Exploring Spinifex Biomass for Renewable Materials Building Blocks

Home , Spinifex (plant), Triodia (plant)

Nasim Amiralian

A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2014 Australian Institute for Bioengineering and Nanotechnology

Abstract

Triodia pungens is one of the 69 species of an Australian native arid grass spinifex which covers approximately 27 % (2.1 million km2) of the Australian landmass. The use of fibrous and resinous components of spinifex has been well documented in traditional indigenous Australian culture for thousands of years. In this thesis the utility of both the cellulosic and resinous components of this abundant biomass for modern applications, and a potential economy for our Aboriginal collaborators, were explored. One part of this study was focused on the optimisation of a resin extraction process using solvent, and the subsequent evaluation, via a field trial, of the potential use and efficacy of the resin as a termiticide. Termiticidal performance was evaluated by re-dissolving the extracted resin in acetone and coating on pine timber blocks. The resin-coated and control blocks (uncoated timber) were then exposed to a colony of Mastotermes darwiniensis termites, which are the most primitive active and destructive species in subterranean areas, at a trial site in northeast Australia, for six months. The results clearly showed that spinifex resin effectively protected the timber from termite attack, while the uncoated control samples were extensively damaged. By demonstrating an enhanced termite resistance, it is shown that plant resins that are produced by arid/semi-arid grasses could be potentially used as treatments to prevent termite attack. In the other part of this study it has been demonstrated that very small diameter and unprecedentedly high aspect ratio cellulose nanofibrils (NFC) can be readily isolated from spinifex biomass using unrivalled mild pulping coupled with minimal mechanical energy by any of three methods; high pressure homogenizer, ultrasonication, and milling. After washing, delignification and bleaching steps, the mechanical defibrillation of fibres was optimized by varying the specific processing conditions of each of these three mechanical treatment methods. Cellulose nanofibres with an extremely low average diameter (below 10 nm) and high aspect ratio (~500) were produced, even after applying very low energy processing. Similarly, acid hydrolysis at lower temperatures and acid concentrations with respect to the published contemporary protocols yielded high aspect ratio cellulose nanocrystals (CNC), for which the average lengths were found to be unaffected by further ultrasonic treatments. We hypothesize that the chemical and morphological origins for this surprisingly low energy input requirement for isolating high aspect ratio nanofibers from Triodia pungens is directly related to the plant’s unique morphology and composition, which contains unusually high amounts of hemicellulose. It is also hypothesised that these unique traits are a result of this species’ evolution, which has been heavily influenced by an extreme semi-arid climate. Polyurethane and spinifex CNC nanocomposites were also prepared via twin-screw reactive extrusion and solvent casting. Twin-screw reactive extrusion offered better CNC dispersion and resulted in superior mechanical properties with respect to the solvent casting method.

Declaration by author

This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the General Award Rules of The University of Queensland, immediately made available for research and study in accordance with the Copyright Act 1968. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis.

iii

Publications during candidature

Peer-reviewed papers

Accepted

 N. Amiralian, P.K. Annamalai, D.J. Martin. Nanocellulose. Patent 2013904527, Australia 2013. p. 23.

 N. Amiralian, P. K. Annamalai, C. Fitzgerald, P. Memmott, D. J. Martin. Optimization of resin extraction from an Australian arid grass ‘Triodia Pungens’ and its potential application as a termite deterrent. Industrial Crops and Products. Vol.59, pp. 241-247 (2014)

* Due to delays in the intellectual property protection process, this important patent caused unavoidable delays in the publication of key data from this thesis.

Under review

 N. Amiralian, P. K. Annamalai, P. Memmott, D. J. Martin. Easily deconstructed, high aspect ratio cellulose nanofibers from Triodia pungens; an abundant Australian arid grass. Prepared manuscript to submit in Chemistry and materials research, 2014.

 N. Amiralian, P. K. Annamalai, P. Memmott, D. J. Martin. Comparing nanofibrillated cellulose obtained from Triodia pungens using different mechanical methods. Prepared manuscript to submit in Cellulose, 2014.

In preparation

 N. Amiralian P. Memmott, D. J. Martin. High aspect ratio cellulose nanocrystal from spinifex. Prepared manuscript to submit in Chemistry and materials research, 2014.

Book chapter

 M. Jorfi, N. Amiralian, M.V. Biyani, D.J. Martin, P.K. Annamalai. Biopolymeric nanocomposites reinforced with nanocrystalline cellulose. In Singha AS, Thakur VK, Biomass

based biocomposites 2013, pages 277-304 Smithers RAPRA, Shawbury UK ISBN: 9781847359803.

Conference presentation

 N. Amiralian, P. K. Annamalai, P. Memmott, D. J. Martin. Unusually high aspect ratio, easily deconstructed cellulose nanofibers from Australian spinifex (Triodia pungens). TAPPI international conference on nanotechnology for renewable materials, 23 - 26th July 2014, Vancouver, Canada .

 N. Amiralian, P. Memmott, G. Edwards, J. Milne, K. Jack, I. Morrow, D. Martin. Extraction microfibrillated cellulose from spinifex grass using high energy milling and chemical pretreatment. ICEAN 2012 (1st International Conference on Emerging Advanced Nanomaterials), 22 - 25th October 2012, Brisbane, Australia.

 N. Amiralian, P. Memmott, D. Martin. Investigation of native spinifex plant resin and fibres as renewable building blocks for composite. 33APS (33rd Australasian Polymer Symposium) 12 - 15th February 2012, Hobart, Australia.

 N. Amiralian, P. Memmott, D. Martin. Investigation of the extraction, properties and processing of Triodia pungens. AIBN Student Conference (Australian Institute for Bioengineering and Nanotechnology Student Conference) 23 - 24th September 2011, Brisbane, Australia.

 N. Amiralian, P. Memmott. Searching for Biomimetic Design Prospects from an understanding of Aboriginal use of spinifex grasses. Invited guest lecturer at University of Southern Cross, Plant Science program, 26th October 2011, Lismore, Australia.

 S. Mondal, P. Memmott, N. Amiralian, G. Edwards, D. Martin. Preparation and properties of bio-composites from spinifex grass fibres and spinifex resin based polyurethane. BIOPOL 2011 (3rd International Conference on Biodegradable and/or Biobased Polymers) 29 - 31th August 2011, Strasbourg, France.

Publications include in this thesis

 N. Amiralian, P. K. Annamalai, D.J. Martin. Nanocellulose. Patent 2013904527, Australia 2013. p. 23. Icorporated as Chapters 5, 6, 7.

Contributor Statements of contribution

Experimental works (Pulping fibre, extraction nanofibrillated cellulose and N. Amiralian cellulose nanocrystals, all the characterizations and analyses): 100 % , preparing the manuscript: 80 %

P.K. Annamalai Assisted with manuscript drafting and data interpretation : 20%

D.J. Martin Supervision, data interpretation, and critical review on manuscript

 N. Amiralian, P.K. Annamalai, C.Fitzgerald, P. Memmott, D.J. Martin. Optimization of resin extraction from an Australian arid grass ‘Triodia pungens’ and its potential application as a termite deterrent. Submitted manuscript in Industrial Crops and Products, 2013. Incorporated as Chapter 4.

Contributor Statements of contribution

Experimental works (Extraction spinifex resin and characterization to optimize N. Amiralian different conditions, data analyses, timber coating): 90 %, preparing the manuscript : 80%

P.K. Annamalai Assisted with critical review on manuscript: 15 %

C.Fitzgerald Assisted with proof reading : 5% , installing samples in Townsville : 50 %

Assisted with providing starting material (spinifex resin), proof reading the P. Memmott manuscript :5 %

D.J. Martin Supervision and critical review on manuscript

Contributions by the others to the thesis

All experiments, data analysis, and the interpretation of these results were performed by Nasim Amiralian, except where acknowledged below:

Darren Martin Significantly contributed to the conception, data interpretation, and critical revision of the work contained within this thesis Paul Memmott Assisted with providing spinifex grass and resin and critical revision of the chapters Pratheep Annamalai Assisted with data interpretation and contributed to the design Isabel Morrow Assisted with TEM (Chapters 6 , 7) and SEM (Chapters 5 , 6) imaging Garry Morgan Assisted with TEM and Cryo-TEM imaging (Chapters 6) John Milne Assisted with milling cellulose (Chapter 6) Nathan Mosier Assisted with chemical analysis of spinifex grass (Chapter 5) Robert Moon Assisted with chemical analysis of spinifex grass (Chapter 5) Grant Edwards Assisted with mechanical testing and milling and reactive extrusion experiment (Chapters 6 , 7) Kevin Jack Assisted with XRD analysis (Chapters 5, 6, 7) Elena Taran Assisted with AFM imaging and AM-FM modulus analysis (Chapter 7) Chris Fitzgerald Assisted with installing timber samples in termite testing site and providing termites (Chapter 4) Assisted with reactive extrusion experiment and also understanding and writing Dr Celine Chaleat this part (Chapter 7) Khairatun Najwa Mohd Assisted with providing reactive extrusion nanocomposite sample and mechanical Amin testing results of the extruded samples (Chapter 7) David Appleton Assisted with ICPOES analysis (Chapter 6) SCION forest nutrition Assisted with chemical analysis of spinifex grass (Chapter 5) and analytical laboratories

Statement of parts of the thesis submitted to qualify for the award of another degree

None.

vii

Acknowledgments

At the end of this great experience, I would like to sincerely thank all the people who in one way or another have contributed to this thesis.

In particular, I wish to express my sincere gratitude to my principle supervisor, Prof Darren Martin for accepting me as his PhD student and for his constant support, encouragement, enthusiastic guidance and constant availability throughout my doctoral studies. Your approachable nature and motivation allowed me to shape my interests and ideas.

My gratitude is also devoted to Prof Paul Memmott, my co-supervisor for accepting me as a member of this great project and providing the opportunity of become familiar with Australian Indigenous environment. I greatly acknowledge your constant support.

I also would like to take this opportunity to convey my appreciation for Dr Pratheep Kumar Annamalai my co-supervisor. Your guidance and direction throughout my project was appreciated.

I would like to thank Dr Isabel Morrow and Dr Gary Morgan who have done a wonderful job in TEM and SEM imaging and Dr Elena Taran who has kindly helped me by AFM and AM-FM analysis. Without your experience and patience would not have been possible to do these analyses.

I would like to extend my gratitude to Dr Kevin Jack, who has assisted me by XRD analysis, Dr Grant Edwards for helping me by mechanical testing and reactive extrusion experiment, Dr Celine Chaleat for helping by reactive extrusion experiment and understanding the procedures, Miss Khairatun Najwa Mohd Amin for sharing data analysis of reactive extrusion with me, Mr John Milne for doing milling, Mr Chris Fitzgerald who has kindly shared his entomology knowledge and assisted with termite testing. Thank you to Prof Rowan Truss and Prof Martin Veidt for being supportive reviewers in each milestone of my study. My special thanks to Dr Phil Nilsson for his great support in commercialization.

I would like to thank Prof Robert Moon and Prof Jeffrey Youngblood at Purdue University for accepting to work in their lab and directing me by useful advice about producing cellulose nanocrystal. I also would like to thank my adopted Colombian family; Gaby and Diego Calderon for your warm-hearted nature and support making my three month visit to Indiana a very enjoyable

viii experience. My three month visit was one of the most rewarding periods during my study, from both an academic and personal level.

I also acknowledge our Aboriginal collaborator, Dugalunji Aboriginal Corporation in Camooweal for the supply of threshed resin and grass and providing insights into the traditional technology of spinifex manufacture.

I would also like to thank all the current and past Martin research and AERC group members for your help, great understanding over these years. Also to the members of the Whittaker and Halley group of Level 4 east, thank you for creating an enjoyable and social working environment.

I would like to greatly acknowledge travel grants through the University of Queensland graduate school international travel award (GSITA) and University of Queensland-Purdue University early career researcher (ECR) mobility scheme grant. This thesis was supported by a University of Queensland international scholarship (UQI), Australian Research Council grant (under ARC Discovery Grant No. DP0877161), and AERC top up-scholarship.

Last, but certainly not least, I would like to express my special thanks to my wonderful parents; Reza and Saeideh who have always given me the strength and wisdom to trust my instinct and follow my dreams, and also my siblings; Nastaran and Saeid. Thank you all for your constant support, unconditional love, and encouragement throughout my studies. In particular, I would like to thank my husband Mehdi for his generous love, constant support, encouragement and selfless understanding and patience over the last two years. Words cannot express how much I love you all and how grateful I am for your support. This journey would have been a lot tougher without these people.

Keywords

Renewable materials, Spinifex, nanocellulose

Australian and New Zealand Standard Research Classifications (ANZSRC)

ANZSRC code, 1007, Nanotechnology: 70% ANZSRC code, 091209 Polymers and Plastics: 20 % ZSRC code, 091202, Composite and Hybrid Materials: 10 %

Fields of Research (FoR) Classification

FoR code: 0912, Materials Engineering: 50 % FoR code: 1007, Nanotechnology: 50 %

“When you go through a hard period, When everything seems to oppose you,

...

When you feel you cannot even bear one more minute, NEVER GIVE UP! Because it is the time and place that the course will divert!”

Rumi, The Essential Rumi

Table of content

List of Figures...... xix

List of Tables...... xxiv

List of Equations...... xxivii

List of Abbreviations...... xxiviii

Chapter 1. Context of study...... 1

1.1 Background and significance of this project...... 1

1.2 Objectives of this research project...... 2

1.3 Thesis outline...... 2

1.4 A note on the exploratory journey of the research...... 4

Bibliography...... 5

Chapter 2. Literature review...... 6

2.1 Materials from renewable resources for sustainable development...... 7

2.2 Spinifex as a potential biomass...... 8

2.2.1 Triodia...... 9

2.2.2.1 Distribution...... 9

2.2.2.2 Ecology...... 10

2.2.3 Triodia pungens group...... 10

2.2.3.1 Distribution...... 10

2.3 Traditional uses by Aboriginal people...... 10

2.4 Contemporary revitalisation of knowledge on spinifex...... 11

2.5 What is known about spinifex resin?...... 11

2.5.1 Chemical analysis...... 12

2.5.2 Spinifex resin sources and applications...... 12

2.6 Sustainable development...... 13

2.7 Termite deterrent...... 13

2.8 Cellulose and nanocellulose...... 15

2.8.1 What is nanofibrillated cellulose (NFC)?...... 16

xii

2.8.2 Properties and applications of nano and microfibrillated cellulose...... 17

2.8.3 Methods of producing nanofibrillated cellulose...... 18

2.8.3.1 Homogenization...... 18

2.8.3.2 Ultrasonication...... 19

2.8.3.3 High energy milling...... 19

2.8.4 Cellulose nanocrystals (CNC)...... 20

2.8.4.1 Properties of cellulose nanocrystals...... 21

2.8.5 Nanocellulose reinforced composites...... 21

Bibliography...... 24

Chapter 3. Materials and methods...... 38

3.1 Materials...... 38

3.1.1 Spinifex grass...... 38

3.1.2 Chemicals...... 38

3.1.3. Aliphatic polyurethane - Tecoflex EG- 80A...... 39

3.1.4. Polyol - Polytetramethylene ether glycol (PTMEG)...... 39

3.1.5. Aromatic diisocyanate - 4,4'-methylene diphenyl diisocyanate (MDI)...... 39

3.1.6. Chain extender - 1,4-butane diol (BDO)...... 39

3.1.7. Catalyst - Dibutyltin dilaurate (DBTDL)...... 39

3.2. Methods...... 40

3.2.1 Resin extraction...... 40

3.2.2 Evaluation of termiticidal performance of spinifex resin...... 40

3.2.3 Chemical analysis of lignocellulosic components in spinifex grass...... 41

3.2.4 Pulping spinifex grass...... 42

3.2.4.1 Washing spinifex grass...... 42

3.2.4.2 Delignification of spinifex grass...... 42

3.2.4.3 Bleaching spinifex grass...... 43

3.2.7 Preparation of spinifex nanofibrillated cellulose (NFC) using a high pressure homogenizer...... 43

xiii

3.2.8 Preparation of spinifex nanofibrillated cellulose (NFC) using ultrasonication...... 44

3.2.9. Preparation of spinifex nanofibrillated cellulose (NFC) using high energy milling...... 45

3.2.10 Preparation of cellulose nanopaper...... 46

3.2.11 Acid treatment with hydrochloric acid (HCl)...... 47

3.2.12 Preparation of spinifex cellulose nanocrystals (CNC) by acid hydrolysing...... 47

3.2.13 Preparation of spinifex CNC-based nanocomposites...... 48

3.2.13.1 Solvent cast...... 48

3.2.13.2 In situ polymerization by twin screw extrusion (Reactive extrusion)...... 48

3.2.13.2.1 Preparation of compression molded samples...... 51

3.3 Characterization of spinifex resin, fibres, nanocellulose and polymer nanocomposites 52

3.3.1 Attenuated total reflectance (ATR) Fourier transform infrared spectroscopy (FTIR)...... 52

3.3.2 Thermogravimetric analysis (TGA)...... 52

3.3.3 Differential scanning calorimetry (DSC)...... 52

3.3.4 Scanning electron microscopy (SEM)...... 52

3.3.5 Transmission electron microscopy (TEM)...... 53

3.3.6 Measurement of diameter and length of spinifex fibre samples...... 53

3.3.6.1 Cryo-TEM...... 53

3.3.6.2 AutoCAD...... 54

3.3.7 Optical microscopy...... 54

3.3.8 X-Ray diffraction...... 54

3.3.9 Measurement of density and porosity...... 55

3.3.10 Mechanical properties...... 55

3.3.10.1 Tensile testing...... 55

3.3.10.2 Amplitude modulation- Frequency modulation (AM-FM)...... 56

3.3.11 Inductively coupled plasma optical emission spectrometry (ICPOES)...... 57

3.4 Summary...... 57

xiv

Bibliography...... 58

Chapter 4. Optimization of resin extraction and its potential application as a anti-termite coating material...... 59

4.1 Introduction...... 60

4.2 Results and discussion...... 60

4.2.1 Resin extraction and characterization...... 61

4.2.1.1 Effect of solvents...... 62

4.2.1.2 Effect of solvent to grass ratio and extraction temperature...... 64

4.2.1.3 Effect of temperature and time of solvent evaporation on the thermal properties of resin...... 67

4.2.2 Evaluation of termite resistance: A field study...... 71

4.3 Summary...... 74

Bibliography...... 75

Chapter 5. Pulping spinifex fibres...... 76

5.1 Introduction...... 77

5.1.1 Delignification and bleaching...... 77

5.1.2 TEMPO-mediated oxidation pretreatment...... 82

5.1.3 Carboxymethylation pretreatment...... 82

5.1.4 Enzymatic pretreatment...... 83

5.2 Results and discussion...... 83

5.2.1 Chemical analysis of lignocellulosic components in spinifex grass...... 83

5.2.2 Effect of delignification and bleaching on chemical structure of spinifex fibre ...... 85

5.2.3 Effect of delignification and bleaching on morphology of spinifex fibres...... 87

5.2.4 Effect of delignification and bleaching on crystallinity of spinifex fibres...... 92

5.2.5 Effect of delignification and bleaching on thermal properties of spinifex fibres...... 93

5.3 Summary...... 95

Bibliography...... 96

Chapter 6. The isolation of high aspect ratio nanofibrillated cellulose from spinifex using mild pulping and ultra-low energy processing...... 101

6.1 Introduction...... 102

6.2 Results and discussion...... 103

6.2.1 The effect of processing on spinifex NFC morphological characteristics...... 103

6.2.1.1 Homogenization...... 103

6.2.1.1.1 Comparison the effect of homogenization on spinifex pulp with commercial MCC...... 108

6.2.1.2 Ultrasonication...... 109

6.2.1.3 High energy milling...... 110

6.2.1.4 Comparison between the effects of the application of different mechanical defibrillation methods on the morphology of spinifex NFC...... 116

6.2.2 Crystallinity of spinifex NFC...... 120

6.2.3 Mechanical properties of spinifex NFC...... 124

6.2.4 Elastic modulus of single spinifex nanofibrils...... 130

6.2.5 Thermal properties of spinifex NFC...... 131

6.2.6 Effect of mild HCl treatment on properties of spinifex pretreated fibre...... 133

6.2.7 The production of NFC from another “hard” spices of Triodia for comparison...... 136

6.3 Summary...... 137

Bibliography...... 138

Chapter 7. Polymer nanocomposites reinforced with high aspect ratio cellulose nanocrystals isolated from Triodia pungens...... 144

7.1 Introduction...... 145

7.2 Results and discussion...... 146

7.2.1 Optimisation of isolation method...... 147

7.2.2 Morphological characterisation of cellulose nanocrystal...... 149

7.2.3 Effect of acid hydrolysis on the chemical structure and composition of spinifex fibres...... 151

7.2.4 Effect of acid hydrolysis on crystallinity of spinifex fibre...... 152

7.2.5 Effect of acid hydrolysis on thermal properties of spinifex fibre...... 154

7.2.6 Comparison of the results with previous published work...... 156

xvi

7.2.7 Tensile properties of solvent cast CNC-TPU nanocomposites...... 159

7.2.8 Tensile properties of reactive extrudes CNC-TPU composites...... 160

7.3 Summary...... 163

Bibliography...... 165

Chapter 8. Conclusion and future recommendation...... 167

8.1 Conclusions...... 168

8.1.1 Spinifex resin and termiticidal property...... 168

8.1.2 Defibrillation of spinifex fibre...... 169

8.1.3 Nanocomposite based spinifex nanocrystal and thermoplastic polyurethane...... 171

8.2 Future research recommendations...... 172

Bibliography...... 175

xvii

List of Figures

Figure 1.1. Thesis outline consisting of investigations of a range of spinifex-based biomaterials; including a renewable polymeric plant resin and novel nanoscale cellulose particles...... 4

Figure 2.1. Spinifex distribution in Australia. Current extend 2...... 8

Figure 2.2. Spinifex hummock grassland...... 8

Figure 2.3. a) Threshing spinifex to extract resin in central Western Australia 18, b) heating threshed resin to make a putty...... 13

Figure 2.4. Schematic showing the hierarchical structure of tree...... 16

Figure 2.5. Comparison between common established procedures for producing MFC (top) and common established procedures for producing NFC (bottom)...... 17

Figure 2.6. Schematic of idealized configurations of the crystalline and amorphous regions of cellulose microfibrils, and the removal of amorphous regions after acid hydrolysis...... 21

Figure 3.1. Flowchart representing the spinifex resin extraction process...... 40

Figure 3.2. Photographs showing; a) alternately arranged resin coated and uncoated timber pieces, and b) the samples covered with plastic sheet and soil at a termite trial site in Townsville, Queensland, Australia...... 41

Figure 3.3. Flowchart summarizing the mild chemical procedures used for pulping spinifex fibres...... 43

Figure 3.4. High pressure homogenizer used for producing NFC from spinifex pulp (EmulsiFlex-C5 Homogenizer)...... 44

Figure 3.5. Ultrasonic probe used for producing NFC from spinifex pulp (Q500 Sonicator)...... 45

Figure 3.6. High energy mill used to produce NFC from spinifex pulp (Netzsch LabStar Laboratory agitator bead mill). This photograph shows the continuous milling configuration...... 46

Figure 3.7. Schematic of the reactive extrusion setup...... 50

Figure 3.8. Twin-screw reactive extruder used to produce spinifex CNC and TPU nanocomposites (Entek Emax twin-screw co-rotating intermeshing extruder)...... 51

Figure 4.1. Schematic representation of the spinifex resin extraction process...... 61

Figure 4.2. DSC spectra of extracted resin before and after drying under vacuum...... 62

Figure 4.3. ATR FTIR spectra of spinifex resin extracted with different solvents (oven dried samples)...... 63

xviii

Figure 4.4. DSC spectra of extracted spinifex resin with different solvents show better thermomechanical stability for the acetone extracted resin (all oven dried samples)...... 64

Figure 4.5. DSC spectra of resin extracted at 30 °C showing the influence of solvent to grass ratio on the thermal transitions of resin...... 65

Figure 4.6. DSC spectra of resin extracted with the solvent to grass ratio of 2:1, showing the influence of extraction temperatures (21 and 30 °C) on the thermal transitions of resin...... 66

Figure 4.7. TGA spectra of resin extracted at different temperatures...... 67

Figure 4.8. DSC spectra of resin showing the improving thermal property of resin by increasing solvent evaporation time at 40 °C...... 68

Figure 4.9. Influence of solvent evaporation temperature on the thermal transitions of resin... 69

Figure 4.10. Influence of conditioning at 65 % relative humidity on the Tg changes of spinifex resin after one week...... 70

Figure 4.11. TGA spectra of spinifex resin dried (first evaporation) at different temperatures. 71

Figure 4.12. a) Timber blocks covered by plastic cover, b) timber blocks in the field, c & d) comparison between resin coated timbers without termite attack and plain wood which was destroyed by termites...... 72

Figure 4.13. a) Spinifex resin-coated and plain wood- there is no evidence of termites or ants, b) note that the pine samples adjacent to spinifex-resin coated samples were still surrounded by termites and other insects...... 72

Figure 4.14. Chemical structure of some major components isolated from spinifex resin 13.... 74

Figure 5.1. Flowchart representing the pulping procedures of spinifex fibres...... 85

Figure 5.2. ATR FTIR spectra of spinifex fibres before and after alkali delignification, organosolve delignification, and bleaching...... 87

Figure 5.3. SEM images of; a) raw fibre, b) water washed fibre, c) fibre subjected to alkali delignification and, d) fibre subjected to organosolve delignification...... 89

Figure 5.4. Optical microscopy images of; a) raw fibre, b) water washed fibre, c) fibre subjected to alkali delignification and, d) fibre subjected to organosolve delignification (the scale bars are 500 μm for image (a)- and 100 μm for images (b, c, d))...... 90

Figure 5.5. Optical microscopy of; a) alkali delignified and bleached, b) organosolve delignified and bleached (the scale bar is 100 nm)...... 91

Figure 5.6. Average diameter of spinifex fibre after washing and grinding, delignification and bleaching...... 91

Figure 5.7. X-Ray diffraction spectra of; a) water washed fibre, b) organosolve delignified fibre, c) alkali delignified fibre, d) organosolve delignified and bleached fibre, e) alkali delignified and bleached fibre...... 92

xix

Figure 5.8. Comparing TGA spectra of spinifex untreated (water washed) with alkali and organosolve treated fibres...... 94

Figure 6.1. The newly-discovered method of producing NFC from spinifex grass ( there is no treatment on the spinifex pulp before mechanical treatments )...... 103

Figure 6.2. TEM images comparing the effect of applied pressure in homogenization of 0.3 % (w/v) pulp dispersion after one single pass, on the morphology of spinifex nanofibrils; a) 105 1500 bar, b) 1000 bar, c) 350 bar (the scale bar is 500 nm)......

Figure 6.3. TEM images comparing the effect of the number of passes in homogenization of 0.3 % (w/v) dispersion of pulp, on the morphology of spinifex nanofibrils when applying 1500 bar pressure; a) 1 pass, b) 5 passes, c) 10 passes, d) 15 passes (the scale bar is 200 nm)...... 106

Figure 6.4. Three dimensional images of spinifex NFC were taken by cryo-TEM...... 107

6.5. TEM images compare the effect of one pass of homogenization at 1500 bar on morphology of; a) MCC, b) spinifex alkali pretreated fibre (the scale bar is 1 μm (a) and 2 μm (b))...... 10 8

Figure 6.6. TEM images of NFC produced from 1 % (w/v) dispersion of spinifex pulp by ultrasonication; a) at 20 % amplitude for 20 minutes, b) at 30 % amplitude for 20 minutes, c) at 60 % amplitude for 5 minutes (the scale bar is 2 μm)...... 110

Figure 6.7. TEM image showing substantial inorganic contamination in the NFC dispersion produced from 0.25 % (w/v) dispersion of organosolve pretreated spinifex fibre by continuous milling for 1 hour using 0.4 mm beads at 32 °C. The speed of milling and pump were 1500 and 65 rpm, respectively (the scale bar is 5 μm)...... 111

Figure 6.8. TEM images showing the effect of pulping on morphology of spinifex NFC; a) alkali treated fibres (milling 0.5 % (w/v) fibre dispersion for 20 minutes at 1000 rpm), b) organosolve treated fibre (milling 0.5 % (w/v) fibre dispersion for 20 minutes at 1000 rpm) (the scale bars are 2 μm)...... 112

Figure 6.9. TEM images showing the effect of concentration of spinifex pulp dispersion on the morphology of NFC; a) 0.5 % (w/v) spinifex fibre dispersion milled for 20 minutes at 1000 rpm, b) 1 % (w/v) spinifex fibre dispersion milled for 20 minutes at 1000 rpm(the scale bar is 2 μm)...... 113

Figure 6.10. TEM images showing higher amount of inorganic contaminations in NFC obtained from milling of 0.5 % (w/v) pulp dispersion at 1000 rpm with increasing milling time; a) 10 minutes, b) 1hour (the scale bar is 2 μm)...... 113

Figure 6.11. TEM image of 1 % (w/v) spinifex pulp dispersion obtained after milling at; a) 1000 rpm for 20 minutes, b) 3000 rpm for 20 minutes (the scale bar in 200 nm)...... 114

Figure 6.12. TEM images and dispersity of diameter of NFCs obtained by; a) homogenization of 0.3 % (w/v) pulp dispersion at 1500 bar only for one pass, b) ultrasonication of 1 % (w/v) pulp dispersion for five minutes at 30 % amplitude, and (c) milling of 1 % (w/v) pulp dispersion for 20 minutes at 1000 rpm (the scale bar is 500 nm)...... 117

Figure 6.13. Parenchyma tissue of Triodia pungens. The fibril bundles are surrounded by mesophyl...... 119

Figure 6.14. SEM micrographs of cross-sections of the raw Triodia pungens; a) global view, b) the bundles of fibrils which show a nodular structure of stroma lamellae covering the bundles, c) stroma lamellae on the surface of microfibrils bundles, d) spongy mesophyll network on the external layer of fibre, e, f, g) flaky mesophyll with the small holes on the surface (the hole size is 1.65± 0.85 μm)...... 120

Figure 6.15. X-Ray diffraction spectra of freeze dried spinifex NFC produced by homogenization, ultrasonication, and high energy milling...... 121

Figure 6.16. Comparing tensile curves for spinifex nanopaper produced from homogenized and milled NFC dispersions and prepared by vacuum filtration and drying in a vacuum oven at 50 °C for 48 hours...... 125

Figure 6.17. Tensile curves of spinifex nanopaper samples with different porosity (P) and thickness (t), produced from homogenized NFC and dried with a hot press at 103 °C for 2 hours...... 127

Figure 6.18. Spinifex nanopaper produced from homogenized NFC after vacuum filtration and drying with hot press at 103 °C for 2 hours; a) folded nanopaper, b) same nanopaper after 12 cycles of folding-unfolding...... 129

Figure 6.19. The optical transparency of spinifex nanopaper produced from homogenized NFC after vacuum filtration and drying with a hot press at 103 °C for 2 hours. The thickness and porosity of this nanopaper sample are 54 μm and 30 %, respectively...... 129

Figure 6.20. Transverse elastic modulus of spinifex nanofibril measured by AM-FM...... 131

Figure 6.21. TGA curves for spinifex NFC produced by homogenization, ultrasonication, and milling. The inset shows lower temperature curves in more detail...... 132

Figure 6.22. ATR FTIR spectra of bleached spinifex fibres and HCl treated fibres...... 134

Figure 6.23. X-ray diffraction spectra of bleached and HCl treated spinifex fibres...... 134

Figure 6.24. Optical microscopy of; a) bleached fibre, b) HCl treated fibre (the scale bar is 100 μm)...... 135

Figure 6.25. TGA spectra, showing the effect of HCl treatment on thermal properties of spinifex bleached fibre...... 135

Figure 6.26. TEM images of homogenized Triodia longiceps after one pass at 1500 bar (the scale bar is 2 μm (a) and 500 nm (b))...... 136

Figure 7.1. Typical procedure for the acid hydrolysis of spinifex grass (a detailed procedure can be found in Chapter 3, Section 3.2.12)...... 146

Figure 7.2. Sulphate groups on the surface of cellulose after hydrolysis with sulphuric acid... 147

Figure 7.3. TEM images of the cellulose nanocrystals obtained via acid hydrolysis from T. pungens comparing the effect of acid hydrolysing conditions on morphology of xxi nanocrystals; a) hydrolysis with 35 % (v/v) acid at 50 °C for 3 hours (the scale bar is 2 μm), b) hydrolysis with 35 % (v/v) acid at 45 °C for 6 hours (the scale bar is 2 μm), c) hydrolysis with 40 % (v/v) acid at 45 °C for 3 hours (the scale bar is 200 nm), d) hydrolysis with 50 % (v/v) acid at 45 °C for 3 hours (the scale bar is 500 nm)(all samples were ultrasonicated at 25 % amplitude for 20 minutes after dialysis)...... 150

Figure 7.4. TEM image of cellulose nanocrystals obtained via 40 % (v/v) sulphuric acid treatment at 45 °C for 3 hours, then dialysis, followed by ultrasonication at 70% amplitude for 20 minutes (the scale bar is 500 nm)...... 151

Figure 7.5. ATR FTIR spectra of bleached spinifex grass and CNCs obtained under different conditions...... 152

Figure 7.6. X-Ray diffraction spectra of spinifex fibre samples after alkali delignification, bleaching, and acid hydrolysis...... 153

Figure 7.7. Effect of sulphuric acid concentration on thermal properties of CNC...... 155

Figure 7.8. Effect of hydrolysis time on thermal properties of CNC...... 156

Figure 7.9. Tensile curves of neat TPU and CNC-TPU nanocomposite s produced by solvent casting...... 160

Figure 7.10. Tensile curves of neat TPU and CNC-TPU nanocomposites produced by reactive extrusion...... 162

Figure 8.1. Flowchart summarizes applied procedures to produce nanofibrils...... 169

Figure 8.2. Predicted structure of spinifex NFC covered by a thin layer of hemicellulose...... 171

xxii

List of Tables

Table 3. 1. List of chemicals used in this work and their supplier...... 38

Table 3.2. Different chemicals and conditions used for extracting spinifex resin from threshed grass...... 40

Table 3.3. List of different homogenization conditions used to produce NFC from spinifex pulp...... 44

Table 3.4. List of different ultrasonication conditions used to produce NFC from spinifex pulp...... 44

Table 3.5. List of different milling conditions used to produce NFC from spinifex pulp...... 46

Table 3.6. List of different drying processes of spinifex NFC nanopaper...... 47

Table 3.7. Different conditions used to produce CNC by acid hydrolysis...... 47

Table 3.8. List of different conditions used to produce spinifex CNC-based nanocomposites...... 48

Table 3.9. Composition used for producing TPU based on blank PTMEG 1000...... 49

Table 3.10. Reagent flow rates at different stoichiometries to produce TPU incorporating CNC...... 49

Table 3.11. Reactive extrusion temperature profiles...... 51

Table 4.1. ATR FTIR peaks and functional groups of extracted resin using different solvents...... 63

Table 4.2. Glass transition (Tg) temperature and extraction yield of extracted spinifex resin with different solvents (drying at 40 °C)...... 64

Table 4.3. Effect of solvent to grass ratio and extraction temperature on yield and Tg of resin (drying at 40 °C for 72 hours and annealing at 35 °C for 24 hours)...... 66

Table 4.4. Thermal property of resin after drying at 40 °C for increasing times...... 68

Table 4.5. Effect of drying temperature on the thermal property of resin...... 69

Table 4.6. Effect of heat treatment (evaporation temperature) on thermal stability of resin.... 70

Table 5.1. Comparing the most common delignification and bleaching processes for cellulosic fibres and wood pulp...... 78

Table 5.2. Chemical analysis of lignocellulosic components of raw and pretreated spinifex grass...... 84

Table 5.3. ATR FTIR peaks and functional groups of spinifex fibres before and after pulping………………...... 87

xxiii

Table 5.4. Effect of pulping on the diameter and crystallinity of the spinifex fibre...... 93

Table 6.1. Comparing the average diameter of nanofibrils produced by homogenization using different dispersion concentration, pressure conditions, and number of passes...... 104

Table 6.2. Comparing the effect of ultrasonication parameters on the average diameter of spinifex nanofibrils (the concentration of dispersion was 1 % (w/v))...... 110

Table 6.3. ICPOES analysis of spinifex fibre and NFC before and after acid treatment...... 115

Table 6. 4. Degree of crystallinity and size of crystalline domains of freeze dried spinifex NFC produced by homogenization, ultrasonication, and milling...... 121

Table 6.5. Comparison crystallinity and crystalline domain size of NFC obtained from different sources and treatments of cellulose with spinifex NFC...... 123

Table 6.6. Mechanical properties of spinifex nanopapers produced from homogenized and milled NFC dispersions and prepared by vacuum filtration and drying in a vacuum oven at 50 °C for 48 hours...... 125 Table 6.7. Mechanical properties of spinifex nanopaper samples with different porosity and thickness, produced from homogenized NFC and dried with hot press at 103 °C for 2 hours. 127

Table 6.8. Comparison the mechanical properties of nanopaper obtained from NFC prepared from the other sources of cellulose with spinifex NFC...... 128

Table 6.9. Thermal degradation temperature of spinifex NFC produced by homogenization, ultrasonication, and milling...... 133

Table 6.10. Comparison between properties of bleached fibre and HCl treated fibre...... 135

Table 7.1. Effect of acid concentration on the yield and appearance of CNC hydrolysed at 45 °C...... 148

Table 7.2. Effect of hydrolysis temperature on the yield and appearance of CNC hydrolysed with 35 % (v/v) sulphuric acid for 3 hours...... 148

Table 7.3. Effect of hydrolysis time on the yield and appearance of CNC hydrolysed with 35 % (v/v) and 45 % (v/v) acid at 45 °C...... 148

Table 7.4. Crystallinity and size of crystalline domain of spinifex fibre after alkali delignification, bleaching, and acid hydrolysis...... 153

Table 7.5. Comparison between CNC obtained from spinifex with the other industry and laboratory sources of cellulose...... 158

Table 7.6. Mechanical properties of neat TPU and CNC-TPU nanocomposites produced by solvent casting...... 160

Table 7.7. Different applied condition of extruder for producing CNC-TPU nanocomposite.. 161

Table 7.8. Mechanical properties of neat TPU and CNC-TPU nanocomposites produced by extrusion...... 163

xxiv

List of Equations

Equation 3.1. Degree of crystallinity...... 55 Equation 3.2. Mean size of crystalline region...... 55 Equation 3.3. Porosity of nanopaper...... 55

xxv

List of abbreviations

AFM Atomic force microscopy AM-FM Amplitude modulation- Frequency modulation ATR FTIR Attenuated total reflectance Fourier transform infrared spectroscopy BDO 1,4-butane diol CNC Cellulose nanocrystal DBTDL Dibutyltin dilaurate DSC Differential scanning calorimetry HEM High energy milling HPH High pressure homogenizer ICPOES Inductively coupled plasma optical emission spectrometry MCC Microcrystalline cellulose MDI 4,4'-methylene diphenyl diisocyanate MFC Microfibrillated cellulose NFC Nanofibrillated cellulose PTMEG Polytetramethylene ether glycol SEM Scanning electron microscopy T.pungens Triodia pungens TEM Transmission electron microscopy TGA Thermogravimetric analysis TPU Thermoplastic polyurethane US Ultrasonication

xxvi

Chapter 1.

Context of the study

1.1 Background and significance of this project This PhD project forms part of a larger Australian Research Council (ARC) project involving a multidisciplinary team of researchers from anthropology, architecture, botany and materials science disciplines that aims to combine Australian Aboriginal traditional knowledge with modern themes of biomimetic and nanotechnology science to explore the future sustainable applications of spinifex grasses as an abundant and renewable bio-resource. Spinifex grasses have been largely ignored as a sustainable resource, despite their widespread distribution throughout Australia, and unique biology that has evolved within harsh environments. As spinifex grasses have had to adapt to changing climates, extremely poor soils, high levels of UV radiation and low rainfall, it is likely that the resultant evolutionary adaptation of these unique plants has necessitated the production of unusual physical structures and/or organic compounds 1. Spinifex resin has been traditionally used by Australian Aborigines for medicines, hafting tools and repairing damaged equipment; the grasses were also used as a cladding in shelter construction 2. However, before commencement of the ARC project and this thesis, no systematic and scientific research had been undertaken into the potential engineering applications of spinifex for modern usage; therefore, our knowledge was limited to nature and Aboriginal people’s traditional knowledge. The research effort presented in this thesis is focused on the replacement of petroleum-based compounds, plastics and composites with more environmentally friendly materials. In addition to investigating a renewable polymeric plant resin and the isolation of novel nanoscale cellulose particles, both derived from native Australian Triodia pungens grass, this project is the only materials science investigation of spinifex-based materials, which also takes anthropological information into consideration. In this project I have identified, isolated and explored some compounds, mixtures or structural building blocks from spinifex grasses that allowed me to prepare renewable-based functional or structural materials of scientific interest. Significantly, this enhanced detailed understanding of spinifex composition, structure, refining and processing, in a materials

science sense, formed the foundation of a provisional patent, “Nanocellulose”, which was lodged on the 22nd of November 2013 3.

1.2 Objectives of this research project

Given the fact that this is the first materials science study involving spinifex components; this PhD project has been unavoidably exploratory in nature. However, in order to focus the research, the primary goal of this thesis is to explore unique compounds, structures and properties of Triodia pungens that may have applications in materials science and engineering. Initial exploratory research on properties has led to three secondary goals which make use of modern instrumentation, methods, and concepts in material science and nanotechnology. Briefly, these are:

1. To optimise spinifex resin extraction and determine its efficacy as a timber coating and termite deterrent; 2. To optimise deconstruction of the cellulosic components of Triodia pungens, and to prepare high quality nanofibrillated cellulose and cellulose nanocrystals; 3. To produce polymer nanocomposites reinforced with renewable spinifex cellulose nanocrystals.

1.3 Thesis outline

This PhD thesis consists of eight chapters;

 Chapter 1: Introduces the research background, significance and objectives of this project.

 Chapter 2: A literature review which covers the subjects of spinifex biomass, its application by Australian Indigenous people, termite deterrents, micro and nanofibrillated cellulose, cellulose nanocrystals, and nanocomposites based on thermoplastic polyurethane and cellulose nanocrystals.

 Chapter 3: Is a methods chapter which summarizes the materials, experimental methods and characterization tools employed to investigate spinifex biomass building blocks.

 Chapter 4: The first part of this chapter involves the optimisation of a resin solvent extraction procedure to achieve reasonably high spinifex resin yields and purity. While a 2

second section presents the systematic evaluation of the termiticidal performance of as- extracted resin mixture, which was applied as a coating to timber blocks and subsequently exposed to termites in the field.

 Chapter 5: This chapter explores the application of different mild chemical pulping to spinifex grass in order to determine the best way to isolate cellulose microfibres in preparation for subsequent mechanical defibrillation. The effects of each step of pulping on spinifex cellulose thermal properties, crystallinity, structure and morphology were also investigated.

 Chapter 6: Forms a substantial part of this thesis and relates to the preparation and characterisation of nanofibrillated cellulose via the systematic applications, and comparison between the following three different mechanical methods; high pressure homogenization, ultrasonication, and high energy milling (or micronizing). Additionally, the mechanical and thermal properties, crystallinity, and morphology of the nanofibrillated cellulose samples obtained were determined. The inorganic contamination in the milled nanofibril dispersions was also quantified, and a treatment to remove this contamination identified and described.

 Chapter 7: In contrast to mechanical methods for spinifex nanocellulose production, this chapter explores the optimal treatment conditions required to produce very high quality cellulose nanocrystals by the application of acid hydrolysis. Polymer nanocomposites based on aliphatic thermoplastic polyurethanes incorporating spinifex cellulose nanocrystals were produced using a solvent casting method in order to demonstrate the reinforcement efficiency and potential of these novel nanoparticles. In addition, a more scalable method of reactive extrusion was employed to successfully prepare aromatic thermoplastic polyurethane nanocomposites from reactive liquid precursors, including a stable polyol-cellulose nanocrystal suspension. Basic mechanical characterization was performed as a precursor to more exhaustive future applied research work and potential nanocomposite polyurethane applications.

 Chapter 8: Summarizes the findings of the thesis and their scientific significance. It also recommends sensible directions and activities for future work, and for shadows potential

applications of spinifex biomass and spinifex-derived nanocellulose additives and materials.

Figure 1.1. Thesis outline consisting of investigations of a range of spinifex-based biomaterials; including a renewable polymeric plant resin and novel nanoscale cellulose particles

1.4 A note on the exploratory journey of the research

As mentioned before, this project was an exploratory study to understand the modern and scientific potential of spinifex resin and grass. In addition to the chapters of this thesis, during the 3.5 years of my PhD, several additional experiments and analyses were performed, especially relating to the spinifex resin, which ultimately did not make their way into the thesis document. It would be fair to say, in hindsight that we underestimated the complexity and variability of spinifex resin. Some of these experiments included: (i) extraction and analyses of the volatile and non- volatile components of resin, (ii) an extensive study on nanocomposites comprised of spinifex resin and different types of nanoclays to try and increase the resin glass transition temperature, (iii) different treatments of spinifex resin in order to improve the thermal and mechanical properties of the resin, (iv) an extensive study on milling of spinifex grass in order to optimize the best conditions, and (v) solvent cast polymer nanocomposites comprised of spinifex nanocrystals and polystyrene. The methodological and scientific findings from some of these incomplete and/or unsatisfactory investigations may be taken up in the course of future research. Some aspects of this will be outlined in Chapter 8.

Bibliography

1. Toon, A.; Crisp, M. D.; Gamage, H.; Mant, J.; Morris, D. C.; Schmidt, S.; Cook, L. G. submitted manuscript at Journal of Biogeography 2014.

2. Memmott, P.; Flutter, N.; Penny, M., Biomimetic design prospects from an understanding of Aboriginal uses of spinifex grasses. In SAHANZ, 2009.

3. Amiralian, N.; Annamalai, P. K.; Martin, D. Provisional patent application, Nanocellulose. 22 November 2013.

Chapter 2 .

Literature review

Cellulose nanofibril Cellulose

Cellulose microfibril

Cellulose fibril

Termite Cell wall layers

Nanocomposite

2.1 Materials from renewable resources for sustainable development

New materials derived from biologically renewable resources have gained a great deal of interest from researchers in both academia and industry as alternatives to petroleum based (synthetic) products due to their potential positive effects on agriculture, environment and economy in terms of sustainability 1. Discovery, innovation and translation of these materials technologies necessitates coordinated research activities to explore new biomass resources. Biomass resources including raw materials (e.g. grass, wood, sugar cane, algae), agricultural crops and their by- products, oil plants (e.g. oil, fat, glycerol), starch plants (e.g. starch, carbohydrates, celluloses), and also waste streams (e.g. from wood, animals, food processing, and bio-waste from households) can provide sustainable development opportunities in the production of bio-resource based fuels, biobased polymers, and chemicals with high “eco-friendliness” values 3-5. Up until c. 1930, the main commercial products from renewable resources were limited to fuels, organic acids, and other basic chemicals, while recently the technology of using biomass has developed rapidly into invention of novel materials, particularly bio-plastics. Vegetable oils are already being applied mainly in plastics, paints, lacquers, and pesticides. Besides ethanol and biogases, the other bio-chemicals like acetone, butanol, ethanol, lactic acid and succinic acid are also being produced biotechnologically on an industrial scale. Many synthetic polymers, fuels and solvents, materials for the construction industry (e.g. sealants, insulants, lacquers, adhesives, binders), paper industry (e.g. coatings, print colours, solvents), textile, food, cosmetics, pharmaceuticals and personal care products could be produced sustainably from biomasses 6. In Australia, in spite of the existence of colossal fossil fuel energy resources, concerns relating to emissions of greenhouse gases and discussions about a “carbon tax” have catalysed increasing innovation towards the use of alternative high quality Australian renewable energy 7 sources such as solar energy, biomass, wave energy and wind energy . The government has a plan to generate 20 % of its total electricity supply from renewable sources by 2020 8, 9. The main source of biomass energy in this country is wood from natural forests. In order to avoid deforestation, it is important to look for alternative and supplementary renewable biomass resources. In this thesis, spinifex biomass, a native and abundant arid Australian grass, is introduced as an alternative renewable resource.

2.2 Spinifex as a potential biomass

In terms of native environmental products, the value of Indigenous traditional knowledge is receiving considerable attention. The modern beneficiaries of this traditional knowledge include groups with not only cultural and environmental interests, but also an interest in developing novel, ecologically-responsible, low-cost biomaterials. The use of plants has been well-documented in traditional Indigenous Australian cultures, and spinifex in particular has been used by Indigenous Australians for many thousands of years. Spinifex (also known as ‘porcupine’ and ‘hummock’grass) is the long-established common name for three genuses which includes genera Triodia, Monodia, and Symplectrodia (not to be confused with the different grass genus named Spinifex that is restricted to coastal dune systems in Australia) 10. With a wide distribution across the semi-arid and arid regions of Australia, spinifex is the main Figure 2.1. Spinifex distribution species in the grassland ecosystems of these in Australia. Current extend 2 regions, and with a very conservative estimate, covers 27 % (roughly 2.1 million km2) of the Australian landmass (Figure 2.1) 11. Spinifex species are perennial evergreen xerophytes (desert plants) that grow on a range of low nitrate soils, across climatic zones with very high temperatures and low water availability 10, 12. They produce highly resilient prickly leaves, growing as hummocks, and often develop into rings providing shelter for animals and other plants (Figure 2.2) 13, 14. Although Indigenous Australians have long used and traded some spinifex species, it was first reported in the written record in 1699, when William Dampier collected a specimen of hummock grass (Triodia danthonioides), from the southwest coast of Western Australia 15. In the 1940s, an anatomical variation of leaf structure was first documented by Nancy Burbidge. She Figure 2.2. Spinifex hummock grassland divided the genuses into two broad categories

8 based on the stiffness of the leaves and production of resin; the ‘hard’ non-resinous species and the ‘soft’ resinous species, respectively 10. Jacobs 16 has shown that these groupings correlate well with leaf anatomy, leaf epidermal characters, and resin production. Mc William and Mison suggested that re-examination of the leaf anatomy indicates that these two forms are likely to have markedly different physiologies relating to water use and photosynthetic activity 13, 17. Lazarides 10 reported over 60 described species of spinifex due to the significant variation in the floral parts. Hard spinifex species have stomata (microscopic pores on the surface of land plants) on both sides of the leaf, while soft spinifex species lack stomata on the outer abaxial leaf side. Species with the hard type anatomy are found throughout arid parts of Australia, with representatives in all mainland states 10, 18. The growth form of spinifex is very restricted in distribution outside Australia, and they appear to be a kind of adaptation to low-nutrient soil and drought 19, 20. While spinifex grasses can tolerate very high moisture stress 21, they are generally absent from the most extreme habitats, such as the mobile dune crests and very low rainfall areas 22, 23.

2.2.1 Triodia

Hummock grassland communities in arid Australia are dominated by spinifex species of the genus ‘Triodia’. There are 69 described species of Triodia, which are long-lived and deep-rooted; the roots can penetrate to depths of tens of metres under the ground 24. They are characterised by their unique drought-adaptive leaf anatomy 10, whereby they have tough, pointed, prickly and longitudinally-folded leaves 25.

2.2.2.1 Distribution

Species of Triodia are endemic to Australia, occurring in all mainland states from coastal islands in Western Australia to the east coast of Queensland. The genus is absent only in cold- temperate and alpine, high-rainfall regions of south-eastern Australia. The greatest numbers of species are in Western Australia (46) and the Northern Territory (34). Victoria has two species, South Australia eleven, New South Wales four and Queensland 15. Many species are widely distributed. Triodia scariosa exists in all mainland States, T. basedowii in all except Victoria, T. longiceps, T. pungens and T. schinzii in all except New South Wales and Victoria 10. T. basedowii is probably the most widespread and common species, along with T. schinzii and T. pungens 26.

2.2.2.2 Ecology

Species of Triodia occur on very extensive areas of sand plains (such as deep siliceous sands, sandy loams and sandy red earths), dune fields, mountains and hills in arid, semi-arid, and seasonally arid (monsoonal) regions of Australia 24.

2.2.3 Triodia pungens group

The soft and resinous Triodia pungens is widely distributed and grows on gravelly and clay sandy soils in semi-arid areas where rainfall can exceed 1000 mm in one wet season but is often less. Due to the resin production in the leaf sheaths, Triodia pungens is also known as the “gummy spinifex” 10. The leaf blades may be flat or flexure, over 30 cm long, and 0.8 - 1.2 mm wide. Triodia pungens forms asymmetrical shaped hummocks, 0.30 m to 1 m high and 0.30 m to 1.5 m diameter 25. The species favours vegetative re-sprouting rather than re-seeding, enabling Triodia pungens to regenerate after fire. Field observations have shown that these soft spinifex plants are deep rooted. They have a number of thin (approximately 1 mm thick) roots that can be found at depths below 30 m. However, in dry regions, lateral roots and root hairs are usually absent at these depths 27.

2.2.3.1 Distribution

Triodia pungens is a densely tufted, permanent, drought resistant grass that grows on both plains and rocky hills over much of northern Australia 25, 26, 28. Its alliance occurs in the highest rainfall region with the highest nutrient levels 26. Different topographical positions and soils in the landscape cause to grow several different morphological forms of T. pungens 29. T. pungens has an extensive distribution throughout north of Lat. 27 °S in Queensland, Northern Territory, Western Australia, and north-western South Australia.

2.3 Traditional uses by Aboriginal people

Spinifex has ancient uses and potentially novel material properties. All parts of this plant have been used by Aboriginal people for different traditional purposes such as use of the resin for hafting stone knives and spear-tips to wooden handles, foliage for shelter-cladding materials, and seed as a food source. The volatile compounds that give rise to the intense aroma released by these resinous grasses have been widely used by Aboriginal people as medicines 30. The non-exhaustive list of Aboriginal uses of spinifex includes:

• Shelter cladding (widespread), composite cladding systems

• Using fibre for making nets, baskets, bags (Western Australia only so far) • Using grass for making coolamons (an Indigenous Australian carrying vessel) to minimize water loss • Using seeds as food (limited evidence, Western Australia arid zone likely) • Fish dams (both static, mobile) • Secreted resin (as an adhesive gum used for hafting tools and plugging holes in water storage vessels 31). More specific functional applications of spinifex resin will be discussed in section 2.5.2.

2.4 Contemporary revitalisation of knowledge on spinifex

This study formed part of a much wider ARC Discovery project (Grant No. DP0877161) conducted in partnership with the Dugalunji Aboriginal Cooperation in Camooweal (western Queensland) focusing on the physical properties and potential modern commercial applications of spinifex resins and grasses. Both fibre and resin have potential uses in building technology as either separate or combined products. The wider ARC project has included ecological aspects which involve understanding optimal plant propagation to develop sustainable harvesting methods, architectural applications including low-tech building construction techniques that draw on traditional practice and have potential for local sustainable contemporary applications, and high- tech, laboratory-manufactured material products that may be of ultimate use in renewable building products and modern architecture. The primary goal of this thesis is to explore whether the unique adaptations of spinifex to extreme environmental conditions have in turn given rise to unique compounds, structures and properties that may have applications in materials science and engineering.

2.5 What is known about spinifex resin?

Resin is a plant product that has evolved frequently throughout higher plants. In spite of there being many different definitions of natural resin, it is commonly accepted as a plant secretion which is translucent, often sticky, and soluble in organic solvents rather than in water. Spinifex resin is a thermoplastic bio-organic polymer, which is a mixture of volatile and non- volatile compounds and secondary metabolites 32. The resin collected from the spinifex plant is an inexpensive, plentiful, renewable material in Australia, and has been widely used by Australian Indigenous people for both functional and traditional purposes.

2.5.1 Chemical analysis

The chemical analysis of spinifex resin is a challenging subject and to my knowledge, while no studies are to be found in journal publications, one study done as a part of the ARC Discovery project and presented in an unpublished conference paper by S. De Silva et al , reported the composition of primary components in the Triodia pungens resin by extraction using various hexane/methanol mixed solvent ratios (flash chromatography), followed by chemical analysis using GC-MS (including use of SPME fibre to adsorb and desorb volatile species), ATR FTIR, 1H and 13C NMR. 38 compounds were identified in her analysis. Among them 3-Hexen-1-ol (3.67 %), α-Pinene (5.71 %), Camphene (3.16 %) Limonene (25.08 %), Decane (9.31 %), α-Terpineol (6.52%), Bornyl acetate (5.1 %), Nerolidol Z, and E (7.18 %) were the main volatile compounds of Triodia pungens. She suggested it was near impossible to identify every molecular component in spinifex resin. The analysis of the non-volatile fraction of spinifex resin by GC-MS revealed that it principally consists of mono and diterpenoid products, some with a labdane-type skeleton. This is certainly an area where more analytical chemistry needs to be performed on these Triodia resins; however a decision was made early on in my candidature that this chemical characterisation would be beyond the scope of my thesis.

2.5.2 Spinifex resin sources and applications

Spinifex resin droplets or crystals can be collected directly from the spinifex plant, and are typically found at the base of the leaf, next to the sheaths, or resin can alternatively be gathered at the base of the spinifex clumps, particularly after the plant has been exposed to fire. Here it is common to find a resin-rich dark mass, which has been concentrated in the nest of the Spinifex ant. In traditional Aboriginal manufacturing, the soft species of spinifex are typically harvested, and firstly smashed and/or threshed on a clean surface using a heavy stick, followed by some sorting and refining by hand to leave the fine particles of the resin separated from the grass. Then the chaff, which is called threshed resin, is heated with a fire stick causing the resin to melt (Figure 2.3) 33, 34. Finally the threshed, heated resin is rolled into a ball and pieces can be used as an adhesive or putty, mainly for attaching stone blades to wooden implements such as spears, knives and axes 24. More heating and working the putty makes it harder, more brittle, and able to remain rigid, even on a hot day in the Australian desert 30-32, 34-38. In addition to its typical use as an adhesive, it was also commonly used as a water-proofing agent for mending cracks and holes in wooden carrying dishes 24, 34. Each plant resin is botanically and geographically specific to the environment in which a particular group of people are living 39. So reportedly the resin was also sometimes reinforced with other materials such as bees wax, fine sand, kangaroo dung and/or plant fibres 40. 12

a b

Figure 2.3. a) Threshing spinifex to extract resin in central Western Australia 18, b) heating threshed resin to make a putty

2.6 Sustainable development

Recently, there has been great interest in producing novel modern materials based on environmentally-abundant, low-cost plant species whereby the resultant product mimics or isolates a particular property or function of the source plant exhibited in its natural habitat. Biomimetic theory and its application, bio-inspired design, derive concepts from natural systems and have the potential to create new types of sustainable materials from renewable resources. In this project I used an approach that links both ethnographic and ecological research with material sciences in order to create and test new spinifex-derived building blocks. More specifically, I investigated selected physico-chemical properties of spinifex grasses to evaluate the feasibility of producing novel and sustainable building technology products. A variety of products were studied, including spinifex resin coatings that have anti-termite capacities, as well as spinifex nanofibrils and nanocrystals, and subsequently spinifex nanocrystal reinforced polymer composites.

2.7 Termite deterrent

Termites serve a great role in recycling plant and woody materials, as they decompose the cellulose and help to break down approximately one third of the annual production of dead wood. Conversely and unfortunately, they are also a highly destructive insects due to their wood eating habits. They destroy structural and non-structural building materials, finished goods, plants and agricultural crops, and also other domestic goods such as paper, cloth, carpets, and other cellulosic materials 41, 42. There are more than 2800 different species of termites, with about 185 considered to be pests 43. In Australia, over 300 species of termites exist, and among them, there are about 15

13 economically important species, which have become particularly well adapted to the urban environment 44. The economic losses associated with termite damage for Malaysia, India, Australia, China, Japan and the United States were 10, 35, 100, 375, 800 and 1,000 million US dollars in 2005, respectively 45. In Australia, in 2005 the annual cost of repair of damage to timber-in-service caused by termites has been estimated by Archicentre (the building advisory service branch of the Australian Institute of Architects) to be $780 million annually, which equates to more than the costs for fire and storm damage combined. It was estimated that during the years 2000 - 2005, 10 % of Australian houses, 650,000 houses, were infested by termites at a cost of about $3.9 billion. The average cost of treatment for a pest manager is $1500, and an average building repair cost is $4,500 44, 46. Termites are thus a serious and expensive threat to building materials and structures. Contemporary protection methods include both physical and chemical approaches. Physical methods include physical barriers, heat, freezing, electricity, and microwaves 43. Physical barriers for the prevention of subterranean termite attack of wooden structures can be toxic and nontoxic. Toxic physical barriers include the use of chemical termiticide in the soil around the structure, and nontoxic physical barriers including substances like sand or gravel aggregates, metal mesh or sheeting that exclude termites, as they are impenetrable 43. For example, TermimeshTM is a stainless steel mesh now commonly employed as a physical termite barrier. Chemical treatment of wood is a common and effective method which is widely used to prevent termite attacks. Several termiticides containing different active ingredients such as bifenthrin (Makhteshim- Agan-Seizure 100 EC), chlorfenapyr (BASF-Phantom), cypermethrin (Effecticide-Spraykill), fipronil (BASF-Termidor), imidacloprid (Bayer–Gaucho) and permethrin (BASF-Coopex TC Termiticide) are registered for termite control around the world under various brand names 43. In spite of many decades of successful termite control through the application of chemical termite deterrent agents, there are serious and growing concerns regarding the use of chemical pesticides leading to environmental pollution and health disorders. To avoid these problems, it is necessary to look for alternatives to minimize the use of synthetic and harmful chemicals 41. Bio-pesticides, which are extracted and/or purified substances from plants and wood are potentially promising replacements due to associated advantages such as their minimal toxicity to mammals, their widespread availability, repellency and/or toxicity to pests, and relatively low cost 47-49 . For example, the crude resins obtained from Dipterocarpus 50, ester fraction of J. curcas and karanjin of P. pinnata oil 51, Guayule resin extracted from arid grass Parthenium argentatum 48, 49, 52, and the leaves of a Brazilian tree (Copaifera langsdorfii) have been known to be significant elements of plant defence against insects and termites 53.

The objectives of Chapter 4 are firstly, the optimisation of a resin solvent extraction procedure, and secondly, to systematically evaluate the termiticidal performance of the as-extracted, non-purified or fractionated resin mixture.

2.8 Cellulose and nanocellulose

Cellulose is the most abundant polymer available worldwide with an estimated annual natural production of 1.5×1012 tons and considered as an almost inexhaustible source of raw materials which has been used for about 150 years 54, 55 . Cellulose is the main constituent of cell wall in lignocellulosic plants, and its content, composition and organisation depends on the plant species, growing environment, position, growth, and maturity. The term nanocellulose generally refers to cellulosic materials having at least one dimension in the nanometer range. The concept of breaking down cellulose fibres into structural components, micro and nanofibrils, has recently become a topic of intense academic and industrial interest. These nano particles are well-known for their unique structure and many useful characteristics. A large number of applications are emerging in multiple industries. The microstructure of plant cell wall includes primary walls (the outer very thin part which is usually less than 1 μm) and secondary wall which consists of three layers composed of microfibrils surrounded in a matrix of hemicelluloses, lignin, and other carbohydrate polymers. The microfibrils consist of monocrystalline cellulose domains linked by amorphous domains 56. Amorphous regions act as structural defects and can be removed via acid hydrolysis, leaving cellulose rod-like nanocrystals, which are also called whiskers, and have a morphology and crystallinity similar to the original cellulose fibres 57, 58. Cellulose, a linear homopolysaccharide composed of β-d- glucopyranose units linked together by β-1–4-linkages (C6nH10n+2O5n+1 ) is predominantly located in the secondary wall 59. The hierarchical structure of cellulose derived from wood is illustrated in Figure 2.4. Functionality, flexibility and high mechanical strength/weight performance of cellulose fibres can be developed by exploiting hierarchical structure design that spans nanoscale to macroscopic dimensions.

Cellulose Cellulose nanocrystal

Cellulose nanofibril

Cellulose microfibril

Cellulose fibril

Biomass

Wood Wood cell Cell wall layers

Figure 2.4. Schematic showing the hierarchical structure of tree

2.8.1 What is nanofibrillated cellulose (NFC)?

Nanocelluloses can be classified to different subcategories on the basis of their dimensions, functions, and preparation methods, which mainly depend on the cellulosic source and processing conditions. Nanofibrillated cellulose (NFC) is a small structural unit of plant fibre, consists of a bundle of stretched cellulose chain molecules with a diameter of 4 -10 nm and a length of 500 - 57 60 61 62 2000 nm . Nanofibrillar cellulose , cellulose nanofiber , cellulose nanofibril and even 63 59 sometimes microfibrillated cellulose (MFC) are all the terms used for nanofibrillated cellulose . It is worth nothing that nanofibrillated cellulose is generally produced through mechanical methods after chemical/mechanical or enzymatic pretreatments of highly purified pulp, while microfibrillated cellulose is commonly produced via mechanical treatment of purified pulp without any further pretreatment and has a diameter in the range of 10 - 100 nm and a length of 0.5 - 10 μm 57, 64 . The isolation of cellulose fibres from cellulose source materials occurs in two stages of; (i) purification and pretreatment of the source material, then (ii) separation of these purified cellulose materials into their nano-sized components 57. As mentioned previously, the microfibrils are embedded in a matrix of hemicellulose and lignin, therefore in order to disintegrate the cell wall into the bundles of microfibrils, a particular pretreatment such as either a chemical 65, mechanical or enzymatic step which also is called pulping is necessary 66-69. Several approaches are used for the second stage to isolate microfibrillated cellulose from the cell wall of wood and lignocellulosic materials, and these include; using very high shear mechanical processes, such as high pressure 16 homogenizers 70-73, grinding, milling or refining 74-78, cryocrushing 79-82, high intensity ultrasonic 83- 85, microfluidization 68, 72, 86 and through a combination of mechanical and chemical treatments 87-89 or enzymatic treatments 90-92 for producing NFC. Figure 2.5 compares two established procedures for producing MFC and NFC. The various mechanical methods consume energy at different levels and lead to different types of micro and nanofibrils, depending on the cellulose raw material 86, so chemical and enzymatic pretreatments can facilitate fibre disintegration into cellulose nanofibrils. It is important to understand the kinetics and factors affecting the degradation tendency of each cellulose source, especially if looking for a scalable method to produce NFC or MFC 69. The MFC and NFC are mostly extracted from bleached kraft pulp 87, 93-97, followed by bleached sulphite pulp 61, 98-100 and other sources such as agricultural crops and their by-products, like sugar beet pulp 101, 102, wheat straw and soy hulls 70, 82, sisal 103, 104, bagasse 105, palm trees 106, ramie 105, carrots 107, cotton 108-110, potato tuber cells 111, banana rachis 112, lemon and maize 113, hemp 110, 114, 115, and pea hull fibre 116.

Figure 2.5. Comparison between common established procedures for producing MFC (top) and common established procedures for producing NFC (bottom)

2.8.2 Properties and applications of nano and microfibrillated cellulose

Among all of the different kinds of nano-sized cellulose particles, NFC and MFC can be prepared more easily with high yields via scalable mechanical methods, which provides the 17 opportunity of using these nano and microfibrils in a number of industrial fields and products 68, 91. Nano and microfibrillated cellulose have revealed interesting and promising properties such as good mechanical properties 91, high stiffness and mechanical resistance along the longitudinal direction, high aspect ratio 68, low-cost, amenability to surface functionalise, biodegradability and renewability 96. Having a theoretical modulus of 145 GPa and the ability of forming dense networks due to interactions between nano-sized elements through strong hydrogen inter-fibrillar bonds or entanglements, making these particles very suitable as reinforcements in polymer nanocomposites 68, 91, 96, 117, 118. It has been reported that NFC can enhance the toughness of composites compared to the pulp fibers 119. The elastic modulus and tensile strength of NFC-based films developed from softwood pulp were respectively found to be 12 and 16 times higher than those of non-woven made of wood pulp 120. The optically transparent composites 121 made of these nanocellulose particles also have good oxygen and water vapour barrier properties due to the relatively high crystallinity 122 and high cohesive energy density, which provide some potential uses in the field of packaging with high barrier properties 123. Nanofibrillated cellulose can also be used as thickeners 68, dispersion stabilizers 118, 124, absorbent sheets 91, antimicrobial films 124, materials for biomedical applications 125 and air and water filtration materials 91. The high water retention capacity of nanocellulose makes it highly desirable as gel-forming agent in the food, pharmaceutical and cosmetic industries 69, 68. It has been found that these particles also are good additives in paper and paperboard manufacture due to their superior mechanical properties such as strength and stiffness and the ability to form thin films 119. In addition, renewability and biodegradability of micro and nanocellulose offer sustainable and viable alternatives to synthetic materials 89, 126, 127. Commercially, the three leading enterprises producing nanocellulose at the time of writing this thesis were CelluForce (Canada) , Innventia (Sweden) and FPInnovations (Canada) who were reportedly producing 1 tonne, 100 and 80 kilograms per day, respectively.

2.8.3 Methods of producing nanofibrillated cellulose

Some of the main mechanical methods of producing NFC, which are also employed in this thesis are; homogenization, ultrasonication, and high energy milling.

2.8.3.1 Homogenization

Treating fibres with a homogeniser involves the preparation of a dilute fibre dispersion with a concentration of lower than 1 - 2 wt% in water in order to delaminate the fibres. Applying a large pressure drop under high shearing forces and impact forces against a valve and an impact ring lead to splitting of the fibres and a reduction of diameters to between 20 and 100 nm, and estimated 18 lengths of several micrometers 117, 128-130. In order to increase the degree of fibrillation, fibers are typically cycled through the homogenizer for approximately 10 - 20 passes 66, 86. It is obvious that with increasing the number of passes, the required energy for disintegration will also increase. The other disadvantage of using homogenization to produce NFC is regular clogging of the system by long fibres, which necessitates disassembly in order to clean and re-start the process. In order to facilitate this process, mechanical refining or some kind of chemical treatment, tends to be beneficial, prior to homogenization 86.

2.8.3.2 Ultrasonication

The ultrasonic technique has been studied as a powerful tool to isolate cellulose nanofibers. This method converts mechanical or electrical energy into high-frequency acoustical energy in the range of approximately 10 - 100 kJ/mol which can isolate cellulose nanofibrils from cellulose fibres 92. The mechanism of this treatment is based on the energy produced by formation, expansion, and collapse of cavities in aqueous solution. The resulted waves on the surfaces of natural fibres, produced during the cavitation, cause erosion of the surface of the fibers and isolate the fibrils 110. The ultrasonic technique has been used to isolate cellulose nanofibrils from different sources of cellulose such as wood , lyocell, wheat straw, cotton, bamboo, ramee, and hemp 83, 84, 92, 110, 131-134. Long nanofibrillated cellulose also obtained by this method from commercial microcrystalline cellulose exhibited very high mechanical properties beneficial for using as a filler to make nanocomposites 132, 133. A disadvantage of this process is that it is less scalable and involves a higher capital expenditure than other methods. It is therefore seen more as a “research interest” more than a fully-industrialisable approach.

2.8.3.3 High energy milling

Mechanical milling is one of the widely used techniques to fabricate a nano-structured material that has unique properties and it is feasible for large-scale production, while being a simple and low-cost technique with high yield. In this (dry or wet) process, often called grinding or micronizing, the starting small particles are trapped between moving balls and the inner surface of the mill vessel and the kinetic energy of the balls can result in breakage of chemical bonds, rewelding, and fragmentation of material, and consequently results in producing fine and dispersed particles in the powder or fluid matrix. Milling of cellulose fibres has mostly been used as a pretreatment of woody biomass in preparation for bio-ethanol production. The structural changes consist of reduction in the crystallinity and size of crystals, increasing the surface area and decreasing the degree of polymerization 135-139. Zhao et al 137 applied this method to decrease the 19 crystallinity as a pretreatment of producing cellulose nanocrystals by a dilute acid hydrolysis. Ago et al 140 demonstrated the crystalline transformation from cellulose I to cellulose II using ball milling in a specific amount of water as a medium. Zhang et al 78 and Liimatainen et al 141 used milling as an instrument to produce nano/microfibrils from the cellulose fibres. The feasibility of using milling is further increased by the possibility of chemical modification of the cellulose material during the milling process, which provides potential for producing minuscule nanocellulose derivatives in commercial scale quantities. A substantial part of this thesis (Chapter 5 and 6) relates to the preparation and characterisation of spinifex-derived nanofibrillated cellulose via the systematic application of these three different mechanical methods, and determination whether an optimum set of processing conditions exists for each approach.

2.8.4 Cellulose nanocrystals (CNC)

Cellulose nanocrystals have been of growing interest because they are biocompatible and biodegradable and can be obtained from a variety of natural sources. CNC are bound by intra- and intermolecular hydrogen bonding and organised into elementary fibrils separated by non-crystalline regions (lignin, pectin, hemicellulose). By defibrillation and/or removing the other amorphous components, the highly crystalline nanofibrils can be recovered 142. Different methods have been applied to prepare cellulose nanocrystals, and each of them leads to different types of nanomaterial (e.g. shape, length, and diameter), depending on the source of cellulose and the deconstruction process employed (e.g. controlled time, temperature and acid concentration), and also applied pretreatments 121, 143-145. The most typical and widespread process of producing cellulose nanocrystals (CNC) involves strong acid hydrolysis under strictly controlled conditions of temperature, agitation, (sometimes) pressure and time to remove amorphous and disordered or paracrystalline regions and consequently isolate crystalline domains with higher resistance to the acid attack (Figure 2.6) 129. Depending on the cellulose source and hydrolytic conditions, cellulose nanocrystals with the diameter range of 3 - 15 nm and length in the range of 50 - 500 nm are isolated 57.

1 nm

Crystalline region Amorphous region 100 nm

Acid hydrolysis

Cellulose nanocrystal

Figure 2.6. Schematic of idealized configurations of the crystalline and amorphous regions of cellulose microfibrils, and the removal of amorphous regions after acid hydrolysis

2.8.4.1 Properties of cellulose nanocrystals

Nano-sized cellulose crystalline substrates display reasonably high aspect ratio, high specific surface area, high crystallinity, high Young’s modulus, high specific strength and flexibility, low density, low thermal expansion, unique optical and specific barrier properties, and also biocompatibility and biodegradability 144-150. These properties help to make a rigid CNC percolating network via hydrogen bonds as the filler in several natural and synthetic polymeric matrices including; natural rubber 151, 152, polystyrene 153, 154 polypropylene 155, poly (vinyl chloride) 156, 157, poly (lactic acid) 158-164, hydroxypropyl cellulose 89, amylopectin-glycerol blends 165, polycaprolactone 166-168, silk fibroin 147, poly(vinyl alcohol) 89, 169-172, polyurethane 147, 173-176, starch 177, 178, chitosan 179, 180, and soy protein 181.

2.8.5 Nanocellulose reinforced composites

Cellulose reinforced composites are characterized by low cost, desirable fibre aspect ratio, low density, high specific stiffness, strength and corrosion resistance 182-185. As a result, they are increasingly being evaluated in various fields such as materials science, electronics, catalysis and biomedicine and also several sectors of engineering such as automotive, aerospace, construction, and sports equipment manufacturing 184, 186. The composites made of cellulose nanoparticles have flexibility during processing with no harm to the equipment, good mechanical properties, enhanced energy recovery, and reduced dermal and respiratory irritation 187. Additionally, their 21 biodegradability and biocompatibility enables cellulose reinforced materials to potentially be used as biomaterials or scaffold in medical device or tissue engineering applications, respectively 188. It has also been demonstrated that with the appropriate surface modification, nanocellulose additives can impart antimicrobial properties to polymeric matrixes 189. One host polymer type which has received a lot of attention in terms of nanocellulose reinforcement is polyurethane 175, 190, 191, where cast elastomer 192, foam 193, 194, and thermoplastic elastomer 195, 196 based nanocomposites have all been reported. Thermoplastic polyurethane (TPU) is a particularly interesting class of polyurethane due to great chemical versatility, and a wide range of properties, service temperatures and hardness options, excellent tear and abrasion resistance, good resistance to nonpolar solvents and high compression and tensile strength 197. These properties make TPUs the polymers of choice in many products such as biomedical devices, automotive components, cables, films, roller systems coatings, foams, adhesives and food processing equipment 198, 199. Typical linear TPU block copolymers consist of alternating flexible “soft” and rigid “hard” segments. The hard segment rich crystalline or paracrystalline phase is composed of a low molecular weight diol or diamine (chain extender) with diisocyanate and engenders stiffness and strength to the TPU while the soft segment, typically composed of a high molecular weight polyester or polyether polyol, provides a rubbery matrix which controls low temperature properties and increases the extensibility of the segmented polyurethane 176. In general, during polymerization the active isocyanate groups in thermoplastic polyurethanes can readily react with hydroxyl groups, so if carefully managed, there is an opportunity to promote a small degree of covalent coupling between the nanocellulose and the polymer matrix, which can help avoid cellulose agglomeration 200, and enhance interfacial stress transfer and mechanical performance of composite. One of the specific challenges in preparing polymer nanocomposites is to improve the balance between stiffness, strength, and strain-to-failure. For example, too much stiffening and a concomitant loss of resilience are not desirable for these TPU thermoplastic elastomers. Several studies have been performed on polyurethane host systems by introducing microfibrillated cellulose (MFC) 173, 201, microcrystalline cellulose (MCC) 201 and cellulose nanocrystals (CNC) 195, 201-205. The cellulose nano/microfibrils can improve the ultimate mechanical properties of the polyurethane nanocomposites. In one experiment an increase of 500 % in the tensile strength was achieved 176 in a polyurethane nanocomposite reinforced with 16.5 wt% of cellulose nanofibrils and prepared by using a compression molding method. Similarly, polycaprolactone-based waterborne polyurethane nanocomposites incorporating CNC have shown a significant increase in the tensile strength, Young’s modulus 206 and tensile modulus 203 . Pei et al 176 synthesized a nanocomposite using in- situ solution polymerization, thereby introducing some covalent bonding between a very low volume fraction of reactive CNC with the hard segments of a polyether based TPU matrix. Only 1 22 wt% addition of cellulose nanocrystals was required to improve the stiffness and toughness of the thermoplastic polyurethane significantly, and most impressively, without reducing its extensibility. Increases in strength, elongation potential and thermal stability have also been reported for bio- nanocomposites made of vegetable oil derived TPU and nanocellulose. Rueda et al 198 , Aranguren et al 191 , Wu et al 201 , and Marcovich et al 202 have all analysed the effect of the addition of cellulose nanocrystals on thermal, morphological and mechanical properties of polyurethane nanocomposites. A strong interaction between matrix and reinforcement was enabled during curing, through a chemical reaction between the OH groups of the cellulose polymer and the isocyanate component. This reaction leads to the increase in the Tg, tensile modulus, Young’s and storage modulus of the prepared PU films. In Chapter 7 of this thesis, aliphatic TPU nanocomposites incorporating spinifex cellulose nanocrystals are produced using the solvent casting method. Furthermore, a more scalable method to manufacture high performance aromatic TPU-spinifex CNC nanocomposites is presented through the use of reactive extrusion of a stable polyol-CNC suspension with diisocyanate and chain extender components.

Bibliography

1. Raquez, J. M.; Deléglise, M.; Lacrampe, M. F.; Krawczak, P. Progress in Polymer Science 2010, 35, (4), 487-509.

2. Anon Australia’s native vegetation: a summary of Australia’s major vegetation; Department of the Environment and Heritage: Canberra, 2007.

3. Demirbaş, A. Energy Conversion and Management 2001, 42, (11), 1357-1378.

4. Narodoslawsky, M. Chemical Engineering Research and Design, (0).

5. Goyal, H. B.; Seal, D.; Saxena, R. C. Renewable and Sustainable Energy Reviews 2008, 12, (2), 504-517.

6. Willke, T.; Vorlop, K. D. Appl Microbiol Biotechnol 2004, 66, (2), 131-142.

7. Yusaf, T.; Goh, S.; Borserio, J. A. Renewable and Sustainable Energy Reviews 2011, 15, (5), 2214-2221.

8. Hasan Chowdhury, S.; Maung Than Oo, A. Renewable and Sustainable Energy Reviews 2012, 16, (9), 6879-6887.

9. Martin, N. J.; Rice, J. L. Renewable Energy 2012, 44, (0), 119-127.

10. B. Rice , M. W. Australian Journal of Ecology 1999, (24), 563-572.

11. Allan, G.; Southgate, R., Fire regimes in the Spinifex landscapes of Australia. In Flammable Australia: The fire regimes and biodiversity of a continent, R. Bradstock, J. W., and M. Gill, Ed. CPU: Cambridge, 2002; pp 145–176.

12. Lazarides, M. Australian Systematic Botany 1997, (10), 381–489.

13. McWilliam, J. R.; Mison, K. Australian Journal of Plant Physiology 1974, 171–175.

14. Jacobs, S. W. L., Spinifex In Arid Australia. In HCogger, E., Ed. Australian Museum: Sydney: 1984; pp 131–142.

15. George, A. S., William Dampier in New Holland. Australia's First Natural Historian. Bloomings Books: Melbourne, 1999.

16. Jacobs, S. W. L., Spinifex (Triodia, Plectrachne, Symplectrodia and Monodia: Poaceae) in Australia. In Desertified Grasslands:Their Biology and Management, Chapman, G. P., Ed. Academic Press: London, 1992; pp 47-62.

17. Craig, S.; Goodchild.D, J. Australian Journal of Botany 1977, 25, 277-290.

18. Powell, O.; Fensham, R.; Memmott, P. Economic Botany 2013, 1-15.

19. Beard, J. S., Vegetation of Central Australia. In Flora of Central Australia, Jessop, J., Ed. A.H. & A.W.Reed: Sydney, 1981; pp xxi–xxvi.

20. Beard, J. S., The vegetation of the Australian arid zone. In Arid Australia Cogger, H. G.; Cameron, E. E., Eds. Australian Museum: Sydney, 1984; pp 113-9.

21. Slatyer, R. O. UNESCO Arid Zone Research 1961, 16, 137-46.

22. Fatchen, T. J.; Barker, S. Transactions of the royal society of south Australia 1979, (103), 113-21.

23. Buckley, R. C. Australian Journal of Ecology 1982, 7, 309-13.

24. Gamage, H. K.; Mondal, S.; Wallis, L. A.; Memmott, P.; Martin, D.; Wright, B. R.; Schmidt, S. Australian Journal of Botany 2012, 60, (2), 114-127.

25. Perry, R. A. Lands of the Alice Springs Area, Northern Territory; 1962; pp 1956–57.

26. Beadle, N. C. W., The Vegetation of Australia. Cambridge University Press: Cambridge, 1981.

27. N. Reid, S. M. H., D. M. Lewis Applied Geochemistry 2008, 23, 76–84.

28. Griffing, F. Journal of Vegetation Science 1990, 1, 435-444.

29. Burbidge, N. T. J. Proc. Roy. Soc. Western Aust 1945, 28, 157-164.

30. Langenhleim, J. H., In Plant resin: chemistry, evolution, ecology, and ethnobotany. Timber Press: Cambridge, 2003.

31. Memmott, P.; Flutter, N.; Penny, M., Biomimetic design prospects from an understanding of Aboriginal uses of spinifex grasses. In SAHANZ, 2009.

32. De Silva, S.; Memmott, P.; Folter, N.; Martin, D., Charectrisation of Spinifex (Triodia pungens) Resin and Fiber. In 11th Pacefic polymer conference Cairns, Australia, 2009.

33. Mondal, S.; Memmott, P.; Wallis, L.; Martin, D. Materials Chemistry and Physics 2012, 133, (2–3), 692-699.

34. Pitman, H.; Wallis, L. Ethnobotany Research & Applications 2012, 10, 109-131.

35. Wassner, D. F.; Ravetta, D. A. Industrial Crops and Products 2005, 21, (2), 155-163.

36. Parr, J. F. Economic Botany 2002, 56, (3), 260-270.

37. Flood, J., Archaeology of the Dreamtime: the Story of Prehistoric Australia and Its People. In Angus & Robertson, S., Ed. New York: 1995.

38. Blee, A. J.; Walshe, K.; Pring, A.; Quinton, J. S.; Lenehan, C. E. Talanta 2010, 82, (2), 745- 750.

39. Lowe, P.; Pike, J., Jilji: Life in the Great Sandy Desert. Magabala Books Broome: Western Australia, 1994.

40. Cribb, A. B.; Cribb, J. W., Useful Wild Plants in Australia. Fontana Books: Sydney, 1982.

41. Seo, S. M.; Kim, J.; Lee, S. G.; Shin, C. H.; Shin, S. C.; Park, I. K. Journal of agricultural and food chemistry 2009, 57, (15), 6596-602.

42. Upadhyay, R. K.; Jaiswal, G.; Ahmad, S.; Khanna, L.; Jain, S. C. Psyche 2012, 2012.

43. Verma, M.; Sharma, S.; Prasad, R. International Biodeterioration & Biodegradation 2009, 63, (8), 959-972.

44. Creffield, J. W. Call for the immediate declaration of all municipalities (metropolitan Melbourne & regional Victoria) as regions where homes, buildings and structures are subject to termite infestation; a joint venture of CSIRO FFP Pty Ltd and Forest Research Australasia Ltd: 2005.

45. Ghaly, A. E.; Edwards, S. American J. of Engineering and Applied Sciences 2011, 4, (2), 187-200.

46. Noller, B. G., Dale. Sadler, Ross.Truss, Rowan. Zalucki, Myron .Connell, Des. Chiswell, Barry. Stewart, Aaron. Rajendran, Sharad. Hasthorpe, Amanda In Urban Australian cities under 26 termite attack, 2nd State of Australian Cities National Conference 2005 (ERA 2010 Rank A), Queensland Conservatiorium, Griffith University, Brisbane, 2005; Griffith University: Queensland Conservatiorium, Griffith University, Brisbane, 2005; pp 1-18.

47. Gupta, A.; Sharma, S.; Naik, S. N. International Biodeterioration & Biodegradation 2011, 65, (5), 703-707.

48. Nakayama, F. S.; Vinyard, S. H.; Chow, P.; Bajwa, D. S.; Youngquist, J. A.; Muehl, J. H.; Krzysik, A. M. Industrial Crops and Products 2001, 14, (2), 105-111.

49. Bultman, J. D.; Gilbertson, R. L.; Adaskaveg, J.; Amburgey, T. L.; Parikh, S. V.; Bailey, C. A. Bioresource Technology 1991, 35, (2), 197-201.

50. Bisset, N. G.; Diaz, M. A.; Ehret, C.; Ourisson, G.; Palmade, M.; Patil, F.; Pesnelle, P.; Streith, J. Phytochemistry 1966, 5, (5), 865-880.

51. Verma, M.; Pradhan, S.; Sharma, S.; Naik, S. N.; Prasad, R. International Biodeterioration & Biodegradation 2011, 65, (6), 877-882.

52. Schloman, W. W.; Hively, R. A.; Krishen, A.; Andrews, A. M. Journal of agricultural and food chemistry 1983, 31, (4), 873-876.

53. Messer, A.; McCormick, K.; Sunjaya; Hagedorn, H. H.; Tumbel, F.; Meinwald, J. J Chem Ecol 1990, 16, (12), 3333-3352.

54. Cao, Y.; Wu, J.; Zhang, J.; Li, H. Q.; Zhang, Y.; He, J. S. Chemical Engineering Journal 2009, 147, (1), 13-21.

55. Klemm, D.; Heublein, B.; Fink, H. P.; Bohn, A. Angew Chem Int Edit 2005, 44, (22), 3358- 3393.

56. Kamel, S. Express Polymer Letters 2007, 1, (9), 546-575.

57. Moon, R. J.; Martini, A.; Nairn, J.; Simonsen, J.; Youngblood, J. Chemical Society Reviews 2011, 40, (7), 3941-3994.

58. Habibi, Y.; Lucia, L. A.; Rojas, O. J. Chemical Reviews 2010, 110, (6), 3479-3500.

59. Abdul Khalil, H. P. S.; Davoudpour, Y.; Islam, M. N.; Mustapha, A.; Sudesh, K.; Dungani, R.; Jawaid, M. Carbohydrate Polymers 2014, 99, (0), 649-665.

60. Ahola, S.; Osterberg, M.; Laine, J. Cellulose 2008, 15, (2), 303-314.

61. Abe, K.; Iwamoto, S.; Yano, H. Biomacromolecules 2007, 8, (10), 3276-3278.

62. Henriksson, M.; Berglund, L. A.; Isaksson, P.; Lindstrom, T.; Nishino, T. Biomacromolecules 2008, 9, (6), 1579-1585.

63. Stenstad, P.; Andresen, M.; Tanem, B. S.; Stenius, P. Cellulose 2008, 15, (1), 35-45.

64. Siqueira, G.; Bras, J.; Dufresne, A. Polymers 2010, 2, (4), 728-765.

65. Saito, T.; Kimura, S.; Nishiyama, Y.; Isogai, A. Biomacromolecules 2007, 8, (8), 2485- 2491.

66. Henriksson, M.; Henriksson, G.; Berglund, L. A.; Lindström, T. European Polymer Journal 2007, 43, (8), 3434-3441.

67. Hassan, M.; Mathew, A.; Hassan, E.; El-Wakil, N.; Oksman, K. Wood Sci Technol 2012, 46, (1-3), 193-205.

68. Alila, S.; Besbes, I.; Vilar, M. R.; Mutjé, P.; Boufi, S. Industrial Crops and Products 2013, 41, (0), 250-259.

69. Suopajärvi, T.; Liimatainen, H.; Niinimäki, J. Cellulose 2012, 19, (1), 237-248.

70. Zimmermann, T.; Bordeanu, N.; Strub, E. Carbohydrate Polymers 2010, 79, (4), 1086-1093.

71. Dufresne, A.; Cavaillé, J.-Y.; Vignon, M. R. Journal of Applied Polymer Science 1997, 64, (6), 1185-1194.

72. Bilbao-Sainz, C.; Bras, J.; Williams, T.; Sénechal, T.; Orts, W. Carbohydrate Polymers 2011, 86, (4), 1549-1557.

73. Rodionova, G.; Lenes, M.; Eriksen, Ø.; Gregersen, Ø. Cellulose 2011, 18, (1), 127-134.

74. Klemm, D.; Kramer, F.; Moritz, S.; Lindström, T.; Ankerfors, M.; Gray, D.; Dorris, A. Angewandte Chemie International Edition 2011, 50, (24), 5438-5466.

75. Siró, .;I Plackett, D. Cellulose 2010, 17, 459.

76. Abe, K.; Yano, H. Carbohydrate Polymers 2011, 85, (4), 733-737.

77. Uetani, K.; Yano, H. Biomacromolecules 2010, 12, (2), 348-353.

78. Zhang , L.; Tsuzuki, T.; Wang, X. Material science Forum Vols 2010, 654-656, 1760-1763.

79. Chakraborty, A.; Sain, M.; Kortschot, M., Reinforcing potential of wood pulp-derived microfibres in a PVA matrix. In Holzforschung, 2006; Vol. 60, p 53.

80. Chakraborty, A.; Sain, M.; Kortschot, M., Cellulose microfibrils: A novel method of preparation using high shear refining and cryocrushing. In Holzforschung, 2005; Vol. 59, p 102.

81. Alemdar, A.; Sain, M. Composites Science and Technology 2008, 68, (2), 557-565.

82. Alemdar, A.; Sain, M. Bioresource Technology 2008, 99, (6), 1664-1671.

83. Chen, W.; Yu, H.; Liu, Y.; Hai, Y.; Zhang, M.; Chen, P. Cellulose 2011, 18, (2), 433-442.

84. Cheng, Q.; Wang, S.; Han, Q. Journal of Applied Polymer Science 2010, 115, (5), 2756- 2762.

85. Johnson, R.; Zink-Sharp, A.; Renneckar, S.; Glasser, W. Cellulose 2009, 16, (2), 227-238.

86. Spence, K.; Venditti, R.; Rojas, O.; Habibi, Y.; Pawlak, J. Cellulose 2011, 18, (4), 1097- 1111.

87. Taipale, T.; Österberg, M.; Nykänen, A.; Ruokolainen, J.; Laine, J. Cellulose 2010, 17, (5), 1005-1020.

88. Zhang, J.; Song, H.; Lin, L.; Zhuang, J.; Pang, C.; Liu, S. Biomass and Bioenergy 2012, 39, (0), 78-83.

89. Zimmermann, T.; Pöhler, E.; Geiger, T. Advanced Engineering Materials 2004, 6, (9), 754- 761.

90. Plackett, D.; Anturi, H.; Hedenqvist, M.; Ankerfors, M.; Gällstedt, M.; Lindström, T.; Siró, I. Journal of Applied Polymer Science 2010, 117, (6), 3601-3609.

91. Minelli, M.; Baschetti, M. G.; Doghieri, F.; Ankerfors, M.; Lindström, T.; Siró, I.; Plackett, D. Journal of Membrane Science 2010, 358, (1–2), 67-75.

92. Chen, W.; Yu, H.; Liu, Y.; Chen, P.; Zhang, M.; Hai, Y. Carbohydrate Polymers 2011, 83, (4), 1804-1811.

93. Iwamoto, S.; Nakagaito, A. N.; Yano, H.; Nogi, M. Appl. Phys. A: Mater. Sci. Process. 2005, 81, 1109.

94. Saito, T.; Kimura, S.; Nishiyama, Y.; Isogai, A. Biomacromolecules 2007, 8, 2485.

95. Saito, T.; Nishiyama, Y.; Putaux, J. L.; Vignon, M.; Isogai, A. Biomacromolecules 2006, 7, 1687.

96. Spence, K. L.; Venditti, R. A.; Habibi, Y.; Rojas, O. J.; Pawlak, J. J. Bioresource Technology 2010, 101, (15), 5961-5968.

97. Spence, K.; Venditti, R.; Rojas, O.; Habibi, Y.; Pawlak, J. Cellulose 2010, 17, (4), 835-848.

98. Ahola, S.; Osterberg, M.; Laine, J. Cellulose 2007.

99. Wang, M.; Olszewska, A.; Walther, A.; Malho, J.-M.; Schacher, F. H.; Ruokolainen, J.; Ankerfors, M.; Laine, J.; Berglund, L. A.; Österberg, M.; Ikkala, O. Biomacromolecules 2011, 12, (6), 2074-2081.

100. Ahola, S.; Salmi, J.; Johansson, L. S.; Laine, J.; Österberg, M. Biomacromolecules 2008, 9, 1273.

101. Leitner, J.; Hinterstoisser, B.; Wastyn, M.; Keckes, J.; Gindl, W. Cellulose 2007, 14, (5), 419-425.

102. Habibi, Y.; Vignon, M. Cellulose 2008, 15, (1), 177-185.

103. Morán, J.; Alvarez, V.; Cyras, V.; Vázquez, A. Cellulose 2008, 15, (1), 149-159.

104. Siqueira, G.; Bras, J.; Dufresne, A. Langmuir 2009, 26, (1), 402-411.

105. Bhattacharya, D.; Germinario, L.; Winter, W. Carbohydrate polymers. 2008, 73, (3), 371- 377.

106. Bendahou, A.; Kaddami, H.; Dufresne, A. European Polymer Journal 2010, 46, (4), 609- 620.

107. Siqueira, G.; Tapin-Lingua, S.; Bras, J.; da Silva Perez, D.; Dufresne, A. Cellulose 2010, 17, (6), 1147-1158.

108. de Morais Teixeira, E.; Corrêa, A.; Manzoli, A.; de Lima Leite, F.; de Oliveira, C.; Mattoso, L. Cellulose 2010, 17, (3), 595-606.

109. Saito, T.; Nishiyama, Y.; Putaux, J.-L.; Vignon, M.; Isogai, A. Biomacromolecules 2006, 7, (6), 1687-1691.

110. Zhao, H. P.; Feng, X. Q.; Gao, H. J. Appl Phys Lett 2007, 90, (7).

111. Dufresne, A.; Dupeyre, D.; Vignon, M. R. Journal of Applied Polymer Science 2000, 76, (14), 2080-2092.

112. Zuluaga, R.; Putaux, J. L.; Cruz, J.; Vélez, J.; Mondragon, I.; Gañán, P. Carbohydrate Polymers 2009, 76, (1), 51-59.

113. Rondeau-Mouro, C.; Bouchet, B.; Pontoire, B.; Robert, P.; Mazoyer, J.; Buléon, A. Carbohydrate Polymers 2003, 53, (3), 241-252.

114. Wang, B.; Sain, M.; Oksman, K. Applied Composite Materials 2007, 14, (2), 89-103.

115. Bhatnagar, A.; Sain, M. Journal of Reinforced Plastics and Composites 2005, 24, (12), 1259-1268.

116. Chen, Y.; Liu, C.; Chang, P. R.; Cao, X.; Anderson, D. P. Carbohydrate Polymers 2009, 76, (4), 607-615.

117. Nakagaito, A. N.; Fujimura, A.; Sakai, T.; Hama, Y.; Yano, H. Composites Science and Technology 2009, 69, (7–8), 1293-1297.

118. Syverud, K.; Stenius, P. Cellulose 2009, 16, (1), 75-85.

119. Nakagaito, A. N.; Yano, H. Appl Phys A 2005, 80, (1), 155-159.

120. Henriksson, M.; Berglund, L. A.; Isaksson, P.; Lindström, T.; Nishino, T. Biomacromolecules 2008, 9, (6), 1579-1585.

121. Krishnamachari, P.; Hashaikeh, R.; Chiesa, M.; El Rab, K. R. M. G. Cell Chem Technol 2012, 46, (1-2), 13-18.

122. Aulin, C.; Ahola, S.; Josefsson, P.; Nishino, T.; Hirose, Y.; Österberg, M.; Wågberg, L. Langmuir 2009, 25, (13), 7675-7685.

123. Aulin, C.; Gällstedt, M.; Lindström, T. Cellulose 2010, 17, (3), 559-574.

124. Andresen, M.; Stenstad, P.; Møretrø, T.; Langsrud, S.; Syverud, K.; Johansson, L.-S.; Stenius, P. Biomacromolecules 2007, 8, (7), 2149-2155.

125. Berglund, L. A.; Mohanty, A. K.; Misra, M.; Drzal, L. T., Natural Fibers, Biopolymers, and their Biocomposites. 2005.

126. Zhang, W.; Liang, M.; Lu, C. Cellulose 2007, 14, (5), 447-456.

127. Taniguchi, T.; Okamura, K. Polym. Int. 1998, 47, 291.

128. Quiévy, N.; Jacquet, N.; Sclavons, M.; Deroanne, C.; Paquot, M.; Devaux, J. Polymer Degradation and Stability 2010, 95, (3), 306-314.

129. Lavoine, N.; Desloges, I.; Dufresne, A.; Bras, J. Carbohydrate Polymers 2012, 90, 735– 764

130. Siró, I.; Plackett, D. Cellulose 2010, 17, (3), 459-494.

131. Li, W.; Zhao, X.; Huang, Z.; Liu, S. J Polym Res 2013, 20, (8), 1-7.

132. Cheng, Q.; Wang, S.; Rials, T. G. Composites Part A: Applied Science and Manufacturing 2009, 40, (2), 218-224.

133. Wang, S.; Cheng, Q. Journal of Applied Polymer Science 2009, 113, (2), 1270-1275.

134. Li, Q.; Renneckar, S. Cellulose 2009, 16, (6), 1025-1032.

135. Fan, L. T.; Lee, Y.-H.; Beardmore, D. H. Biotechnology and Bioengineering 1980, 22, (1), 177-199.

136. Suganuma, S.; Nakajima, K.; Kitano, M.; Yamaguchi, D.; Kato, H.; Hayashi, S.; Hara, M. Journal of the American Chemical Society 2008, 130, (38), 12787-12793.

137. Zhao, H.; Kwak, J. H.; Wang, Y.; Franz, J. A.; White, J. M.; Holladay, J. E. Energy & Fuels 2005, 20, (2), 807-811.

138. Teramoto, Y.; Tanaka, N.; Lee, S.-H.; Endo, T. Biotechnology and Bioengineering 2008, 99, (1), 75-85.

139. Yu, Y.; Wu, H. AIChE Journal 2011, 57, (3), 793-800.

140. Ago, M.; Endo, T.; Hirotsu, T. Cellulose 2004, 11, (2), 163-167.

141. Liimatainen, H.; Sirviö, J.; Haapala, A.; Hormi, O.; Niinimäki, J. Carbohydrate Polymers 2011, 83, (4), 2005-2010.

142. Jorfi, M.; Amiralian, N.; Biyani, M.; Martin, D. J.; Annamalai, P. K., Biopolymeric nanocomposites reinforced with nanocrystalline cellulose. In Biomass-based Biocomposites, Singha, A.; Thakur, V., Eds. Rapra publishing: Shawbury UK 2013; pp 277-304

143. Brinchia, L.; Cotanaa, F.; Fortunatib, E.; Kenny, J. M. Carbohydrate Polymers 2013, 94, 154– 169

144. Yue , Y.; Zhou , C.; French , A. D.; Xia , G.; Han , G.; Wang , Q.; Wu, Q. Cellulose 2012, 19, 1173–1187.

145. Peng, B. L.; Dhar, N.; Liu, H. L.; Tam, K. C. The Canadian journal of chemical engineering 2011, 89.

146. Trovatti , E.; Fernandes , S. C. M.; Rubatat , L.; Perez , D. d. S.; Freire , C. S. R.; Silvestre , A. J. D.; Neto, C. P. Composites Science and Technology 2012, 72, 1556–1561.

147. Gao, Z.; Peng, J.; Zhong, T.; Sun, J.; Wang, X.; Yue, C. Carbohydrate Polymers 2012, 87, 2068– 2075.

148. Fan, J. s.; Li, Y. h. Carbohydrate Polymers 2012, 88, 1184-1188.

149. Pan, H.; Song, L.; Ma, L.; Hu, Y. Ind. Eng. Chem. Res. 2012, 51, 16326-16332.

150. Mathew, A. P.; Oksman, K.; Pierron, D.; Harmand, M. F. Carbohydr. Polym. 2012, 87, 2291-2298.

151. Azizi Samir, M. A. S.; Alloin, F.; Dufresne, A. Biomacromolecules 2006, 6, 612-628.

152. Bras, J.; Hassan, M. L.; Bruzesse, C.; Hassan, E. A.; El-Wakil, N. A.; Dufresne, A. Industrial Crops and Products 2010, 32, (3), 627-633.

153. A. Boldizar, C. K., J. Kubát, P. Näslund, P. Sáha. Int J Polym Mater 1987, 11, 229-262.

154. Rojas, O. J.; Montero, G. A.; Habibi, Y. Journal of Applied Polymer Science 2009, 113, (2), 927-935.

155. Ljungberg, N.; Cavaillé, J. Y.; Heux, L. Polymer 2006, 47, 6285-6292.

156. Chazeau, L.; Cavaille, J. Y.; Perez, J. Journal of Polymer in Science: Polymer in Physics 2000, 38, 383–392.

157. Chazeau, L.; Cavaille, J. Y.; Canova, G.; Dendievel, R.; Boutherin, B. Journal of Applied Polymer Science, Applied Polymer Symposium 1999, 71, 1797-1808.

158. Pandey, J. K.; Kim, C. S.; Chu, W. S.; Choi, W. Y.; Ahn, S. H.; Lee, C. S. Journal of Composite Materials 2012, 46, (6), 653-663.

159. Fortunati, E.; Armentano, I.; Zhou, Q.; Puglia, D.; Terenzi, A.; Berglund, L. A.; Kenny, J. M. Polymer Degradation and Stability 2012, 97, (10), 2027-2036.

160. Fortunati, E.; Peltzer, M.; Armentano, I.; Torre, L.; Jiménez, A.; Kenny, J. M. Carbohydrate Polymers 2012, 90, (2), 948-956.

161. Xiang, C.; Joo, Y. L.; Frey, M. W. Journal of Biobased Materials and Bioenergy 2009, 3, (2), 147-155.

162. Bondeson, D.; Oksman, K. Composites Part A: Applied Science and Manufacturing 2007, 38, (12), 2486-2492.

163. Fortunati, E.; Puglia, D.; Monti, M.; Santulli, C.; Maniruzzaman, M.; Kenny, J. M. Journal of Applied Polymer Science 2013, 128, (5), 3220-3230.

164. Suryanegara, L.; Nakagaito, A.; Yano, H. Compos Sci Technol 2009, 69, 1187-1192.

165. Svagan, A. J.; Samir, M. A. S. A.; Berglund, L. A. Biomacromolecules 2007, 8, (8), 2556- 2563.

166. Habibi, Y.; Dufresne, A. Biomacromolecules 2008, 9, (7), 1974-1980.

167. Habibi, Y.; Goffin, A. L.; Schiltz, N.; Duquesne, E.; Dubois, P.; Dufresne, A. Journal of Materials Chemistry 2008, 18, (41), 5002-5010.

168. Zoppe, J. O.; Peresin, M. S.; Habibi, Y.; Venditti, R. A.; Rojas, O. J. ACS Applied Materials and Interfaces 2009, 1, (9), 1996-2004.

169. Lu, J.; Wang, T.; Drzal, T. Composites: Part A 2008, 39, 738-746.

170. Peresin, M. S.; Habibi, Y.; Zoppe, J. O.; Pawlak, J. J.; Rojas, O. J. Biomacromolecules 2010, 11, (3), 674-681.

171. Paralikar, S. A.; Simonsen, J.; Lombardi, J. Journal of Membrane Science 2008, 320, (1-2), 248-258.

172. Li, W.; Wu, D.; Pan, F. Discrete Mathematics 2012, 312, (2), 479-485.

173. Özgür Seydibeyoğlu, M.; Oksman, K. Composites Science and Technology 2008, 68, (3–4), 908-914.

174. Auad, M. L.; Richardson, T.; Hicks, M.; Mosiewicki, M. A.; Aranguren, M. I.; Marcovich, N. E. Polymer International 2012, 61, (2), 321-327.

175. Marcovich, N. E.; Auad, M. L.; Bellesi, N. E.; Nutt, S. R.; Aranguren, M. I. Journal of Materials Research 2006, 21, (4), 870-881.

176. Pei, A.; Malho, J. M.; Ruokolainen, J.; Zhou, Q.; Berglund, L. A. Macromolecules 2011, 44, (11), 4422-4427.

177. Cao, X.; Chen, Y.; Chang, P. R.; Muir, A. D.; Falk, G. Express Polymer Letters 2008, 2, (7), 502-510.

178. Cao, X.; Chen, Y.; Chang, P. R.; Stumborg, M.; Huneault, M. A. Journal of Applied Polymer Science 2008, 109, (6), 3804-3810.

179. Hosokawa, J.; Nishiyama, M.; Yoshihara, K.; Kubo, T.; Terabe, A. Ind Eng Chem Res 1991, 30, 788-792.

180. Fernandes, S. C. M.; Freire, C. S. R.; Silvestre, A. J. D.; Pascoal Neto, C.; Gandini, A.; Berglund, L. A.; Salmén, L. Carbohydrate Polymers 2010, 81, (2), 394-401.

181. Wang, Y.; Cao, X.; Zhang, L. Macromolecular Bioscience 2006, 6, (7), 524-531.

182. Sdrobiş, A.; Darie, R. N.; Totolin, M.; Cazacu, G.; Vasile, C. Composites Part B: Engineering 2012, 43, (4), 1873-1880.

183. Floros, M.; Hojabri, L.; Abraham, E.; Jose, J.; Thomas, S.; Pothan, L.; Leao, A. L.; Narine, S. Polymer Degradation and Stability 2012, 97, (10), 1970-1978.

184. Masoodi, R.; El-Hajjar, R. F.; Pillai, K. M.; Sabo, R. Materials & Design 2012, 36, (0), 570- 576.

185. Ul-Islam, M.; Khan, T.; Park, J. K. Carbohydrate Polymers 2012, 89, (4), 1189-1197.

186. Yue, Y.; Zhou, C.; French, A.; Xia, G.; Han, G.; Wang, Q.; Wu, Q. Cellulose 2012, 19, (4), 1173-1187.

187. John, M. J.; Thomas, S. Carbohydrate Polymers 2008, 71, (3), 343-364.

188. Müller, F. A.; Müller, L.; Hofmann, I.; Greil, P.; Wenzel, M. M.; Staudenmaier, R. Biomaterials 2006, 27, (21), 3955-3963.

189. Kenawy, E.-R.; Worley, S. D.; Broughton, R. Biomacromolecules 2007, 8, (5), 1359-1384.

190. Seydibeyoglu, M. O.; Misra, M.; Mohanty, A.; Blaker, J. J.; Lee, K. Y.; Bismarck, A.; Kazemizadeh, M. Journal of Materials Science 2013, 48, (5), 2167-2175.

191. Aranguren, M. I.; Marcovich, N. E.; Salgueiro, W.; Somoza, A. Polymer Testing 2013, 32, (1), 115-122.

192. Cherian, B. M.; Leao, A. L.; de Souza, S. F.; Costa, L. M. M.; de Olyveira, G. M.; Kottaisamy, M.; Nagarajan, E. R.; Thomas, S. Carbohydrate Polymers 2011, 86, (4), 1790-1798.

193. Li, Y.; Ren, H. F.; Ragauskas, A. J. Nano-Micro Lett 2010, 2, (2), 89-94.

194. Ali, E. S.; Ahmad, S. Compos Part B-Eng 2012, 43, (7), 2813-2816.

195. Pei, A. H.; Malho, J. M.; Ruokolainen, J.; Zhou, Q.; Berglund, L. A. Macromolecules 2011, 44, (11), 4422-4427.

196. Gao, Z. Z.; Peng, J.; Zhong, T. H.; Sun, J.; Wang, X. B.; Yue, C. Carbohydrate Polymers 2012, 87, (3), 2068-2075.

197. Ummartyotin, S.; Juntaro, J.; Sain, M.; Manuspiya, H. Industrial Crops and Products 2012, 35, (1), 92-97.

198. Rueda, L.; Saralegui, A.; Fernández d’Arlas, B.; Zhou, Q.; Berglund, L. A.; Corcuera, M. A.; Mondragon, I.; Eceiza, A. Carbohydrate Polymers 2013, 92, (1), 751-757.

199. Sapuan, S. M.; Pua, F.-l.; El-Shekeil, Y. A.; Al-Oqla, F. M. Materials & Design 2013, 50, (0), 467-470.

200. Boldizar, A.; Klason, C.; Kubát, J.; Näslund, P.; Sáha, P. Int J Polym Mater 1987, 11, (4), 229-262.

201. Wu, Q.; Henriksson, M.; Liu, X.; Berglund, L. A. Biomacromolecules 2007, 8, (12), 3687- 3692.

202. Marcovich, N. E.; Auad, M. L.; Bellesi, N. E.; Nutt, S. R.; Aranguren, M. I. Journal of Materials Research 2006, 21, (04), 870-881.

203. Auad, M. L.; Contos, V. S.; Nutt, S.; Aranguren, M. I.; Marcovich, N. E. Polymer International 2008, 57, (4), 651-659.

204. Auad, M. L.; Contos, V. S.; Nutt, S.; Aranguren, M. I.; Marcovich, N. E. Proceedings of COMAT 2005 2005, 35-36.

205. Rueda, L.; Fernández d'Arlas, B.; Zhou, Q.; Berglund, L. A.; Corcuera, M. A.; Mondragon, I.; Eceiza, A. Composites Science and Technology 2011, 71, (16), 1953-1960.

206. Cao, X.; Dong, H.; Li, C. M. Biomacromolecules 2007, 8, (3), 899-904.

Chapter 3 . Materials and methods

3.1 Materials

This chapter describes the methods and materials used to prepare, treat, process and measure the properties of spinifex derived materials to address the three thesis objectives introduced earlier.

3.1.1 Spinifex grass

Spinifex grass and threshed resin were collected directly from Triodia pungens plants growing around Camooweal, Queensland, Australia. Timber blocks of Baltic Pine used in the termite study (200 × 75 × 25 mm MGP-10 grade), were purchased from a local hardware store (Bunnings). Microcrystalline cellulose Avicel PH-101 NF (Farm grade) was purchased from FMC biopolymer, Philadelphia, United States.

3.1.2 Chemicals

The general chemicals which were used in this research, and the corresponding suppliers, are summarized in Table 3.1.

Table 3. 1. List of chemicals used in this work and their supplier

Chemicals Supplier Acetone (AR 99.5 %) Thermo Fisher Scientific(Australia) N,N-Dimethylformamide EMD chemicals (Saudi Arabia) Ethanol (ACS reagent grade) Merck (Australia) Ethyl acetate (anhydrous, 99.8%) Sigma-Aldrich (Castle Hill, Australia) Glacial acetic acid Ajax Finechem (via Thermo Fischer Pty Ltd, Scoresby, Australia) Hydrochloric acid 32% Ajax Finechem (via Thermo Fischer Pty Ltd, Scoresby, Australia)

Sodium chlorite (Technical Grade, 80%) Sigma-Aldrich (Castle Hill, Australia) Sodium hydroxide Ajax Finechem (via Thermo Fischer Pty Ltd, Scoresby, Australia) Sulphuric acid 98% RCI Labscan (Bangkok , Thailand)

3.1.3. Aliphatic polyurethane - Tecoflex EG- 80A

The thermoplastic aliphatic polyether polyurethane with the specific gravity of 1.04 g/cm3 was purchased from Lubrizol (Lubrizol Advanced Materials, Cleveland, Ohio, United States) and used as-received.

3.1.4. Polyol - Polytetramethylene ether glycol (PTMEG)

PTMEG is a waxy, low crystalline solid that melts near room temperature and is produced by polymerization of tetrahydrofuran. Compared to polycaprolactones and adipate-polyesters, it has lower crystallinity, a lower melting point and lower viscosity. This polyol is the most used polyether in thermoplastic polyurethane (TPU) production. In this thesis PTMEG with the molecular weight of 1000 was purchased from Invista (Invista, Wichita, Kansas, United States).

3.1.5. Aromatic diisocyanate - 4,4'-methylene diphenyl diisocyanate (MDI)

MDI is obtained from the condensation of aniline with formaldehyde to produce methylene dianiline (MDA), which is in turn reacted with phosgene to form MDI. Commercial MDI consists of over 98% 4,4´-MDI with small amounts of 2,4´-isomer. The pure MDI was purchased from Huntsman (Huntsman Polyurethanes, Victoria, Australia).

3.1.6. Chain extender - 1,4-butane diol (BDO)

Any short-chain could be used as a chain extender, but 1,4-butanediol is the most frequently used in polyurethane production due to the optimum size of hard segments that it produces. 1,4- buthanediol was purchased from Sigma-Aldrich (Castle Hill, Australia).

3.1.7. Catalyst - Dibutyltin dilaurate (DBTDL)

Dibutyltin dilaurate was bought from Sigma-Aldrich (Castle Hill, Australia).

3.2. Methods

3.2.1 Resin extraction

Spinifex resin was extracted from threshed grass using different solvents (acetone, ethyl acetate and ethanol) by vigorous stirring for 24 hours with varying solvent-to-resin ratios and extraction temperatures. Solutions were filtered using Whatman filter paper (GF/C with particle retention of 1.2 µm) and evaporated in two steps; first in a nitrogen purging oven (to slowly dry and minimize the oxidation of resin) at different temperatures and times, and then in a vacuum oven for 24 hours at 35 C. All of the different conditions employed for resin extraction are described in Table 3.2, and the extraction flowchart is shown in Figure 3.1.

Table 3.2. Different chemicals and conditions used for extracting spinifex resin from threshed grass

Solvents Acetone , Ethyl acetate, Ethanol Temperature of extraction (°C) 30, Room temperature (21) Solvent : grass ratio (v/w) 1:1, 2:1, 3:1, 4:1

Time of solvent evaporation under N2 (hours) 24 , 48, 72

Temperature of solvent evaporation under N2 (°C) 40, 60, 80, 100

Figure 3.1. Flowchart representing the spinifex resin extraction process

3.2.2 Evaluation of termiticidal performance of spinifex resin

The termite resistance property of spinifex neat (dried at 40 °C) and heat-treated (dried at 100 °C) resins were evaluated by exposing the uncoated and resin-coated timber blocks to subterranean termites aggregated in a trench. A 30 % (w/v) solution of resin in acetone was applied to the Baltic Pine (Picea abies) blocks using a brush. The coated timber blocks were allowed to dry at room temperature for four days. The coating efficiency was gravimetrically determined and the weight

40 increase after coating was approximately 4 - 5 grams. Then, the coated and uncoated (control) timber blocks were alternately arranged (Figure 3.2 (a)) and the whole package wrapped in corrugated cardboard and secured with tape ready for installation in the termite trench. The package was installed on pine wooden rails (as an additional food source for the termites) on the surface of the trench (where termites were already active) then covered with a plastic container and black plastic sheeting (Figure 3.2 (b)). The trial site was located at the Rowes Bay Golf Club, Townsville (19°12’S, 146°38, E) in North Queensland where the giant northern termite Mastotermes darwiniensis is a significant pest species (this is a regular site for evaluating the termiticidal performance of different treatments utilized by staff of the Agri-Science Queensland, Department of Agriculture, Fisheries and Forestry). After six months exposure, the timber blocks were removed and the extent of damage to each timber block was assessed using a visual rating scale combined with a quantification estimate according to the following damage scale: 0 = no apparent damage; 1 = light damage; 2 = moderate damage; 3 = heavy damage; and 4 = specimen missing (disintegrated).

3.2.3 Chemical analysis of lignocellulosic components in spinifex grass

TAPPI standard methods were used to characterize the components present in spinifex grass samples before and after pulping. At first spinifex water washed grass and pretreated fibres were ground to create 60 mesh fibre size through small Wiley mill. Then the ground fibre samples were extracted with ethanol in a Soxhlet apparatus (Tecator Soxtec System Model HT 1043, from Foss,

Denmark) for one hour followed by rinsing with water for another hour. The total lignin content was determined using the standard methods (TAPPI, Acid-insoluble lignin in wood and pulp, modified method based on Test Method T-222 om-88, 1988; TAPPI, Acid-soluble lignin in wood and pulp, Useful Method UM-250, 1991). Monomeric sugars also were determined by ion chromatography 1.

3.2.4 Pulping spinifex grass

In order to remove the non-cellulosic components from the grass and isolate the pure cellulose to produce nanofibrillated cellulose and cellulose nanocrystals, spinifex grass was treated in three main steps of; washing, delignification, and bleaching. The flowchart in Figure 3.3 summarizes the procedures used for pulping spinifex grass in this study.

3.2.4.1 Washing spinifex grass

Spinifex grass was washed with hot water under vigorous mechanical stirring three times to remove impurities and waxy substances covering the external surface of the fibres. After drying, the grass was chopped to an approximate length in the range of 0.3 - 7 mm and an average diameter of 63 ± 38 μm (the size of fibres was measured from SEM images by Image J software).

3.2.4.2 Delignification of spinifex grass

Two different delignification processes were used to remove lignin;  Organosolve : In this treatment, chopped spinifex grass was treated with a 40 % (v/v) ethanol solution (25:10 ml/g solvent-to-grass ratio) at 185 °C for 2 hours under 8 bar pressure in a Teflon-lined autoclave then washed with 1 M sodium hydroxide (NaOH) solution and water, respectively. These procedures were repeated once more in order to ensure the extraction of the residual lignin existing between the fibrils.  Alkali : For the alkali treatment method, spinifex fibre was first treated with an alkali solution of 2 % (w/v) sodium hydroxide (NaOH) with 10:1 solvent-to-grass ratio at 80 °C for 2 hours, and then washed with water.

3.2.4.3 Bleaching spinifex grass

In order to remove the residual lignin remaining between the fibrils and loose lignin adhering on the surface of the fibre after delignification, spinifex fibre was treated with an acidic solution of 1 % (w/v) sodium chlorite at 70 °C for an hour with a 30:1 solvent-to-grass ratio and a pH of 4, whereby adjustment of the pH was achieved with the gradual addition of glacial acetic acid. The bleaching process was repeated one more time until the coloured substances were completely removed from the grass.

Figure 3.3. Flowchart summarizing the mild chemical procedures used for pulping spinifex fibres

3.2.7 Preparation of spinifex nanofibrillated cellulose (NFC) using a high pressure homogenizer

The 0.1 - 0.7 % (w/v) dispersion of spinifex pulp was passed through a high pressure homogenizer (Model EmulsiFlex-C5.Homogenizer, from Avestin Inc, Ottawa, Canada). The homogeniser rapidly reduces the size of fibre from micron to nanometer scale based on the principle of dynamic high pressure homogenisation. The different homogenization conditions employed for producing nanofibrils are summarized in Table 3.3, and Figure 3.4 shows the homogenizer that was used in this study. The resulting NFC material was either freeze dried or vacuum filtered for further testing.

Table 3.3. List of different homogenization conditions used to produce NFC from pretreated spinifex fibre

Homogenization conditions Number of passes 1, 5, 10, 15 Concentration of dispersion (% w/v) 0.1, 0.3, 0.7 Applied pressure (bar) 350, 1000, 1500 Delignification Organosolve, alkali

Figure 3.4. High pressure homogenizer used for producing NFC from spinifex pulp (EmulsiFlex- C5 Homogenizer)

3.2.8 Preparation of spinifex nanofibrillated cellulose (NFC) using ultrasonication

A 1 % (w/v) dispersion of alkali pretreated spinifex fibre in deionized water was treated with an ultrasonication probe (Model Q500 Sonicator, from QSonica, Newtown, United States - Figure 3.5) for different time intervals and amplitudes at the frequency of 20 kHz and the outpour energy of 500W(Table 3.4). Treated NFC was freeze dried from dilute aqueous dispersion and stored for further analysis.

Table 3.4. List of different ultrasonication conditions used to produce NFC from spinifex pulp Ultrasonication conditions Time (minutes) 5, 20 Amplitude (%) 25, 30, 60

Figure 3.5. Ultrasonic probe used for producing NFC from spinifex pulp (Q500 Sonicator)

3.2.9. Preparation of spinifex nanofibrillated cellulose (NFC) using high energy milling

Two different high energy milling configurations were employed to producing NFC from spinifex pulp. A Netzsch Laboratory agitator bead mill (LabStar via Netzsch, Selb/Bavaria - see Figure 3.6) was used in either continuous (recirculating milling setup with an external reservoir) or batch milling mode (with a closed milling chamber). In the continuous setup, a dispersion of spinifex fibre in water was subjected to continuous, recirculating milling at 32 °C with variations in the total milling time. The speed of milling and pump was 1500 and 65 rpm respectively. In the batch milling setup, two different concentrations of fibre dispersion in water were milled at 25 °C for different times and different speeds. Table 3.5 summarizes all of the different milling conditions applied in this study to produce NFC from spinifex. It is worth noting that all of the preliminary milling results were based on a continuous, recirculating milling setup. As a second stage, in an effort to reduce both the amount of energy consumption as well as the degree of inorganic contamination from the milling vessel and pot, batch milling was employed instead of continuous milling. The resulting NFCs obtained from both setups were either freeze dried or vacuum filtered for further analysis.

Table 3.5. List of different milling conditions used to produce NFC from spinifex pulp

Continuous milling Time 30 minutes, 1 hour, 2 hour, 3 hour Milling bead size (mm) 0.1, 0.4, 1

Batch milling (using 1mm bead size) Time 10 minutes, 20 minutes, 40 minutes, 1 hour Speed (rpm) 1000, 3000 Dispersion concentration (% w/v) 0.5, 1 Delignification Organosolve, Alkali

Figure 3.6. High energy mill used to produce NFC from spinifex pulp (Netzsch LabStar Laboratory agitator bead mill). This photograph shows the continuous milling configuration

3.2.10 Preparation of cellulose nanopaper

Spinifex cellulose nanopaper was produced by the application of a vacuum filtration step to a NFC dispersion using a Büchner funnel fitted with cellulose acetate membrane filter (pore size: 0.45 μm, diameter: 47 mm via Advantec, Dublin, Canada). The filtration was continued until the wet sheet of NFC was formed, then the wet sheet was carefully dispatched from the filter paper and dried with three different drying processes listed in Table 3.6.

Table 3.6. List of different drying processes of spinifex NFC nanopaper

Drying methods Time and temperature Vacuum drying 50 oC , 48 hours Hot press drying 103 oC , 2 hours Freeze drying -80 °C, 48 hours

3.2.11 Acid treatment with hydrochloric acid (HCl)

In order to eliminate non-cellulosic components such as inorganic ions from spinifex pulp, the sample was treated with 2 M hydrochloric acid solution at 80 °C for 2 hours 2 before washing with de-ionised water several times until the pH=7 obtained.

3.2.12 Preparation of spinifex cellulose nanocrystals (CNC) by acid hydrolysing

Different concentrations of sulphuric acid solution at 45 °C and 50 °C for different hydrolysis times were used to digest amorphous parts of spinifex fibres in order to liberate cellulose nanocrystals. The dispersion was then centrifuged four times with a speed of 4750 rpm for 20 minutes to remove excess aqueous acid and dissolved amorphous components. The resultant precipitate was rinsed and dialyzed against water for one week and then re-suspended in water using ultrasonication at 25 % and 75 % amplitude for 20 minutes. To avoid the agglomeration of CNC in host polymer (e.g. TPU), cellulose nanocrystals were freeze dried from dilute aqueous dispersion and re-dispersed in N,N-Dimethylformamide (DMF) at different solid contents.

Table 3.7. Different conditions used to produce CNC by acid hydrolysis

Acid hydrolysis conditions Acid concentration (% v/v) 35, 40, 45, 50 Hydrolysis time (hour) 3, 6 Hydrolysis temperature (°C) 45, 50

3.2.13 Preparation of spinifex CNC-based nanocomposites

3.2.13.1 Solvent cast

In order to minimize the moisture content, spinifex CNC and TPU were vacuum-dried at 70 °C for 24 hours and TPU polymer was subsequently dissolved in dimethylformamide (DMF) at room temperature by stirring. A dispersion of freeze-dried CNC in DMF was stirred for 1.5 hours then subjected to ultrasonication at 25 % amplitude for 5 minutes, whereby the spinifex gel was formed (using the Q500 Sonicator). This procedure was repeated three times until the stable dispersion of CNC in DMF was obtained. The cellulose dispersion was subsequently added to the TPU polymer solution at different target percentages (Table 3.8) and these combined dispersions were then mixed by stirring overnight at room temperature with a magnetic stirrer. To ensure a high level of mixing prior to casting, the composite solutions were mixed for a further 5 minutes with an ultrasonic probe at 25 % amplitude, followed by magnetic stirring for a further 2 hours. Immediately prior to casting, the solution was left to stand, unstirred, for a few minutes in order to degas. They were then cast into a glass mold and oven dried at 60 °C under a nitrogen gas purge. Drying time for the CNC-TPU composites was about 72 hours. It was important to ensure that the moisture was carefully excluded during casting; otherwise this can result in low-quality cloudy films with inferior mechanical properties. The solvent cast TPU films were then annealed under vacuum at 70 °C for 6 hours to ensure complete removal of any residual solvent.

Table 3.8. List of different conditions used to produce spinifex CNC-based nanocomposites

Spinifex CNC and thermoplastic polyurethane solvent cast nanocomposite Concentration of CNC Concentration of TPU Concentration of CNC dispersion (mg/ml) dispersion (mg/ml) in composite (wt%)

5 100 0 0.1 0.25 0.5 1

3.2.13.2 In situ polymerization by twin screw extrusion (Reactive extrusion)

In order to reduce the effect of moisture, PTMEG was dried using thin/wiped film evaporator (VTA, Niederwinkling, Germany) achieving water content below 300 ppm, and purged with

48 nitrogen gas before storage in an air-tight bottle until required for reactive extrusion. The dispersion of spinifex CNC in PTMEG was prepared using 0.83 % (w/w) CNC using a proprietary mixing process. Before doing the extrusion, PTMEG and MDI both were melted at 55 ºC overnight and the chain extender (BDO) was dried using molecular sieve in a bottle, then all of these precursors were sealed with an air-tight lid and nitrogen gas purge.

Table 3.9. Composition used for producing TPU based on blank PTMEG 1000

Hard segment Isocyanate Stoichiometry Polyol MDI (wt %) BDO ratio ratio (wt %) (wt %) 0.44 1 1 56 36.06 7.94

Based on these contents and a target throughput rate of 3.21 kg/hr, the following mass flow rates were used to produce a blank TPU at a stoichiometry of 1: - PTMEG 1000: 1797.6 g/hr - MDI: 1157.6 g/hr - BDO: 254.8 g/hr

After adding CNC to the polyol, the polyol flow rate was adjusted to compensate for the mass of CNC to 1812.5 g/hr. Table 3.10 summarises the flow rates used to produce TPU incorporating CNC nanocomposite at different stoichiometries. The higher stoichiometries required for nanocomposite formulations due to the additional hydroxyl functionality on the CNC nanofillers which consumed isocyanate. This was determined in preliminary trials which are beyond the scope of this thesis.

Table 3.10. Reagent flow rates at different stoichiometries to produce TPU incorporating CNC

Stoichiometry Polyol flow rate (g/h) MDI flow rate (g/h) BOD flow rate (g/h)

1 1812.5 1157.6 254.8 1.01 1812.5 1169.1 254.8 1.02 1812.5 1189.7 254.8 1.03 1812.5 1192.3 254.8

Dispensing the reagents 50 ppm of DBTDL (catalyst) was added to the PTMEG just prior to the reactive extrusion experiments, then the mixture of PTMEG-DBTDL, MDI, and BDO were fed directly to the extruder’s main hopper using gear pumps. Each reagent line was fitted with a Bronkhorst Coriolis mass flow meters allowing very precise flow rate readings and therefore facilitating fine adjustments of flow on each of the gear pumps. This enables unparalleled control of ultimate TPU and nanocomposite compositions.

Description of the extruder setup Reactive extrusion of the blank TPU and CNC-TPU composites were conducted on a small- scale Entek Emax twin-screw co-rotating intermeshing extruder (via Entek, Lebanon, Pennsylvania, United States- Figure 3.8) with a screw diameter of 27 mm and a length to diameter ratio (L/D) of 40:1. As illustrated in Figure 3.7, the extruder was divided into twelve separately controlled zones; 10 barrel zones, a die adapter zone and a die block zone. The first barrel zone was not electrically heated but was cooled with water. The 9 other barrel zones were electrically heated with cartridge heaters and cooled by water flowing through channels in the barrel. The die consisted of three circular openings of 3 mm diameter. Table 3.11 shows the details temperature profile employed for reactive extrusion of blank TPU and TPU incorporating CNC. It was observed that a 5 ºC increase of the die temperature setting was necessary when processing the CNC composite. The throughput rate was 3.2 kg/hr with a screw speed of 90 rpm. A water bath was used at the end of the line to cool down the final product. It needs to be mentioned that when nanocellulose was added to the polymer, it increased the viscosity, so in order to decrease the die pressure, we had to increase the temperature of these zones by 5 °C in order to optimise processing conditions.

Figure 3.7. Schematic of the reactive extrusion setup

Table 3.11. Reactive extrusion temperature profiles

Zone Z1 Z2 Z3 Z4 Z5 Z6 Z7 Z8 Z9 Z10 Z11 Z12

Set temperature (ºC) for blank 130 190 185 185 185 185 185 180 185 180 175 175 TPU Set temperature (ºC) for TPU 130 190 185 185 185 185 185 180 185 180 180 180 + CNC * The actual operating temperature of the first non-heated zone (Z1) usually varies between 105 C and 115 C

Figure 3.8. Twin-screw reactive extruder used to produce spinifex CNC and TPU nanocomposites (Entek Emax twin-screw co-rotating intermeshing extruder)

3.2.13.2.1 Preparation of compression molded samples

The extruded, pelletized and dried (vacuum oven at 70 °C overnight) TPU and CNC nanocomposite materials were put on a pair of Teflon (mold release) sheets and compressed between a pair of metal plates in a hydraulic hot press apparatus. The mold has a rectangular cavity of 85 mm × 55 mm where the control and nanocomposite sheets were melted inside the cavity. The molds were heated at 180 ºC, then TPU pellets were pre-pressed at 4.5 KPa pressure between the plates for 20 seconds and then pressure was released. The temperature of molds was maintained at 180 °C and the next pressure was applied at 7 KPa for 20 seconds, then molds were cooled down using water jacket cooling for 20 seconds before removing the samples. Composite films were

51 annealed at 80 °C for 12 hours under vacuum then allowed to age at room temperature for at least a week prior to testing.

3.3 Characterization of spinifex resin, fibres, nanocellulose and polymer nanocomposites

3.3.1 Attenuated total reflectance (ATR) Fourier transform infrared spectroscopy (FTIR)

In order to understand the chemical composition and functionalities of the spinifex resin and fibre samples, Fourier transform infrared spectroscopy was performed on a Thermo-Nicolet 5700 ATR FTIR spectrometer equipped with a Nicolet Smart Orbit single bounce, diamond ATR accessory (thermo electron Crop., Waltham, United States). Spectra were recorded in the wave number range of 4000 to 500 cm-1 and at a resolution of 4 cm-1 for 128 scans. Samples were directly pressed onto the diamond internal reflection element of the ATR accessory.

3.3.2 Thermogravimetric analysis (TGA)

The stability of spinifex resin and fibre to thermal degradation was measured using a TGA/DSC thermogravimetric analyser (Mettler Toledo, Tennyson, Australia). An approximately 5- 7 mg sample was placed in a 40 μl aluminium pan and the temperature was increased from 25 to 500 °C with a constant heating rate of 10 °C/min under nitrogen atmosphere.

3.3.3 Differential scanning calorimetry (DSC)

DSC measurements were carried out using the Mettler Toledo DSC 1 star (Mettler Toledo, Tennyson, Australia). Approximately 5 - 6 mg of spinifex resin was put in a 40 μl aluminium pan and scanned over the temperature range of -100 °C to 250 °C, and at a constant heating rate of 10 °C/min.

3.3.4 Scanning electron microscopy (SEM)

Spinifex fibre sample was mounted on a sticky carbon tab adhered to a SEM stub and gold coated using a SPI-Module sputter coater (SPI supplies, Pennsylvania, United States). The sample was imaged using a Neoscope JCM-5000 (JEOL, Tokyo, Japan) operating at 10 kV.

3.3.5 Transmission electron microscopy (TEM)

A 0.04 mg/ml dispersion of spinifex fibre sample in water was sonicated at 25 % amplitude for 5 minutes using Q500 Sonicator. 1µl of dispersion was spotted onto a formvar-coated 200 mesh copper/palladium grid (ProSciTech, Queensland, Australia) and allowed to dry at room temperature. The sample was then stained with a 2 % uranyl acetate aqueous solution (UA) for 10 minutes in the absence of light. Then, after removing the excess UA with a paper tip, the grid was allowed to dry at room temperature. Finally the grid was examined on a JEOL 1011 TEM (JEOL Pty Ltd., Frenchs Forest, Australia) at 100 KV and images were captured on a SIS Morada 4K CCD camera system.

3.3.6 Measurement of diameter and length of spinifex fibre samples

The average diameter of water-washed, delignified, bleached, chemical and mechanical treated fibres was determined using digital image analysis (Image J). For each sample, 250 measurements of diameter were randomly selected and measured from several TEM images with the same magnification. For measuring the length of spinifex water-washed delignified, and bleached fibres, and also short cellulose nanocrystals, digital image analysis (Image J) was used. For measurement of the length of long and curly spinifex NFC two different methods were employed; (a) cryo-TEM, a 3D tomography, and (b) measurement from TEM images by AutoCAD software.

3.3.6.1 Cryo-TEM

Plunge freezing protocol 4 µl of NFC dispersion in water was transferred onto TEM holey carbon grids (C-flat and lacey carbon), in an FEI Vitrobot Mark 3 (FEI Company, Eindhoven, the Netherlands), while the chamber was set to 100 % humidity at room temperature (~22 ºC). Optimal blot time was 3 - 5 seconds, and then the sample was plunged into liquid ethane.

Electron microscopy Frozen/vitrified samples were viewed on a Tecnai F30 TEM (FEI Company) operating at 300kV, and imaged at 23,000x magnification with a Direct Electron LC1100 4k x 4k camera (Direct Electron, San Diego, United States), using low-dose mode of SerialEM image acquisition software (http://bio3d.colorado.edu/SerialEM/). The reason that samples were subjected to low-dose conditions is the extreme sensitivity of the unstained cellulose nanofibres to beam damage. This consisted of using spot size five, making focus and exposure adjustments outside of the image

53 capture area, creating a map of grid locations at very low magnification where area selection was based on the quality of vitreous ice rather than sample morphology, and performing subsequent high magnification imaging via an automated batch imaging function in SerialEM, where the total electron dose was limited to 130 electrons/Å² or less. The tilt range was +/-60º with an increment of ~1.5º to 2.5º. Image processing and analysis 125 2D images were captured in this instance and the raw image data was then processed using IMOD processing and modeling software (http://bio3d.colorado.edu/imod). This program allows contours to be manually drawn following the non-linear path of each cellulose nanofibril in xy space and contains tools for the subsequent calculation of contour length.

3.3.6.2 AutoCAD

Similar to the use of the IMOD software, the small contours were manually drawn along the individual nanofibril or the bundles of nanofibrils in several adjacent high-resolution TEM images by using AutoCAD, then the software was used to calculate the sum of contours length automatically. It is easier to measure the length of a bundle of nanofibrils in the lower magnification TEM images because the microscopist can readily locate the ends.

3.3.7 Optical microscopy

A drop of dilute dispersion of spinifex fibre sample (0.1 % (w/v)) was put on a clean glass slide and dried at 50 °C in vacuum oven for 5 hours then mounted on the stage of optical microscopy. An optical microscope (model Alpha 300R microscope via Witec, Germany) was used to observe and photograph the samples with 10 and 20x magnification.

3.3.8 X-Ray diffraction

XRD diffraction analysis was conducted on a Bruker D8 Advance x-ray diffractometer (Bruker, Karlsruhe, Germany) with a 0.2 mm slit. Graphite-filtered Cu-Ka radiation was generated at 30 kV and 20 mA. A sample was put in the sample holder and scanned over a range of 2θ = 5° - 40° at a scan speed of 1°/min. The degree of crystallinity of each sample was measured using the intensity of XRD peaks. Although this method is sensitive to instrumental inaccuracies, it has been widely applied as one of the best ways to measure crystallinity of plant fibres 3. Abraham et al 4 believed that it is not possible to accurately determine the degree of crystallinity of cellulosic fibres

54 as the crystalline portions are imperfect and not entirely ordered. The degree of crystallinity of the spinifex sample was measured using equation (3.1):

I200 – Iam Cr = ( ) × 100 (3.1) I200

Where I200 represents the intensity of the both amorphous and crystalline regions and Iam is the intensity of amorphous parts in XRD spectra. The mean size of crystalline regions is another parameter which was used to evaluate the effect of different treatments on the crystalline structure of fibre, and was calculated by the equation (3.2) 5 :

κλ D = (3.2) βcosθ

Where K is the shape factor and is approximately = 0.89 , β is the line width in radians at half the maximum intensity of I200 , λ is the wave length of the radiation (0.154 nm) and θ is the scattering angle of the peak (200).

3.3.9 Measurement of density and porosity

The density of NFC nanopaper was calculated by measuring the weight of paper and dividing it by its volume calculated from the thickness by digital calliper and its area. The corresponding porosity was estimated using the following equation (3.3):

ρNFCpaper Porosity = 1 − ( ) (3.3) ρcellulose

where ρNFC paper and ρcellulose represent density of the obtained NFC film and neat cellulose (1460 kg/m3), respectively 6.

3.3.10 Mechanical properties

3.3.10.1 Tensile testing

Mechanical properties of porous spinifex NFC nanopaper and CNC based nanocomposite with thermoplastic polyurethane were measured at room temperature using an Instron model 5543

55 universal testing machine (Instron Pty Ltd., Melbourne, Australia) with a capacity of 500 N load cell. A total of five replicates of each NFC nanopaper sample with 25 mm in length and 6 mm in width were tested at a cross-head speed of 1 mm/min and a gauge length of 10 mm. The nanocomposite samples were punched using an ASTM D 638 M 3 die to obtain a bumble shape specimen. Five replicates of each solvent cast nanocomposite were tested at cross-head speed of 150 mm/min and a gauge length of 10 mm and five replicates of extruded nanocomposite were tested at cross-head speed of 50 mm/min and a gauge length of 14 mm. Young’s modulus which reflects the film stiffness, was determined from the slope of the initial linear region of the stress- strain curves. Maximum tensile strength is the largest stress that a film is able to sustain against applied tensile stress before the film tears. Elongation at break is the maximum percentage change in the original film length before breaking, and work to fracture represents the tensile toughness which is measured as the area under the stress-strain curve. The results were reported as mean ± standard deviation.

3.3.10.2 Amplitude modulation- Frequency modulation (AM-FM)

Height images All topography images have been obtained from a dried drop of very dilute dispersion of nanocrystal on mica using a Cypher S AFM (Asylum Research/Oxford Instruments, Santa Barbara, CA) by employing the taping mode of the AFM in air. The cantilevers used were Tap 300 (Budget Sensors, Bulgaria) with nominal resonance frequency of 300 kHz and nominal spring constant of 40 N/m.

Elastic modulus Elastic modulus of individual cellulose nanocrystal was measured by AM-FM (Amplitude Modulation - Frequency Modulation). All experiments have been done using a Cypher S AFM (Asylum Research/Oxford Instruments, Santa Barbara, Canada) in AM-FM viscoelastic mapping mode employing Tap 300 probes (Budget Sensors, Bulgaria) with nominal resonance frequency fN of 300 kHz and nominal spring constant kN of 40 N/m. All probes have been mounted in a high frequency/ low noise AM-FM cantilever holder. For the AM pass, probes were actuated to a free air amplitude of 1.5 V, at the nominal resonant frequency and engaged at a 800 mV setpoint. The second pass was done actuating for the second harmonic at approximately 2 MHz for a free air amplitude of 100 mV, while maintaining phase to 90 degrees. Sample engagement was done so that probe tapping was in repulsive mode at a phase less than 50 degrees for the AM pass.

3.3.11 Inductively coupled plasma optical emission spectrometry (ICPOES)

This technique was used for detection of inorganic components in spinifex fibre samples. Sample was digested using a Milestone microwave digester (Model Ethos-1, Milestone Srl via distributed through in vitro, Victoria, Australia) at 1500 W power. Digested samples were analysed on a Varian Vista Pro radial ICPOES (Varian inc, Melbourne, Australia) instrument at 1000 W with the plasma argon flow of 15 L/min, auxiliary argon flow of 1.5 L/min, nebuliser argon flow of 0.75 L /min and sample intake rate of 2 ml /min for three replicates.

3.4 Summary

In order to be concise, direct reference will be made to the various materials and methods described in this chapter in the forthcoming results chapters.

Bibliography

1. Pettersen, R. C.; Schwandt, V. H. Journal of wood chemistry and technology 1991, 11, (4), 495-501.

2. Zuluaga, R.; Putaux, J. L.; Cruz, J.; Vélez, J.; Mondragon, I.; Gañán, P. Carbohydrate Polymers 2009, 76, (1), 51-59.

3. Thygesen, A.; Oddershede, J.; Lilholt, H.; Thomsen, A. B.; Ståhl, K. Cellulose 2005, 12, (6), 563-576.

4. Abraham, E.; Deepa, B.; Pothan, L. A.; Jacob, M.; Thomas, S.; Cvelbar, U.; Anandjiwala, R. Carbohydrate Polymers 2011, 86, (4), 1468-1475.

5. Avolio, R.; Bonadies, I.; Capitani, D.; Errico, M. E.; Gentile, G.; Avella, M. Carbohydrate Polymers 2012, 87, (1), 265-273.

6. Sehaqui, H.; Zhou, Q.; Ikkala, O.; Berglund, L. A. Biomacromolecules 2011, 12, (10), 3638- 3644.

Chapter 4 . Optimization of resin extraction and its potential application as an anti-termite material

4.1 Introduction

Plant resins are defined as predominately lipid-soluble mixtures of volatile and non-volatile terpeniod and phenolic secondary compounds that are usually secreted in specialized structures located either internally, or on the surface of the plants. Some physical properties of the resin such as fluidity and viscosity are determined by the ratio of volatile to non-volatile compounds 1. Fresh resins have been used for several purposes, including as adhesives, lubricants, perfumes, fuels, incense, medicines and wine additives throughout human history. Phytochemical studies on the resin of the North American G. camporum showed that non- volatile compounds are formed by two fractions: acidic and neutral 2. Twelve labdane-type diterpene acids were isolated from the acidic fraction that accounted for more than 90 % of the total, whereas the neutral fraction was composed of a mixture of methyl-esters of grindelic acids, flavonoids, sterols, and waxes 3. According to classification, the most widespread and abundant form of resinites are derived from resins based on polymers of labdane carboxylic acids, in particular communic, ozic and zanzibaric acids. These labdanes may contain conjugated diene compounds that can be readily polymerized by a free radical mechanism initiated by light and oxidation. Other classes of resinites are based on polymers of candinene, polystyrene, or cedrane. Maturation over time induces further cross-linking, isomerisation and cyclization reactions, among others 4. In general, plant derived resins are heterogeneous mixtures of many compounds which complicate characterisation and thereby obscure the identification of suitable applications. Recently due to public concern about the risks associated with chemical pesticides and insect deterrents, the promising potential of plant resins in controlling pests and insects has been developed. They are locally available, cheap, not harmful to the environment, and also regulate insect numbers rather than eliminate them, such that the benefits provided by these insects and ecosystems are not lost. The objectives of this chapter are firstly to optimise the solvent extraction and drying conditions required to achieve a high spinifex resin yield, and secondly, to perform the subsequent evaluation, via field trials, to demonstrate the potential use and efficacy of this resin as a natural anti-termite coating material.

4.2 Results and discussion

The schematic in Figure 4.1 shows the resin extraction process employed in this study in order to investigate resin extraction with different organic solvents as well as the effects of different

extraction parameters on the resin glass transition temperature, functional groups, thermal degradation properties, and also extraction yield (the full details of the resin extraction process are provided in Chapter 3, Section 3.2.1).

Figure 4.1. Schematic representation of the spinifex resin extraction process

4.2.1 Resin extraction and characterization

Threshed Triodia pungens grass was first dissolved in acetone with the 2:1 (v/w) solvent to grass ratio at 21 C for 24 hours while stirring. After filtration, the drying process was performed in two steps of solvent evaporation under nitrogen flow at 40 °C for 72 hours and drying under vacuum for 24 hour at 35 °C. Figure 4.2 shows the DSC spectra of the extracted resin before and after vacuum drying. A 28 C increase in glass transition temperature after vacuum drying can be observed. This result is thought to be caused by a combination of solvent evaporation and removal of volatile components from the resin, which both plasticise the resin and therefore affect its thermomechanical properties.

-1.0

-1.5

-2.0

Heat flow (mW) flow Heat -2.5 Undried Vacuum dried -3.0 -50 0 50 100 Temperature (°C)

Figure 4.2. DSC spectra of extracted resin before and after drying under vacuum

4.2.1.1 Effect of solvents

In order to find the most suitable solvent for high yield extraction, three common organic solvents with different polarity were used. Figure 4.3 shows the ATR FTIR spectra of resin extracted using acetone, ethyl acetate, and ethanol. The spectral patterns of the resin differ slightly depending on the solubility of metabolites in the solvent. Table 4.1 summarises the peak values from ATR FTIR spectra and possible chemical units in the obtained resins using different solvents. The broad peak around 3200 - 3500 cm-1 which is assignable to the O-H groups confirms the presence of alcoholic, phenolic and /or carboxylic acid compounds in the resin. The peak in the carbonyl region (1750 - 1600 cm-1) indicates the presence of carbonyl compounds such as carboxylic acid (-CO-O-H) and esters (-CO-O-R). The peaks from 1480 to 1330 cm-1 may be assigned to aromatic ring structures (C=C-C) and bending vibrations of hydrocarbons (CH3). The resin extracted with ethanol shows sharp peaks at 3250 cm-1, 2100 cm-1, 1160 cm-1 and 500 cm-1 which can be attributed to the polymeric OH, alkynes or substituted aromatic compounds.

0.25 Acetone Ethyl acetate 0.20 Ethanol

0.15

0.10

Absorbance 0.05

0.00

4000 3500 3000 2500 2000 1500 1000 Wave number (cm-1)

Figure 4.3. ATR FTIR spectra of spinifex resin extracted with different solvents (oven dried samples)

Table 4.1. ATR FTIR peaks and functional groups of extracted resin using different solvents

Peak (cm-1) Functional group Responsible units 3200 - 3570 O-H (polymeric O-H, Alcohols, phenols, carboxylic acids (broad peak) hydrogen bonding, associated O-H) 2800 - 3000 C-H stretching Stretching of SP2 hybridisation 2100 C C Alkyne compounds 1630 - 1710 C=O Carbonyl compounds (ester, ketone, aldehyde)

1446 - 1560 C=C-C and CH2 rocking Aromatic compounds

1378 - 1380 CH3 bending 1240 - 1190 P-O-C Aromatic phosphates 1200 - 1220 C-O-C stretching Carboxylic acid esters 1160 - 1230 Aromatic ring Aromatic hydrocarbons 1095 - 1075 Si – O- Si Orangano siloxane 1000 - 1030 Aliphatic ring C-H Cyclohexane units, terpenic compounds 500 - 430 S-S Polysulfides or aryl sulphides

Figure 4.4 shows the DSC spectra of spinifex resin extracted at 30 C with different solvents. In comparison, a higher glass transition temperature was observed for the resin extracted using acetone. This suggests that the resin may contain higher molecular weight polymeric compounds which exhibit reduced mobility under heating at a fixed rate.

Table 4.2. Glass transition (Tg) temperature and extraction yield of extracted spinifex resin with different solvents (drying at 40 °C)

Solvent Polarity index Yield (%) Tg (°C) Acetone 5.1 35 30 Ethyl acetate 4.4 27 11 Ethanol 5.2 17 16

o Tg ~ 16 oC Tg~30 C -1

Tg~11 oC -2

Heat flow (mW) flow Heat Acetone Ethyl acetate Ethanol -3 -60 -40 -20 0 20 40 60 80 100 Temprature (°C)

Figure 4.4. DSC spectra of extracted spinifex resin with different solvents show better thermomechanical stability for the acetone extracted resin (all oven dried samples)

4.2.1.2 Effect of solvent to grass ratio and extraction temperature

In order to optimize the extraction process and determine the highest yield using the minimum amount of solvent, the solvent to grass ratio and also the extraction temperature (21 °C and 30 °C) were systematically varied. Figures 4.5 and 4.6 show the thermal transitions of the obtained resin

after 24 hours drying in a vacuum oven at 35 C, while Table 4.3 summarises the yield (wt%) and glass transition temperature of the resin extracted using acetone. It can be seen that with increasing solvent to grass ratio, the yield of extracted resin increased from 12 to 33 % and 15 to 36 % at 21 C and 30 C, respectively. With increasing solvent to grass ratio, the glass transition temperature of resin is maximised when a 2:1 solvent to grass ratio is employed. A lower Tg achieved using other ratios may be due to either the higher amount of polymeric compounds which can be extracted from grass with higher amount of solvent, or alternatively due to the plasticisation effects of residual solvent. Considering the aim of achieving the highest yield with minimal use of solvent, the 2:1 ratio of solvent and grass was employed in the larger scale extraction for preparing timber coatings for subsequent termite field trials.

-1

-2

S:G=1:1

Heat flow (mW) flow Heat -3 S:G=2:1 S:G=3:1 S:G=4:1 -4 -75 -50 -25 0 25 50 75 100 Temperature (°C)

Figure 4.5. DSC spectra of resin extracted at 30 °C showing the influence of solvent to grass ratio on the thermal transitions of resin

0.0

-0.5

-1.0

-1.5

Heat flow (mW) flow Heat -2.0 T= 21 oC T= 30 oC -2.5 -50 0 50 100 Temperature (°C)

Figure 4.6. DSC spectra of resin extracted with the solvent to grass ratio of 2:1, showing the influence of extraction temperatures (21 and 30 °C) on the thermal transitions of resin

Table 4.3. Effect of solvent to grass ratio and extraction temperature on yield and Tg of resin (drying at 40 °C for 72 hours and annealing at 35 °C for 24 hours)

Solvent : grass Yield (%) of Tg (C) of resin Yield (%) of Tg (C) of resin (v/w) resin extracted extracted at 21 resin extracted at extracted at 30 °C at 21C C 30°C 1:1 12 20 15 13 2:1 28 25 35 30 3:1 29 23 35 10 4:1 33 17 36 6

The TGA spectra in Figure 4.7 show that resin samples extracted at different temperatures exhibit almost identical mass loss curves. It can be seen that the degradation occurs over a wide range of temperatures (150 to 450 C), where initial degradation will be due to the emission of low molecular weight volatile compounds leading to the gradual formation of a more crosslinked resin structure via the formation of primary and secondary bonds. Under the influence of thermal energy, in addition to the evaporation of low molecular weight volatiles, thermal degradation of the resin progresses through the breakage of some C-H and C-C bonds, and the formation of gaseous 5, 6 oxidation products like CO2 and H2O, which results in a further reduction in weight . The

majority of mass loss occurs between 250 and 400 °C. Initially, decomposition was due to breaking the molecular chains, side-reactions, and cyclization reactions, but at higher temperatures this is followed by the advanced fragmentation of the previously formed chains, dehydrogenation, gasification, and decomposition of the char formed in the previous steps 7. The residual content after full thermal decomposition of the resin at 500 °C is about 4 to 6 wt%, which may be due to the contamination of resin by some remnant inorganic elements (such as Al, Fe, Mg, Mn, Ca, P , S, Si) which are known to be absorbed as micro-nutrients from the soil by spinifex roots 6.

100

Weight (%) Weight 20 T= 21 oC 0 T= 30 oC 100 200 300 400 500 Temperature (oC)

Figure 4.7. TGA spectra of resin extracted at different temperatures

4.2.1.3 Effect of temperature and time of solvent evaporation on the thermal properties of resin

The drying process firstly involved solvent evaporation at different temperatures and times followed by drying under vacuum at 35 C for 24 hours. Figure 4.8 and Table 4.4 show the influence of solvent evaporation time at 40 °C on the glass transition temperature. The results indicate that at least 72 hours of solvent evaporation under nitrogen flow is required to remove the low molecular weight volatile components and residual acetone solvent completely. Evaporation periods of less than 72 hours exhibited lower thermal transitions (Tg), indicating the presence of solvent molecules which may increase the chain mobility of the polymer, and consequently plasticise the resin.

Table 4.4. Thermal property of resin after drying at 40 °C for increasing times Time (hours) Glass transition temperature (°C)

24 13 48 27

72 30

T ~30 °C g -1 T ~27 °C g

T ~13 °C g -2

Heat flow (mW) flow Heat t= 24 hours -3 t= 48 hours t= 72 hours -75 -50 -25 0 25 50 75 100 Temperature (°C)

Figure 4.8. DSC spectra of resin showing the improving thermal property of resin by increasing solvent evaporation time at 40 °C

To investigate the influence of evaporation temperature on the thermal properties of resin, the solvent was evaporated at 40 °C, 60 °C, 80 °C, and 100 °C under flow of the nitrogen for 72 hours. Table 4.5 summarizes changes in thermal properties as a function of drying temperature. The DSC spectra in Figure 4.9 indicate that an increasing temperature of evaporation is associated with increase in resultant resin Tg. This may be due to the removal of moisture, solvent and low molecular weight volatile components, and also due to enhanced crosslinking or secondary interactions between functional groups, or molecular sequences, and perhaps inorganic minerals in the resin 6. It is noteworthy that storage conditions influence the thermal transition temperatures. From Figure 4.10, it can be observed that conditioning of dried resin at 65 % humidity for one week has decreased the glass transition temperature by about 12 C. This indicates that the functional

(hydroxyl) groups in the resin have an affinity for moisture, and the absorbed water molecules can re-induce plasticization of the resin.

Table 4.5. Effect of drying temperature on the thermal property of resin

Drying temperature (°C) Tg (°C) after drying Tg (°C) after one week conditioning 40 30 - 60 20 - 80 36 - 100 54 42

-0.5

T ~30 oC -1.0 g

-1.5 T ~20 oC g T ~36 oC g -2.0 T= 40 (°C) T ~54 oC Heat flow (mW) flow Heat T= 60 (°C) g -2.5 T= 80 (°C) T= 100 (°C) -3.0 -75 -50 -25 0 25 50 75 100 125 Temperature (°C)

Figure 4.9. Influence of solvent evaporation temperature on the thermal transitions of resin

-1.0

-1.5

-2.0 Tg~54 °C Tg~42 °C

Heat flow (mW) flow Heat -2.5 After conditioning After drying -3.0 -75 -50 -25 0 25 50 75 100 125 Temperature (°C)

Figure 4.10. Influence of conditioning at 65% relative humidity on the Tg changes of spinifex resin after one week

Table 4.6. Effect of heat treatment (evaporation temperature) on thermal stability of resin

Evaporation Temperature (°C) Onset degradation temperature (°C) 40 140 60 115 80 180 100 200

The TGA spectra of resin in Figure 4.11 and Table 4.6 show the improvement in thermal stability of resin after heat treatment. The un-treated resin (solvent evaporation temperature was at 40 °C) started to degrade at about 140 °C while the samples with higher evaporation temperatures showed improvements in thermal stability up to 200 °C.

100

100 90

80 60 100 150 200 250 300

Weight (%) Weight T= 40 (°C) 20 T= 60 (°C) T= 80 (°C) 0 T= 100 (°C) 100 200 300 400 500 600 700 Temperature (°C)

Figure 4.11. TGA spectra of spinifex resin dried (first evaporation) at different temperatures

4.2.2 Evaluation of termite resistance: A field study

The termite resistance of the neat (untreated) and heat-treated resins was evaluated by exposing the resin coated timbers and uncoated timbers in a termite field. After 22 weeks (April to September 2013) of exposure to the mastotermites at Townsville, timber pieces were collected from the field (Figure 4.12 (a, b)). It is important to mention that while collecting the spinifex resin- coated samples, no residual infestation of termites or any other insects was observed in our samples, whereas the other samples (from separate parallel studies) installed adjacent to them were covered by termites and other insects (Figure 4.13). The samples were cleaned by brush to remove mud and other dirt particles. A visual observation of the samples (Figure 4.12 (c, d)) indicated that the resistance to Mastotermites was higher for coated samples with both neat and heat-treated resins than the control timber pieces. The resin-coated specimens were well-preserved during this field trial whereas all of the uncoated control wood blocks were heavily damaged. No trace of exploratory “nibbling” by termites or fungal colonization on the resin-coated timber specimens was observed.

The resistance of resin coated timbers to termite attack seems to be due to the presence of some active components in the spinifex resin, which perhaps relate to the resin’s natural defence functions in spinifex. Other researchers have investigated this potential and extracted the active components of various plant resins and consequently tested them against termite attack. The most active compounds against Southeast Asian termites (Neotermes spp) obtained from crude resins of four Dipterocarpus species proved to be alloaromadendrene, humulene, and caryophyllene. The investigators in this study concluded that these sesquiterpenes, serve an essential role in defence 8 against insects . Bultman et al extracted the components of Parthenium argentatum (Guayule) including polyphenols, cinnamyl derivatives, and a variety of terpenoids, which provided antitermitic and antifungal properties 9-11. Caryophyllene and other sesquiterpenes in the leaves of a Brazilian tree (Copaifera langsdorfii) have also been shown to be significant elements of plant defence against insects 12. Pinus produces a resin which contains ɑ-Pinene, and is well known for its antimicrobial and insecticidal properties 12. Based on the only previous spinifex resin analytical chemistry work performed in our team by De Silvia et al 13, extractions from spinifex resin also contain some of these common termite repellent components. Although it is a very difficult task to identify every molecular component in these plant resins, the analysis of the volatile fraction of spinifex resin showed that the main components in spinifex resin are; 3-hexen -1-ol, α-pinene, camphene, limonene, α-terpineol, bornyl acetate, caryophyllene, alloaromadendrene, mono and diterpinoid, labdane and communic acid based structures. Figure 4.14 shows the structure of some chemicals isolated from spinifex resin.

3-Hexen -1-ol α-Pinene Camphene Limonene

α-Terpineol Bornyl acetate Caryophyllene Alloaromadendrene

Diterpinoid Labdane Communic acid

Figure 4.14. Chemical structure of some major components isolated from spinifex resin 13

4.3 Summary

In this study, I have reported the optimised process for extracting resin from Triodia pungens by varying the extraction parameters (such as solvent, drying temperature and time) and studied their influence on resin glass transition and degradation temperatures. For a high yield of resin with minimal use of solvent and higher product glass transition temperature, the optimised extraction conditions involved the use of acetone, with solvent to grass ratio of 2:1, at 30 C, followed by drying firstly in a nitrogen purged oven at 100 C for 72 hours, and then in a vacuum oven at 35 C for 24 hours. A preliminary termite trial was successfully carried out by exposing resin-coated and control timbers to the Mastotermes darwiniensis. The results indicated strong termite resistance activity of the resin extracted from spinifex. The details of the active ingredients and the full protective mechanisms are still unclear. By demonstrating higher resistance of both neat (dried at 40 C) and heat-treated (dried at 100 C) resins towards the termites, it can be reported that plant resins which are produced by arid and semi-arid grasses could be potentially used as anti-termite protective coating materials.

Bibliography

1. Langeheim, J. H. J. Chem. Ecol 1994, 20, 1223–1280.

2. Hoffmann, J. J.; Kingslover, B.; McLaughlin, S. P.; Timmermann, B. N., Production of resins by arid-adapted Astereae. In Phytochemical Adaptations to Stress, B.N. Timmermann, C. S., F. Loewus, Ed. Plenum Press: New York, 1984; pp 251–270.

3. Zhaobang, S. Center for International Forestry Research. Occasional Paper 1995, 6, 1–17.

4. D.F. Wassner, D. A. R. Industrial Crops and Products 2005, 21, 155–163.

5. Liao, Y. H.; Rao, M. M.; Li, W. S.; Yang, L. T.; Zhu, B. K.; Xu, R.; Fu, C. H. Journal of Membrane Science 2010, 352, (1–2), 95-99.

6. Mondal, S.; Memmott, P.; Wallis, L.; Martin, D. Materials Chemistry and Physics 2012, 133, (2–3), 692-699.

7. Peng, Z.; Kong, L. X. Polymer Degradation and Stability 2007, 92, (6), 1061-1071.

8. Bisset, N. G.; Diaz, M. A.; Ehret, C.; Ourisson, G.; Palmade, M.; Patil, F.; Pesnelle, P.; Streith, J. Phytochemistry 1966, 5, (5), 865-880.

9. Nakayama, F. S.; Vinyard, S. H.; Chow, P.; Bajwa, D. S.; Youngquist, J. A.; Muehl, J. H.; Krzysik, A. M. Industrial Crops and Products 2001, 14, (2), 105-111.

10. Bultman, J. D.; Gilbertson, R. L.; Adaskaveg, J.; Amburgey, T. L.; Parikh, S. V.; Bailey, C. A. Bioresource Technology 1991, 35, (2), 197-201.

11. Schloman, W. W.; Hively, R. A.; Krishen, A.; Andrews, A. M. Journal of Agricultural and Food Chemistry 1983, 31, (4), 873-876.

12. Messer, A.; McCormick, K.; Sunjaya; Hagedorn, H. H.; Tumbel, F.; Meinwald, J. J Chem Ecol 1990, 16, (12), 3333-3352.

13. De Silva, S.; Memmott, P.; Folter, N.; Martin, D., Charectrisation of Spinifex (Triodia pungens) Resin and Fiber. In 11th Pacific polymer conference Cairns, Australia, 2009.

Chapter 5 .

Pulping spinifex fibres

5.1 Introduction

In order to facilitate the defibrillation process of cellulose fibres to obtain cellulose nanocrystals and nanofibrils, while ideally decreasing energy and chemical consumption during the mechanical or chemical treatments 1, 2 , several different methods of pretreatment have been proposed on delignified and bleached fibres( pulp). These methods may be either physical such as refining by valley beater, PFI mill, 3, 4 and ultrasonication 5, or chemical such as delignification and bleaching with chemicals 3, 6-11, acid hydrolysis 2, 12, carboxymethylation 13-15, and 2,2,6,6- tetramethylpiperidinyl-1-oxyl (TEMPO)-mediated oxidation 16-18 or enzyme-treatment 1, 3, 16, 19, 20 . The steam explosion process is also another efficient pretreatment method for pretreatment of lignocellulosic biomass, with the final aim of separating nanofibers 6, 21. Here at first some common delignification and bleaching methods are summarized in a table in order to compare with the spinifex established pulping methods then followed by a brief explanation of common pretreatment methods on pulp of different cources of cellulose.

5.1.1 Delignification and bleaching

The first step of producing nanoparticles from cellulosic fibres is the removal of pectins, hemicellulose, and lignin by chemical methods which are similar to the techniques that are used in the pulp and paper industries. The processes start with chopping long fibres into manageable particle sizes, followed by delignification with an alkali solution such as sodium hydroxide or potassium hydroxide, or alternatively delignification with organic solvents like ethanol, acetone or methanol. Secondly, a bleaching process must be carried out with an oxidizing agent such as oxygen or sodium chlorite to solubilise the residual lignin 6. Table 5.1 summarizes some different methods for delignification and bleaching. Treating lignocellulosic fibres with sodium hydroxide is an effective and common procedure for removing waxy and cement-like hydrocarbon components from the fibres. To the best of our knowledge, and based on all current scientific and patent literature information, this thesis reports, for the first time, that cellulose micro/nanofibrils can be prepared from spinifex grass following merely a mild ethanol solution treatment, similar to that more commonly used for pulping wood.

Table 5.1. Comparing the most common delignification and bleaching processes for cellulosic fibres and wood pulp

Sources Delignification Bleaching Ref

Sisal Fibre Treating chopped fibres with 4 wt% sodium Using acetate buffer of chlorite solution (1.7 22 hydroxide solution at 80 °C for 2 hours under wt%) at 80 °C for 4 hours under mechanical mechanical stirring (repeating for 3 times) stirring (repeating for 4 times)

Grass Zoyasia Soaking fibre in alkali solution (5 % w/w) for 2 Using acetate buffer of chlorite solution (1.7 23, 24 days then boiling fibre for 1h in 2 % (w/w) sodium wt %) at 70 °C hydroxide solution and de-waxing with equal proportion of ethanol and water

Coconut fibre, Cladoded, Stirring fibres in 2 % sodium hydroxide solution Using sodium chlorite and glacial acetic acid 25-27 Sugar beet for 2 hours at 80 °C (repeating for 2 times) solution at 60 - 70 °C for 1 hour under stirring for 1 - 4 times, then treating with 0.05 N nitric acid solution for 1 hour at 70 °C

Banana fibre Treating fibre with 2 % sodium hydroxide solution Using mixture of sodium hydroxide and acetic 28 in autoclave with 20 lb pressure at 110 - 120 °C acid and sodium hypochlorite solution for two for 1 hour 1 hour (repeating for 3 times)

Ramie Fibre Treating fibre with 2 % sodium hydroxide solution - 29 at 80 °C for 2 hours

Sugar bagasse - Using a mixture of 5 % sodium hydroxide 30, 31 solution and 11 % hydrogen peroxide solution at 55 °C for 90 minutes

Wood flour De-waxing fibre in toluene/ethanol solution for 6 Sodium chlorite treatment then alkali treatment 32 hours with 5 % potassium hydroxide solution

Wood powder Treating wood in toluene/ethanol solution for 6 Using acidified sodium chlorite solution at 70 33 hours °C for 1 hour, treating with 6 wt% potassium hydroxide solution at room temperature for 78

overnight then at 80 °C for 2 hours

Wood pulp - Using sodium chlorite solution under an acidic 34 condition for 1 hour at 80 °C (repeating for 10 times)

Canola straw Treating fibre with sodium hydroxide and Bleaching in 3 steps: 35 anthraquinone solution at 170 °C for 3 hours -Sodium chlorite solution (2%) at 60 °C for 1 hour -A mixture of sodium hydroxide (2 %) and peroxide (2 - 3 %) solution at 70 °C for 1 hour -Sodium chlorite solution (2 %) at 80 °C for 1 hour then treating with potassium hydroxide solution (10 %) at 80 °C for 2 hours

Eucalyptus glubulus Treating fibre with ethanol/methanol solution at - 36 different temperatures and time in autoclave under 41 lb pressure

Mixed soft woods Treating wood with 40 - 60 % (w/w) ethanol - 31 solution and sulphuric acid as catalyst (PH=2 - 3.4) at 185 - 195 °C for 30 - 60 minutes then washing with aqueous ethanol (70 % w/w)

Wheat straw Treating fibre with ethanol solution (55 % v/v) at - 37 195 °C for 2 hours in autoclave then washing 4 times with same solution at 145 °C for 30 minutes in autoclave

Rice straw Cooking fibre with ethanol solution (65 %) and - 38 sodium hydroxide solution at 195 °C for 180 minutes then washing with warm water for 240 minutes

Wheat straw Treating fibre with 2 different solutions and - 39 different ratio: - 60 % ethanol (ethanol + acetone) solution at 160 °C for 90 minutes - 55 % (ethanol + acetone/ethanol + acetone + water) solution at 160 °C for 90 minutes

Prairie sandreed, cord Treating fibres with 14 % active alkali and 20 % - 40 grass, big bluestem, switch sulphidity solution at 160 °C for 1 hour grass

Eucalyptus glubulus Treating fibre with ethanol water mixture (30 - 70 - 41 % w/w) under the pressure of 4 lb at different time and temperature then washing with 0.1 M sodium hydroxide solution flax, hemp, jute, leaves of Treating fibres with an aqueous solution of sodium Using sodium chlorite solution (1.5%) at pH 4 11 sisal and abaca hydroxide (16 - 17 wt%) at 165 °C and 6 bar

Hemp and flax bast Treating fibres with sodium hydroxide solution of - 42 17.5 % (w/w) for 2 hours, washing with distilled water, treating with 1M hydrochloric acid solution at 60 - 80 °C, neutralized with distilled water. Then treating with 2 % w/w sodium hydroxide solution for 2 hours at 60 - 80 °C

Soybean pods Treating fibre with sodium hydroxide solution of Using a chlorine dioxide solution (PH 2.3) for 7 17.5 % (w/w) for 2 hours, wash with distilled 1 hour at 50 °C water, treating with 1M hydrochloric acid solution at 80 °C, neutralizing with distilled water. Then treating with 2 % (w/w) sodium hydroxide solution for 2 hours at 80 °C

Wood fibre Treating fibre with acidified sodium chlorite - 6 solution at 75 °C for 1 hour (repeating for 5 times), treating in 3 wt% potassium hydroxide solution at 80 °C for 2 hours, and then in 6 wt% potassium hydroxide solution at 80 °C for 2 hours

5.1.2 TEMPO-mediated oxidation pretreatment

Using the 2,2,6,6-tetramethylpiperidine-1-oxyl radical (TEMPO) to oxidize the surface of cellulose fibres before breaking down the size of fibres into nanoscale objects is one of the most effective and well-documented ways to prepare high quality nanocellulose, causing conversion of the primary hydroxyl groups to carboxylate and aldehyde functional groups in aqueous and mild conditions 43. The water-soluble ɑ-1,4-linked polyglucuronic acid (cellouronic acid) presents the advantage of disintegrating fibres into microfibrils with smaller widths, while using a much lower energy input 2, 17, 44, 45. This pretreatment has been successfully applied to the majority of different cellulose sources such as wood pulp, cotton linters, tunicate, bacterial cellulose, ramie, and even spruce holocellulose 44, 46. Moreover, it was found that the TEMPO oxidation pretreatment resulted in a decrease in the subsequent energy consumption required for successful high pressure homogenization and production of MFC (e.g. from 700 - 1400 MJ/kg to less than 7 MJ/kg) 47, 48. Three different TEMPO-mediated oxidation approaches have been reported. One process consists of oxidation of cellulose fibers via the addition of sodium hypochlorite (NaClO) to aqueous suspensions of cellulose in 2,2,6,6 tetramethyl-1-piperidinyloxy (TEMPO) and sodium bromide (NaBr) at pH 10 - 11 at room temperature 46, 49. The second approach is based on the same process as the previous one, except that the primary oxidant is sodium chlorite (NaClO2) instead of NaClO, and NaClO replaces NaBr. This treatment takes place at neutral pH 47, 48, 50-52. The third approach, a TEMPO electro-mediated reaction, has been developed as a new sustainable method to produce MFC that has carboxylate and aldehyde groups on its surface, and it appears to be a promising replacement for the first two approaches. In this last process, the electro-mediated oxidation has been performed with two slight variations; firstly TEMPO at pH=10, and secondly 4-acetamido- TEMPO at pH = 6.8 in a buffer solution were applied to cellulose fibres and yielding subtly- different results 47, 48. The first variant produced oxidized cellulose with a small amount of aldehyde groups, while the second method resulted in no aldehyde group on the cellulose surface after exposure to the longer reaction time performed at a higher temperature.

5.1.3 Carboxymethylation pretreatment

Another chemical pretreatment is carboxymethylation. This treatment involves the partial carboxymethylation of cellulose pulp, and results in an increased number of anionic charges on the surface of the fibres, subsequently leading to electrostatic repulsion between the fibrils 15, 16, 53. This charging makes the fibrils easier to liberate and also limits their agglomeration 14. Taipale et al 54 reported 3.2 MWh/t decrease in the net specific energy consumption for processing following this

82 treatment in comparison with producing microfibrillated cellulose without pretreatment. In this treatment, never dried cellulose fibres were washed with ethanol, and then soaked in monochloroacetic acid and isopropanol solution. The fibers were subsequently added to a solution of sodium hydroxide, methanol and isopropanol. This carboxymethylation reaction was continued for one hour at just below its boiling temperature. The final steps involved treatment with acetic 13 acid, and lastly sodium bicarbonate (NaHCO3) .

5.1.4 Enzymatic pretreatment

Enzymatic hydrolysis is a less aggressive pretreatment method which has advantages from an environmental point of view, and with respect to the other chemical methods, since it does not involve solvents or chemical reactants 20, 55. For example, using endoglucanase enzyme works selectively to hydrolyse amorphous cellulose regions and facilitates the mechanical disintegration and reduced energy consumption because of the increased swelling of fibres in water 19, 20, 56 . The microfibrillated cellulose (MFC) obtained after using this treatment, showed a higher aspect ratio, longer and highly entangled nanoscale fibrils in comparison with acid treated fibres. This pretreatment method could also be a very promising method for pilot production, as a favourable MFC structure results through a low energy consumption method allowing further evaluation of these high quality nanofibrils for industrial applications 2, 43. In this thesis, in order to produce nanoparticles from spinifex grass, the organosolve treatment was firstly adapted as a relatively milder treatment, and subsequently compared with an alternative mild alkali treatment in terms of efficiency to remove lignin, hemicellulose, and the other waxy components, as well as the quality and characteristics of the resultant NFCs and CNCs in later sections.

5.2 Results and discussion

5.2.1 Chemical analysis of lignocellulosic components in spinifex grass

Lignocellulose, the fibrous material that forms plant cell walls, is a complex typically inedible plant material consisting of mainly three constituents; cellulose, hemicelluloses and lignin. TAPPI standard methods were used to characterize lignin and neutral carbohydrates and anhydrosugars present in the water washed, alkali and organosolve delignified and bleached spinifex fibres (details of analyses are in Chapter 3, Section 3.2.3). Table 5.2 shows that in spinifex grass, cellulose which consists of high molecular weight polymers of glucose, accounts for 29 % (w/w) of the lignocellulose. Hemicellulose consists of shorter polymers of various sugars that glue the cellulose bundles together, and accounts for 38.5 % (w/w) of the lignocelluloses in spinifex, and lignin, the 83 component that confers strength to the plant’s structure and consists of a tridimensional phenolic polymer that is imbedded in and bound to the hemicelluloses, accounts for 20 % (w/w) in spinifex.

Table 5.2. Chemical analysis of lignocellulosic components of raw and pretreated spinifex grass

Sample Cellulose (% w/w) Hemicellulose (% w/w) Lignin (% w/w) Water washed 29 38.5 20 Alkali delignified 27 38 23 Alkali delignified and 48 37 2.5 bleached Organosolve delignified 35 30 23 Organosolve delignified 51 30 7 and bleached

In lignocellulosic plants lignin and hemicellulose are typically deposited between the microfibrils to give an interrupted lamellar structure, so removal of these non-cellulosic components is generally necessary for enabling the subsequent separation and production of cellulose nanoparticles. Alkali and organosolve treatments in addition to remove a certain amount of lignin, hemicellulose, pectin, resin, wax and oils which covering the external surface of the fibre cell wall, also depolymerise the native cellulose structure, and defibrillate the external cellulose microfibrils (see details of delignification procedures in Chapter 3, Sections 3.2.4.2). Table 5.2 shows that after alkali and organosolve delignification, higher amount of lignin were observed in the mass balances of delignified grass compared to water washed grass. Similar observations were reported by Leschinsky et al and Borrega et al 57-59. They believed that after delignification, some parts of the soluble lignin, especially those with higher molecular mass will precipitate during the cooling process and form a condensed insoluble fraction. Increasing the mass balance of lignin is likely due to the attaching degraded carbohydrates to condensed lignin fragments in the insoluble lignin fraction 57-59. The amount of hemicellulose after alkali treatment did not change significantly while organosolve treatment reduced almost 8 % (w/w) of hemicellulose content of fibres. Bleaching delignified fibres is also necessary for complete elimination of the remaining lignin from the fibrous structure (details are in Chapter 3, Section 3.2.4.3). Bleaching fibres after alkali and organosolve delignifications reduced 2.5 and 7 % (w/w) lignin content, respectively. The flowchart in Figure 5.1 summarizes the resultant changes in spinifex fibre colour from dark yellow to dark brown after delignification, indicative that some residual free lignin remained on the surface

84 of the fibres after alkali and organosolve delignifications. A white dispersion was obtained when delignified fibres were then treated with sodium chlorite solution in the bleaching process.

Figure 5.1. Flowchart representing the pulping procedures of spinifex fibres

5.2.2 Effect of delignification and bleaching on chemical structure of spinifex fibre

Cellulose, hemicellulose and lignin are mainly composed of alkanes, esters, aromatics, ketones and alcohols, with different oxygen-containing functional groups 60. Figure 5.2 compares the ATR FTIR spectra of water washed spinifex fibres with delignified and bleached fibres, and Table 5.3 summarizes the peak values from ATR FTIR spectra and assigns possible chemical units of fibres before and after pulping. It can be seen that delignification and bleaching yielded more cellulose percentage by removing hemicellulose, especially lignin, and other waxy components from the structure of fibres. The ATR FTIR spectra for water washed and chemically pretreated spinifex fibres are associated with a main broad peak within 3000 - 3650 cm-1 which confirms stretching vibrations of OH groups as the principal functional group in lignocellulosic materials. The absorbance peak with a maximum around 2900 cm-1 (responsible for C-H stretching) also decreased after delignification and bleaching, indicating the removal of hydrophobic lignin and other waxy components 61. A decrease in the peak observed around 1734 cm-1 (C=O stretching) after delignification and bleaching can be attributed to the breakage of the ester linkages of carboxylic groups of lignin and/or hemicellulose 62 and oxidation of the terminal glucopyranose unit. After delignifications, fibres still contain residual lignin left on the surface and between the fibrils (based on chemical analysis of delignified fibre explained in Section 5.2.1). So the 85 disappearance of C=O stretching band after chemical treatments does not necessarily indicate the complete removal of all hemicellulose and lignin from the fibres, as the ether bonds between lignin and hemicellulose will not be affected by this treatment. Another possibility is that carboxyl or aldehyde absorption could be arising from the opened terminal glycopyranose rings or oxidation of the C-OH groups 60. The other band around 1240 cm-1, which corresponds to the axial asymmetric strain of =C-O- in ether, ester, and phenol groups of lignin, is also seen to decrease for the bleached fibers. The peaks in the region of 950 - 1200 cm-1 may be assigned to C-O stretching bands, and an increase in the intensity of the band at 896 cm-1 after chemical treatments can be attributed to the typical structure of cellulose 63, 64. The water washed fibre showed characteristic peaks in between 1730 - 1740 cm-1 and 1200 - 1300 cm-1. These peaks are mainly responsible for the lignin components, which were absent in the final bleached cellulose fibre. It is worth nothing that cellulose fibre strongly adsorbs moisture which makes the quantitative ATR FTIR analysis difficult, therefore all of the specimens were dried in a vacuum oven before testing.

1.0 Water washed Alkali delignified 0.8 Bleached

C-O-C 0.6 -C-O O-H C-H C=O O-H 0.4

Absorbance

0.2

0.0 4000 3500 3000 2500 2000 1500 1000 Wave number (cm-1)

1.0 Water washed Organo-solve delignified 0.8 Bleached C-O-C 0.6 -C-O O-H C-H C=O O-H 0.4

Absorbance

0.2

0.0 4000 3500 3000 2500 2000 1500 1000 Wave number (cm-1)

Figure 5.2. ATR FTIR spectra of spinifex fibres before and after alkali delignification, organosolve delignification, and bleaching

Table 5.3. ATR FTIR peaks and functional groups of spinifex fibres before and after pulping

Peak (cm-1) Functional group Responsible units 3650 - 3000 (broad O-H Alcohol/phenol O-H stretch peak) 2800 - 3000 C-H stretching Vibrations in methyl and methylene groups 1730 - 1740 C=O Carbonyl compounds (ester, acetyl , carboxylic groups)

1446 - 1600 C=C-C and CH2 rocking Aromatic compounds 1200 - 1300 =C-O- axial asymmetric strain Ether, ester, and phenol groups 950 - 1200 C-O stretching

5.2.3 Effect of delignification and bleaching on morphology of spinifex fibres

Spinifex grass is the same as the other plants in that it has outer layers consisting of hemicellulose, lignin and other substances which protect the cellulose structures in the grass from being damaged by microorganisms in the environment. In Figure 5.3, the SEM images of untreated and chemically pretreated spinifex fibers show removal of the surface impurities along with evidence of some partial defibrillation with treatment.

Before water washing, the surface of the fibres were covered with impurities, dust and also cell wall components such as lignin, hemicellulose and the other waxy materials. Water washing (Figures 5.3 (a and b)) partly removed these substances. In Figures 5.3 (c and d) it can be observed that pulping successfully removed non-fibrous components at the surface and partly into the bulk of the fibres. The altered morphology of the fibre after delignification reveals a very unusual morphology. Elementary fibrils are intertwined and arranged to form microfibers, and these connect perpendicularly with hollow tube-like channels called stroma lamellae, which are structures responsible for photosynthesis during day time and the storage of biosynthesized carbohydrates 65, 66. Desert plants must be able to absorb large quantities of water in short periods, and they need to retain the absorbed water under unfavourable conditions. The leaves of spinifex are open like "normal" grass up until they experience their first dry spell, and then the leaves roll in on themselves and remain rolled for the remaining life of the plant. This rolling of the leaves gives drought tolerance to the spinifex plant in several ways 65, 66 such as reducing the amount of water lost to the atmosphere through open stomatas, which are inside the rolled leaf, thereby decreasing the rate of water lost by transpiration when the wind is blowing. Also the grass will lose less water due to the reduction in leaf area exposed to the hot sun by this rolling or curling of the leaf structure in response to drought.

Figure 5.3. SEM images of; a) raw fibre, b) water washed fibre, c) fibre subjected to alkali delignification and, d) fibre subjected to organosolve delignification

Figure 5.4 compares the optical microscopy images of water washed, alkali and organosolve delignified spinifex fibres. It needs to be mentioned that an average diameter of the chopped raw fibre (before washing and grinding) is 1033 ± 524 μm, while after washing the size of fibres was reduced by grinding, so in order to understand the effect of delignification and bleaching on the fibre morphology, the diameter of water washed samples after grinding was reported for comparison. An average diameter of water washed fibres was measured to be 63 ± 38 μm, with an approximate length distribution in the range of 0.3 - 7 mm. In comparison, after the delignification processes, an average diameter was 16.3 ± 12.6 μm and 12 ± 4.7 μm for alkali and organosolve treated fibres, respectively (Figure 5.6). It appears that after delignification, dissolved lignin was precipitated on the surface of the fibres. Organosolve treated fibres displayed a darker colour and smaller diameter, which indicated a higher degree of precipitation of dissolved dark lignin on to the surface of fibres and lower amount of hemicellulose in fibres structure. This precipitated lignin was subsequently removed by rinsing fibres with mild alkali solution (see details of delignification procedures in Chapter 3, Section 3.2.4.2). The larger diameter of alkali treated fibres confirms the existence of higher content of hemicellulose remaining between the fibrils. The chemical analysis 89 also confirmed the existence of a higher amount of hemicellulose components in the spinifex fibres after alkali delignification.

The residual lignin between the fibrils and dissolved lignin remaining on the surface of the delignified fibres were removed in the subsequent bleaching process. Figures 5.5 and 5.6 show that after bleaching, the average diameter of delignified fibres which had been treated either with alkali solution or ethanol solution, were almost identical. Long and white cellulose fibres with an average diameter of approximately 12 μm were prepared for the next steps of producing cellulose nanocrystals and nanofibrillated cellulose.

Figure 5.5. Optical microscopy of; a) alkali delignified and bleached, b) organosolve delignified and bleached (the scale bar is 100 nm)

Organosolve delignified and bleached

Alkali delignified and bleached

Organosolve delignified

Alkali delignified

Water washed

0 10 20 30 40 50 60 70 80 Averege diameter of fibres (m)

Figure 5.6. Average diameter of spinifex fibre after washing and grinding, delignification, and bleaching

Delignification and bleaching are important and necessary steps prior to mechanical and chemical treatments as they result in partial defibrillation and an opening or loosening of the fibre bundles, and this consequently enables less energy consumption during the next stage of cellulose

disassembly and production of cellulose nanofibres.

5.2.4 Effect of delignification and bleaching on crystallinity of spinifex fibres

Cellulosic fibers consist of the three main components of lignin, hemicelluloses, and ɑ- celluloses, of which cellulose is crystalline in nature, while lignin and hemicellulose are amorphous. So the crystallinity of the fibers should increase after pulping due to the removal of non-cellulosic components surrounding the crystalline cellulose fibre. The observed XRD peaks at around 2θ=16 and 22.6 in Figure 5.7 are representative of a typical structure of cellulose I. Treated fibres show a higher degree of order as evidenced by a higher diffraction intensity at 2θ = 22.6° . The broad peaks of the water washed spinifex fibre are due to the amorphous nature of the lignin, while the higher peak intensity of the chemically treated fibers indicates the removal of non-cellulosic components due to dissolution in delignification and bleaching solvents. The XRD diffraction spectra of untreated and pretreated spinifex fibres indicate that by removing the non-cellulosic components of the fibres, the degree of crystallinity and size of crystal domains are altered. Delignification and bleaching of fibres change the surface topography and the crystallographic structure. The degree of crystallinity values for each sample was calculated and listed in Table 5.4 (details of measuring the crystallinity are in Chapter 3, Section 3.3.8). It can be seen that both delignification and bleaching processes increase the degree of crystallinity due to the removal of lignocellulosic materials which exist in the amorphous regions, and their removal enables realignment of the intermediate ordered regions. The increase in crystallinity after chemical treatments also has been reported by several authors 6, 67-69. Furthermore, after chemical treatments the thickness of crystalline domains increased.

1500

1000

Alkali delignified and bleached

Intensity 500 Organosolve delignified and bleached Alkali delignified Organosolve delignified Water washed 0 10 20 30 40 2Theta (degree)

Table 5.4. Effect of pulping on the diameter and crystallinity of the spinifex fibre

Water Alkali Alkali Organosolve Organosolve washed delignified delignified delignified delignified fibre and bleached and bleached Crystallinity (% ) 52 71 74 72 75

Crystal domain 2.94 4 4 3.82 3.92 size (nm)

5.2.5 Effect of delignification and bleaching on thermal properties of spinifex fibres

Thermal decomposition parameters were determined from the TGA spectra at a heating rate of 10 °C/min. The small weight loss in the range of 40 - 150 °C shown in the TGA spectra in Figure 5.8 was due to the evaporation of the absorbed moisture and low molecular weight compounds were remaining on the surface of the fibres after treatment procedures. Due to the differences in the chemical structures between hemicellulose, cellulose and lignin, they usually decompose at different temperatures 70. The earlier decomposition occurred around 270 °C due to the low decomposition temperature of hemicellulose and preceded by the second stage which was associated with the pyrolysis of cellulose process at around 320 °C for bleached fibres and about 357 °C, 347 °C and 358 °C for water washed, alkali and organosolve delignified fibres, respectively. The thermal degradation temperature of organosolve delignified fibre is higher due to removal of more hemicellulose after treatment. The maximum decomposition temperature of cellulose for water washed and delignified samples were higher than the samples which treated with sodium chlorite due to elimination of more thermal resistant chemical groups present in lignin structure after bleaching 44. The residual content of samples at 500 °C could be residual lignin, hemicellulose, and inorganic components taken up by the roots of grass as nutrients and micronutrients in the soil 71. There is also another reason that has been reported by Austen et al 60 about an increase in residual char formation and decrease in degradation temperature during the pyrolysis. They suggested that this might be caused by an increase rate of formation of free radicals which are stabilized by condensed carbon ring formation in the char.

100

Weight (%) Weight Water washed 20 Alkaline delignified Bleached 0 100 200 300 400 500 Temperature (°C)

100 90 80 70 60 50

Weight (%) Weight 40 Water washed 30 Organosolve delignified 20 Bleached

100 200 300 400 500 Temperature (°C)

Figure 5.8. Comparing TGA spectra of spinifex untreated (water washed) with alkali and organosolve treated fibres

It seems that the thermal stability of spinifex fibre was decreased when it was subjected to alkalization due to removal of lignin components with higher degradation temperature while the amount of hemicellulose with lower degradation temperature was almost same. The wide decomposition temperature range observed during the lignin decomposition for the water washed and delignified fibres is attributed to the different activities of chemical groups present in the lignin structure 44. The decrease in residual content of delignified and bleached fibres in comparison with the water washed fibres indicates the lower amount of lignin left on the fibres after pulping.

5.3 Summary

The aim of this chapter is to compare a simpler alkali treatment with milder organosolve treatment for the extraction of cellulose from spinifex grass. Chemical analyses based of TAPPI standard methods showed that spinifex grass, same as the other grasses, contains high amount of hemicellulose about 38.5 % (w/w), 29 % (w/w) cellulose, and 20 % (w/w) lignin. After two different applied delignification procedures, alkali and organosolve, the amount of these three main components (cellulose, hemicellulose, and lignin) was not same in grass mass balance. Alkali treatment was more effective in term of removing lignin while the amount of hemicellulose remained after alkali delignification and subsequent bleaching was showed only 2.5 % decrease in comparison with the raw grass. After organosolve delignification, the lignin content remained in fibres structure was decreased to almost 7 % (w/w) and the hemicellulose content was decreased to 30 % (w/w). ATR FTIR, X-Ray, TGA and morphological analysis with optical microscopy and SEM were indicated that both of the delignification treatments and subsequent bleaching caused partly defibrillation of fibres, along with isolation of crystalline cellulose fibres with an average diameter of about 12 μm. The crystallinity of the fibre specimens are increased following each of these treatments and a thermogravimetric study revealed that the thermal stability of pretreated spinifex fibres is higher than the respective raw fibres. The presence of lignin in fibre is believed to significantly affect the defibrillation of micro/nanofibrils. For producing NFC and CNC, obtaining cellulose of high-purity is pre-requisite, which generally necessitates multistep treatments with harsh chemicals (acid/alkaline) or sometimes enzymes 72. Two different applied pulping methods in this thesis are milder than the other reported pulping methods used for producing NFC and CNC. The organosolve treatment which was adapted here is commonly used for pulping purposes and has never been successfully used as a very mild pulping prior to bleaching and subsequent mechanical or chemical separation of nanofibrils or nanocrystals (i.e normally more aggressive bases, acids and/or high temperatures are required). Also the alkali treatment is milder in comparison to the other reported publications for producing nanocellulose which higher concentration of alkaline was used or alkali treatment was followed by more intense acidic hydrolysis for fibre pretreatment 29.

Bibliography

1. Jonoobi, M.; Mathew, A. P.; Oksman, K. Industrial Crops and Products 2012, 40, (0), 232- 238.

2. Siqueira, G.; Bras, J.; Dufresne, A. Polymers 2010, 2, (4), 728-765.

3. Suopajärvi, T.; Liimatainen, H.; Niinimäki, J. Cellulose 2012, 19, (1), 237-248.

4. Spence, K. L.; Venditti, R. A.; Habibi, Y.; Rojas, O. J.; Pawlak, J. J. Bioresource Technology 2010, 101, (15), 5961-5968.

5. Johnson, R. K.; Zink-Sharp, A.; Renneckar, S. H.; Glasser, W. G. Cellulose 2009, 16, (2), 227-238.

6. Chen, W.; Yu, H.; Liu, Y.; Chen, P.; Zhang, M.; Hai, Y. Carbohydrate Polymers 2011, 83, (4), 1804-1811.

7. Wang, B.; Sain, M. Composites Science and Technology 2007, 67, (11–12), 2521-2527.

8. Wang, B.; Sain, M. Polymer International 2007, 56, (4), 538-546.

9. Abraham, E.; Deepa, B.; Pothen, L. A.; Cintil, J.; Thomas, S.; John, M. J.; Anandjiwala, R.; Narine, S. S. Carbohydrate Polymers 2013, 92, (2), 1477-1483.

10. Moon, R. J.; Martini, A.; Nairn, J.; Simonsen, J.; Youngblood, J. Chemical Society Reviews 2011, 40, (7), 3941-3994.

11. Alila, S.; Besbes, I.; Vilar, M. R.; Mutjé, P.; Boufi, S. Industrial Crops and Products 2013, 41, (0), 250-259.

12. Zimmermann, T.; Pöhler, E.; Geiger, T. Advanced Engineering Materials 2004, 6, (9), 754- 761.

13. Wagberg, L.; Decher, G.; Norgren, M.; Lindstrom, T.; Ankerfors, M.; Axnas, K. Langmuir 2008, 24, (3), 784-795.

14. Siró, I.; Plackett, D.; Hedenqvist, M.; Ankerfors, M.; Lindström, T. Journal of Applied Polymer Science 2011, 119, (5), 2652-2660.

15. Eyholzer, C.; Bordeanu, N.; Lopez-Suevos, F.; Rentsch, D.; Zimmermann, T.; Oksman, K. Cellulose 2010, 17, (1), 19-30.

16. Plackett, D.; Anturi, H.; Hedenqvist, M.; Ankerfors, M.; Gällstedt, M.; Lindström, T.; Siró, I. Journal of Applied Polymer Science 2010, 117, (6), 3601-3609.

17. Saito, T.; Kimura, S.; Nishiyama, Y.; Isogai, A. Biomacromolecules 2007, 8, (8), 2485- 2491.

18. Besbes, I.; Vilar, M. R.; Boufi, S. Carbohydrate Polymers 2011, 86, (3), 1198-1206.

19. Pääkkö, M.; Ankerfors, M.; Kosonen, H.; Nykänen, A.; Ahola, S.; Österberg, M.; Ruokolainen, J.; Laine, J.; Larsson, P. T.; Ikkala, O.; Lindström, T. Biomacromolecules 2007, 8, (6), 1934-1941.

20. Henriksson, M.; Henriksson, G.; Berglund, L. A.; Lindström, T. European Polymer Journal 2007, 43, (8), 3434-3441.

21. Cherian, B. M.; Leão, A. L.; de Souza, S. F.; Thomas, S.; Pothan, L. A.; Kottaisamy, M. Carbohydrate Polymers 2010, 81, (3), 720-725.

22. Siqueira, G.; Bras, J.; Dufresne, A. Biomacromolecules 2008, 10, (2), 425-432.

23. Pandey, J. K.; Chu, W. S.; Kim, C. S.; Lee, C. S.; Ahn, S. H. Composites Part B: Engineering 2009, 40, (7), 676-680.

24. Pandey, J.; Lee, J.-W.; Chu, W.-S.; Kim, C.-S.; Ahn, S.-H.; Lee, C. Macromolecular Research 2008, 16, (5), 396-398.

25. Malainine, M. E.; Dufresne, A.; Dupeyre, D.; Mahrouz, M.; Vuong, R.; Vignon, M. R. Carbohydrate Polymers 2003, 51, (1), 77-83.

26. Rosa, M. F.; Medeiros, E. S.; Malmonge, J. A.; Gregorski, K. S.; Wood, D. F.; Mattoso, L. H. C.; Glenn, G.; Orts, W. J.; Imam, S. H. Carbohydrate Polymers 2010, 81, (1), 83-92.

27. Dinand, E.; Chanzy, H.; Vignon, M. R. Cellulose 1996, 3, (1), 183-188.

28. Deepa, B.; Abraham, E.; Cherian, B. M.; Bismarck, A.; Blaker, J. J.; Pothan, L. A.; Leao, A. L.; de Souza, S. F.; Kottaisamy, M. Bioresource Technology 2011, 102, (2), 1988-1997.

29. Habibi, Y.; Goffin, A. L.; Schiltz, N.; Duquesne, E.; Dubois, P.; Dufresne, A. Journal of Materials Chemistry 2008, 18, (41), 5002-5010.

30. Teixeira, E. d. M.; Bondancia, T. J.; Teodoro, K. B. R.; Corrêa, A. C.; Marconcini, J. M.; Mattoso, L. H. C. Industrial Crops and Products 2011, 33, (1), 63-66.

31. Pan, X.; Arato, C.; Gilkes, N.; Gregg, D.; Mabee, W.; Pye, K.; Xiao, Z.; Zhang, X.; Saddler, J. Biotechnology and Bioengineering 2005, 90, (4), 473-481.

32. Nogi, M.; Iwamoto, S.; Nakagaito, A. N.; Yano, H. Advanced Materials 2009, 21, (16), 1595-1598.

33. Abe, K.; Iwamoto, S.; Yano, H. Biomacromolecules 2007, 8, (10), 3276-3278.

34. Uetani, K.; Yano, H. Biomacromolecules 2011, 12, (2), 348-353.

35. Yousefi, H.; Faezipour, M.; Nishino, T.; Shakeri, A.; Ebrahimi, G. Polymer journal 2011, 43, (6), 559-564.

36. Oliet, M.; Garcı́a, J.; Rodrı́guez, F.; Gilarrranz, M. A. Chemical Engineering Journal 2002, 87, (2), 157-162.

37. Xu, Y.; Li, K.; Zhang, M. Colloids and Surfaces A: Physicochemical and Engineering Aspects 2007, 301, (1–3), 255-263.

38. Navaee-Ardeh, S.; Mohammadi-Rovshandeh, J.; Pourjoozi, M. Bioresource Technology 2004, 92, (1), 65-69.

39. Jiménez, L.; Pérez, I.; López, F.; Ariza, J.; Rodrı́guez, A. Bioresource Technology 2002, 83, (2), 139-143.

40. Madakadze, I. C.; Radiotis, T.; Li, J.; Goel, K.; Smith, D. L. Bioresource Technology 1999, 69, (1), 75-85.

41. Gilarrahz, M. A.; Oliet, M.; Rodriguez, F.; Tijero, J. The Canadian journal of chemical engineering 1998, 76, (2), 253-260.

42. Bhatnagar, A.; Sain, M. Journal of Reinforced Plastics and Composites 2005, 24, (12), 1259-1268.

43. Lavoine, N.; Desloges, I.; Dufresne, A.; Bras, J. Carbohydrate Polymers 2012, 90, 735– 764 98

44. Saito, T.; Nishiyama, Y.; Putaux, J.-L.; Vignon, M.; Isogai, A. Biomacromolecules 2006, 7, (6), 1687-1691.

45. Besbes, I.; Alila, S.; Boufi, S. Carbohydrate Polymers 2011, 84, (3), 975-983.

46. Saito, T.; Okita, Y.; Nge, T. T.; Sugiyama, J.; Isogai, A. Carbohydrate Polymers 2006, 65, (4), 435-440.

47. Isogai, T.; Saito, T.; Isogai, A. Cellulose 2011, 18, (2), 421-431.

48. Isogai, A.; Saito, T.; Fukuzumi, H. Nanoscale 2011, 3, (1), 71-85.

49. Eichhorn, S. J.; Dufresne, A.; Aranguren, M.; Marcovich, N. E.; Capadona, J. R.; Rowan, S. J.; Weder, C.; Thielemans, W.; Roman, M.; Renneckar, S.; Gindl, W.; Veigel, S.; Keckes, J.; Yano, H.; Abe, K.; Nogi, M.; Nakagaito, A. N.; Mangalam, A.; Simonsen, J.; Benight, A. S.; Bismarck, A.; Berglund, L. A.; Peijs, T. Journal of Materials Science 2010, 45, (1), 1-33.

50. Hirota, M.; Tamura, N.; Saito, T.; Isogai, A. Cellulose 2010, 17, (2), 279-288.

51. Ishii, D.; Saito, T.; Isogai, A. Biomacromolecules 2011, 12, (3), 548-550.

52. Saito, T.; Hirota, M.; Tamura, N.; Kimura, S.; Fukuzumi, H.; Heux, L.; Isogai, A. Biomacromolecules 2009, 10, (7), 1992-1996.

53. Siro, I.; Plackett, D. Cellulose 2010, 17, (3), 459-494.

54. Taipale, T.; Österberg, M.; Nykänen, A.; Ruokolainen, J.; Laine, J. Cellulose 2010, 17, (5), 1005-1020.

55. Henriksson, M.; Berglund, L. A.; Isaksson, P.; Lindström, T.; Nishino, T. Biomacromolecules 2008, 9, (6), 1579-1585.

56. Engström, A. C.; Ek, M.; Henriksson, G. Biomacromolecules 2006, 7, (6), 2027-2031.

57. Leschinsky, M.; Sixta, H.; Patt, R. Bioresources 2009, 4, (2), 687-703.

58. Borrega, M.; Nieminen, K.; Sixta, H. Bioresources 2011, 6, (2), 1890-1903.

59. Leschinsky, M.; Weber.K, H.; Patt, R.; Sixta, H. Lenzinger Berichte 2009, 87, 16-25.

60. Abraham, E.; Deepa, B.; Pothan, L. A.; Jacob, M.; Thomas, S.; Cvelbar, U.; Anandjiwala, R. Carbohydrate Polymers 2011, 86, (4), 1468-1475.

61. Avolio, R.; Bonadies, I.; Capitani, D.; Errico, M. E.; Gentile, G.; Avella, M. Carbohydrate Polymers 2012, 87, (1), 265-273.

62. Sheltami, R. M.; Abdullah, I.; Ahmad, I.; Dufresne, A.; Kargarzadeh, H. Carbohydrate Polymers 2012, 88, (2), 772-779.

63. Xiao, B.; Sun, X. F.; Sun, R. Polymer Degradation and Stability 2001, 74, (2), 307-319.

64. Zuluaga, R.; Putaux, J. L.; Cruz, J.; Vélez, J.; Mondragon, I.; Gañán, P. Carbohydrate Polymers 2009, 76, (1), 51-59.

65. Habibi, Y.; Mahrouz, M.; Vignon, M. R. Food chemistry 2009, 115, (2), 423-429.

66. Dinand, E.; Chanzy, H.; Vignon, M. R. Food Hydrocolloids 1999, 13, (3), 275-283.

67. Alemdar, A.; Sain, M. Bioresource Technology 2008, 99, (6), 1664-1671.

68. Li, R. J.; Fei, J. M.; Cai, Y. R.; Li, Y. F.; Feng, J. Q.; Yao, J. M. Carbohydrate Polymers 2009, 76, (1), 94-99.

69. Sonia, A.; Priya Dasan, K. Carbohydrate Polymers 2013, 92, (1), 668-674.

70. Yang, H.; Yan, R.; Chen, H.; Lee, D. H.; Zheng, C. Fuel 2007, 86, (12–13), 1781-1788.

71. Mondal, S.; Memmott, P.; Wallis, L.; Martin, D. Materials Chemistry and Physics 2012, 133, (2–3), 692-699.

72. Hubbe, M. A.; Rojas, O. J.; Lucia. A. , L.; Sain, M. BioResources 2008, 3, (3), 929-980.

100

Chapter 6 . The isolation of high aspect ratio nanofibrillated cellulose from spinifex using mild pulping and ultra-low energy processing

101

6.1 Introduction

Mechanical methods such as homogenisation, ultrasonication, milling, or combinations of these methods are widely used for isolating microfibrillated cellulose (MFC) particles from cellulose fibres, where these MFC particles have a diameter in the range of 20 - 100 nm and a length in the range of 0.5 - 10’s of μm 1. In order to separate the next smallest constituents, called nanofibrillated cellulose (NFC) with a diameter in the range of 4 - 20 nm and a length in the range of 500 - 2000 nm 1, the application of a significantly higher amount of mechanical energy is typically required. In reported methods, chemical or enzymatic pretreatments are usually claimed to be beneficial for reducing mechanical energy consumption and fibre diameter. Applying intense amounts of mechanical energy causes the cellulose fibres to fracture, thereby reducing their length and aspect ratio. In order to avoid this issue, the cellulosic fibres can be treated by employing multiple, but more gentle mechanical processing treatments to facilitate the gradual breakdown, or defibrillation, of the fibre to its nanoscale fibrils, while hopefully maintaining aspect ratio and not damaging the fibrils. For example, cellulosic feedstock materials may be passed through apparatus such as high pressure or high shear homogenisers or disc refiners several times (up to 20 or 30, or even 150 passes in some examples 2, 3). In current commercial processes, the multi-step processing results in high energy costs, long processing times, and consequently reduces the commercial attractiveness of both process and product. In the process of defibrillation of spinifex fibre, the established procedures of producing MFC were applied to produce NFC with a very small average diameter and high aspect ratio. This chapter details my breakthrough findings with respect to the isolation of high aspect ratio nanofibrillated cellulose following very mild pulping, and followed by ultra-low energy processing. Figure 6.1 shows how significantly less energy was used to produce nanofibrillated cellulose from spinifex grass. This work formed the foundation of a provisional patent, “Nanocellulose” 4, which was lodged on the 22nd of November in 2013.

102

Figure 6.1. The newly-discovered method of producing NFC from spinifex grass ( there is no treatment on the spinifex pulp before mechanical treatments )

6.2 Results and discussion

High aspect ratio nanofibrillated cellulose can be isolated from spinifex pulp via low energy mechanical treatments. We hypothesise that the lower energy consumption or milder conditions required to deconstruct the fibrils could be attributable to the unique structural morphology of the grass, which typically grows in extreme and fluctuating arid conditions, where adaptations of the plant to the typically dry climatic conditions would be expected.

6.2.1 The effect of processing on spinifex NFC morphological characteristics

6.2.1.1 Homogenization

Table 6.1 details the different conditions applied for the homogenization of bleached spinifex fibres after alkali delignification, in order to optimize the procedure (details are in Chapter 3, Section 3.2.7). During the preparation of NFC, it was found that high pressure homogenization has a noticeable effect on reducing the diameter of fibres, and produces very long, thin nanofibrils with a complex and web-like structure. After systematically applying different pressures, different dispersion concentrations, and also varying the number of passes through the homogenizer, it was surprising to observe that the resulting NFCs produced exhibited almost the same fibre diameter (less than 7 nm), and similar length (several microns). In the case of homogenization at higher pressures, more effective defibrillation was observed (Figure 6.2). Increasing the number of passes through the homogenization at 1500 bar caused more entanglement and aggregation of the nanofibrils (Figure 6.3). This may be due to the high surface energy rendered by excessive defibrillation with increasing the number of passes.

103

A significant finding from this study is that it is easy to defibrillate spinifex pretreated fibres into nanoscale fibrils after just one pass using the minimum 350 bar pressure setting, and without any clogging issues. This is in stark contrast to the most directly-relevant published studies, where a minimum of 3 passes were necessary for NFC preparation, and where treatment with acid/alkaline /polyelectrolyte was also usually performed to reduce the number of passes, which is crucial in terms of minimising the energy consumption of the process 5-8. The high amount of hemicellulose in the spinifex pulp (about 37 % (w/w)) decrease the fibres cell wall cohesion, making cell wall delamination easier and consequently avoid of blocking the constriction chamber of the homogenizer and decrease the required energy consumption.

Table 6.1. Comparing the average diameter of nanofibrils produced by homogenization using different dispersion concentration, pressure conditions, and number of passes

0.1 % (w/v) 0.3 % (w/v) 0.3 % (w/v) 0.3 % (w/v) 0.3 % (w/v) 0.7 % (w/v) , , , , , , 1 pass 1 pass 5 passes 10 passes 15 passes 1 pass

1500 bar 3.2 ± 0.7 3.5 ± 0.8 3.2 ± 0.7 3.3 ± 0.8 3.2 ± 0.8 3.7 ± 0.7

1000 bar 3.5 ± 0.6 - - - - -

350 bar 3.7 ± 1 - - - - -

104

Figure 6.2. TEM images comparing the effect of applied pressure in homogenization of 0.3 % (w/v) pulp dispersion after one single pass, on the morphology of spinifex nanofibrils; a) 1500 bar, b) 1000 bar, c) 350 bar (the scale bar is 500 nm)

105

The length-to-thickness ratio of these nanofibrils is unusually higher than the reported NFCs from other methods 9-12, however this high aspect ratio primarily results from the unusually low diameter of nanofibrils (e.g. from 1 - 7 nm, and averaging around 3.5 nm). The high hemicellulose content (discussed in the Chapter 5, Section 5.2.1) imparts flexibility to the fibres, so the individual nanofibrils are curled and entangled with each other, and the main orientation appears to be random-in-the plane. Upon drying the aqueous nanocellulose suspensions, it is well-known that the strong secondary inter-fibril interactions such as hydrogen bonding encourage agglomeration and make the characterization of these materials very difficult 13. However the longer curled morphology of our spinifex-derived NFCs prevents close packing to some extent, thereby making it easier to measure NFC particle dimensions from the lower magnification TEM images, albeit typically for bundles of nanofibers rather than primary, discrete nanofibrils. These nanofiber bundles display an average diameter of approximately 10 nm, which can be clearly seen at that magnification. The average aspect ratio of individual fibrils produced from homogenization of 0.3 106

% (w/v) fibre dispersion for only one pass at 1500 bar pressure is 527 ± 185 and ranging from 266 to 958, with an average diameter of 3.2 ± 0.8 nm and average length of 1686 ± 591 nm, (as measured from TEM images taken at a higher magnification and cryo-TEM using methods described in Chapter 3, Section 3.3.6.2 and 3.3.6.1, respectively). The average aspect ratio of a bundle of several nanofibrils from the same sample is 540 ± 166 and ranging from 305 to 727, with an average diameter of 10.7 ± 3.9 nm and an average length of 5773 ± 1700 nm (as measured from TEM images taken at low magnification to enable observation of the whole long bundle in a single image. Details of these methods are provided in Chapter 3, Section 3.3.6.2). Figure 6.4 shows the three dimensional images of NFC which were taken by cryo-TEM. The length of individual nanofibrils was measured from this type of images.

Figure 6.4. Three dimensional images of spinifex NFC were taken by cryo-TEM

Homogenization of organosolve treated spinifex fibre also resulted in production of very high aspect ratio NFC. It was found that a minimum of three passes through the homogenizer was necessary to produce nanofibrils with the same diameter as nanofibrils obtained from alkali treated fibres after only one pass at the same pressure (1500 bar) through the homogenizer. At one pass we could see still some bundles of nanofibrils with an average diameter above 10 nm. Alkali treatment has enhanced the defibrillation due to the elimination of less hemicellulose and removal of more 107

lignin, while the residual hemicellulose and lignin content in organosolve treated fibres was respectively, 7 wt % and 5.5 wt % higher than alkali treated fibres (see Chapter 5, Section 5.2.1).

6.2.1.1.1 Comparison the effect of homogenization on spinifex pulp with commercial MCC

Microcrystalline cellulose (MCC) has been a successful product since its discovery in 1955 and has been commercialized under the trade name of Avicel. MCC is commonly used for applications in the pharmaceutical and food industries as a thickening or emulsifying agents e.g. in a number of food formulations, and also as a common starting material to prepare cellulose nanocrystals (CNC) in several laboratories 14, 15. The common method for producing MCC is acid hydrolysis of wood and plant fibres, followed by neutralization with alkali, and spray-drying. The resulting aggregate bundles of multi-sized cellulose microfibrils with a diameter in the range of 10- 50 μm, are porous, have a high cellulose content, and a high crystallinity 1. In order to compare the spinifex NFC with another common source of cellulose, homogenization of an aqueous dispersion of MCC with a concentration of 0.7 % (w/v) was performed at a pressure of 1500 bar for one pass. TEM images in Figure 6.5 compare the effect of homogenization on the morphology of MCC and spinifex NFC obtained using identical processing. Homogenization of MCC resulted in a significant reduction in its particle size and produced regular rod shaped cellulose nanocrystals with an average diameter of 5.6 ± 1.4 nm and an average length of 216 ± 70nm. It can be seen that although the diameter of MCC nanocrystals and spinifex NFC are almost identical, the length of spinifex nanofibrils still is strikingly higher than the MCC nanocrystal.

6.5. TEM images compare the effect of one pass of homogenization at 1500 bar on morphology of; a) MCC, b) spinifex alkali pretreated fibre (the scale bar is 1 μm (a) and 2 μm (b)) 108

In comparison with published aspect ratio values for NFCs obtained via homogenization in combination of enzymatic hydrolysis 11, TEMPO oxidation 9, 10, and carboxymethylation 12, the aspect ratio of spinifex NFCs obtained via homogenization without any further treatment is still very high averaging at values of approximately 500.

6.2.1.2 Ultrasonication

The use of ultrasonic energy has been already proven to defibrillate or deconstruct the cellulose fibers into long nanofibrils (from bamboo 16) and nanocrystals (from cotton 17) depending on the amount of applied energy. In order to investigate the effects of ultrasonication on defibrillation of pretreated fibres, two key parameters of amplitude and ultrasonication time were varied (See Chapter 3, Section 3.2.8). It was observed that the ultrasonication of spinifex alkali pretreated fibre dispersion at low amplitude (20 %) for either 5 or 20 minutes, produced a dispersion of high aspect ratio nanofibrils with an average diameter of approximately 4 and 7 nm, respectively and a length of several microns (Figure 6.6 and Table 6.2). A slight increase in the ultrasonication amplitude (30 %) and time (20 minutes) enhanced the defibrillation to obtain NFC with an average diameter of approximately 5 nm. A further increase in ultrasonication energy (60 % amplitude and 5 minutes) also resulted in long nanofibrils with slight entanglement (as seen in Figure 6.6 (c)). In the case of spinifex pulp, further increases in ultrasonication energy (above 20 minutes for 20 and 30 % and above 5 minutes for 60 % amplitude) resulted in more agglomerated and entangled fibres. The mechanism for this re-agglomeration is still unclear. It may be attributed to excessive vibrational energy leading to the higher mobility of fibrils as well as increased polar (hydrogen bonding) surface area after defibrillation. This suggests that a shorter period of ultrasonication at lower energy is enough to produce high aspect ratio NFC from pretreated spinifex fibre. It should be taken into account that ultrasonication is not considered the most convenient and scalable method to treat spinifex fibres uniformly with a high yield. In fact, only a small amount of cellulose fibres break down to nanofibrils 18 and remain suspended as supernatant, while the large untreated particles settle out. As far as ideal shear force and scalability are considered, high pressure homogenisation still remains the best method for defibrillation of spinifex pretreated fibres.

109

Table 6.2. Comparing the effect of ultrasonication parameters on the average diameter of spinifex nanofibrils (the concentration of dispersion was 1 % (w/v))

Time of 20 % amplitude 30 % amplitude 60 % amplitude ultrasonication 5 minutes 4.1 ± 0.9 3.62 ± 0.9 14 ± 3.8 20 minutes 6.8 ± 1.4 5.3 ± 1.8 -

6.2.1.3 High energy milling

The first attempt at exploring the utility of continuous high energy milling was performed on bleached spinifex fibres after organosolve delignification. All of the preliminary milling results were based on a continuous, re-circulating milling setup (described in Chapter 3, Section 3.2.9). As

110

displayed in Figure 6.7, these first spinifex NFC dispersions produced by continuous milling showed evidence of substantial inorganic contamination in the TEM images. Others have also reported a high degree of contamination, for example when rice straw pulp was processed in a similar manner 19.

As a second stage, and in an effort to reduce both the amount of energy consumption as well as inorganic contamination, batch milling was employed, and the resulting NFC specimens were compared with the NFCs obtained from homogenization and ultrasonication to give a global comparison of the three mechanical defibrillation approaches. The milling conditions; milling time, dispersion concentration, and milling speed, and also different delignifications; alkali and organosolve, were systematically varied to identify an optimal process window, where NFCs with a high aspect ratio are produced (see methods in Chapter 3, Section 3.2.9). From the TEM images shown in Figure 6.8, the NFCs obtained from milling of 0.5 % (w/v) concentration of different pretreated spinifex fibres (alkali and organosolve) for 20 minutes at 1000 rpm, and Figure 6.9, the NFC produced by milling of different concentrations of alkali pretreated fibre dispersion for 20 minutes at 1000 rpm, are not clear enough to measure the diameter of NFCs due to the entanglement of fibrils and also the existence of substantial inorganic contamination.

111

As was the case with high pressure homogenization, the alkali treated spinifex fibres showed superior defibrillation after milling in comparison with the organosolve treated fibres, so all further milling experiments were done on alkali pretreated fibres. However post-milling TEM characterization to determine the effect of different milling conditions on the morphology of NFC was found to be difficult for these particular samples due to the higher degree of NFCs agglomeration and the impurities present. According to Balaz et al 20, the milling time is the most important parameter in the high energy milling process, and as such has the most influence on the morphology of micronized samples. Figure 6.10 shows that increasing the milling time results in a higher amount of contamination from the vessel and also probably from the milling beads in the NFC dispersion. However, using optimized milling speed and milling time may effectively reduce the contamination.

112

In order to determine the type and degree of these contaminations in the dispersion of spinifex samples, ICPOES analysis was performed on milled NFC. ICPOES results in Table 6.3 show that spinifex NFC milled for 20 minutes at 1000 rpm, contains a significant amount of inorganic ions such as Ca, Al, K, Mg, Na, S, Si, Cu and also a small amount of Ba, Cr, Mn, P, Sr, and Zn .This contamination may be attributed to the highly-abrasive nature of cellulose in the high energy mill, combined with the strong interfacial adhesion of hydroxyl groups on the surface of NFC with these mineral components. It has been reported that treating cellulose fibre with mild hydrochloric acid solution removes the inorganic components from the surface of the fibres so a mild acid treatment of bleached fibres was applied in order to partial exchange of metal ions with H+ 21 (see the

113

procedure of acid treatment in Chapter 3, Section 3.2.11), then ICPOES analysis was performed for the HCl treated bleached fibre and HCl treated milled NFC. Treating spinifex bleached fibre with mild HCl solution before milling also showed the same inorganic ions in ICPOES analysis but in lower contents. Presumably these contaminations, as in the case with spinifex resin 22, due to uptake from the soil through the roots as nutrients and micronutrients during growth. Again milling the acid treated spinifex fibres at the identical conditions showed an increase in the inorganic ion content in ICPOES analysis which confirms that the contaminations also come from the milling pot and vessels or even trace contaminants leaching into the water via the several sealing systems operating on the mill. In the next step, in order to understand the effect of milling speed on the morphology of NFC, a 1 % (w/v) dispersion of HCl treated spinifex bleached fibre milled for 20 minutes at a speed of 1000 and 3000 rpm, respectively. TEM images in Figure 6.11 show that milling fibres at a higher speed resulted in a higher milling intensity, more breakage of fibres, and consequently a shorter average NFC length. Milling at 3000 rpm produced short and straight cellulose nanocrystals having an average diameter of 8 ± 2 nm and an average length of around 341 ± 100 nm, while the nanofibrils obtained by milling at 1000 rpm had an average diameter of around 8.7 ± 4.8 nm and a length of few microns (details of the employed measurement method are provided in Chapter 3, Section 3.3.6). It also can be observed that with reducing the amount of contamination by mild acid treatment, it is easier to determine the effect of milling speed on morphology of NFCs based on the dimension of nanofibrils. Detailed analysis about the effect of HCl treatment on the properties of pretreated fibre will explain in Session 6.2.6.

Figure 6.11. TEM image of 1 % (w/v) spinifex pulp dispersion obtained after milling at; a) 1000 rpm for 20 minutes, b) 3000 rpm for 20 minutes (the scale bar in 200 nm)

114

Table 6.3. ICPOES analysis of spinifex fibre and NFC before and after acid treatment

Sample treatment Inorganic components (mg/kg)

Al Ba Ca Cr Cu Fe K Mg Mn Na P S Si Sr Bleaching  HCl 213 0 43 12 4 111 85 47 8 24 1 152 9734 16 treatment Bleaching  993 6 5945 7 270 528 2533 1999 111 10963 2 2196 2294 103 Milling BleachingHCl 716 23 2752 78 14 636 77 263 12 61 1199 355 21811 119 treatmentMilling

115

6.2.1.4 Comparison between the effects of the application of different mechanical defibrillation methods on the morphology of spinifex NFC

Figure 6.12 compares the transmission electron microscopy images of NFCs produced by homogenizer, ultrasonication, and high energy milling and the histograms corresponding to 250 measurements of NFCs diameter based on the method provided in Chapter 3, Section 3.3.6. The geometrical average diameter of bleached fibres after alkali delignification was around 11.45 ± 4.31 μm and a length ranging from several microns up to almost 7 mm. Generally speaking, the NFCs obtained via the three different mechanical treatments (at the most optimal conditions) all displayed filament-like nanofibrils with average diameters of below 10 nm (the diameter of their bundles is below 40 nm) and a length of several microns. Homogenization of a 0.3 % (w/v) pulp dispersion at 1500 bar and only for one pass through the homogenizer, exhibited NFCs with a diameter in the range of 1.5 - 7 nm and an average of 3.2 ± 0.75 nm. The NFCs obtained from ultrasonication of alkali delignified and bleached fibre dispersion for just 5 minutes at 30 % amplitude had a diameter in the range of 1.7 - 6.5 nm and an average of 3.62 ± 0.9 nm, with a length of several microns. It is important to mention that whilst nanofibres with a very similar high aspect ratio prepared from the other sources of cellulose have been reported, they required TEMPO pretreated fibre and significantly longer ultrasonication times 23-25. NFC and its bundles (which also can be called MFC) produced by milling of 1 % (w/v) pulp dispersion for 20 minutes at 1000 rpm, showed an average diameter in the range of 2.2 - 33 nm and an average around 8.7 ± 4.82 nm. After a relatively short period of milling when the fibre size diminished, the adhesion and interactions between nanofibrils is enhanced as a consequence of ‘van der waals’ and ‘hydrogen’ bonding. The presence of very thin nanofibrils along with relatively larger fibrils greatly promotes the aggregation which causes a broader range of nanofibrils and their bundles sizes 20. Among these three methods, high pressure homogenisation was found to be the most efficient and effective in producing high quality nanofibrils from spinifex grass. The lowest applied pressure required in order to produce nanofibrils with high aspect ratio without any clogging issue was 350 bar. To our best knowledge, this is superior in comparison with all published results in terms of the number of passes (and overall applied energy) via homogenization after any cellulose chemical pretreatments (carboxymethylation, TEMPO oxidation and polyelectrolyte swelling) 26-28 and enzymatic hydrolysis in combination in mechanical refining for obtaining nanofibrils from wood pulp 29, 30. Isolation nanofibrils from non-wood sources has also been reported, but these examples

116

all still required either an acid pretreatment 2, 29, 31-36 or a substantially higher number of passes through the homogenizer 37-40.

Homogenized NFC 120

100

Number of measurements of Number 20

0 1-2 nm 2-3 nm 3-4 nm 4-5 nm 5-6 nm Nanofibril diameter (nm)

Ultrasonicated NFC 100

Number of measurement of Number 20

0 2-3 nm 3-4 nm 4-5 nm 5-7 nm Nanofibril diameter(nm)

80 Milled NFC 70

Number of measurments of Number 10

0 2-5nm 5-7 nm 7-10 nm 10-15 nm 15-33 nm Nanofibril diameter (nm)

Figure 6.12. TEM images and dispersity of diameter of NFCs obtained by; a) homogenization of 0.3 % (w/v) pulp dispersion at 1500 bar only for one pass, b) ultrasonication of 1 % (w/v) pulp dispersion for 5 minutes at 30 % amplitude, and (c) milling of 1 % (w/v) pulp dispersion for 20 minutes at 1000 rpm (the scale bar is 500 nm)

117

The results from the defibrillation via different mechanical treatments suggest that unusual high aspect ratio nanofibrillated cellulose with a high yield can be produced from Triodia pungens using a very mild chemical pulping followed by low mechanical energy processing. This has apparent beneficial implications with respect to the consumption of chemicals and energy in the manufacture of high quality nanofibrillated cellulose. The peculiar behavior of spinifex appears to be related to structural morphology, composition and leaf anatomy of the grass which has evolved through adaptation to the hot and dry environments. In general plants form two types of cell wall which are different in function and composition. Primary cell wall is generally a thin, flexible and extensible layer formed while the cell is growing. The cell starts to produce the secondary cell wall when the primary cell wall is complete and the cell has stopped expanding. The much thicker and stronger secondary wall provides additional protection to cells and rigidity and strength to the larger plant. Both primary and secondary walls contain cellulose, hemicellulose and pectin in different proportions. The cellulose fibrils are embedded in a network contains hemicellulose and lignin, whereby the amount of hemicellulose in grasses is higher than the other sources 41. Similar to other grasses, spinifex grass also has these two walls and several cell types in the leaf epidermis including epidermal cells and stomata, resin producing cells, fibre, mesophyl, vascular tissue and multi-cellular hairs and unicellular papillae. 42, 43 Spinifex grasses exhibit a C4 leaf anatomy which possess two type of cells ; outer mesophyl cells and inner spongy bundle sheath cells (another type of mesophyl) which are arranged in a circular manner like a necklace. According to the light microscopy images of the cross-section of spinifex fibre 44, in Triodia, the bundle of sheath cells seems to extend beyond the vascular bundle and is surrounded by mesophyl tissue (photosynthetic parenchyma cells that lie between the upper and lower epidermis layers of a leaf). Triodia pungens possesses a higher percentage of mesophyl tissues that are predominantly found in the primary cell wall 39, 40. In Figure 6.13, it can be observed that the bundles of fibrils are interconnected by the soft spongy-like mesophyl tissues. It should be pointed out that there are also some holes with an average size of 1.65 ± 0.85 μm on the surface of flaky mesophyl which have a ventilation and metabolis function 45 (Figure 6.14 (e, f, g)). So the peculiar “deconstruction” behaviour of spinifex biomass can be traced back, in part, to this particular morphology of spinifex, which mainly consists of parenchyma tissue. This tissue is slightly fragile and the cellulose microﬁbrils are organized in a looser network embedded in an abundant matrix consisting of hemicelluloses rather than a strong and tight lignin network 46, 47 (also of importance, the chemical analysis of grass confirms the higher amount of hemicellulose than lignin (Chapter 5, Section 5.2.1)), which is the main key contributing factor in this easier deconstruction behaviour. 118

Figure 6.13. Parenchyma tissue of Triodia pungens. The fibril bundles are surrounded by mesophyll

Another reason which facilitates the efficient defibrillation of spinifex fibre is shown in the SEM micrographs of cross-sections of T. pungens (shown in Figures 6.14 (b, c)). The bundles of cellulose fibrils are separated by a nodular structure on the surface, indicating the slack assembly of fibres, which is generally observed in C4-leaftype grasses. These hollow tube-like channels are stroma lamellae and are responsible for photosynthesis during daytime and the storage of biosynthesized carbohydrates. Because of the slack microﬁbrillar interaction with the matrix in the parenchyma tissue, micro and nanoﬁbrils can easily be separated from each other after a mild chemical treatment, by a significantly low energy mechanical treatment with respect to other reports in this technology space. In fact, applying excessive or intense chemical and/or mechanical treatments appears to cause damage to the fibrils and results in nanofibres with a shorter length, and consequently a lower aspect ratio.

119

Figure 6.14 . SEM micrographs of cross-sections of the raw Triodia pungens; a) global view, b) the bundles of fibrils which show a nodular structure of stroma lamellae covering the bundles, c) stroma lamellae on the surface of microfibrils bundles, d) spongy mesophyll network on the external layer of fibre, e, f, g) flaky mesophyll with the small holes on the surface (the hole size is 1.65± 0.85 μm)

6.2.2 Crystallinity of spinifex NFC

Cellulose fibres are comprised of crystalline and amorphous regions. The crystallinity of cellulose can be affected dramatically by different calculation methods because of the overlapping and broad diffraction peaks of cellulose 48, so the most common and accepted method for measuring the crystallinity of plant fibre was applied for all NFC samples. The XRD diffraction spectra of the NFC samples produced by homogenization, ultrasonication and milling in Figure 6.15 show that spinifex-based NFC is crystalline in nature. The observed XRD peaks at around 2θ =15.6 and 22.6 for freeze dried NFC samples obtained from milling (milling of 1% (w/v) pulp dispersion for 20 minutes at 1000 rpm) and homogenization (homogenization of 0.3 % (w/v) pulp dispersion at 1500 bar for one pass) are representative of a typical structure of cellulose I, while in contrast, treating 1% (w/v) pulp dispersion with ultrasonication for 5 minutes at a 30 % amplitude resulted in peaks transformation to 2θ =12.56, 15.23, and 22.15 due to a different molecular rearrangement 18, 49. Ago et al 50 , suggested that this transformation could be as a result of high molecular motion of the cellulose chains during the application of intense vibrational energy. The degree of crystallinity and 120

crystalline domain size for NFCs produced by different mechanical treatments were calculated and are listed in Table 6.4 (details of this calculation are provided in Chapter 3, Section 3.3.8). The degree of crystallinity of NFC samples showed that after all mechanical treatments, the crystallinity of NFCs slightly decreased. This may be due to the damage of the nanofibril cell walls upon the application of mechanical energy (as mentioned in Chapter 5 Section 5.2.3, the degree of crystallinity of bleached fibre before mechanical treatment, is 74 %). Another parameter which evaluates the effect of mechanical treatments on cellulose structure is the mean size of crystalline domains. Interestingly, after mechanical treatments, the thickness of crystalline domains increased due to intermolecular rearrangement during these mechanical treatments (the crystalline domain size of bleached fibre is 4 nm- Chapter 5, Section 5.2.3).

Table 6. 4. Degree of crystallinity and size of crystalline domains of freeze dried spinifex NFC produced by homogenization, ultrasonication, and milling

Sample Degree of crystallinity% Size of crystalline domains (nm) Homogenized NFC 69 4.32 Ultrasound NFC 63 4.1 Milled NFC 69 4.05

500 Milled NFC 450 Homogenized NFC 400 Ultrasonicated NFC 350 300 250 200 150

Intensity(a.u) 100 50 0 10 20 30 40 50 2Theta(degree)

Figure 6.15. X-Ray diffraction spectra of freeze dried spinifex NFC produced by homogenization, ultrasonication, and high energy milling

121

Table 6.5 compares the crystallinity and crystalline domain size of spinifex homogenized NFC with the NFC and MFC obtained from the other sources of cellulose. Applying intense mechanical treatment on cellulose fibre for producing NFC resulted in a significant decrease in crystallinity and size of crystalline domain. For the samples which preserved the crystalline size and showed improvement in crystallinity, a relatively mild treatment was performed which resulted in the production of MFC instead of NFC 16, 38, 51. The degree of crystallinity of spinifex NFC did not change significantly after homogenization and the size of crystalline domains increased (from 4 to 4.32 nm) due to intermolecular rearrangement during treatments.

122

Table 6.5. Comparison crystallinity and crystalline domain size of NFC obtained from different sources and treatments of cellulose with spinifex NFC

Source of cellulose Mechanical treatment NFC/MFC Crystallinity Crystallinity Crystallinity Crystalline Crystalline Ref Diameter of raw fibre of pretreated of NFC/MFC domain size domain size (%) fibre (%) (%) of pretreated of NFC (nm) fibre (nm) /MFC(nm) Spinifex Homogenization 1.7 - 6.5 52 74 69 4 4.32 Bleached eucalyptus pulp Microfluidization 4.7 - 31 - 55 44 - - 30 Bamboo fibre Ultrasonication 30 - 80 53.39 66.91 61.25 - - 16 Bleached wood pulp Homogenization 21 - 70.5 57.6 - - 52 Sugar-beet pulp Ultrasonication and 2 -15 - 13 11 - - 53 homogenization Sugar beet pulp Homogenization 20 - 70 39.96 69.62 77.89 - - 38 Abaca Homogenization 20 - 75 60 - - 28 Hemp Homogenization 30 - 50 - 86 78 - - 28 Bleached eucalyptus fibre Ultrasonication and 1000 - - 16.22 15.50 - - 51 homogenization 5000 TEMPO oxidized wood Ultrasonication 1 73.5 72 52 4.25 2.79 54 pulp Arbocel BWW40 Milling 3.4 - 4 - 53 15 3.97 - 55 Lemon peel Homogenization 3 -10 - 51 55 3.5 3.5 56

123

6.2.3 Mechanical properties of spinifex NFC

Figure 6.16 presents the typical tensile stress-strain curve for spinifex NFC “nanopaper” produced by vacuum filtration of homogenized NFC (homogenization of 0.3 % (w/v) pretreated fibre dispersion for one pass through the homogenizer at a pressure of 1500 bar) and milled NFC (milling of 0.5 % (w/v) fibre dispersion for 20 minutes at a milling speed of 1000 rpm), dried in a vacuum oven at 50 °C for 48 hours (see details of drying procedures in Chapter 3, Section 3.2.10). Table 6.6 summarizes the corresponding average values for basic tensile mechanical properties of these nanopaper samples (it needs to be mentioned that after ultrasonication, due to the existence of untreated fibre, the obtained film was not strong enough for mechanical testing, so only the mechanical properties of homogenized and milled samples were investigated). The mechanical properties of NFC nanopaper not only depend on the fibrils’ modulus, and tensile strength, but also on the orientation and degree of interaction between nanoﬁbrils within the paper, which are in-turn affected by the preparation method (vacuum filtration and drying). Nanopaper produced from homogenized NFC is stronger than nanopaper produced by milled NFC, and showed a tensile stress of approximately 48 ± 2.1 MPa in comparison with the milled NFC nanopaper, which registered a tensile stress of 39 ± 2.4 MPa. The milled spinifex NFC became contaminated with significant amounts of inorganic particles from the milling media and chamber. These particles could reduce the hydrogen bonding between the nanofibrils and act as stress raisors, and can thus adversely affect the strength of the nanopaper. Hassan et al 19 also reported the existence of silica contaminants in the dispersion of rice straw fibrils, which adversely effected the mechanical properties of the film produced from rice straw fibrils.

124

10 Tensile stress (MPa) stress Tensile Homogenized NFC Milled NFC 0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 Tensile strain (%)

Figure 6.16. Comparing tensile curves for spinifex nanopaper produced from homogenized and milled NFC dispersions and prepared by vacuum filtration and drying in a vacuum oven at 50 °C for 48 hours

Table 6.6. Mechanical properties of spinifex nanopapers produced from homogenized and milled NFC dispersions and prepared by vacuum filtration and drying in a vacuum oven at 50 °C for 48 hours Porosity Thickness Young's Tensile Tensile Work at (% ) (mm) Modulus strain stress fracture (GPa) (%) (MPa) (MJ m-3) Homogenized 76 0.055 4.7 ± 0.4 3.2 ± 0.5 48 ± 2.3 1.05 ± 0.6 NFC Milled NFC 40 0.059 2.5 ± 0.5 3.3 ± 0.2 39 ± 2.5 0.88 ± 0.07

In order to understand the effect of drying methods and conditions on the physical and mechanical properties of nanopaper samples, NFC nanopaper formed from vacuum filtration of a homogenized NFC dispersion was dispatched from filter paper and placed on a pair of Teflon sheets and compressed between a pair of metal platens in a hydraulic hot press instrument with no significant force at 103 °C for 2 hours (details of drying procedures are in Chapter 3, Section 3.2.10). The effect of preparation methods on the properties of resulting nanopaper samples was compared by Sehaqui et al 57. They suggested that the mechanical properties of cellulose nanopaper are very sensitive to orientation distribution of the nanofibers. The high modulus and ultimate strength will be obtained when the applied drying procedure leads to good in-plane orientation of

125

the nanofibers, and possibly also less porosity. Figure 6.17 and Table 6.7 compare the stress-strain curves and property values for NFC nanopaper samples obtained with different porosity and thickness values. A comparison between different drying methods indicates that the nanopaper samples which were dried with the hot press under low-force compression showed less porosity and better mechanical properties than vacuum dried samples due to the higher density and corresponding number of interactions between the nanofibrils. It seems that drying rate and compression may also be critical for the degree of bonding between the nanofibrils and consequently for the mechanical properties of nanopaper samples. The tensile stress of the nanopaper mainly depends on the degree of bonding between the fibrils. An increased drying temperature under compression resulted in nanopapers with the less porosity and better mechanical properties. The overall toughness of spinifex derived NFC (area under the tensile curve) is impressive and higher than that from the other sources (Table 6.8)58-62. Clearly the mechanisms at play are very complex, however we suggest that this could be associated with interfibril debonding, bending and plasticity of nanofibrils, slippage of high aspect ratio and well-entangled nanofibrils, and ultimately, high tensile fracture of nanofibrils enabling quite a high plastic deformation before breakage 13. Despite a relatively high porosity between 20 and 30 %, the nanopaper samples obtained from spinifex NFC strained to a larger value in comparison with the other sources of cellulose (Table 6.8) 27, 61, 63. This could be due to the smaller and more homogeneously distributed voids, reasonably strong interfibril adhesion, and the strength of individual nanofibrils 13. One very important differentiating factor for spinifex NFC is the unusually-high hemicellulose content (as presented in Chapter 5, Section 5.2.1) and the flexibility and toughness that this imparts to nanofibrils through strong but “forgiving” and labile interfiber bonding, which reportedly is beneficial for imparting toughness and flexibility to NFC nanopaper 64 65.

126

100

40 P=22 %, t=0.089 mm 20 P=32 %, t=0.063 mm

Trensile stress (MPa) stress Trensile P=35 %, t=0.113 mm P=30 %, t=0.124 mm 0 0 5 10 15 Tensile strain (%)

Figure 6.17. Tensile curves of spinifex nanopaper samples with different porosity (P) and thickness (t), produced from homogenized NFC and dried with a hot press at 103 °C for 2 hours

Table 6.7. Mechanical properties of spinifex nanopaper samples with different porosity and thickness, produced from homogenized NFC and dried with hot press at 103 °C for 2 hours

Porosity Thickness Young's Tensile strain Tensile stress Work at (% ) (mm) Modulus (%) (MPa) fracture (GPa) (MJ m-3) 22 0.089 3.2 ± 0.2 18 ±0.2 84 ± 5 12.3 ± 2 30 0.124 3.6 ± 0.14 7 ± 0. 3 86 ± 2.3 4 ± 0.7 32 0.062 3 ± 0.7 12 ± 0.3 67 ± 0.13 6 ± 0.2 35 0.113 3.1 ± 0.54 9 ± 0.9 63 ± 3.7 4 ± 1.2

127

Table 6.8. Comparison the mechanical properties of nanopaper obtained from NFC prepared from the other sources of cellulose with spinifex NFC

Source of cellulose Mechanical treatment Young's Modulus Tensile strain Tensile stress Work at fracture Ref (GPa) (%) (MPa) (MJ m-3) Spinifex Homogenization 3.2 ± 0.2 18 ± 0.2 84 ± 5 12.3 ± 2 Sodium form washed wood pulp Homogenization 11.2 7.2 230 10.55 27 TEMPO mediated wood pulp Homogenization 1.4 16.6 83 7.8 61 Bleached soft wood Homogenization 6.67 ± 0.6 Tensile index(NM/g) 5.67 ± 2.1 66 105.3 ± 19 Bleached hard wood Homogenization 6.3 ± 0.6 Tensile index(NM/g) 5.8 ± 3.2 66 91.7 ± 25 Soft wood pulp with the DP=1100 Microﬂuidizer 14.7 6.9 205 9.8 67 Beech-wood pulp Homogenization 7.5 5 152 - 68 Bleached soft wood Homogenization 2.3 11 80 - 3 Bleached hard wood Homogenization 1 2 20 - 3

128

In order to demonstrate the toughness with high strain and relatively low modulus, the flexibility and durability of nanopaper were further tested by repeated bending (180° folding) as shown in Figure 6.18. It can be observed that similar to aerogels 69 and hydrogel 61, spinifex nanopaper has retained in its original structure without any visible fracture after repeated the folding-unfolding for 12 cycles (Figure 6.19 (b)).

The optical transparency of spinifex NFC nanopaper is shown in Figure 6.19. Despite a porosity of 30 % the paper is still translucent, indicating that the nanopaper structure has a low extent of nanofibril aggregation 70 and also a relatively small pore size.

129

6.2.4 Elastic modulus of single spinifex nanofibrils

In order to gain a better understanding about the mechanical properties of spinifex NFC, especially if rigid nanocomposite applications are considered, the transverse elastic modulus of individual nanofibrils were measured using AM-FM (Amplitude Modulation - Frequency Modulation) a relatively new method performed using AFM. In all reported methods, the force- displacement curves were acquired in AFM and then fitted by several different models to estimate the transverse elastic modulus. In this study the AM-FM method was applied to measure the transverse elastic modulus of spinifex nanofibrils directly from the AFM image. AM-FM viscoelastic mapping combines the features and benefits of normal tapping mode with quantitative, high sensitivity frequency modulation mode, as well as fast scanning. A non-invasive, high quality image was obtained in normal tapping mode when the AFM cantilever applied a small load at the surface of the NFC sample. The frequency produced by the cantilever was adjusted to keep the phase at 90 degrees, on resonance. This resonant frequency is a sensitive measure of the tip-sample interaction. The stiffer the tip-sample interaction resulted the higher the frequency shifts of the second mode. This frequency shift then was converted into a quantitative modulus measurement through the Hertz-Sneddon mechanical model 71. AFM image and modulus spectra in Figure 6.20 show that the elastic modulus of spinifex NFC with an average diameter of 3.5 ± 0.8 nm is in the range of 19 - 24 GPa. This elastic modulus range is a little lower than most reported values for CNC (refer to values), which is consistent with our findings that the spinifex NFCs have a relatively high amorphous hemicellulose content (transverse elastic modules about 18 - 50 GPa for wood-derived CNC 72, 24.8 and 17.7 GPa for wood and cotton CNCs respectively 73, and 2 - 25 GPa for tunicate CNC 74, 75), giving them more toughness and flexibility, rather than stiffness, as expected for CNCs. This elastic modulus range is still excellent, and indicates the existence of strong inter and intramolecular hydrogen bonding, and also can be explained by high crystallinity and large crystalline domain values for our spinifex NFC, despite the unusually high hemicellulose content.

130

2.0

GPa 1.5 35

1.0 µm 20

15 0.5 10

5 0.0 0.0 0.5 1.0 1.5 2.0 µm

23 Transverse (GPa) elastic modulus Transverse 0 25 50 75 100 125 150 μm

Figure 6.20. Transverse elastic modulus of spinifex nanofibril measured by AM-FM

6.2.5 Thermal properties of spinifex NFC

Thermo gravimetric analysis (TGA) was performed in order to evaluate the amount of absorbed moisture in freeze dried spinifex NFC samples, as well as the relative thermal stability of the NFC samples as a function of mechanical treatment (details of analysis are in Chapter 3, Section 3.3.2). Figure 6.21 compares the thermal degradation process of spinifex NFC produced by homogenization, ultrasonication, and milling and Table 6.9 summarizes the degradation temperatures of the various NFC samples. The weight loss of NFC samples in the range of 30 - 150 °C corresponds to the evaporation of absorbed water. All spinifex NFC samples showed less than 7 % weight loss due to loss of moisture. At a higher temperature range (above 150 °C), the 131

degradation behaviours of NFC obtained through the ultrasonication and homogenization were almost identical, while milled NFC sample started to degrade earlier due to existence of inorganic contaminations, which perhaps prohibit some degree of hydrogen bonding between the nanofibrils, but which may also catalyze thermal degradation. Degradation of NFC samples also showed two separate processes. The earlier degradation occurred around 270 °C and is due to the low decomposition temperature of hemicellulose which remained on the surface of nanofibrils, and preceded the second stage which was associated with a maximum decomposition process at around 320 °C attributed to the pyrolysis of cellulose. The residual solid mass at 500 °C for the homogenized and ultrasonicated NFCs was about 26 wt % (same as the residual mass for bleached fibre at this temperature. See the TGA spectra in Chapter 5, Section 5.2.4) while the residual mass for the NFC produced by milling was increased to 40 wt%. As mentioned before in Chapter 5, this could be due to the existence of inorganic components taken up by the roots of grass as nutrients and micronutrients in the soil and also for the milled NFC attributed to the contamination of milling vessel and beads 50 (see the ICPOES analysis results in Section 6.2.1.3).

100

99 100 98 97

96 90 95

80 93

92 40 60 80 100 120 140 70

Mass loss ( %) ( loss Mass Ultrasonicated NFC 30 Homogenized NFC 20 Milled NFC

100 200 300 400 500 Temperature (°C)

Figure 6.21. TGA curves for spinifex NFC produced by homogenization, ultrasonication, and milling. The inset shows lower temperature curves in more detail

132

Table 6.9. Thermal degradation temperature of spinifex NFC produced by homogenization, ultrasonication, and milling Sample First decomposition Second decomposition temperature (°C) temperature (°C) Homogenized NFC 272 328 Ultrasonicated NFC 275 321 Milled NFC 272 324

6.2.6 Effect of mild HCl treatment on properties of spinifex pretreated fibre

To investigate the effect of mild hydrochloric acid treatment on fibres structure, comparison between the ATR FTIR spectra of bleached spinifex fibre and HCl treated bleached fibre in Figure 6.22 confirms that both samples showed the same functional groups. The XRD spectra in Figure 6.23 also indicate the same crystalline structure for bleached and acid treated fibres. Optical microscopy images in Figure 6. 24 show that the morphology of fibres slightly changed after acid treatment and also thermal property of acid treated fibre showed improvement in TGA spectra in Figure 6.25. Slight decrease in the fibres diameter and improvement of thermal property might be due to removing the inorganic components which were prohibited from hydrogen bonding between the cellulose nanofibrils and also partly removing the hemicellulose with the lower degradation temperature 76. Table 6.10 summarizes the average diameter of fibres, crystallinity and degradation temperature of spinifex bleached and HCl treated fibres.

133

0.3 Bleached fibre HCl treated fibre

0.2

0.1

Absorbance

0.0 4000 3500 3000 2500 2000 1500 1000 Wave number (cm-1)

Figure 6.22. ATR FTIR spectra of bleached spinifex fibres and HCl treated fibres

700 Bleached fibre 600 HCl treated fibre

500

400

300

Intensity (a.u) Intensity 200

100

10 20 30 40 2 Theta(degree)

Figure 6.23. X-ray diffraction spectra of bleached and HCl treated spinifex fibres

134

Figure 6.24. Optical microscopy of; a) bleached fibre, b) HCl treated fibre (the scale bar is 100 μm)

Table 6.10. Comparison between properties of bleached fibre and HCl treated fibre

Bleached fibre HCl treated fibre Crystallinity (% ) 75 75 Average diameter of fibres (μm) 11 9 Degradation temperature (°C) 230 260

110 Bleached fibre 100 HCl treated fibre 90

Mass loss ( %) ( loss Mass 40

100 200 300 400 500 Temperature (°C)

Figure 6.25. TGA spectra, showing the effect of HCl treatment on thermal properties of spinifex bleached fibre

135

6.2.7 The production of NFC from another “hard” spices of Triodia for comparison

As mentioned before in Chapter 2, spinifex forms the dominant Australian vegetation over approximately 27 % of the continent. In this study our main focus was only on Triodia pungens, a “soft” species collected from North-west Queensland in a region with an average annual rainfall of 400 mm, while there are a further 68 spinifex species that we have not explored yet. So in order to understand which species are the most suitable, and enable larger, more targeted harvesting trials, I applied our established methods of producing NFC to another species, Triodia longiceps, a hard non-resinous spinifex. As a preliminary trial, pretreated Triodia longiceps (alkali delignified and bleached fibre, details of procedures are same as Triodia pungens and can be find in Chapter 3, Section 3.2.4) was homogenized at 1500 bar pressure for one pass in order to compare with Triodia pungens. Figure 6.27 show the TEM images of NFC obtained from Triodia longiceps, and the diameter of nanofibrils were measured based on the same methods described in Chapter 3, Section 3.3.6. It can be observed that the homogenization of Triodia longiceps also produced high aspect ratio NFC with an average diameter of 15.5 ± 4 nm and a length of several microns. The bundle of NFC also has an average diameter of 32.4 ± 8.9 nm. It appears that a higher number of passes is required to fully defibrillate Triodia longiceps and produce similar NFC as that obtained from Triodia pungens, but in only one pass through the homogenizer. More studies on chemical analysis of Triodia longiceps and optimization of homogenization parameters will be outlined in the recommended future work outline in Chapter 8.

Figure 6.26. TEM images of homogenized Triodia longiceps after one pass at 1500 bar (the scale bar is 2 μm (a) and 500 nm (b))

136

6.3 Summary

Different mechanical methods have been applied to prepare NFC from spinifex pulp which all of them clearly demonstrated unusually high aspect ratio nanofibrils due to high content of hemicellulose. Obtaining a homogeneous dispersion of nanofibres within a few number of passes through the homogenizer with 100 % yield or using high energy milling, or ultrasonication for short time and lower energy all indicated that long nanofibrils with very small diameter can be obtained by very mild chemical treatments and significant lower energy consumption compared to the other type of cellulose sources. Applying different pressure, different slurry concentration and also varying the number of passes through the homogenizer exhibited almost the same result on the nanofibrils diameter and length. In defibrillation process of spinifex grass, homogenisation of pretreated fibres produces very high aspect ratio NFC even at only one pass through the homogenizer without any clogging issues. In comparison with the other source of cellulose, it was easier to defibrillate spinifex fibre into nanoscale fibres at first pass. Ultrasonication of spinifex pretreated fibre even at low energy and short time produced nanofibre with the diameter of below 10 nm and several micron length. However it seems that this treatment can not be a scalable method to treat spinifex fibres uniformly with a high yield. As far as ideal shear force and scalability are considered, high pressure homogenisation still remained the best method for defibrillation of spinifex fibres. Extraction of cellulose nanofibrils from spinifex grass using a high energy milling also resulted in nanofibrils with very high aspect ratio while the crystallinity of obtained nanofibrils and structure of cellulose did not change significantly. High energy milling can provide good opportunity to scale-up the production of NFC if nanocellulose with high purity is not considered. The NFC obtained from spinifex has crystallinity about 69 % and thermal stability up to 320 °C. Based on the tensile stress-strain curves, the spinifex NFC nanopaper has interesting mechanical properties. At 22 % porosity; Young’s modulus, tensile strain, tensile stress, and toughness (work at fracture), are 3.2 GPa, 18 %, 84 MPa, and 12 MJ m-3 respectively. These favourable properties are due to the high aspect ratio nanofibrils orientation, network structure and high hemicellulose content. It seems that the chemical and morphological origins for this surprisingly low energy input requirement for isolating unusual high aspect ratio nanofibres from Triodia pungens is directly related to the plant’s unique morphology and evolution, which have been heavily influenced by an extreme arid climate 44.

137

Bibliography

1. Moon, R. J.; Martini, A.; Nairn, J.; Simonsen, J.; Youngblood, J. Chemical Society Reviews 2011, 40, (7), 3941-3994.

2. Lavoine, N.; Desloges, I.; Dufresne, A.; Bras, J. Carbohydrate Polymers 2012, 90, (2), 735- 764.

3. Stelte, W.; Sanadi, A. R. Industrial & Engineering Chemistry Research 2009, 48, (24), 11211-11219.

4. Amiralian, N.; Annamalai, P. K.; Martin, D. Provisional patent application, Nanocellulose. 22 November 2013.

5. Österberg, M.; Vartiainen, J.; Lucenius, J.; Hippi, U.; Seppälä, J.; Serimaa, R.; Laine, J. ACS Applied Materials & Interfaces 2013, 5, (11), 4640-4647.

6. Swerin. Nordic Pulp & Paper Research Journal 1990, 05, (04), 188-196.

7. Junka, K.; Filpponen, I.; Johansson, L.-S.; Kontturi, E.; Rojas, O. J.; Laine, J. Carbohydrate Polymers, (0).

8. Chinga-Carrasco, G.; Kuznetsova, N.; Garaeva, M.; Leirset, I.; Galiullina, G.; Kostochko, A.; Syverud, K. Journal of Nanoparticle Research 2012, 14, (12), 1-10.

9. Saito, T.; Nishiyama, Y.; Putaux, J.-L.; Vignon, M.; Isogai, A. Biomacromolecules 2006, 7, (6), 1687-1691.

10. Saito, T.; Kimura, S.; Nishiyama, Y.; Isogai, A. Biomacromolecules 2007, 8, (8), 2485- 2491.

11. Pääkkö, M.; Ankerfors, M.; Kosonen, H.; Nykänen, A.; Ahola, S.; Österberg, M.; Ruokolainen, J.; Laine, J.; Larsson, P. T.; Ikkala, O.; Lindström, T. Biomacromolecules 2007, 8, (6), 1934-1941.

12. Wagberg, L.; Decher, G.; Norgren, M.; Lindstrom, T.; Ankerfors, M.; Axnas, K. Langmuir 2008, 24, (3), 784-795.

138

13. Henriksson, M.; Berglund, L. A.; Isaksson, P.; Lindström, T.; Nishino, T. Biomacromolecules 2008, 9, (6), 1579-1585.

14. Dinand, E.; Chanzy, H.; Vignon, R. M. Food Hydrocolloids 1999, 13, (3), 275-283.

15. Brinchia, L.; Cotanaa, F.; Fortunatib, E.; Kenny, J. M. Carbohydrate Polymers 2013, 94, 154– 169

16. Chen, W.; Yu, H.; Liu, Y. Carbohydrate Polymers 2011, 86, (2), 453-461.

17. Capadona, J. R.; Shanmuganathan, K.; Trittschuh, S.; Seidel, S.; Rowan, S. J.; Weder, C. Biomacromolecules 2009, 10, (4), 712-716.

18. Wang, S.; Cheng, Q. Journal of Applied Polymer Science 2009, 113, (2), 1270-1275.

19. Hassan, M.; Mathew, A.; Hassan, E.; El-Wakil, N.; Oksman, K. Wood Sci Technol 2012, 46, (1-3), 193-205.

20. Balaz, P., Mechanochemistry in nanoscience and minerals engineering. 1.ed.08th ed.; Springer Berlin Heidelberg: New York, NY, 2008.

21. Zuluaga, R.; Putaux, J. L.; Cruz, J.; Vélez, J.; Mondragon, I.; Gañán, P. Carbohydrate Polymers 2009, 76, (1), 51-59.

22. Mondal, S.; Memmott, P.; Wallis, L.; Martin, D. Materials Chemistry and Physics 2012, 133, (2–3), 692-699.

23. Cheng, Q. Z.; Wang, S. Q.; Han, Q. Y. Journal of Applied Polymer Science 2010, 115, (5), 2756-2762.

24. Li, Q.; Renneckar, S. Cellulose 2009, 16, (6), 1025-1032.

25. Saito, T.; Kuramae, R.; Wohlert, J.; Berglund, L. A.; Isogai, A. Biomacromolecules 2012, 14, (1), 248-253.

26. Junka, K.; Filpponen, I.; Johansson, L.-S.; Kontturi, E.; Rojas, O. J.; Laine, J. Carbohydrate Polymers 2014, 100, (0), 107-115.

139

27. Osterberg, M.; Vartiainen, J.; Lucenius, J.; Hippi, U.; Seppala, J.; Serimaa, R.; Laine, J. ACS Applied Materials & Interfaces 2013, 5, (11), 4640-4647.

28. Alila, S.; Besbes, I.; Vilar, M. R.; Mutjé, P.; Boufi, S. Industrial Crops and Products 2013, 41, (0), 250-259.

29. Siqueira, G.; Tapin-Lingua, S.; Bras, J.; da Silva Perez, D.; Dufresne, A. Cellulose 2010, 17, (6), 1147-1158.

30. Qing, Y.; Sabo, R.; Zhu, J. Y.; Agarwal, U.; Cai, Z. Y.; Wu, Y. Q. Carbohydrate Polymers 2013, 97, (1), 226-234.

31. Cherian, B. M.; Leao, A. L.; de Souza, S. F.; Costa, L. M. M.; de Olyveira, G. M.; Kottaisamy, M.; Nagarajan, E. R.; Thomas, S. Carbohydrate Polymers 2011, 86, (4), 1790-1798.

32. Abraham, E.; Deepa, B.; Pothen, L. A.; Cintil, J.; Thomas, S.; John, M. J.; Anandjiwala, R.; Narine, S. S. Carbohydrate Polymers 2013, 92, (2), 1477-1483.

33. Sonia, A.; Priya Dasan, K. Carbohydrate Polymers 2013, 92, (1), 668-674.

34. Abraham, E.; Deepa, B.; Pothan, L. A.; Jacob, M.; Thomas, S.; Cvelbar, U.; Anandjiwala, R. Carbohydrate Polymers 2011, 86, (4), 1468-1475.

35. Deepa, B.; Abraham, E.; Cherian, B. M.; Bismarck, A.; Blaker, J. J.; Pothan, L. A.; Leao, A. L.; de Souza, S. F.; Kottaisamy, M. Bioresource Technology 2011, 102, (2), 1988-1997.

36. Cherian, B. M.; Leão, A. L.; de Souza, S. F.; Thomas, S.; Pothan, L. A.; Kottaisamy, M. Carbohydrate Polymers 2010, 81, (3), 720-725.

37. Ferrer, A.; Filpponen, I.; Rodriguez, A.; Laine, J.; Rojas, O. J. Bioresource Technology 2012, 125, 249-255.

38. Li, M.; Wang, L.-j.; Li, D.; Cheng, Y.-L.; Adhikari, B. Carbohydrate Polymers 2014, 102, (0), 136-143.

39. Dufresne, A.; Dupeyre, D.; Vignon, M. R. Journal of Applied Polymer Science 2000, 76, (14), 2080-2092.

40. Wang, B.; Sain, M. Composites Science and Technology 2007, 67, (11-12), 2521-2527.

140

41. Sun, R. C. Bioresources 2009, 4, (2), 452-455.

42. Gamage, H. K.; Mondal, S.; Wallis, L. A.; Memmott, P.; Martin, D.; Wright, B. R.; Schmidt, S. Australian Journal of Botany 2012, 60, (2), 114-127.

43. McWilliam, J. R.; Mison, K. Australian journal of plant physiology 1974, 1, (1), 5.

44. Watson, L.; Dallwitz, M. J., The grass genera of the world: descriptions, illustrations, identification, and information retrieval; including synonyms, morphology, anatomy, physiology, phytochemistry, cytology, classification, pathogens, world and local distribution, and references. In http://delta-intkey.com, 1992 onwards; Vol. Version: 18th December 2012.

45. Liu, R.; Yu, H.; Huang, Y. Cellulose 2005, 12, (1), 25-34.

46. Habibi, Y.; Mahrouz, M.; Vignon, M. R. Food chemistry 2009, 115, (2), 423-429.

47. Dinand, E.; Chanzy, H.; Vignon, M. R. Food Hydrocolloids 1999, 13, (3), 275-283.

48. Thygesen, A.; Oddershede, J.; Lilholt, H.; Thomsen, A. B.; Ståhl, K. Cellulose 2005, 12, (6), 563-576.

49. Sèbe, G.; Ham-Pichavant, F.; Ibarboure, E.; Koffi, A. L. C.; Tingaut, P. Biomacromolecules 2012, 13, (2), 570-578.

50. Ago, M.; Endo, T.; Hirotsu, T. Cellulose 2004, 11, (2), 163-167.

51. Urruzola, I.; Serrano, L.; Llano-Ponte, R.; Ángeles de Andrés, M.; Labidi, J. Chemical Engineering Journal 2013, 229, (0), 42-49.

52. Han, J.; Zhou, C.; Wu, Y.; Liu, F.; Wu, Q. Biomacromolecules 2013, 14, (5), 1529-1540.

53. Agoda-Tandjawa, G.; Durand, S.; Berot, S.; Blassel, C.; Gaillard, C.; Garnier, C.; Doublier, J. L. Carbohydrate Polymers 2010, 80, (3), 677-686.

54. Li, Q. Q.; Renneckar, S. Biomacromolecules 2011, 12, (3), 650-659.

55. Avolio, R.; Bonadies, I.; Capitani, D.; Errico, M. E.; Gentile, G.; Avella, M. Carbohydrate Polymers 2012, 87, (1), 265-273.

141

56. Rondeau-Mouro, C.; Bouchet, B.; Pontoire, B.; Robert, P.; Mazoyer, J.; Buléon, A. Carbohydrate Polymers 2003, 53, (3), 241-252.

57. Sehaqui, H.; Liu, A.; Zhou, Q.; Berglund, L. A. Biomacromolecules 2010, 11, (9), 2195- 2198.

58. Sehaqui, H.; Allais, M.; Zhou, Q.; Berglund, L. A. Composites Science and Technology 2011, 71, (3), 382-387.

59. Svagan, A. J.; Samir, M. A. S. A.; Berglund, L. A. Biomacromolecules 2007, 8, (8), 2556- 2563.

60. Ferrer, A.; Quintana, E.; Filpponen, I.; Solala, I.; Vidal, T.; Rodriguez, A.; Laine, J.; Rojas, O. J. Cellulose 2012, 19, (6), 2179-2193.

61. Sehaqui, H.; Zhou, Q.; Ikkala, O.; Berglund, L. A. Biomacromolecules 2011, 12, (10), 3638- 3644.

62. Henriksson, M.; Berglund, L. A. Journal of Applied Polymer Science 2007, 106, (4), 2817- 2824.

63. Jonoobi, M.; Mathew, A. P.; Oksman, K. Industrial Crops and Products 2012, 40, (0), 232- 238.

64. Li, L.; Lee, S.; Lee, H. L.; Youn, H. J. Bioresources 2011, 6, (1), 721-736.

65. Bourree, G.; Herring, W.; Jack, D., Cellulose pulp having increased hemicellulose content. In Google Patents: 2004.

66. Spence, K.; Venditti, R.; Rojas, O.; Habibi, Y.; Pawlak, J. Cellulose 2010, 17, (4), 835-848.

67. Henriksson, M.; Berglund, L. A.; Isaksson, P.; Lindstrom, T.; Nishino, T. Biomacromolecules 2008, 9, (6), 1579-1585.

68. Wolfgang, G.-A.; Stefan, V.; Michael, O.; Christian, T.; Jozef, K., High-Modulus Oriented Cellulose Nanopaper. In Functional Materials from Renewable Sources, American Chemical Society: 2012; Vol. 1107, pp 3-16.

142

69. Paakko, M.; Vapaavuori, J.; Silvennoinen, R.; Kosonen, H.; Ankerfors, M.; Lindstrom, T.; Berglund, L. A.; Ikkala, O. Soft Matter 2008, 4, (12), 2492-2499.

70. Chinga-Carrasco, G. Nanoscale Res Lett 2011, 6, (1), 1-7.

71. Bertolazzi, S.; Brivio, J.; Radenovic, A.; Kis, A.; Wilson, H.; Prisbrey, L.; Minot, E.; Tselev, A.; Philips, M.; Viani, M.; Walters, D.; Proksch, R. MicroscopyandAnalysis 2013, 27, (3), 21-24 (AM).

72. Lahiji, R. R.; Xu, X.; Reifenberger, R.; Raman, A.; Rudie, A.; Moon, R. J. Langmuir 2010, 26, (6), 4480-4488.

73. Pakzad, A.; Simonsen, J.; Heiden, P. A.; Yassar, R. S. Journal of Materials Research 2012, 27, (3), 528-536.

74. Wu, X. W.; Moon, R. J.; Martini, A. Cellulose 2013, 20, (1), 43-55.

75. Wagner, R.; Moon, R.; Pratt, J.; Shaw, G.; Raman, A. Nanotechnology 2011, 22, (45).

76. Zhuang, J. P.; Liu, Y.; Wu, Z.; Sun, Y.; Lin, L. Bioresources 2009, 4, (2), 674-686.

143

Chapter 7.

Polymer nanocomposites reinforced with high aspect ratio cellulose nanocrystals isolated from Triodia pungens

144

7.1 Introduction

One of the most industrialised applications of nanoparticles is to use them for reinforcing polymers in order to enhance properties and performance of the materials. The degree of reinforcement effect in polymer nanocomposites mainly depends on polymer-nanoparticle interfacial interactions, the nanoparticle specific surface area, aspect ratio and the mechanical properties of individual nanoparticles 1. Earlier studies have suggested that nanoparticles with a high aspect ratio give better reinforcement properties due to the formation of a percolating network of fillers at very low loading, which enhances stress transfer from the matrix to the fibres 2. In recent decades, many publications and patents on isolating cellulose nanocrystals from different sources like wood pulp, cotton and lignocellulosic fibers and developing their nanocomposites have appeared. Most of the methods have relied on acid hydrolysis to isolate rod-shaped crystals with a diameter in the range of 3 - 15 nm, and a length in the range of 50 - 500 nm to then go ahead and make nanocomposites 3, 4. A typical procedure of producing cellulose nanocrystals involves acid hydrolysis using corrosive acids, followed by centrifuging, dialysing, ultrasonication and drying (the flowchart of a typical process is shown in Figure 7.1). High aspect ratio cellulose nanocrystals (with an aspect ratio of 70 - 100) can be obtained from rare marine animals called tunicates (urochordates) 4. Due to their relatively rare abundance, their use is much restricted to academic research. Therefore, producing low cost nanocrystals from plant materials with a higher aspect ratio, or closer to that of CNCs derived from tunicates (t-CNCs) remains a high priority challenge. This chapter aims to outline the results of optimisation of the acid hydrolysis process for the isolation of high aspect ratio cellulose nanocrystals from T. pungens and the subsequent evaluation of their reinforcement potential in an industrially-relevant polymer matrix, i.e. thermoplastic polyurethane (TPU). It should be mentioned that the term ‘nanocrystal’ is used in this chapter based on the traditional CNC isolation method which uses acid hydrolysis. Commonly the nanocrystals obtained via acid hydrolysis have a lower aspect ratio and can also be called ‘nanowhiskers’, ‘whiskers’, or ‘nanocrystals’ 5, while the long spinifex nanofibres that I have produced in this project using acid hydrolysis could legitimately be called ‘nanofibrils’ or nanofibrillated cellulose’ based on the dimensions and aspect ratio that was serendipitously obtained here.

145

Figure 7.1. Typical procedure for the acid hydrolysis of spinifex grass (a detailed procedure can be found in Chapter 3, Section 3.2.12)

7.2 Results and discussion

Typical procedures employed for the production of cellulose nanocrystals consist of hydrolysing cellulose fibres with a strong acid under strictly controlled conditions of temperature, time, and agitation. So far, sulphuric acid has been extensively used as a hydrolysing agent; it reacts with hydroxyl groups at the surface of cellulose to give a negative sulphate charge that promotes the dispersion of CNC in the water (Figure 7.2). These hydrolysis conditions are known to affect the properties of the resulting cellulose nanocrystal and also the yield of CNC 6. Therefore the concentrations of sulphuric acid and hydrolysis temperature and time were taken into account in the process of acid treatment (all samples were ultrasonicated for 20 minutes at 25 % amplitude after dialysis).

146

Figure 7.2. Sulphate groups on the surface of cellulose after hydrolysis with sulphuric acid

7.2.1 Optimisation of isolation method

In a typical procedure of producing cellulose nanocrystals by acid hydrolysis, the acid concentration varies from 35 to 65 % and the temperature can range from room temperature up to 70 °C 7, 8. The corresponding hydrolysis time depends on the hydrolysis temperature that has been varied from 30 minutes to overnight 9. Spinifex bleached fibres were treated at 45 °C with four different sulphuric acid concentrations. Table 7.1 summarizes the yield and appearance of dried CNC obtained from acid hydrolysis, and Tables 7.2 and 7.3 summarize the effect of hydrolysis temperature for fibres treated with a 35 % (v/v) acid solution for 3 hours, and also the effect of hydrolysis time for the treatment with 35 and 45 % (v/v) acid at 45 °C, respectively. As the concentration of sulphuric acid increased, the colour of the CNC product changed from white to yellow, and significantly, using an acid concentration above 45 % (v/v) resulted in degradation of cellulose. The yield of CNC increased with increasing acid concentration up to 40 %, at which the highest yield was obtained. Increasing only 5 °C of hydrolysis temperature (from 45 °C to 50 °C) while at a 35 % (v/v) sulphuric acid concentration for 3 hours resulted in increased CNC yield, but also a change in the CNC colour to light yellow. The colour of CNC also changed with increasing the hydrolysis time, from white to light yellow when the fibre was treated with 35 % (v/v) acid at 45 °C for 3 hours and 6 hours, respectively. We assume that a slight change in the colour of obtained CNCs from white to yellow can be due to the degradation of hemicellulose covering the surface of pretreated fibres (as 147

mentioned before in Chapter 5, Section 5,2,1, chemical analysis showed that the bleached fibre contains about 37 % (w/w) hemicellulose). Hydrolysis at higher acid concentration (above 45 % (v/v)) for 3 hours results in burning of the cellulose fibres. Based on these results as well as early feedback from our collaborators at Purdue University, we surmised that spinifex has irregular crystalline regions that hydrolyse more readily, and which therefore require careful control of the hydrolysis time, particularly when higher acid concentration are used.

Table 7.1. Effect of acid concentration on the yield and appearance of CNC hydrolysed at 45 °C

Acid concentration and Yield (%) Appearance hydrolysis time 35 % (v/v), 3 hours 33 White 40 % (v/v), 3 hours 42 White 45 % (v/v), 1 hour 8 Deep yellow 50 % (v/v), 1 hour < 2 Brown

Table 7.2. Effect of hydrolysis temperature on the yield and appearance of CNC hydrolysed with 35 % (v/v) sulphuric acid for 3 hours

Hydrolysis temperature Yield (%) Appearance 45 ºC 33 White 50 ºC 40 Light yellow

Table 7.3. Effect of hydrolysis time on the yield and appearance of CNC hydrolysed with 35 % (v/v) and 45 % (v/v) acid at 45 °C

Hydrolysis time Yield (%) Appearance

35 % (v/v) acid 3 hours 33 White 6 hours 43 Light yellow

45 % (v/v) acid 1 hour 8 Deep yellow 3 hours - Acid burnt the cellulose

148

With spinifex grass, use of an acid concentration above 45 % (v/v) and a temperature above 50 C resulted in extreme and destructive hydrolysis, either through charring or by complete hydrolysis into low molecular weight sugars.

7.2.2 Morphological characterisation of cellulose nanocrystal

Figure 7.3 compares the TEM images of spinifex cellulose nanocrystals produced using different conditions. The nanocrystals obtained from spinifex grass have a relatively high length and small diameter. The average diameter of acid treated fibres (using a 40 % (v/v) acid concentration at 45 °C for 3 hours and ultrasonication at 25 % amplitude for 20 minutes after dialysis) is 3.45 ± 0.75 nm. A measurement of the length of short fibres after acid treatment (i.e. where it was possible to find the start and end points of CNCs in single TEM images) yielded measured dimensions of 497 ± 106 nm. This gives an aspect ratio of about 144. As it can be observed from the TEM images (Figure 7.3), the nanocrystals obtained from spinifex grass are longer and thinner than those reported for CNC derived from many other plant sources 2. The high aspect ratio of cellulose nanocrystals at low acid concentration (below 45 %) can be related to the high amount of hemicellulose. As far as we are aware, the production of such high aspect ratio CNC derived from plant sources was not possible prior to this project, and so this has presented us with a unique opportunity to evaluate these nanoparticles as “functional nanofillers” in polymer nanocomposites.

149

Figure 7.3. TEM images of the cellulose nanocrystals obtained via acid hydrolysis from T. pungens comparing the effect of acid hydrolysing conditions on morphology of nanocrystals; a) hydrolysis with 35 % (v/v) acid at 50 °C for 3 hours (the scale bar is 2 μm), b) hydrolysis with 35 % (v/v) acid at 45 °C for 6 hours (the scale bar is 2 μm), c) hydrolysis with 40 % (v/v) acid at 45 °C for 3 hours (the scale bar is 200 nm), d) hydrolysis with 50 % (v/v) acid at 45 °C for 3 hours (the scale bar is 500 nm)(all samples were ultrasonicated at 25 % amplitude for 20 minutes after dialysis)

It was observed that the optimised condition for isolating cellulose nanocrystals with high yield from spinifex grass relied on a combination of low temperature and low acid concentration. For example, 40 % (v/v) sulphuric acid at 45 °C, for 3 hours gave a high yield of 42 % CNC. It can be seen from the TEM images (Figure 7.3), that the application of more intense conditions, such as a sulphuric acid concentration above 45 % (v/v) (for comparison, the most widely-used acid concentration in the literature to produce CNC is 64 % 7, 8) and a temperature above 50 ºC, results in spinifex-derived plant material degradation into low molecular weight sugars.

150

To the best of my knowledge, the combination of mild acid concentration and low treatment temperature has not previously been reported as a suitable method to produce high aspect ratio cellulose nanocrystals from a plant resource. Even if the spinifex sample after acid hydrolysis (40 % (v/v) sulphuric acid at 45 °C for 3 hours) was exposed to the intense ultrasonic energy (at 70 % amplitude for 20 minutes), the very high aspect-ratio cellulose nanocrystal structure was maintained (Figure 7.4). Typically, with other nanocellulose production methods, intense chemical treatment conditions along with much more intense ultrasonication are required in order to sufficiently separate the cellulose fibres into fibres with nanometre scale widths. However, such harsh conditions also tend to cause breakage of the fibres and consequently result in a severe reduction of average CNC length. For instance, Annamalai et al 10 reported that the CNC obtained from acid hydrolysis of cotton filter paper using 64 % sulphuric acid at 50 °C for 3.5 hours and ultrasonication for 16 after dialysis, resulted in an average diameter and length of 34.5 ± 6.1 nm and 365 ± 80 nm, respectively (the approximate aspect ratio based of the average diameter and length is 10.58).

Figure 7.4. TEM image of cellulose nanocrystals obtained via 40 % (v/v) sulphuric acid treatment at 45 °C for 3 hours, then dialysis, followed by ultrasonication at 70 % amplitude for 20 minutes (the scale bar is 500 nm)

7.2.3 Effect of acid hydrolysis on the chemical structure and composition of spinifex fibres

Figure 7.5 compares the ATR FTIR spectra of CNC obtained by acid hydrolysis using different conditions with bleached spinifex fibre. The absorbance band in the range of 3000 and 3600 cm-1, reflects the strong hydrogen bonding of hydroxyl groups (O-H). The peak around 2908 -1 -1 cm is assigned to C-H vibration from -CH2 groups and the peak around 1430 cm confirms the 11 -1 -1 CH2 bending . The peaks at 1161 cm and 897 cm which are assigned to the β-glucosidic

151

linkage for cellulose I 12 can be observed in all samples except the sample hydrolysed using 50 % (v/v) sulphuric acid. No absorbance was observed at 1400 - 1600 cm-1 and around 1200 cm -1 due to sulphate groups for the acid treated fibre. This was a similar finding to previous reports by Hamad et al 13 and Yiying Yue et al 14 who believed that the degree of sulphonation in the CNC samples was too low (less than 7 sulphate groups per 100 anhydroglicose) to be detectable by ATR FTIR 13, 14.

0.7

0.6

0.5

0.4 50 % (v/v) acid, 1 hour, 45 °C 45 % (v/v) acid, 1 hour, 45 °C 0.3 35 % (v/v) acid, 3 hours, 50 °C 40 % (v/v) acid, 3 hours, 45 °C 0.2 35 % (v/v) acid, 6 hours, 45 °C 0.1 35 % (v/v) acid, 3 hours, 45 °C Bleached fibre 0.0 1000 2000 3000 4000 Wave number (cm-1)

Figure 7.5. ATR FTIR spectra of bleached spinifex grass and CNCs obtained under different conditions

7.2.4 Effect of acid hydrolysis on crystallinity of spinifex fibre

In order to understand the effect of different acid treatment conditions on the crystalline structure of spinifex fibre, the crystallinity of nanocrystals was measured by X-ray diffraction (XRD). Figure 7.6 compares the XRD diffraction patterns for freeze-dried CNC samples with alkali delignified and bleached fibres, and Table 7.4 summarizes the crystallinity values of CNC samples and the corresponding size of crystalline domains.

152

3000 o 2500 50 v/v% acid,1hour,45 C

2000 1500

1000

500

4000 0

3500 10 20 30 40

3000

2500

o 2000 45 % (v/v) acid, 1 hour, 45 C o 1500 40 % (v/v) acid, 3 hours, 45 C o Intensity 35 % (v/v) acid, 3 hours, 50 C 1000 35 % (v/v) acid, 6 hours, 45 oC 500 35 % (v/v) acid, 3 hours, 45 oC 0 Bleached

10 20 30 40 2 Theta (degree)

Figure 7.6. X-Ray diffraction spectra of spinifex fibre samples after alkali delignification, bleaching, and acid hydrolysis

Table 7. 4. Crystallinity and size of crystalline domain of spinifex fibre after alkali delignification, bleaching, and acid hydrolysis Sample Crystallinity (%) Crystalline domain size (nm) Alkali delignified fibre 71 4 Bleached fibre 72 4 Hydrolysed CNC (35 % (v/v) acid, 3 hours, 45 °C) 72 4.3 Hydrolysed CNC (35 % (v/v) acid, 6 hours, 45 °C) 71 4.2 Hydrolysed CNC (35 % (v/v) acid, 3 hours, 50 °C) 77 4.7 Hydrolysed CNC (40 % (v/v) acid, 3 hours, 45 °C) 73 4.7 Hydrolysed CNC (45 % (v/v) acid, 1 hour, 45 °C) 78 5.7

All X-ray diffraction spectra of acid hydrolysed fibre produced using a lower than 45 % (v/v) sulphuric acid concentration, showed characteristic peaks at around 2θ = 18.6° and 22.4° which are indicative of the typical structure of cellulose I. The crystallinity of CNC samples were measured by

153

determining the relative intensity of amorphous and crystalline peaks of each sample (details of measurement are in Chapter 3, Section 3.3.8). There are no major differences between the crystallinity of CNC samples treated at milder acid conditions with the alkali delignified and bleached fibres. Again it was observed that using a lower than 45 % (v/v) acid concentration at a temperature below 50 °C did not impart any major morphological changes. Although hydrolysing using higher acid concentration (45 % (v/v)) or higher temperature (50 °C) resulted in the deconstruction of more cellulose nanocrystals registering a higher crystallinity due to the removal of amorphous domains (specially hemicellulose), the over hydrolysing of smaller fibres caused them to burn and changed the CNC colour. Change in the size of crystalline domains also showed a similar tendency. The crystalline domain size for the nanofibres hydrolysed with 45 % (v/v) sulphuric acid was higher (about 5.4 nm), while the CNC samples produced under milder hydrolysis conditions showed almost the same crystalline domain sizes of about 4.3 - 4.7 nm, which were slightly greater than those of delignified and bleached fibres. The results indicate that the crystalline structure of native cellulose was not destroyed during the isolation process when mild acid hydrolysis conditions were applied. When spinifex pretreated fibres hydrolysed using higher acid concentration (45 % (v/v)) or higher temperature (50 °C), the crystallinity slightly increased (77 % for hydrolysed CNC using 35 % (v/v) acid for 3 hours at 50 °C, and 78 % for hydrolysed CNC using 45 % (v/v) acid for one hour at 45 ºC). This was attributed to the degradation of less stable amorphous regions and less oriented crystalline domains during these hydrolysis conditions. Using an acid concentration of higher than 45 % (v/v) might cause the degradation of cellulose, as indicated by the diffraction pattern for the hydrolysed sample with 50 % (v/v) acid (see inset in Figure 7.5) which did not show the characteristic diffraction spectra anymore.

7.2.5 Effect of acid hydrolysis on thermal properties of spinifex fibres

Thermogravimetric (TGA and DTA) analyses were used to evaluate the thermal stability of spinifex CNC as a function of hydrolysing time and acid concentration. As mentioned before in Chapter 5, the decomposition process of bleached fibre (original cellulose) was a one-step pyrolysis reaction with the maximum weight loss at about 320 °C (Figure 5.8) while degradation of acid hydrolysed cellulose was a multi-step pyrolysis process in which pyrolysis occurred in two temperature ranges of 100 - 150 °C and 150 - 400 °C. As found for the other sources of cellulose 15, 16, acid hydrolysis decreased the thermal stability of CNCs. The weight loss of acid treated samples below 150 °C was more than 10% which could be due to the degradation of more accessible and highly sulphated amorphous regions. P. Lu et al 17 suggested that this gradual mass loss of cellulose

154

nanocrystal samples could be a direct solid-to-gas phase transitions decomposition which are catalysed by surface sulphate groups. Furthermore, decomposition at lower temperatures might signify the faster heat transfer in CNC samples in comparison with the raw fibres 17. At the temperature range of 150 to 400 °C, the second step of thermal degradation corresponds to the decomposition of glycosyl units that was followed by the formation of a charred residue 18. Figure 7.7 shows the effect of acid concentration on the thermal stability of CNC. The main degradation temperature of the fibre hydrolysed with 35 % (v/v) and 40 % (v/v) acid for 3 hours was almost same (about 184 °C), while a comparison of the two CNC samples produced by 45 % (v/v) and 50 % (v/v) sulphuric acid for 1 hour shows a significant decrease in thermal properties of CNC with increased acid concentration due to an increase in sulphate groups. The DTA spectra of treated fibre with 50 % (v/v) acid also showed a very low thermal stability indicated by the sharp degradation peak at about 130 °C due to enhanced access to the crystal interior through the ends of sulphated crystals. In addition, the amount of charred residue at 500 °C for the CNC obtained with 50 % (v/v) sulphuric acid was almost 20 % more than the other samples.

110 35 % (v/v) acid, 3 h, 45 °C 0.00 100 40 % (v/v) acid, 3 h, 45 °C 90 -0.05 80 -0.10 70 -0.15 60 50 -0.20

Mass loss ( %) ( loss Mass 40 -0.25

30 (mg/min) lose mass of Rate 35 % (v/v) acid, 3 h, 45 °C -0.30 40 % (v/v) acid, 3 h, 45 °C 20 100 200 300 400 500 100 200 300 400 500 Temperature (°C) Temperature (°C)

110 45 % (v/v) acid, 1 h, 45 °C 0.00 100 50 % (v/v) acid, 1 h, 45 °C 90 -0.02 80 70 -0.04 60 -0.06 50

Mass loss (%) loss Mass -0.08 40 Rate of mass loss (mg/min) loss mass of Rate 45 % (v/v) acid, 1 h, 45 °C 30 -0.10 50 % (v/v) acid, 1 h, 45 °C 20 100 200 300 400 500 100 200 300 400 500 Temperature (°C) Temperature (°C)

Figure 7.7. Effect of sulphuric acid concentration on thermal properties of CNC 155

Figure 7.8 shows that the results of investigations on the effect of hydrolysis time on thermal properties of CNC was in agreement with the previous observation 18 that higher hydrolysing times resulted in CNC samples with a lower thermal stability because of the higher amount of sulphate groups on the surface of the nanocrystals. The CNC sample which was treated for 1 hour with 45 % (v/v) sulphuric acid showed the highest degradation temperature ( about 263 ºC), despite the use of a higher concentration of acid.

110 35 % (v/v) acid, 3 h, 45 °C 0.00 100 35 % (v/v) acid, 6 h, 45 °C 90 -0.05 80

70 -0.10 60 -0.15 50

Mass loss (%) loss Mass 40 -0.20

30 (mg/min) loss mass of Rate 35 % (v/v) acid, 3 h, 45 °C 35 % (v/v) acid, 6 h, 45 °C 20 -0.25 100 200 300 400 500 100 200 300 400 500 Temperature (°C) Temperature (°C)

Figure 7.8. Effect of hydrolysis time on thermal properties of CNC

7.2.6 Comparison of the results with previous published work

CNC can be extracted from almost any cellulosic material including plants, animals (such as tunicates), bacteria and algae. The production of CNC from wood, owing to its natural abundance, has been introduced as one of the key sources of cellulose. Commercially available MCC and filter paper, due to their purity and ready availability in the laboratory, are the other common sources of cellulose for basic experiments. However as mentioned before, tunicate has been a favoured source of CNC because of its high aspect ratio and crystallinity, and as such has provided an industry benchmark. Table 7.5 compares the CNC obtained from spinifex with the CNC obtained from the other industry and laboratory sources of cellulose. The diameter of spinifex CNC is in the lowest range, while the length of this nanocrystal is certainly at the higher end of the scale. Perhaps even more importantly from a composites reinforcement perspective, the aspect ratio of spinifex CNC (144) is higher than the aspect ratio of tunicate (83 19). It is worth nothing that the average length of spinifex CNC was measured from the samples of shorter crystals which fitted into the single TEM images, and so we are confident that our value is very conservative because the longest length fraction could not be included in our data set. The degree of crystallinity of spinifex CNC is also in 156

the high range. The acid concentration used for producing CNC from spinifex is lower than the other sources, presumably due to the loose fibre structure which is relatively easy to defibrillate using milder conditions.

157

Table 7.5. Comparison between CNC obtained from spinifex with the other industry and laboratory sources of cellulose

Source Hydrolysis condition Diameter (nm) Length (nm) Aspect ratio Crystallinity Crystalline Ref (%) domain size (nm)

Spinifex 40 % H2SO4 at 45 °C for 3 hours 3.45 ± 0.75 497 ± 106 177 73 4.7 20 Sisal 65 % H2SO4 at 50 °C for 40 minutes 5 ± 1.5 115 ± 21 23 81 - 21 Sisal 65 % H2SO4 at 55 °C for 30 minutes 5 ± 1.5 215 ± 67 43 53 - 22 Sisal 65 % H2SO4 at 60 °C for 15 minutes 4 ± 1 250 ± 100 62.5 - - 20 Ramie 65 % H2SO4 at 55 °C for 30 minutes 6.5 ± 0.7 185 ± 25 28.5 88 - 20 Cotton 65 % H2SO4 at 55 °C for 30 minutes 14.3 ± 2 140 ± 15 10 88 - 17 Cotton 60 - 65 % H2SO4 at 45 °C for 1 hour <10 200 - 400 - - - 23 Mulberry 64 % H2SO4 at 60 °C for 30 minutes 20 - 40 400 - 500 - 73.4 - 24 Wheat straw 64 % H2SO4 at 30 °C for 60 minutes 31.3 200 nm - 1mm - 77 - 25 Rice straw 64 - 65 % H2SO4 at 45 °C for 45 minutes 8 - 14 117 ± 39 - 91.2 8.33 26 Cotton linters 65 % H2SO4 at 45 °C for 30 minutes 27 141 5.2 - - 26 Avicel 65 % H2SO4 at 72 °C for 30 minutes 12 105 8.75 - - 26 Tunicin 48 % H2SO4 at 55 °C for 13 hours 28 1073 38.3 - - 19 Tunicate 55 % H2SO4 at 60°C for 1 hour 30 ± 5 2500 ± 1000 83.3 - 27 Raw Flax 64 % H2SO4 at 45 °C for 4 hours 21 ± 7 327 ± 108 15.6 - 10 Whatman ﬁlter paper 64 % H2SO4 at 50°C for 3.5 hours 34.5 ± 6.1 365 ± 80 10.6 - - 28 Softwood 64 % H2SO4 at 45 °C for 45 minutes 4.5 ± 0.3 105 ± 4 23.3 - - 6 Kenaf 65 % H2SO4 at 45 °C for 40 minutes 12.0 ± 3.4 158.4 ± 63.6 13.2 81.8 - * Aspect ratio of CNCs was calculated based on average diameter and length

158

7.2.7 Tensile properties of solvent cast CNC-TPU nanocomposites

The use of cellulose nanocrystal as a reinforcement in composite materials was reported for the first time in 1995 by Favier et al who investigated the percolation of nanocrystals extracted from tunicates 29. Since then a large number of groups have reported using cellulose nanoparticles as fillers in composites due to enhanced mechanical properties in comparison with the traditional microscale cellulose fibres. In this study, nanocomposite films were prepared from a clear and stable dispersion of strong and high aspect ratio spinifex CNC (CNC was obtained from acid hydrolysis using 40 % (v/v) acid for 3 hours at 45 °C) in a medium hardness aliphatic thermoplastic polyurethane (TPU) using the solvent casting method (see details in Chapter 3, Section 3.2.13). The nanocomposite films with different contents of CNC were then tested under uniaxial extension at room temperature (details of testing are in Chapter 3, Section 3. 3.10.1) and the results of mechanical properties including ultimate tensile stress, tensile strain at break, Young’s modulus and work at fracture (toughness) are presented in Figure 7.9 and Table 7.6, with the neat TPU as a reference. The neat TPU film possessed a typical high elongation at break of about 1268 %, a high tensile strength of 61 MPa, and a Young’s modulus of 7 MPa. The Young’s modulus of this host TPU was increased from 7 MPa to 11.3 MPa by adding 1 wt% CNC due to some stiffening of the elastomer through reinforcement and load transfer by the cellulose crystals network, and the toughness was increased, not insignificantly in such a tough elastomer, by about 20%. The increases in the modulus and toughness of the composite can be due that the CNCs preferentially interacted with the more polar hard segments of the host polyurethane rather than the more hydrophobic soft segments, consequently avoiding the undesired stiffening of the soft microdomains and thereby retaining the large and desirable elongation of polyurethane nanocomposites 30. The improvement in tensile strength was low for these solvent cast composites, noting that 61 MPa is already very strong for this class of materials. This can be attributed to the self-aggregations of the high aspect ratio CNCs, which could reduce the interfacial area and resulting reinforcement efficiency between the CNC and the polymer matrix relative to the overall surface area of CNC, and consequently result in decreased hydrogen bonding and deficiency in stress transferral 31. It also has been reported that the nanofibre with the aspect ratio smaller than 50 and higher than 100 would not have any major improvement in mechanical properties of nanocomposite 2 . In order to optimise these CNC-TPU nanocomposite systems, further exploration of ideal CNC surface modifications will indeed be necessary.

159

Table 7.6. Mechanical properties of neat TPU and CNC-TPU nanocomposites produced by solvent casting Young's Tensile strain Tensile stress Work at fracture Modulus (%) (MPa) (MJ m-3) (MPa) TPU 7 ± 0.1 1268 ± 9 61 ± 1.4 255 ± 21

TPU, 0.1wt % CNC 8 ± 0.2 1219 ± 25 56 ± 3.2 252 ± 13

TPU, 0.25 wt% CNC 9 ± 0.3 1207 ± 16 63 ± 1.2 275 ± 24

TPU, 0.5 wt% CNC 10 ± 0.5 1176 ± 11 59 ± 3.6 271 ± 9

TPU, 1 wt% CNC 11.3 ± 0.3 1191 ± 23 67 ± 1.5 299 ± 19

70 TPU TPU, 0.1 wt% CNC 60 TPU, 0.25 wt% CNC 50 TPU, 0.5 wt% CNC TPU, 1 wt% CNC 40

Tensile stress (MPa) stress Tensile 10

0 0 200 400 600 800 1000 1200 1400 Tensile strain (%)

Figure 7. 9. Tensile curves of neat TPU and CNC-TPU nanocomposite s produced by solvent casting

7.2.8 Tensile properties of reactively extruded CNC-TPU composites

In this case, the nanocomposites were prepared via reactive extrusion of a stable polyol-CNC suspension with dimethyl diphenyl diisocyanate (MDI) and 1,4-butanediol (BDO). Table 7.7 summarizes the amount of each components used for making blank TPU and its nanocomposites with CNC (CNC was obtained from acid hydrolysis using 40 % (v/v) acid for 3 hours at 45 °C).

160

Table 7.7. Conditions applied during reactive extrusion of CNC-TPU nanocomposite

Sample Polyol type CNC Isocyanate Hard Stoichiometry Polyol MDI flow BDO flow Torque Die content index segment flow rate rate (g/h) rate (g/min) (%) pressure (%) ratio (g/h) (bar)

X1 PTMEG1000 - 1 0.44 1 1797.6 1157.6 254.8 26-27 15

X2 PTMEG1000 + CNC 0.465 1 0.44 1 1812.5 1157.6 254.8 26 16

X3 PTMEG1000 + CNC 0.465 1 0.44 1.01 1812.5 1169.1 254.8 30-31 21-22

X4 PTMEG1000 + CNC 0.465 1 0.44 1.02 1812.5 1180.7 254.8 39-40 32-33

X5 PTMEG1000 + CNC 0.465 1 0.44 1.03 1812.5 1192.3 254.8 43 37-38

161

The nanocomposites prepared from the polyol and CNC suspension with different stoichiometric ratios of isocyanate were tested under tension. Figure 7.10 compares the tensile curves of the obtained nanocomposite with the neat TPU and Table 7.8 summarizes the mechanical properties values of blank TPU and CNC-TPU nanocomposites. The tensile strain of composite samples was decreased slightly with increasing the amount of isocyanate while the tensile stress at break showed improvement by adding both CNC and employing a higher content of isocyanate. This may be due to the increase in the urethane linkage formation between isocyanates and both polyol and hydroxyl functional groups available for reaction on the surface of cellulose nanocrystals. For example, about a 43 % increase in tensile stress was observed for the nanocomposite which contained 0.46 wt % CNC with a stoichiometry of 1.03 (see Table 7.7).

60 X 1 X 50 2 X 3 40 X 4 X 5 30

Tensile stress (MPa) stress Tensile 10

0 0 200 400 600 800 1000 1200 Tensile strain (%)

Figure 7.10. Tensile curves of neat TPU and CNC-TPU nanocomposites produced by reactive extrusion

162

Table 7.8. Mechanical properties of neat TPU and CNC-TPU nanocomposites produced by reactive extrusion Samples Young's Modulus Tensile strain Tensile stress Work at fracture (MPa) (%) (MPa) (MJ m-3)

X1 14.5 ± 1.2 1124 ± 31 41 ± 3 237 ± 10

X2 16.6 ± 0.3 1115 ± 41 43 ± 1 237 ± 6

X3 14.5 ± 0.4 1098 ± 21 48 ± 1.2 257 ± 6

X4 14.5 ± 0.6 1023 ± 11 54 ± 2 250 ± 9

X5 16 ± 0.7 1012 ± 25 59 ± 3.2 256 ± 14

These mechanical properties results of CNC-TPU nanocomposites indicate that the processing via reactive extrusion, with the extra shear and in-situ polymerisation, has resulted in nanocomposites with higher performance. Further future investigations involving more detailed characterisation will give a better understanding of the mechanical property profiles and performance possible in these complex TPU nanocomposite systems.

7.3 Summary

This chapter aims to optimize the acid hydrolysis process for the isolation of high aspect ratio cellulose nanocrystals from T. pungens, and the evaluation of reinforcement efficiency of spinifex CNC incorporated in thermoplastic polyurethane nanocomposites. Hydrolysis of spinifex pretreated fibre at lower temperature and acid concentrations than the regular reported protocols yielded high aspect ratio cellulose nanocrystals (CNCs). The average lengths were found to be unaffected by intense ultrasonic treatments. Using 40 % (v/v) sulphuric acid at 45 °C for 3 hours was found to be an optimum procedure for producing CNC from spinifex with higher yield and the crystallinity of 73 % and thermal degradation temperature of about 184 °C. The nanocomposites processed via a solvent casting method showed a slight increase in Young’s modulus and toughness. On the other hand, the nanocomposites processed via reactive extrusion with CNC dispersed polyol, exhibited a significant improvement in mechanical properties including tensile stress at break, even at very low loadings (0.46 wt%) of spinifex CNC. This improvement in mechanical properties for reactively extruded nanocomposites can be related to the increased interactions between cellulose nanocrystals and polyurethane matrix, which may involve a small degree of covalent crosslinking with CNC through the formation of urethane linkages. The 163

materials produced were still thermoplastic and could be compression moulded, so this did not affect CNC-TPU nanocomposite processability. The significance of these findings is that spinifex nanocrystals provide a new, comparatively economic and clean source for the cellulose industry and a new benchmark in terms of aspect ratio. There is clearly potential for many superior industrial applications.

164

Bibliography

1. Jorfi, M.; Amiralian, N.; Biyani, M.; Martin, D.; Annamalai, P., Biopolymeric nanocomposites reinforced with nanocrystalline cellulose. In Biomass based biocomposites Singha. AS, T. V., Ed. Smithers RAPRA: Shawbury UK 2013; pp 277-304

2. Eichhorn, S. J.; Dufresne, A.; Aranguren, M.; Marcovich, N. E.; Capadona, J. R.; Rowan, S. J.; Weder, C.; Thielemans, W.; Roman, M.; Renneckar, S.; Gindl, W.; Veigel, S.; Keckes, J.; Yano, H.; Abe, K.; Nogi, M.; Nakagaito, A. N.; Mangalam, A.; Simonsen, J.; Benight, A. S.; Bismarck, A.; Berglund, L. A.; Peijs, T. Journal of Materials Science 2010, 45, (1), 1-33.

3. Brinchia, L.; Cotanaa, F.; Fortunatib, E.; Kenny, J. M. Carbohydrate Polymers 2013, 94, 154– 169

4. Moon, R. J.; Martini, A.; Nairn, J.; Simonsen, J.; Youngblood, J. Chemical Society Reviews 2011, 40, (7), 3941-3994.

5. Abdul Khalil, H. P. S.; Davoudpour, Y.; Islam, M. N.; Mustapha, A.; Sudesh, K.; Dungani, R.; Jawaid, M. Carbohydrate Polymers 2014, 99, (0), 649-665.

6. Krishnamachari, P.; Hashaikeh, R.; Chiesa, M.; El Rab, K. R. M. G. Cell Chem Technol 2012, 46, (1-2), 13-18.

7. Saralegi, A.; Gonzalez, M. L.; Valea, A.; Eceiza, A.; Corcuera, M. A. Composites Science and Technology 2014, 92, (0), 27-33.

8. He, X.; Xiao, Q.; Lu, C.; Wang, Y.; Zhang, X.; Zhao, J.; Zhang, W.; Zhang, X.; Deng, Y. Biomacromolecules 2014.

9. Habibi, Y.; Lucia, L. A.; Rojas, O. J. Chemical Reviews 2010, 110, (6), 3479-3500.

10. Annamalai, P. K.; Dagnon, K. L.; Monemian, S.; Foster, E. J.; Rowan, S. J.; Weder, C. ACS Applied Materials & Interfaces 2013, 6, (2), 967-976.

11. Rueda, L.; Fernández d'Arlas, B.; Zhou, Q.; Berglund, L. A.; Corcuera, M. A.; Mondragon, I.; Eceiza, A. Composites Science and Technology 2011, 71, (16), 1953-1960.

12. Yue , Y.; Zhou , C.; French , A. D.; Xia , G.; Han , G.; Wang , Q.; Wu, Q. Cellulose 2012, 19, 1173–1187. 165

13. Hamad, W. Y.; Hu, T. Q. The Canadian journal of chemical engineering 2010, 88, (3), 392- 402.

14. Araki, J.; Wada, M.; Kuga, S.; Okano, T. Colloids and Surfaces A: Physicochemical and Engineering Aspects 1998, 142, (1), 75-82.

15. Tonoli, G. H. D.; Teixeira, E. M.; Correa, A. C.; Marconcini, J. M.; Caixeta, L. A.; Pereira- da-Silva, M. A.; Mattoso, L. H. C. Carbohydrate Polymers 2012, 89, (1), 80-88.

16. Teixeira, E. D.; Correa, A. C.; Manzoli, A.; Leite, F. L.; de Oliveira, C. R.; Mattoso, L. H. C. Cellulose 2010, 17, (3), 595-606.

17. Lu, P.; Hsieh, Y.-L. Carbohydrate Polymers 2010, 82, (2), 329-336.

18. Roman, M.; Winter, W. T. Biomacromolecules 2004, 5, (5), 1671-1677.

19. Jorfi, M.; Roberts, M. N.; Foster, E. J.; Weder, C. ACS Applied Materials & Interfaces 2013, 5, (4), 1517-1526.

20. Habibi, Y.; Hoeger, I.; Kelley, S. S.; Rojas, O. J. Langmuir 2009, 26, (2), 990-1001.

21. Siqueira, G.; Bras, J.; Dufresne, A. Biomacromolecules 2008, 10, (2), 425-432.

22. Garcia de Rodriguez, N. L.; Thielemans, W.; Dufresne, A. Cellulose 2006, 13, (3), 261-270.

23. Li, R. J.; Fei, J. M.; Cai, Y. R.; Li, Y. F.; Feng, J. Q.; Yao, J. M. Carbohydrate Polymers 2009, 76, (1), 94-99.

24. Rahimi, M.; Behrooz, R. International Journal of Polymeric Materials and Polymeric Biomaterials 2011, 60, (8), 529-541.

25. Lu, P.; Hsieh, Y.-L. Carbohydrate Polymers 2012, 87, (1), 564-573.

26. Elazzouzi-Hafraoui, S.; Nishiyama, Y.; Putaux, J.-L.; Heux, L.; Dubreuil, F.; Rochas, C. Biomacromolecules 2007, 9, (1), 57-65.

27. Cao, X.; Dong, H.; Li, C. M. Biomacromolecules 2007, 8, (3), 899-904.

28. Beck-Candanedo, S.; Roman, M.; Gray, D. G. Biomacromolecules 2005, 6, (2), 1048-1054.

166

29. Favier, V.; Chanzy, H.; Cavaille, J. Y. Macromolecules 1995, 28, (18), 6365-6367.

30. Pei, A.; Malho, J. M.; Ruokolainen, J.; Zhou, Q.; Berglund, L. A. Macromolecules 2011, 44, (11), 4422-4427.

31. Gao, Z.; Peng, J.; Zhong, T.; Sun, J.; Wang, X.; Yue, C. Carbohydrate Polymers 2012, 87, 2068– 2075.

167

Chapter 8. Conclusions and future recommendations

8.1 Conclusions

Triodia pungens is a soft resinous spinifex with a leaf size of over 30 cm long, and 0.8 - 1.2 mm wide 1. It mainly grows in regions north of Lat. 27 °S in Queensland, the Northern Territory, Western Australia, and north-western South Australia 2. The resin obtained from Triodia pungens is a mixture of several different volatile and non-volatile components mainly mono and diterpenoid products, some with a labdane type skeleton 3. The use of fibrous and resinous components of spinifex has been well-documented in traditional indigenous Australian culture for thousands of years. Our wider ARC discovery project was formed with the aim of applying biomimetic theory and producing practical outcomes by observing and respecting Aboriginal traditional knowledge, and engineering new materials from spinifex building blocks in order to evaluate spinifex properties in the context of novel biomimetic materials. The primary goal of this study was to discover the unique structure and properties of Triodia pungens and its potential applications in material science and engineering. In order to systematically achieve this goal, work was separated into the three secondary goals of (i) optimization of resin extraction and evaluation of its termiticidal properties, (ii) optimization of the deconstruction of spinifex fibre via mechanical and chemical methods, and (iii) an ultimate application of spinifex derived nanocrystal as a reinforcement for making high quality nanocomposites. During my PhD, the properties and sustainable applications for spinifex in materials science using innovative methodologies were examined which led to the discovery of a unique, very high grade of cellulose nanofibrils and nanocrystals which can be readily and cost-effectively produced. An application for the spinifex resin as a termiticide was also explored in this study. I shall summarize each of these findings below.

8.1.1 Spinifex resin and termiticidal properties

The first secondary goal of this study was focused on the investigations on spinifex resin by optimization of spinifex resin extraction with different organic solvents and the subsequent application of resin as a termiticide by coating the extracted and dissolved resin on the surface of

168 the timber. The optimized conditions for resin extraction with the best thermal properties and highest yield were obtained using acetone as a solvent and extraction for 24 hours at 30 °C and subsequent drying and annealing at 100 °C for 72 hours and at 35 °C for 24 hours, respectively. The resin products resulting from several different extraction conditions were analysed using ATR- FTIR, DSC, and TGA and it was found that the thermal properties of the resin were significantly improved by heat treatment. It was also found, however, that the resin suffers from moisture sensitivity and that is rapidly plasticised by the absorption of water from the atmosphere. In field trials, it was shown that the application of acetone extracted resin onto timber blocks showed that spinifex resin coatings successfully provide effective termite resistance against the very destructive termite species of Mastotermes darwiniensis.

8.1.2 Defibrillation of spinifex fibre

Another secondary goal was defibrillation of spinifex fibres which entailed the discovery that spinifex grass has unique attributes that enable its easy deconstruction into very high quality and low-cost cellulose nanofibrils. Figure 8.1 summarizes the applied procedures to produce nanofibrils, and the properties of the different cellulosic products obtained at each stage of processing or treatment.

Figure 8.1. Flowchart summarizes applied procedures to produce nanofibrils

169

The preliminary study on pulping spinifex fibre involved the application of two different methods of delignification: (a) organosolve and (b) alkali, and subsequent bleaching using milder conditions than the other reported pulping methods used for producing NFC and CNC. The purified cellulose fibres were analysed by TAPPI standard methods to determine different components and characterized with different analysis methods such as; ATR FTIR, XRD, TGA, SEM, and TEM. The results of chemical analysis of spinifex grass before and after pulping indicated that spinifex grass contains an unusually high amount of hemicellulose (almost 40 % (w/w)) which even after delignification and bleaching remains on the surface of the cellulose. X-ray results showed that spinifex fibre is crystalline in nature and has the structure of cellulose I. Delignification and bleaching of spinifex resulted in an increase in crystallinity of almost 30 % in comparison to the raw fibre. The thermal stability of delignified and bleached fibres also showed an improvement after these treatments were performed. In order to produce cellulose nanofibrils from spinifex grass, pretreated fibres were subsequently processed with three different mechanical methods; homogenization, ultrasonication, and high energy milling. All three techniques yielded nanofibrils of very small diameters (below 40 nm) and long lengths (several microns). Homogenization of pretreated fibres produced an unprecedentedly high aspect ratio NFC, even after only a single pass through the homogenizer at 350 bar, and without any clogging issues. Ultrasonication of spinifex pretreated fibre also resulted in the production of high aspect ratio NFC at short ultrasonication times and low energy. It should be taken into account that the scalability of defibrillation via ultrasonication to obtain a high yield of relatively monodisperse nanofibrils is still questionable. Using high energy milling for defibrillation of pretreated fibre also produced long and thin NFC. Milling at high speed caused the breakdown the NFC into smaller cellulose nanocrystals (CNC), however contamination and re-agglomeration of the product was evident using this processing route. To summarize, the amount of energy and chemicals required to produce NFC from spinifex biomass is very low when spinifex NFC is benchmarked against the other leading academic and commercially available nanofibrils, and it has the highest aspect ratio with respect to these other reported cellulose nanofibrils. The high amount of hemicellulose facilities defibrillation process and leads the use of lower energy processing, and very mild pulping. A cartoon showing what we believe to be the structure for the cellulose nanofibrils obtained from spinifex is illustrated in Figure 8.2. Crystalline and amorphous parts of nanofibrils are covered by a thin layer of hemicellulose which works as a “glue” to prevent breakage of amorphous parts and results in very long, tough and flexible nanofibrils. The XRD, TGA, and mechanical analysis showed that spinifex NFC has a crystallinity of about 69 %, with a thermal degradation temperature of about 320 °C and a tensile stress and toughness (for vacuum filtered nanopaper) of about 84 MPa and 12 MJm-3 respectively. The transverse elastic modulus of 170 individual nanofibrils is in the range of 19 - 23 GPa. It seems that spinifex NFC can potentially provide different applications in multiple industries including membranes and filtration 4, 5, electronics such as separators for batteries, supercapacitors and flexible electronics 6-8, and biomedical applications such as wound dressings and tissue engineering scaffolds 9, 10.

Figure 8.2. Predicted structure of spinifex NFC covered by a thin layer of hemicellulose

Similarly, acid hydrolysis at lower acid concentration and temperature (using 40 % (v/v) acid at 45 °C) than the regular reported protocols, yielded high aspect ratio cellulose nanocrystals (CNC). The average length was found to be unaffected by further intense ultrasonic treatments. The crystallinity of the spinifex CNC sample was about 73 % and thermal degradation temperature was about 184 °C. The remarkable traits of spinifex appear to trace back to the evolution of the grass

(combination of a modified C4 leaf anatomy, and a unique structural morphology with high hemicellulose content) and its adaptation to the extremely hot and dry conditions in semi-arid Australia, where spinifex forms the dominant vegetation over approximately 27 % of the continent 11, 12. More fundamental work is required in order to elucidate the relationship between spinifex species (69 exist), plant evolution, plant physiology, effects of environmental gradients and ease of deconstruction into the various nanocellulose products.

8.1.3 Nanocomposite based spinifex nanocrystal and thermoplastic polyurethane

The solvent cast TPU nanocomposites reinforced with spinifex CNC only showed moderate improvements in mechanical properties with increasing CNC loading due to agglomeration of the high-aspect ratio CNC in the TPU host polymer matrix, while the CNC-TPU nanocomposite processed via polyol pre-dispersion and reactive extrusion exhibited significant improvements in tensile strength and toughness due to enhanced dispersion and a small amount of covalent reactions between cellulose nanocrystals, diisoyanate, and polyol during high shear in-situ polymeristaion. This class of nanocomposites appears to be a very promising direction for further research and development.

171

8.2 Future research recommendations

As a materials science thesis, this work has significantly contributed to the understanding of (a) spinifex resin and its application as a termiticide, and (b) spinifex fibre and its derivative cellulose nanofibrils and nanocrystals. Consequently, these studies will form the basis of future investigations. As mentioned before the outstanding finding of this study resulted in a patent 13 application which has already provided a new funding opportunity (2014 UQ collaboration and industry engagement fund (CIEF) - 2013002412) to perform in-depth investigations in the following areas:

 Investigation the derivability of NFC from the other widely distributed spinifex species, and the effects of growth conditions and plant structural elements on NFC yield and quality; The material properties and processing of further spinifex species will be systematically evaluated. This strategy ensures the identification of the best biomass starting materials required to achieve a given NFC property profile. It is expected there will be variation between species, especially hard and soft types, of nanofibril and nanocrystal properties.

 Elimination of inorganic components from spinifex suspension ; Spinifex grass takes up mineral inorganic ions from the soil and even after mild hydrochloric acid treatment, these ions will remain on the surface of nanofibres. Some different methods such as hydrochloric acid treatment after washing the grass (before grinding and pulping), or adding hydrochloric acid in the bleaching solvent instead of glacial acetic acid can be applied to remove the inorganic components. As part of these studies it will be necessary to measure the degree of polymerization (DP) of these samples after each treatment in order to monitor and minimise molecular weight reduction.

 Development of techniques for the direct, unambiguous determination of the sulphate groups on the surface of CNC after acid hydrolysis; Our team’s future research will explore the use of conductometric titration as a possible technique to measure the amount of sulphate groups on the surface of cellulose nanocrystals. Among other things, sulphate groups on the surface of the CNC diminish the thermal stability of composite materials in which the CNC are used as a filler, so it is important to optimize the hydrolysis conditions with sulphate measurement when CNC is being used to make nanocomposites 14.

172

 A detailed understanding of the full spectrum of mechanical properties of nanopaper and individual nanofibrils using Instron and AM-FM Further investigations will be done on increasing the strength of interfibril bonding of NFC nanopaper in order to achieve the most attractive strength-stiffness-toughness balance for particular applications such as filtration media or electronic spacers. Our collaboration with Purdue as well as with Asylum (AFM instrument manufacturer) needs to continue to develop and refine the AM-FM based method in order to measure the elastic modulus of individual NFC. Developing a very fast and valuable tool for assessing NFC quality will be important for many investigators working on these materials.

 Measuring the specific surface area using Brunauer-Emmett-Teller (BET), and the degree of polymerization estimate based on the intrinsic viscosity; The optical, mechanical, and permeability properties of NFC samples can be influenced by specific surface area. Thus the measurement of specific surface area could be helpful to understand the physical properties of nanofibril networks and its reinforcement effects in various host polymers 15, 16. The degree of polymerization (DP) is an important parameter for evaluating the length and branching of cellulose chains, and is frequently used to assess NFC products. Since the mechanical properties of NFC-based materials are strongly dependent on fibre length, evaluation of DP could provide valuable information about subsequent comparisons of NFC films 17.

 Chemical analysis of spinifex CNC The amount of different components in spinifex CNC after acid hydrolysis will be determined by TAPPI standard methods. After acid treatment the amount of hemicellulose will decrease, but we believe that in case of spinifex CNC, still some remnant hemicellulose will be present covering the surface of CNC which assists in production of long nanocrystals. Further quantification of this will be required as a function of spinifex species, plant component (eg leaf tip, stem, root, runner), environmental gradient, etc.

 Resin bio-refining Determination of the composition of spinifex resin can be done by analysis of the secondary metabolites from the resin by extracting these in an organic solvent 18, 19. This extract would be largely free of primary metabolites (such as amino acids and sugars) and biological polymers which

173 will hopefully allow the identification of the main classes of compounds present, and perhaps identify a few of the major aromatic components. With fresh plant material, the first step often involves extraction with a nonpolar solvent such as hexane in order to remove hydrocarbons and waxes, as these materials interfere with subsequent characterisation of the more polar compounds. Steam distillation of the plant material is also commonly performed in order to remove and/or identify volatile materials commonly known as “essential oils” 20-22. Once again, these oils would influence the characterisation of extract and make extraction more difficult. The approach that we propose would involve these preliminary steps. The subsequent steps would involve further successive extraction with solvents of increasing polarity in order to further fractionate the material and make characterisation easier at the same time it will provide material suitable for further applications. There are several possible choices of solvents but we would need to establish the most suitable ones by experimental trials. A summary of this approach is:

Phase one;  Distillation using atmospheric pressure steam in order to isolate and characterise any essential oil fraction.  Characterisation of the volatile material by combined Gas Chromatography-Mass Spectrometry (GC-MS)

Phase two;  Extraction using a nonpolar solvent such as hexane with soxhlet technique. This procedure would be carried out both on the original extract and on the residue from the steam distillation. Partial characterisation of this extract would be attempted by Nuclear Magnetic Resonance (NMR) spectroscopy and/or GC-MS depending on the composition of the extract.

174

Bibliography

1. Perry, R. A. Lands of the Alice Springs Area, Northern Territory; 1962; pp 1956–57.

2. Beadle, N. C. W., The Vegetation of Australia. Cambridge University Press: Cambridge, 1981.

3. De Silva, S.; Memmott, P.; Folter, N.; Martin, D., Charectrisation of Spinifex (Triodia pungens) Resin and Fiber. In 11th Pacefic polymer conference Cairns, Australia, 2009.

4. Ma, H. Y.; Burger, C.; Hsiao, B. S.; Chu, B. Biomacromolecules 2012, 13, (1), 180-186.

5. Sehaqui, H.; Morimune, S.; Nishino, T.; Berglund, L. A. Biomacromolecules 2012, 13, (11), 3661-3667.

6. Gao, K. Z.; Shao, Z. Q.; Li, J.; Wang, X.; Peng, X. Q.; Wang, W. J.; Wang, F. J. J Mater Chem A 2013, 1, (1), 63-67.

7. Nogi, M.; Iwamoto, S.; Nakagaito, A. N.; Yano, H. Adv Mater 2009, 21, (16), 1595-+.

8. Guo, B.; Chen, W.; Yan, L. ACS Sustainable Chemistry & Engineering 2013, 1, (11), 1474- 1479.

9. Chang, C.-W.; Wang, M.-J. ACS Sustainable Chemistry & Engineering 2013, 1, (9), 1129- 1134.

10. Shahid-ul-Islam; Shahid, M.; Mohammad, F. Ind Eng Chem Res 2013, 52, (15), 5245-5260.

11. Gamage, H. K.; Mondal, S.; Wallis, L. A.; Memmott, P.; Martin, D.; Wright, B. R.; Schmidt, S. Australian Journal of Botany 2012, 60, (2), 114-127.

12. Toon, A.; Crisp, M. D.; Gamage, H.; Mant, J.; Morris, D. C.; Schmidt, S.; Cook, L. G. submitted manuscript at Journal of Biogeography 2014.

13. Amiralian, N.; Annamalai, P. K.; Martin, D. Provisional patent application, Nanocellulose. 22 November 2013.

14. Roman, M.; Winter, W. T. Biomacromolecules 2004, 5, (5), 1671-1677.

15. Beck, S.; Bouchard, J.; Berry, R. Biomacromolecules 2012, 13, (5), 1486-1494.

175

16. Henrique, M. A.; Silvério, H. A.; Flauzino Neto, W. P.; Pasquini, D. Journal of Environmental Management 2013, 121, (0), 202-209.

17. Zimmermann, T.; Bordeanu, N.; Strub, E. Carbohydrate Polymers 2010, 79, (4), 1086-1093.

18. Wang, L.; Weller, C. L. Trends in Food Science & Technology 2006, 17, (6), 300-312.

19. Turkmen, N.; Sari, F.; Velioglu, Y. S. Food chemistry 2006, 99, (4), 835-841.

20. Boutekedjiret, C.; Bentahar, F.; Belabbes, R.; Bessiere, J. M. Flavour and Fragrance Journal 2003, 18, (6), 481-484.

21. Masango, P. Journal of Cleaner Production 2005, 13, (8), 833-839.

22. Chemat, F.; Lucchesi, M. E.; Smadja, J.; Favretto, L.; Colnaghi, G.; Visinoni, F. Analytica Chimica Acta 2006, 555, (1), 157-160.

176