Oxidative 8, 42, 129, 131, 133, 162, 167, 202, 226, 343 degradation 167 stability 343 Oxidised 128, 140-141, 335 Oxygen 38, 43, 50-51, 123, 132, 156, 158-159, 162, 167, 200-202, 224, 240, 309-313, 322-323, 327-328, 331, 335, 341, 351, 357, 399 Ozone 159, 322, 334-335

P Packaging 7, 13-15, 91, 109, 136, 153, 170, 175, 177, 181, 193, 325, 401, 403404, 414-415, 417-420, 427, 436, 438, 442 Paint 142-14, 173, 195, 403, 427 Pan 39, 44, 150, 280, 287, 453 Paper 6, 59, 98, 100, 109, 117, 121, 123, 127, 129-130, 134, 141, 143-145, 235, 408, 420, 422, 435, 437, 441 industry 123 Particle 45, 61, 63, 76, 137, 142, 174, 208, 210, 216, 223, 267, 321, 337-338, 392 size 321 Pattern 28, 41-42, 44, 46, 414, 432, 442 PDMS 311, 327, 329-330, 335-336, 338, 343-345, 348, 350-356, 453

483

Biotechnology in Biopolymers Developments, Applications & Challenging Areas PE 93, 403, 453 Peat 141, 408 PEEK 157, 453 PEG 17, 24-26, 335, 356, 453 Penetration 99, 123, 133, 143-144, 161, 243, 257, 293, 295, 344, 352, 395, 413, 430 Performance 3, 5-6, 17, 37, 39, 41, 43, 45, 47, 49, 51, 53, 59, 65, 76, 93, 95, 101, 108-109, 112, 114, 117, 131, 134, 144, 196, 220, 242, 249, 325, 337, 339-340, 351, 358, 362, 364, 371, 378, 410-411, 414, 428 Permanent 14, 58, 60, 64, 259, 269, 416 Permeability 5, 56, 90-91, 136, 236, 254, 297, 322, 339, 355, 357, 414-415, 427 Permeation 136, 172, 241, 451 Peroxide 66, 95, 198-199, 201, 317-318 PET 158, 180, 454 Petrochemical 403, 427 industry 427 pH 8, 33, 74-75, 159, 163-164, 166-167, 172, 183, 194-196, 199-200, 202-204, 207-208, 212-213, 218-219, 223, 247, 252, 267, 315, 355-356, 399 Pharmacology 285, 392, 403 Phase separation 48, 56, 155 transition 196 Phenol-formaldehyde 134-135, 139, 143 Phenylene oxide 157, 454 Phosphorylation 142, 162 Photo-oxidation 170 Photoinitiator 169, 329, 332 Physical properties 60, 96, 126, 132, 175, 179, 241, 321, 382, 414 Physics 33, 54, 79-80, 82, 84-87, 151, 186, 227-228, 230, 307, 330, 360-362, 391, 435 Pipe 13-14, 404, 406, 411 PLA 16-17, 77, 159, 179, 253, 395, 447, 454 Plant 37, 59, 99, 102-104, 106-111, 113, 116, 133, 142, 145, 175, 179, 235, 404, 406-407, 410-412, 414, 419, 422, 441, 446 Plasma 93, 95, 327-328, 332-335, 337, 343, 354, 358, 361-362, 454 treatment 328, 332 Plastic 13, 42, 77, 91, 95, 98, 114, 140, 153, 169, 172, 175, 177-181, 183, 189, 191, 200-201, 264, 275, 278, 283-284, 339, 347, 365, 402-403, 406, 408, 410, 416, 418, 456 ilm 406 484

Index Plasticisation 132-133, 135-136, 139, 144 Plate 204, 212 Platform 128, 357 PMMA 24, 326, 350, 403, 454 Polar 5, 93, 132, 141-142, 288-290, 311 solvent 141, 289 Polarisation 26-27, 223 Polarity 93, 140, 288 Polyacetals 325 Polyamide 37, 105, 134, 164, 167, 325 Polycaprolactone 16, 77, 156, 265, 395-396, 407, 447, 453 Polycarbonate 180, 350, 395, 403 Polycondensation 24, 316, 427 Polydimethylsiloxane 311, 322-323, 327, 343, 355, 453 Polyester 46, 89, 93, 100-101, 108, 132, 135, 154, 161, 164, 176, 180, 259, 398399, 414, 453-454, 456 resin 101, 108 Polyethylene 13, 15, 17, 24, 37, 43, 45, 93, 105, 132-135, 137, 139, 166, 169170, 175, 180, 325, 338, 345, 353, 356, 363, 395, 400, 403-405, 407, 409, 411, 414, 417, 451-453, 455 glycol 17, 43, 45, 132, 135, 338, 345, 363, 453 oxide 45, 132, 135, 169, 338, 453 terephthalate 180 Polyimide 166 Polylactide 37, 136, 139 Polymer 1-3, 5-8, 10-12, 14, 16, 18-19, 25, 27-29, 31-35, 38, 42-44, 46-50, 5256, 58-60, 64, 66-72, 74, 77-80, 82-85, 91-96, 101, 103, 105, 107-108, 112, 114, 116-121, 131-132, 134-137, 139-141, 143-144, 147-148, 150-151, 153160, 162-175, 183-190, 194-195, 202, 208-210, 226-231, 236-237, 242-245, 263, 272, 284, 287-301, 303-307, 312, 317, 320, 324-325, 327-328, 330-331, 334-335, 337-339, 344, 348, 350-351, 354-358, 360-363, 366, 369, 371, 383384, 386, 388-389, 395-396, 398, 400, 403-404, 408-409, 413-414, 419-423, 427, 444-445, 447-448, 450-451 backbone 42, 290 Polymeric 1, 7-8, 10-11, 13-15, 17-18, 26, 38-39, 44, 46, 48, 51-52, 56-57, 59, 66, 72, 76, 78, 91, 93, 95, 143-144, 153, 155, 157, 159-167, 169-173, 175-177, 179, 181, 183, 185, 187, 189, 191, 196, 217, 233-234, 241-242, 244-246, 248, 250-251, 266-267, 287-289, 291, 293, 296, 306, 310, 325, 332, 334, 337, 339340, 343, 350-351, 355, 392, 395, 397-404, 414-417, 451, 453

485

Biotechnology in Biopolymers Developments, Applications & Challenging Areas Polymerisation 23, 43, 124, 129, 133, 144, 155, 203, 208, 210, 245, 273, 316, 327, 331-332, 334-335, 427, 432 Polyoleins 50 Polypropylene 37, 89, 93, 105-106, 109-115, 133-136, 139, 141, 180, 259, 325, 452, 454 Polystyrene 68, 99, 112-113, 132, 134-135, 180, 325, 352-353, 451, 455 Polyurethane 50, 133-135, 166-167, 170, 266, 325, 343, 345, 398-400, 455 Polyvinyl alcohol 7, 16-17, 37, 105, 132-135, 155, 383, 395, 414, 455 Polyvinyl chloride 37, 114, 132, 135-136, 180, 395, 407, 455 Pore 161, 243-245, 261, 352, 354, 383 size 261, 352, 383 Porosity 142, 243-244, 254, 261, 352, 414, 417 Porous 90, 131, 233, 241, 244-246, 253, 257, 301-302, 354, 383 Potential 6, 8, 12-13, 15, 17, 55, 59, 90-91, 93, 97, 114, 116, 123, 137-138, 140, 175-176, 194, 196-197, 199-203, 220-223, 237, 239-240, 242, 244-245, 248, 251, 255-260, 263, 265-266, 269, 300-303, 329, 335, 356, 379-380, 382, 384386, 398, 400-401, 410, 415-416, 418, 425-426, 428 Potting 319 Powder 27, 134, 164, 454 Power 104, 114, 212, 329-330 PP 93, 106-107, 109-113, 180, 423, 452, 454 Precipitation 140, 381-382 Precision 194, 235, 249 Precursor 9, 124, 137, 207, 291 Preparation 16, 143, 183, 196, 207, 210, 217, 249, 269, 315, 383, 396 Press 31, 35, 52, 54, 79, 81, 122, 145, 184, 186, 188-189, 226, 272, 306, 358360, 362-364, 420, 422, 444-446 Pressure 10, 40, 57, 109, 157, 236, 238, 243, 297-298, 301-302, 315, 351, 354, 357, 368, 407, 411, 416, 442, 455 sensitive adhesive 368 Pretreatment 96, 183, 327-328, 332 Price 3, 59, 76, 83, 283, 392 Probability 68, 253 Probe 199, 351 Procedure 40, 96, 140, 204, 347 Process 20, 25, 38, 42-43, 48, 59, 64, 70-73, 78, 90, 96, 103-105, 109, 112, 123, 126-128, 130-131, 133-134, 138, 143-145, 156, 159, 161-162, 171, 181-182, 187, 191, 195, 201, 203, 207, 210, 217, 220, 223, 239, 242, 245-246, 250-252, 260, 262, 265, 271, 293, 309, 315-316, 320, 324, 329, 349, 355, 379, 381, 404, 418, 425, 429, 431 486

Index Processing 3, 5-6, 8, 51, 54, 58, 66, 79, 89, 92-93, 95, 98, 102, 110, 117, 119, 131, 133, 141, 143, 149, 156, 182, 184, 195, 197, 234-235, 239, 317, 323, 325, 378, 395, 421, 427-428 conditions 6, 131, 141 Producer 98, 106, 117, 175, 419, 427, 431, 437, 448 Product 5-6, 10, 38, 40-41, 95-96, 100, 109, 117, 131, 140, 144, 159, 162, 181, 189, 196-198, 201, 221, 264, 378, 404, 413-414, 443 Production 9, 12-16, 37, 43, 55, 59, 89-91, 93, 95, 97-99, 101-109, 111-115, 117-119, 121, 129, 171-173, 175, 179, 221, 238, 252, 254, 269, 324, 371, 378379, 382, 384-385, 395-396, 398, 402-404, 406-407, 409-412, 414-416, 418419, 422, 424-425, 427-428, 432, 435, 442-444 cost 114 Proile 50, 112, 143, 349 Proliferation 78, 233, 241, 243, 251, 253, 255, 257, 263, 265, 335, 352, 380, 382-384, 441 Promoter 138 Propagation 159 Properties 1-3, 5-10, 12-14, 16, 18, 24, 30, 37-38, 43, 46, 48-50, 55-65, 67, 69, 71-73, 75-79, 81, 83, 85, 87, 89-96, 98-102, 105-111, 114-115, 117, 123, 126, 131-134, 136-138, 140-141, 143-144, 154-156, 161, 164, 168-169, 172, 175, 177, 179-180, 184, 188, 193-196, 206-208, 210, 212, 217-220, 223, 226, 233236, 239-245, 247, 249, 253-254, 258-260, 263, 265, 268-269, 283, 311, 318, 320-323, 325-328, 332, 338-341, 344, 352, 355, 357-358, 372, 378-382, 384386, 396, 398-400, 403-405, 414-415, 417, 421, 427-428 Propylene 133 Protection 37, 104, 106, 146, 177, 257, 262-263, 346, 351, 402, 407, 442, 450 Protective coating 78 Protein 12-13, 18-19, 29-30, 42-43, 45-46, 56, 60, 63, 66, 77-78, 111, 153-154, 177, 238, 241-242, 244, 249, 253, 258, 262, 266, 328, 335, 337-338, 358, 371, 373, 375-376, 378-388, 390, 398, 453 synthesis 12, 381, 385 Protocol 64, 181 Proton 24-26, 132 Protonated 21, 59, 75, 133, 214, 291-292 Prototype 233 PS 180 PTFE 290, 455 PU 134, 343-344, 455 Pump 340, 344

487

Biotechnology in Biopolymers Developments, Applications & Challenging Areas Puriication 128, 224, 237, 241, 378, 381, 388, 398 Purity 44, 51 PVC 114, 135, 180, 344, 395, 455 Pyrolysis 43, 52, 129, 140-141, 147, 159

Q Quality 13-14, 17, 44, 104, 109, 175, 218, 234, 238, 262, 373, 378, 404, 409, 412, 416, 442 control 378 Quaternary 42, 181

R Radiation 38, 57, 80, 87, 134, 157, 195, 227, 320, 327, 329-331, 360-361, 406, 410, 453 Radical 23, 43, 127, 141, 145, 158-159, 170, 205-206, 327, 331, 425 Radius 61, 414 Ramp 64 Rate constant 214 Ratio 8, 14, 19, 61, 98, 133-134, 136, 206, 218, 260, 403-404, 410, 432-433 Raw material 91, 104, 398, 425 Reach 65, 72, 260 Reaction 19, 25, 30, 38, 40, 43-45, 94, 131, 137, 142, 144, 159, 164, 194, 196199, 201-203, 207, 213-214, 220, 223-225, 245, 255, 257, 266-267, 294, 310, 314-318, 320, 328, 330, 344, 396 conditions 131, 144 mechanism 45 mixture 245 temperature 315 time 223, 225 Reactivity 132, 134 Reactor 129, 199, 315, 328 Reconstituted 64 Recovery 5, 59, 76, 123, 145, 424 Recycle 7, 90, 105, 110, 112, 114, 122, 150, 161, 168, 171, 175, 177-181, 191, 410, 417, 427, 455 Reduction 4, 24, 46, 63, 66, 91, 94, 131, 178, 194, 201-202, 214, 220, 222, 287, 292-293, 309, 314, 335, 339, 342, 346, 351, 399, 414, 418, 428, 455 Relectance 136, 202 Refractive index 72 Regenerated cellulose 50

488

Index Regeneration 9, 78, 88, 155, 166, 176, 209, 233-234, 239-243, 251, 255-258, 260, 262-263, 265-266, 337 Regulation 252, 262, 383 Reinforcement 6, 91, 94-95, 98-99, 101, 112, 116-117, 137-138, 141, 144, 155 agent 355 Relative 60, 70, 200, 303, 338, 384, 406, 455 humidity 60, 70, 406, 455 Reliability 37, 340 Renewable resource 395 Replacement 59, 115, 140, 166, 175, 250, 257, 259-260, 323-326, 340-341, 343344, 355, 365, 403 Reproducibility 5, 117 Research 6, 9, 12, 14-16, 32-33, 51, 53-55, 78, 80, 82, 87, 111, 113-114, 117, 121, 124, 128, 142, 145, 149, 184, 186-188, 190, 192, 221, 230, 234, 246, 250, 253, 256-257, 259-260, 262, 269-278, 280-285, 306, 309, 332, 344, 348, 352, 359-361, 363-364, 368, 379-380, 382-383, 385, 389, 391-392, 395, 403404, 414-415, 418, 421-423, 425-426, 428-431, 434, 437, 439-442, 444-446, 455 Residue 39, 41-43, 112 Resin 3, 44, 66, 92-93, 99, 101, 104, 108, 128, 134-135, 137, 139, 141-143, 151, 180, 291, 354-355, 395, 397-398, 413, 437 Resist 96 Resolution 4, 24, 27, 198, 290 Resonance 21, 24, 31, 33-34, 105, 210-211, 452 Reuse 105, 181-182, 191 Review 8, 52-53, 82-83, 85, 128-130, 138, 142, 164, 188, 191, 196, 231, 234, 265, 271, 287, 358, 371, 386, 422, 424 RH 70-71, 455 Rheology 55, 63 Rheumatism 366 Rigidity 46, 59, 111, 124, 143, 248, 260, 354 Ring 43, 129-130, 235, 261, 375 Ring-opening polymerisation 43 Risk 93, 180, 253, 326, 418, 432 assessment 418 Roadmap 441, 446 Rods 55, 176, 248 Room temperature 207-208, 212, 319, 331, 455 Rotation 46, 290, 311, 322

489

Biotechnology in Biopolymers Developments, Applications & Challenging Areas Roughness 172, 243 Rubber 49, 58, 60, 72-74, 84-85, 91, 112, 133, 153, 240, 317, 319-320, 322, 329, 331-335, 338-342, 344, 351-353, 355, 357, 359-361, 364, 419

S Safety 93, 146, 338, 411 Salinity 416 Salmonella 379 Sample 22, 24, 27, 39-42, 44, 46-48, 74, 105, 166, 173, 175, 183, 197-198, 212, 295, 336, 399 Saturated 50, 200-201, 413-414 Saw 249, 396 Scaffold 9-10, 77-78, 166-167, 172-173, 233, 235-236, 238-239, 241-245, 251253, 255, 257-263, 265, 268-269, 337, 351, 383, 452 Scales 238, 242 Scattering 22, 27, 35, 172, 290, 382 Screening 128, 182 Secretion 167, 255, 265-266, 376 Sedimentation 182 Segment 49, 64, 69 Selectivity 199, 220-221, 225 SEM 102-103, 161, 167-168, 172-173, 208, 210, 214, 216, 256, 293, 295-296, 337, 384, 386, 397, 399-401, 455 Semicrystalline 27, 51, 58 Sensitivity 43, 50, 59, 66, 198-199, 212, 220, 224, 381 Sensor 70, 76, 86, 138, 206, 227, 229, 302, 350-351 Separation 40, 48, 56, 142, 155, 218, 221, 248, 290, 431 Serum 26, 46, 242 Services 437, 441 Shape 7-8, 14, 39, 48, 67, 69, 76, 172, 194-196, 218, 220, 248, 290, 302, 347, 375, 396, 402-403 Shear 64, 68, 70-71, 280 yielding 70 Sheet 265, 325, 382, 398-399 Shell 142, 210, 236 Shortage 181, 254 Shrinkage 102 Side chain 129, 318 Sign 38, 289

490

Index Silicone 2, 12, 40-41, 261, 309-315, 317-323, 325-327, 329-349, 351-355, 357359, 361, 363-365, 367, 369, 452, 455 Simulation 172, 296, 339 Size 8, 12, 14, 18, 27, 74, 167, 172, 178, 194-195, 212, 218, 220, 245, 250, 261, 288, 290, 321, 346, 352, 375, 379, 383, 385, 396, 402-403, 437, 441, 455 Skeletal 349 Soaking 354 Soft 57, 61, 64, 72, 77, 82, 140, 178, 237, 257, 327, 339, 344, 349, 354, 357, 383 Software 39 Sol 207, 212, 290, 292, 296, 368 Solid 2, 24, 26-27, 34, 48, 60-61, 81, 105, 143, 149, 154, 158, 191, 199, 236, 246, 289-290, 294, 315, 348-349, 418 state 24, 26, 34, 105, 191, 199, 236 waste 143, 418 Soluble 28, 55, 59, 131, 134, 140-142, 144, 183, 194, 236, 238, 322, 331, 355356, 376-377, 380-381, 413, 456 Solution 4, 18, 24, 27, 55, 60, 71-72, 75, 136, 143-144, 155, 167, 177, 179, 183, 198-201, 204, 207-208, 212-213, 218, 225, 245-246, 254, 288-289, 291-293, 325, 330, 335, 348-349, 353, 376, 378, 413, 417 Solvent 10, 22, 24, 28, 132-134, 136, 141-142, 144, 166, 245, 256, 289, 291, 297 Sorption 94 Speciic surface 328 Speciicity 128, 162, 183, 212 Spectra 2, 18-20, 23-26, 33, 208, 215, 223, 290 Spectrometer 40 Spectroscopy 1-2, 18, 20, 22-27, 31-34, 43, 131, 133, 137, 142, 173, 214, 231, 359, 450, 452, 456 Speed 15, 19, 24, 442 Spherical 61, 63, 76, 179, 382, 402 Spontaneous 315 Spreading 212, 329, 352, 418 Square 220, 222 Stabiliser 78, 133, 203 Stability 3, 5, 30, 32, 37, 42-43, 45-47, 49-53, 55, 66, 78, 80, 93, 104, 111-112, 114, 117, 133-134, 136-137, 144, 147-148, 153, 184-187, 206, 208, 238, 245, 255, 289-290, 311, 322, 326-327, 339-340, 343, 351-352, 357, 420 Stacking 109, 137 Staining 10, 267

491

Biotechnology in Biopolymers Developments, Applications & Challenging Areas Standard 4, 61, 63, 76, 183, 225, 241, 244, 291, 296, 396, 424, 432, 442, 453 deviation 225 Static 82, 297, 339 Statistics 108, 121 Steady state 298 Steps 42-43, 143, 156, 163, 179, 225, 246, 292, 314, 427 Sterilisation 328, 352, 355, 456 Stiffness 43, 64, 102, 137, 236, 243, 260, 296, 298, 352, 381 Stimulation 209, 243, 254, 302, 341 Stirring 207 Storage 26, 48-50, 196, 235, 240, 252, 402, 407, 415 modulus 48-50 Strain 4, 27, 48, 57, 61, 63-65, 67, 72, 74, 76, 137-138, 157, 174, 208, 243, 302, 455 Strategy 424 Strength 3, 5, 8, 14, 16-17, 30, 56, 71-72, 76-77, 90, 94-96, 98-99, 101-102, 105109, 111, 113-115, 131, 133-134, 137-138, 141, 155-156, 166, 169-170, 172, 174-175, 181, 193, 195-196, 208, 235, 242-244, 253-254, 266, 289, 291, 309, 322-324, 329, 358, 372-373, 378, 380-381, 384, 396, 398-400, 403-404, 414 Stress 2, 4, 48, 51, 57, 60-66, 69-70, 72, 76, 93, 110, 133, 157, 175, 194, 196, 253, 257, 412 relaxation 63-65 Stress-strain curve 4, 57 Stretch 4, 19-21, 67-68, 72, 74, 195, 214 Structural modiication 128 Structure 8, 12, 18-19, 23-24, 27, 30, 33-34, 42-44, 46, 51-52, 55-56, 60, 66, 69-70, 72, 90-91, 98-99, 105, 124-125, 129, 132-133, 136-137, 144, 146, 156157, 160, 162, 164-165, 167, 173, 188, 193, 195-196, 206, 210, 216, 218, 220, 235-241, 243-245, 247-249, 251-252, 263, 265, 289-292, 311-313, 322-323, 331, 340, 344, 354, 375-376, 378-379, 381, 385, 396, 421 Substitution 20, 59, 129, 164, 194, 208, 291, 313, 431 Substrate 77, 128, 170, 196, 201-202, 212-213, 224, 252, 255, 266, 328, 330, 337, 351-353, 399, 413 Sugar 97, 103-105, 116, 129, 133, 235-236, 427 Sulfonated 128 Sulfur 317, 413 Sunlight 126, 322, 417 Supply 14, 175, 202, 240, 243, 249, 351, 416, 429, 444

492

Index Surface 5-6, 14, 22, 54, 93-94, 99, 101, 115, 117-118, 133-134, 136-137, 141, 147, 161, 166-167, 172-173, 177, 201, 204, 206, 208, 211-214, 216, 218-221, 223, 225, 241-243, 254, 257, 262, 265, 271, 292-295, 311, 321, 323, 326-335, 337-338, 340-341, 344-345, 351-355, 357, 360, 362, 366, 372, 382, 396-397, 399-401, 403-404, 410, 417 activity 353 chemistry 242, 328 coating 355 free energy 332 resistivity 293 tension 311, 323, 357 Surfactant 155, 169 Survey 37, 250, 422 Suspension 129-130, 137 Sustainable 7, 17, 76, 90, 123, 418, 428 Swelling 59, 194, 195-196, 208, 243-244, 289, 301 Symmetric 21, 46 Symposium 32, 230, 307, 358-359, 420 Synergistic 128-129, 175, 207, 398 Synthesis 1, 12, 25, 123, 155, 170-171, 203-204, 206, 210, 220, 245-246, 248, 253, 255, 314, 317, 358, 373, 381, 385 Synthetic resin 142

T Table 20-21, 94-95, 130, 135-136, 138-139, 159-160, 179-181, 198, 211, 312313, 325-326, 376-377, 399-400, 434-437, 440 Tank 182, 411 Technical 91, 189, 234, 371, 403, 425, 428 TEM 7, 337, 384, 456 Temperature 2, 8, 37-44, 46-49, 55-56, 60, 72-73, 92, 95-96, 102, 109, 131-132, 134, 136-138, 156, 158-159, 163-164, 167, 172-173, 175, 183, 194-196, 198, 201-202, 207-208, 212, 221, 262, 293, 301, 309-311, 314-315, 317, 319, 322, 331, 340, 357, 378, 404-406, 409-410, 413, 451-452, 455-456 range 46, 311 Template 210, 248, 265 Tensile properties 99, 105-106, 131, 133, 136, 372, 405 strength 96, 101, 105-106, 111, 113, 115, 131, 134, 138, 141, 172, 322, 378, 380-381, 384, 398-400 testing 50

493

Biotechnology in Biopolymers Developments, Applications & Challenging Areas Tension 64, 68, 74, 302, 311, 323, 357 Termination 381, 385 Tertiary 66, 126, 181-182 Test 2, 4, 50, 54, 70, 120, 137, 142, 156, 171-174, 183, 190, 201-202, 255, 357358, 384, 396, 398-401, 404, 421, 436, 449 Tg 39-40, 44, 73, 112 TGA 40-43, 46-48, 51, 208, 456 Theory 61, 141, 160, 296 Thermal analysis 2, 37, 39, 41, 43, 45-49, 51-54, 121, 148, 186-187, 450 conductivity 134 degradation 5, 38, 42, 50, 93, 95, 133, 142, 158-159, 163-164 properties 2, 38, 46, 48, 94, 108, 131, 134, 136, 138, 175, 384 stability 5, 43, 45, 47, 49-51, 93, 104, 112, 114, 117, 133-134, 136-137, 144, 208, 289, 322, 357 treatment 49, 141, 143 Thermodynamic 2, 46 Thermogram 28, 39-40, 42 Thermogravimetric analysis 39, 42, 134, 456 Thermogravimetry 43 Thermoplastic 55, 58, 91, 102, 105, 112, 118, 120, 143, 158, 178, 235, 240, 402403 Thermoset 58, 95, 102, 104-105, 133, 137, 141, 178 resin 137, 141 Thickness 27, 210, 262, 265-266, 300, 302, 304-305, 331, 353, 413-414 Thin ilm 212, 335-336 Three-dimensional 18, 24, 58, 77, 162, 233, 248, 251, 260, 290-291, 351, 449 Time 4, 39, 42, 46-48, 61, 64-65, 72, 76, 90, 107, 114, 127, 131, 144-145, 153, 156, 169, 176, 185, 202, 207-208, 220, 223, 225, 234, 238, 242, 245-246, 249, 255, 262, 301, 319, 322-324, 326, 337-340, 343, 356-357, 372, 378, 412, 428, 442 Tip 74, 298, 344 Tissue 5, 9, 12, 17, 31, 63, 77-78, 88, 90, 124, 166, 176, 233-239, 241-273, 275277, 279-285, 323, 325, 327, 336-337, 339, 342-343, 348-349, 351-352, 357, 378-380, 382-384, 386, 434 Tool 1, 3, 5, 7, 9, 11-13, 15, 22, 24, 27, 76, 114, 131, 171, 175, 194, 235, 248249, 256, 332, 402 Tough 4-5, 90, 180, 372-373, 378, 385 Toxic 55, 123, 142, 176, 178, 248, 253, 257, 323, 337, 347, 381, 383, 417 Trade 108, 237, 402-403

494

Index Trademark 430, 456 Transfer 8, 40, 43, 66, 78, 93, 110, 157-158, 214, 218, 220-221, 223-224, 226, 253, 287, 355, 454 reaction 214 Transformation 13, 20, 170, 179 Transition 5, 29, 39, 44-45, 47-49, 56, 60, 68, 70-72, 90, 132, 134, 136-137, 158, 164, 194, 196, 288, 322, 382, 456 Transmission 242, 267, 337, 357, 406, 456 electron microscopy 267, 337, 456 Transmittance 19, 23, 128, 211 Transparency 8, 14, 138, 266, 354, 403-404, 407 Transportation 166, 177, 266, 352, 376 Transverse 64, 199, 405 Trend 344, 431 Trivalent 142 Tube 40, 74, 195, 344-346, 374 Twisting 302 Two-component 319 Two-dimensional 449 Two-phase 210, 290 Two-step 42

U Ultrasonic 66, 160, 163, 174-175, 293 Ultraviolet 126, 134, 170, 262 Uncured 317 Uniformity 137 Unmodiied 27-28, 335, 338 Unplasticised polyvinyl chloride 132 Unsaturated 50, 93, 95, 108, 141, 159, 456 polyester resin 108 Unstable 158 Untreated 29-30, 101, 103, 105, 257, 321 Upper 96, 116, 183, 263 UV 38, 123, 134, 157, 170, 175, 208, 402

V Vacuum 303 Validation 355 Van 68, 80, 82-84, 272, 276, 282, 359, 364, 392, 405, 443

495

Biotechnology in Biopolymers Developments, Applications & Challenging Areas Vapour 40, 133, 246, 413 Vector 13 Velocity 67-68 Vessel 317, 344 Vibration 20-21, 214-215 Viscoelasticity 81 Viscometry 171 Viscosity 2, 28, 63, 65, 72, 137, 142-143, 354 VMQ 318, 456 Void 244, 414 Volatile 40, 43, 95, 171 Voltage 11, 195, 202, 208, 213, 299, 301 Volume 39, 46, 72, 80, 187, 206, 218, 225, 243-244, 270, 322, 355, 364-365, 412, 414 Volume resistivity 322

W Wall 100, 126, 259, 406 Washing 399-400 Waste disposal 416 Water 5, 12, 14, 18, 21, 27-28, 42-43, 45-46, 48, 50, 55, 59, 74, 90, 93-94, 102, 104, 110, 123-124, 126, 128, 136-138, 141, 143-144, 157, 167, 169, 173, 175, 179, 181, 192, 204, 212, 236-238, 244, 246, 256, 289-291, 297, 299, 315-317, 327, 331, 336, 356-357, 395-396, 402, 410-414, 416-418, 421, 427, 450 content 46, 50, 102, 244, 357 uptake 356 vapour 246, 413 Wavelength 18, 27, 410 Web 372-373, 375 Weight 8, 14, 38, 40-42, 47-48, 50, 55, 59-60, 66, 91, 126, 128-129, 137-138, 140-141, 144, 155-156, 159, 171-173, 179, 262, 316-317, 319, 323, 355-356, 377, 384-385, 399, 402-403, 407, 416 loss 40-42, 137, 173, 407 ratio 8, 14, 403 Well 2-3, 6, 10, 12, 14-15, 20, 24-27, 30, 38, 44, 55, 64, 68, 71, 76, 89, 96, 103, 105-106, 109, 114, 123, 126, 131, 140, 143, 166, 184, 196, 213, 218, 220, 233-234, 236-239, 241-242, 244, 248-250, 253-255, 259, 265, 267-268, 287, 290-291, 293, 300, 309, 317, 323, 325-328, 337-338, 340, 344-345, 352, 357358, 371-372, 378-379, 397-398, 402-403, 407, 417, 422, 425, 431, 442 Wetting 157, 242, 323, 355

496

Index Wide-angle 290 Width 108, 245 Wind 195, 405 Window 95, 108 Wood 6, 95, 105, 110, 114, 116, 123-124, 127-129, 131, 135, 140-143, 145, 150, 181, 194, 408

X X-ray diffraction 1, 27-29, 35, 337, 382, 456 XRD 27-30, 173, 456

Y Yield 69, 141, 202, 373, 375, 378-379, 402-403, 409, 411-412, 415, 417 Yielding 70, 133, 416 Young’s modulus 61-63, 76, 91-92, 96, 138, 169, 298

Z Zwitterionic 289

497

Biotechnology in Biopolymers Developments, Applications & Challenging Areas

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498

Published by Smithers Rapra Technology Ltd, 2012

This comprehensive book provides up-to-date information on the developments in the field of biopolymers. It offers an introduction to progress in the field and outlines the various applications of biopolymers. Different methods and techniques of synthesis and characterisation are detailed as individual chapters. Various modes and mechanisms of degradation of materials are discussed. There is a dedicated chapter on industrially available biopolymers and their applications, as well as a chapter detailing ongoing research, current trends and future challenges. This book is essential for students who are interested in biotechnology and polymer research. Each chapter explains the science and technology from inception to the most advanced, state-of-the-art developments available. This book will also be useful for those involved in high-tech research as it offers the most up-to-date information available in this field.

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