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Isolation of Cellulose Nanofibres: Elucidation of a Novel Approach
Utilizing Fungal Pretreatment
By
Sreekumar Janardhnan
A thesis submitted in conformity with the requirements
for the degree of Doctor of Philosophy
Graduate Department of Chemical Engineering and Applied Chemistry
University of Toronto
© Copyright by Sreekumar Janardhnan (2012)
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Abstract
Isolation of Cellulose Nanofibres: Elucidation of a Novel Approach Utilizing Fungal Pretreatment Doctor of Philosophy, 2012 Sreekumar Janardhnan Graduate Department of Chemical Engineering and Applied Chemistry University of Toronto
In plant cell wall, cellulose chains are organized into perfect stereoregular configuration
called microfibrils through a regular network of inter and intramolecular hydrogen bonds.
The cellulose microfibril along with hemicellulose chains that tether to the cellulose
microfibrils and other polysaccharides through hydrogen bonding forms the cell wall
structural framework. Isolation and application of cellulose nanofibres is expanding rapidly
due to their environmental benefits and specific strength properties, especially in nano-
biocomposite area. Currently, cellulose nanofibres are isolated from natural fibres through a
combination of high energy refining and high pressure homogenization or through a
combination of biological and mechanical process that involves fibre treatment with
hydrolytic enzymes followed by high pressure homogenization and all of these processes are
very energy intensive. In this research, a fungal pre-treatment for wood fibres is investigated
which can bring about internal defibrillation in the fibres through reduction of hydrogen
bonds and cleavage of hemicellulose chains tethering the cellulose microfibrils together. The
pre-treatment of wood fibres with Ophiostoma Ulmi, a causative agent of Dutch elm disease
in Elm trees, has found to reduce the energy requirement to isolate cellulose nanofibres. The
treatment has found to bring about internal defibrillation in the fibres by disrupting the
hydrogen bonding that holds the hemicellulose - cellulose microfibril network and the
cellulose chain in the cellulose microfibrils together. The effect of bio-treatment on hydrogen
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bonding density and their nature in the fibre cell wall was investigated using FT-IR and 13C
NMR and its effect on cellulose structure using FT-IR and X-ray crystallography. The treated
fibres showed a decrease in the intra-molecular hydrogen bonding density and crystallinity
and also a decrease in the hemicellulose content. The net energy required to isolate cellulose
nanofibres from bio-treated fibres was estimated at 2,000 kWh/T compared to 16,000 kWh/T
required for isolating nanofibres from untreated fibres. The isolation of cellulose nanofibres
from treated and untreated fibres by refining in disk refiner tends to obey the Rittinger’s law.
These observations confirm the fact that Ophiostoma ulmi treatment of fibres can
significantly reduce the energy required to isolate cellulose nanofibres from wood pulp
fibres.
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ACKNOWLEDGEMENT
It is my utmost pleasure to thank the many people who made this thesis possible.
I would like to express my sincere gratitude to my advisor Prof. Mohini M Sain for his
continuous guidance, generous support and valuable advice throughout my research.
I am deeply grateful to Prof. Ramin Farnood, Department of Chemical Engineering and
Applied Chemistry, University of Toronto, Prof. Dinesh Christendat, Department of Botany,
University of Toronto and Prof. Sally Krigstin, Faulty of Forestry, University of Toronto, for
their detailed and constructive support throughout this work.
I wish to express my warm and sincere thanks to my fellow students and other department
staff for their help and continued support to this stage of my research.
A special note to my wife Suhara and my kids Abhinav, Tejas and Rithik - Thank you for all
the support and encouragement and with so much of happiness let me say that this effort is
dedicated to you all.
Finally, to my parents (late), your love and thoughts have been my strength and inspiration
and would always remain.
Thank you all.
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Table of Content
Abstract ....................................................................................................................................................... ii
Chapter 1 Introduction ....................................................................................................................... 1
1.1 Cellulose and Cellulose Microfibrils ................................................................................. 1
1.2 Cellulose Nanofibres .......................................................................................................... 1
1.3 Why Cellulose Nanofibres? ............................................................................................... 2
1.4 Applications ....................................................................................................................... 3
1.5 Cellulose Nanofibre Isolation and Application Challenges ............................................... 8
1.6 Isolation of Cellulose Nanofibres ................................................................................... 10
1.7 Research Significance ...................................................................................................... 18
Chapter 2 Scientific Background ..................................................................................................... 20
2.1 Structure of Cellulose ...................................................................................................... 20
2.2 Biodegradation of Cellulosic Materials ........................................................................... 36
2.3 Research Rationale ........................................................................................................... 47
2.4 The Choice of Ophiostoma Ulmi ..................................................................................... 49
Chapter 3 Research Hypothesis and Objectives ............................................................................. 51
3.1 Hypothesis........................................................................................................................ 51
3.2 Research Objectives ......................................................................................................... 51
Chapter 4 Approach .......................................................................................................................... 52
Chapter 5 Experimental .................................................................................................................... 55
5.1 Materials .......................................................................................................................... 55
5.2 Methods............................................................................................................................ 55
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Chapter 6 Results and Discussion .................................................................................................... 70
Bio-treatment of Natural Fibres: Effect on Fibres and Yield of Cellulose Nanofibres ............... 70
6.1 Introduction ...................................................................................................................... 70
6.2 Bleached Kraft Fibre Characterization ............................................................................ 70
6.3 Effect of Ophiostoma Ulmi Bio-treatment of Fibres on Cellulose Nanofibres Yield and
Fibre Diameter Distribution ............................................................................................. 71
6.4 Conclusions ...................................................................................................................... 82
Chapter 7 Results and Discussion .................................................................................................... 83
Bio-Treatment of Natural Fibres: Impact of Pre-refining of Fibres on Bio-treatment
Efficiency and Nanofibre Yield ....................................................................................... 83
7.1 Introduction ...................................................................................................................... 83
7.2 Pre-Refining of Wood Fibres and Its Effect on Bio-treatment ........................................ 83
7.3 Cellulose Nanofibres Overall Yield and Diameter Distribution ...................................... 85
7.4 Conclusions ...................................................................................................................... 90
Chapter 8 Results and Discussion .................................................................................................... 91
Bio-treatment of Natural Fibres: Effect on Hydrogen Bonding Network ................................... 91
8.1 Introduction ...................................................................................................................... 91
8.2 Determination of Relative Intensity of Hydrogen Bonding by FT-IR ............................. 92
8.3 Effect of Bio-treatment on the Hydroxyl Chemistry of Cellulose Fibres ........................ 95
8.4 Decrease in the Relative Intensity of Hydrogen Bond Network ...................................... 97
8.5 Solid State 13C Nuclear Magnetic Resonance Spectroscopy ......................................... 107
8.6 Isolation and Identification of Fungal Protein Secreted by Ophiostoma Ulmi .............. 112
8.7 Conclusions .................................................................................................................... 117
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Chapter 9 Results and Discussion .................................................................................................. 119
Bio-treatment of Natural Fibres: Effect on Cellulose Structure and Mechanical Properties. .... 119
9.1 Introduction .................................................................................................................... 119
9.2 FT-IR Bands in 1400 – 800 cm-1 Region ....................................................................... 119
9.3 X-Ray Diffraction Crystallography ............................................................................... 121
9.4 Fibre Length and Aspect Ratio of Cellulose Nanofibres ............................................... 124
9.5 Mechanical Strength Properties of Cellulose Nanofibres .............................................. 125
9.6 Conclusions .................................................................................................................... 128
Chapter 10 Results and Discussion .................................................................................................. 130
Bio-treatment of Natural Fibres: Process Scale-up and Estimation of Energy Requirement for
the Isolation of Cellulose Nanofibres. ........................................................................... 130
10.1 Introduction .................................................................................................................... 130
10.2 Theory of Refining ......................................................................................................... 130
10.3 Overview of Fibre Refining Process for Cellulose Nano-Fibre Isolation ...................... 132
10.4 Disk Grinder................................................................................................................... 133
10.5 Specific Edge Load ........................................................................................................ 137
10.6 Rittinger’s Law .............................................................................................................. 141
10.7 Energy Estimation .......................................................................................................... 142
10.8 Rittinger’s Constant Calculation for Treated and Untreated Fibres .............................. 144
10.9 Quantification of Energy Reduction .............................................................................. 149
10.10 Conclusions .................................................................................................................... 151
Chapter 11 Research Conclusions.................................................................................................... 153
12. References ................................................................................................................................... 156
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13. Appendix ..................................................................................................................................... 180
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List of Figures
Figure 1.1 SEM of mechanically isolated cellulose nanofibres.
Figure 1.2 Pictogram – filed patents for nanocellulose application.
Figure 1.3 Production of cellulose nanofibres.
Figure 1.4 PFI Mill and rotor.
Figure 2.1 Structure of glucose.
Figure 2.2 Structure of cellobiose.
Figure 2.3 Structure of a cellulose molecule.
Figure 2.4 Cellobiose molecules and hydrogen bonds.
Figure 2.5 Cellulose biosynthesis: rosette and newly synthesized microfibrils.
Figure 2.6 Microfibril structure.
Figure 2.7 Plant cell wall structure.
Figure 2.8 Polysaccharide network in lignocellulosic matrix.
Figure 2.9 Typical structures of hemicelluloses in wood.
Figure 2.10 Hybrid model for action of Expansin.
Figure 2.11 Structure of Expansin.
Figure 2.12 A model for Expansin’s wall-loosening action.
Figure 2.13 Mechanisms of enzymatic hydrolysis of the glycosidic bond.
Figure 2.14 A sketch of fungal cellulase in action.
Figure 4.1 Working principle of PFI Mill.
Figure 6.1 Yield of cellulose microfibrils with different Ophiostoma Ulmi treatment
conditions.
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Figure 6.2 Weight loss of fibre with different Ophiostoma Ulmi treatment conditions.
Figure 6.3 Cellulose content of fibre after different Ophiostoma Ulmi treatment
conditions.
Figure 6.4 Hemicellulose and the extractable hemicellulose content of fibres after
different Ophiostoma Ulmi treatment conditions.
Figure 6.5 Effect of Ophiostoma Ulmi treatments on number averaged diameter
distribution of nanofibres after PFI refining for 4 days treatment.
Figure.6.6 SEM of Ophiostoma Ulmi treated fibre treated fibre before PFI refining.
Figure 6.7 Effect of Ophiostoma Ulmi fibre treatments on internal defibrillation.
Figure 6.8 Effect of Ophiostoma Ulmi treatments of fibres on the yield and distribution
of cellulose microfibrils after refining and cryocrushing.
Figure 6.9 TEM of cellulose microfibrils isolated from Ophiostoma Ulmi treated fibres
through PFI refining and cryocrushing.
Figure 6.10 TEM of cellulose microfibrils isolated from untreated fibres through PFI
refining and cryocrushing.
Figure 7.1 Cellulose content and weight loss of pre-sheared fibres after different
Ophiostoma Ulmi treatment condition.
Figure 7.2 Effect of pre-refining of fibres on the effectiveness of bio-treatment.
Figure 7.3 TEM images of cellulose microfibrils isolated from cellulose fibres with
various levels of pre-refining followed by bio-treatment and 10 pass
homogenization.
Figure 7.4 TEM images of cellulose fibres with various levels of pre-refining followed
by bio-treatment and 5 pass homogenization.
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Figure 7.5 TEM images of pre-refined cellulose fibre end showing extensive
defibrillation after biotreatment and 5 pass homogenization.
Figure 8.1 FT-IR spectra of cellulose fibre with various peaks selected in 2nd derivative
mode.
Figure 8.2 Normalized FT-IR of cellulose wood fibres subjected to different levels of
enzyme treatment.
Figure 8.3 FT-deconvoluted OH bands (3600 to 3200 cm-1) of treated and un-treated
cellulose fibres.
Figure 8.4 Curve fitting and peak assignments for OH stretching regions.
Figure 8.5 FT-deconvoluted OH bands (3600 to 3200 cm-1) of 4 days treated and un-
treated cellulose fibres.
Figure 8.6 13C CP-MAS spectra of cotton linters.
Figure 8.7 13C CP-MAS spectra of untreated cellulose fibres.
Figure 8.8 13C CP-MAS spectra of bio-treated cellulose fibre.
Figure 8.9 Protein concentration on treated fibres.
Figure 8.10 Characterization of proteins isolated from treated fibres based on a biological
processes classification system.
Figure 8.11 SEM of treated and untreated fibres after sonication with glass beads.
Figure 9.1 IR absorption characteristics of cellulose fibres in the 800 to 1400 cm-1 region.
Figure 9.2 X-ray diffraction patterns of treated and un-treated cellulose fibres.
Figure 9.3 TEM of cellulose nanofibres.
Figure 9.4 Stress-strain curves of cellulose nanofibre films prepared with nanofibres
from bio-treated and un-treated cellulose fibres.
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Figure 10.1 Geometrical parameters of disk wet mill refiner.
Figure 10.2 Schematics of stator and rotor disk on a disk wet mill.
Figure 10.3 TEM of cellulose nanofibres obtained from bio-treated fibres after single pass
through the disk refiner.
Figure 10.4 TEM of cellulose nanofibres obtained from un-treated fibres after single pass
through the disk refiner.
Figure 10.5 TEM of cellulose nanofibres obtained from un-treated fibres after eight pass
through the disk refiner.
Figure 10.6 Schematics of mechanical treatment of fibres between stator and rotor inside a
refiner.
Figure 10.7 Stress – strain curve of hemp fibre.
Figure 10.8 Diameter distributions of cellulose nano-fibres from treated and untreated
fibres for single pass through disk refiner.
Figure 10.9 Variation in Rittinger’s constant with specific energy of refining.
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List of Tables
Table 1.1 Mechanical properties of cellulose.
Table 2.1 Hemicellulose characteristics in soft wood.
Table 6.1 Composition of bleached Kraft pulp fibre.
Table 6.2 Average degree of polymerization and chain length for treated and untreated
fibres.
Table 8.1 Bands assignment of the OH band of cellulose from literature.
Table 8.2 Change in IR index of cellulose fibres with enzymatic treatment.
Table 8.3 IR index of D2O saturated cellulose fibres with enzymatic treatment.
Table 9.1 Crystallinity index of biotreated and untreated cellulose fibres.
Table 9.2 Average mechanical properties of films prepared from cellulose nanofibres
isolated from bio-treated fibres and that isolated from untreated fibres.
Table 10.1 Rittinger’s constant for various passes of treated and untreated fibres.
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Appendix
Appendix 1 Fibre quality analysis data
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Scientific Publications
1. J. Sreekumar, M. Sain, (2008), “Isolation of cellulose microfibrils utilizing enzymatic
pre-treatment approach”, BioResources 1(1), 1-5.
2. Janardhnan, S., and Sain, M., (2011). "Targeted disruption of hydroxyl chemistry and
crystallinity in natural fibres for the isolation of cellulose nano-fibres via enzymatic
treatment," BioRes. 6(2), 1242-1250.
3. Sreekumar Janardhnan and Mohini Sain (2011), “Isolation of Cellulose Nanofibres:
Effect of Biotreatment on Hydrogen Bonding Network in Wood Fibres,” International
Journal of Polymer Science, Volume 2011, Article ID 279610.
4. Sreekumar Janardhnan and Mohini Sain (2011), “Bio-Treatment of Natural Fibres in
Isolation of Cellulose Nanofibres: Impact of Pre-Refining of Fibres on Bio-Treatment
Efficiency and Nanofibre Yield,” Journal of Polymer and the Environment, Journal:
10924, Article: 312.
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Chapter 1 Introduction
The introduction briefly highlights the scientific concept, benefits and application of
cellulose nanofibres followed by a brief description of various challenges associated with
their isolation, modification and utilization. Also, a clear emphasis will be placed on the
potential positive scientific and industrial implications of this research keeping in mind the
overall direct and consequential environmental benefits.
1.1 Cellulose and Cellulose Microfibrils
Cellulose is the most abundant bio-polymer on earth and is mainly found in the secondary
cell wall of a plant cell. Cellulose microfibrils are a self-assembly of cellulose chains that
occurs during the biosynthesis of cellulose in the plant cell wall. Cellulose microfibrils
provide the structural framework in a plant cell wall into which all other polysaccharides like
hemicellulose, lignin and pectin are deposited during the plant cell growth (O’Sullivan 1997).
Hydrogen bonding forms the major binding force between the cellulose and hemicellulose in
the plant cell wall and provides the structural strength to the cell wall (Fengel 1984).
1.2 Cellulose Nanofibres
Currently, in most of the literature reviewed, there has not been any clear definition or
distinction between cellulose microfibrils and cellulose nanofibres and is often used
interchangeably. For the purpose of this research, nanofibre is defined as a single microfibril
or a group of microfibrils with diameter in the 0 to 100 nm range and with an aspect ratio of
greater than 100.
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1.3 Why Cellulose Nanofibres?
The elementarization of natural fibres into its elementary cellulosic constituents like
nanofibres has gained considerable attention due to their potential economic and
environmental benefits.
a. high strength and stiffness (Tashiro and Kohayashi 1991, Nishino et al. 1995)
b. high reinforcing potential (Burglund 2004)
c. their biodegradability and renewability
(a). Cellulose nanofibre suspension (b). SEM of Cellulose nano-fibre
Figure 1.1 Cellulose nanofibres (Koskinen, 2011)
Quest for value added products, especially in the pulp and paper industry, is the main
motivation in this line of research. However, its infiltration into other areas of application
was instantaneous as there is tremendous pressure in all fields of engineering to minimize
environmental foot print. Nano-biocomposite and pharmaceutical science stands to benefit
hugely from this specific area of research with reported use of cellulose nanofibres as
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reinforcing agents in nano-biocomposites that has load bearing engineering application as
well as in bio-engineering applications like scaffolds for bone and ligament substitution.
Potential areas of application of cellulose nanofibres are detailed in Section 1.4.
The literature differentiates between microfibrillated cellulose (MFC) obtained through a
mechanical homogenization (Herrick et al. 1983) and microcrystalline cellulose (MCC) that
is generated by chemical treatment of various plant fibres. MFC has an aspect ratio around 50
to 100 and are extensively investigated for its reinforcing potential while MCC has an aspect
ratio of about 3. Cellulose nanofibres isolated through various chemical and mechanical
processes tend to be more individualized and with certain types of isolation processes an
aspect ratio greater than 4000 is attainable (Koskinen, 2011).
1.4 Applications
Isolation and application of cellulose nanofibres are expanding rapidly in various scientific
and industrial communities for economic and environmental reasons. Numerous high-end
potential applications for cellulose microfibrils are currently being explored (Tashiro and
Kohayashi 1991, Berglund 2004, Nakagaito and Yano 2004, Saito et al. 2006).
Cellulose nanofibres can be used to make ultra-light materials (e.g. film) the plasticity
(elasticity) of which is possible to regulate, e.g. by addition of starch. Microfibrils are being
used to reinforce biopolymers like polylatic acid and starch based polymers for packaging
materials that are 100% biodegradable. Conductive cellulose based materials can be made
reinforcing conductive polymers with cellulose microfibrils. It is also possible to coat
microfibrils with a thin layer of titanium dioxide, which makes the material
4
photocatalytically active. Titanium dioxide coated microfibrillar cellulose could be used, for
instance, in solar cells and applications in which self-cleaning surfaces are needed, such as
filters. Cellulose nanofibres give considerable toughness and strength to traditional paper
products even in small quantities. Some of the potential applications identified in research
publications and verified on a laboratory scale are
Paper and paperboard applications
- Dry strength agent
- Surface strength agent
- Nano-coating
- Surface barriers
Nano-biocomposites
Food applications
Cosmetics and creams
Medical and bio-medical applications
Absorbent application
Advanced building products
Optical films
In addition to using microfibrils as the reinforcing agent, there is an increasing trend at
present to isolate pure cellulose crystallites, commonly referred to as cellulose “whiskers”,
and to use them in preparing composites with superior mechanical performance. Such highly
crystalline cellulose whiskers have been isolated from a variety of sources like the animal
cellulose tunicin (Favier et al. 1995), chitin (Morin and Dufresne 2002), bacterial cellulose
5
(Grunert and Winter 2002), and microcrystalline cellulose (Oksman et al. 2006). Reinforcing
with these high crystallinity cellulose chains in a variety of polymers through solution casting
demonstrated improved mechanical properties compared to the base polymers. Some of the
potential and demonstrated application and their current extent are identified in Figure 1.2.
Figure 1.2 Filed patents for applications for cellulose nanofibres in different filed (Koskinen
2011).
Figure 1.2 is a survey by Aalto University detailing the current trends in the field of cellulose
nanofibres and its application. Bio-composites and bio-nanocomposites are considered to be
the green materials for the next generation. A biocomposite is a material formed by a matrix
(resin) and a reinforcement of natural fibres (usually derived from plants or cellulose). The
bio-based nanocomposites can be produced from renewable resource-based polymers in
combination with nano-filler reinforcement, such as cellulose nanocrystals and nanofibres.
Composite materials (38%) Nonwovens, adsorbent webs (18%) Paper and board (16%) Food products (13%) Paper and board coatings (8%) Cosmetics and toiletry (3%) Filter materials (4%)
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The market for these bio-based nanocomposites has the potential for dramatic growth with a
green concept in recent years. Nanocomposites of this category are expected to possess
improved strength and stiffness with small reduction in toughness, reduced gas/water vapor
permeability, a lower coefficient of thermal expansion, and an increased heat deflection
temperature (Ray and Okamoto 2003, Samir et al. 2004, Zimmermann et al. 2004). Bio-based
nanocomposites have the greatest market growth potential in industries, where
biocompatibility and environmentally responsible design and constructions are required
(Oksman et al. 2009).
The tensile properties of the filler material are an important indication of its reinforcing
potential. In this regard, the tensile modulus of microcrystalline cellulose has been studied.
Microcrystalline cellulose is prepared by acid hydrolysis by eliminating the amorphous
regions of cellulose (Yu and Atalla 1998). The product, cellulose nanocrystals, have a
diameter in the range of 10-30 nm (Batitista 1975), and aggregate randomly through
hydrogen bonds. Microcrystalline cellulose is a useful reinforcing agent because of its very
high strength and stiffness and its low abrasiveness. Eichhorn and Young (2001) measured
the mechanical properties of microcrystalline cellulose, in accordance to a method devised by
Sakurada et al. (1962), for the experimental determination of the elastic modulus of the
crystalline regions of oriented polymers. In this work, a value of 25 ± 4 GPa was estimated
for the Young’s modulus of microcrystalline cellulose. However, despite the high stiffness of
microcrystalline cellulose, they are present as powders, and their reinforcing potential in
composites is expected to be higher with greater aspect ratio (length/diameter) of the filler
material. This indicates the strong advantage of the use of cellulose fibres with high aspect
ratio as reinforcing agents. Application of cellulose nanofibres in other fields like, pulp and
7
paper, absorbent, food, and cosmetics had also shown similar interest as the potential for
growth is enormous considering the specific functional, economic, and environmental befits
this can bring.
Table 1.1 Mechanical properties of cellulose / Cellulose nanofibres; Fellers et al. (1983),
Mikael and Lindström (2009)
Pulp Stress at break
MPa
Young’s Modulus
GPa
Strain at break
%
Nanocellulose 200 10 - 20 6 - 12
UKP 64 5 4
BKP – SW 54 5 5
BKP – HW 34 4 4
Newsprint 16 2 2
Ground wood 6 1 1
UKP: unbleached Kraft pulp / BKP – SW: bleached Kraft pulp soft wood / BKP – HW:
bleached Kraft pulp hard wood
The major step in isolation of cellulose nanofibres is the process of delamination of the fibres
from the cell wall. This research focus on addressing the key issue of high energy
consumption for the defibrillation of cell wall, therefore, the challenges and methods of
fibrillation are discussed in detail in the following sections.
8
1.5 Cellulose Nanofibre Isolation and Application Challenges
Some of the main challenges faced by the research community in the isolation and
application of cellulose nano-fibres that could hamper the current momentum in the direction
of industrial scale application are
a) Poor economics due to high-energy requirement
b) Achieving proper dispersion of cellulose nanofibres in polymer matrix for bio-
composite applications
c) Interfacial compatibility, especially in composite and medical application
d) Handling – low suspension concentration and converting to dry form.
Minimization of energy required to isolate cellulose nanofibres from plant cell wall is one of
most researched topic in past decade and is also one of the most critical challenge facing the
scientific community today (Chakraborty et al. 2005, Hayashi et al. 2005, Henriksson et al.
2007, Mikael 2007, Abe et al. 2007, 2009, Abe and Yano 2009, 2010, Nogi et al. 2009,
Cheng et. al. 2007, 2009,2010, Zhao et al. 2007, Chen et al. 2011). Various methods
developed to isolate cellulose nanofibres are limited to the laboratory scale with few attempts
to scale up to a plant. The results of research in the direction of minimizing energy had been
quite encouraging. The energy required to isolate cellulose nanofibres has shown significant
reduction in the past decade, an estimate of 80,000 kWh/T in the late 90’s to 40,000 kWh/T
in mid-2000 and now claims as low as 5 to 6,000 kWh/T (Mikael and Lindström, 2009). A
transition from a purely mechanical process to bio-mechanical process has resulted in
substantial reduction in energy consumption. However, most of the identified processes are
still a combination of high shear refining and high pressure homogenization followed by
9
enzymatic hydrolysis, where a cellulolytic enzyme is used to disrupt the structure of fibre
structure with a noted disadvantage of cellulose breakdown.
In addition to the challenges faced with isolation, hydrophilicity of cellulose nanofibres is
another challenge faced by researchers in fully exploiting its properties, especially in bio-
composites. However, there are numerous ways to overcome the inherent chemical and
surface characteristics of the cellulose nanofibres and subsequently extend their scope of
application. Cellulose microfibrillar surface chemistry can provide potential for surface
modification using well established carbohydrate chemistry (Orts et al. 2005). Chemical
modifications of microfibrillated Cellulose (MFC) could be achieved in both aqueous and
organic solvents (Stenstad et al. 2008). The surface modifications used were the grafting of
hexamethylene diisocyanate, succinic acid, and maleic acid. Alkali treatment was used to
enhance the toughness of microfibrillated cellulose (NFC)-reinforced phenolic composites
(Nakagaito and Yano 2008). The improvement was attributed to the transformations in the
amorphous regions along the cellulose microfibrils. To summarize, some of the structural and
surface modification being considered and investigated are (Koskinen 2011)
Functionalization of NFC using polymers
Chemical modification of NFC surface
Functionalization using nanoparticles
Nanocellulose modified with inorganics and surfactants
Biochemical modification
Enabling drying & dispersing
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1.6 Isolation of Cellulose Nanofibres
In cellulose, microfibrils are joined laterally by means of hydrogen bonding (Brown et al.
1976). As the microfibres are generated, they were found to coalesce laterally through
interfibrillar hydrogen bond to form bundles. As stated by the authors, “the bundles associate
with neighboring bundles to produce a composite ribbon of cellulose microfibres”. The
glucose and cellobiose structures show the presence of several hydroxyl groups in the
cellulose chain and all these hydroxyl groups participate in hydrogen bonding (Nissan and
Batten 1990). The interfibrillar hydrogen bonding energy has to be overcome in order to
separate the microfibres into individual entities. More than one type of H-bond is present in
cellulose - intermolecular and intramolecular, so a range of values can be used to quantify the
hydrogen bond strength. This energy (U) for cellulose ranges between 19 and 21 MJ/kg mol
(Nissan et al. 1985).
Young’s modulus (E) of a hydrogen bond-dominated solid such as paper has given quantified
by (Nissan et al. 1985) as follows:
E = <kR>N1/3, where
R = the total H-bond length
<kR> = the average value of the force constant for stretching R by a unit distance
N = the effective number of H-bonds per unit volume involved in taking up strain
under uniaxial stress conditions.
Microfibrils are more flexible and agglomerate less in the presence of water. Fengel (1974)
indicated that intensive disintegration in a homogenizer could split even the elementary
11
fibrils and microfibres down to few molecular diameters of cellulose chain. However, the
process is extremely energy intensive, therefore, less rigorous defibrillation methods like
steam explosion, refining (disk and PFI) were used for generating microfibres on the fibre
surface.
Research efforts are increasingly being focused on the expanded possibility of the use of
microfibrils with micron-sized diameters and very high aspect ratios, as well as cellulose
nanocrystals with high crystallinity but relatively low aspect ratio, as reinforcing agents.
Microfibrils are generated in the laboratory through a combination of high energy refining in
a PFI mill, and subsequent cryocrushing under the presence of liquid nitrogen (Chakraborty
et al. 2005). Some of the current approach adopted keeping energy consumption in focus
are:
a) Fibre disintegration through high pressure homogenization (Herrick et al, 1983,
Nakagaito & Yano 2004, 2005, 2008, Turbek 1983).
b) Mechanical refining and cryocrushing (Chakraborty et al. 2005)
c) Chemi - mechanical process (Wang and Sain 2007)
d) Fibre high shear refining - cellulolytic enzymatic treatment – high pressure
homogenization (Hayashi et al. 2005, Henriksson et al. 2007, Mikael 2007)
e) Wet mill refining of fibres (Abe et al. 2007, 2009, Abe and Yano, 2009, 2010,
Nogi, et al. 2009).
f) High intensity ultrasonification followed by chemical pre-treatment (Cheng et al.
2007, 2009, 2010, Zhao et al. 2007, Chen et al. 2011).
12
All of the above processes for isolating cellulose nanofibres in the sequence presented are an
improvement over the predecessor with respect to the energy consumption, process
complexity, environmental foot print and the characteristics of the isolated cellulose
nanofibres. Fibre disintegration in a high pressure homogenizer to nanofibre level required
several passes and hence very high energy requirement. Chemical pre-treatment of fibres
followed by mechanical shearing did not reduce the energy requirement significantly (Wang
and Sain 2007) and use of chemical treatment had a negative environmental impact. The
wet mill grinding process for isolating cellulose nanofibres developed by Yano et al. (2008)
is a single step process; however, the raw fibres used were in powdered form that required
either refining or ball milling. Two generally used procedure to isolate cellulose nanofibres
from plant fibres are identified in Figure 1.2.
Ultrasonication approach (Chen et al. 2011) also required extensive chemical pre-treatment
in order to achieve cellulose nanofibre isolation. In this process, lignin and hemicellulose are
initially removed through chemical treatment. Ultrasound energy is transferred to cellulose
chains through a process called cavitation, which refers to the formation, growth and violent
collapse of cavities in water. The energy provided by this means is in the range of 10 – 100
kJ/mol which is within the hydrogen bond energy scale (Tischer et al. 2010). This research
work is a step forward in this direction and focus on understanding and developing
fungal/enzymatic fibre treatment chemistry with the objective of facilitating the isolation of
cellulose microfibrils.
13
(a) (b)
Figure 1.3 Production of cellulose nanofibres, (a) Conventional method. (b) Enzymatic
method.
Chakraborty (2006) had successfully extended the application of cellulose nanofibres in the
direction of 100% bio-degradable biopolymer composites with focus on composite
transparency and strength. Sain et al. (2011) patented the process to produce modified
thermoplastic starch from polysaccharide via fungal treatment. The modified starch when
reinforced with cellulose nanofibres produced a film with better mechanical properties and
optical properties.
Bleached Softwood Pulp
PFI Refiner
Cryo‐crushing or Homogenizig
Filtration 80 mesh
Nanofibre
Bleached Softwood
Enzymatic Treatment
Refining & Homogenizig
Filtration 80 mesh
Nanofibres
14
The production of microfibrils from non-wood resources has been reported by Dufresne et al.
(1997). These microfibrils were produced by chemically separating the pectin from the
cellulose chains, and subsequent pressurization in a laboratory homogenizer. Production of
microfibrils through similar methods from sugar beets has been reported by Dinand et al.
(1999). Microfibrils from wood pulp fibres have been reported in 1996 by Taniguchi. In this
work, a supermass colloider was used to shear apart the fibres to nanofibres diameters in the
range of 100 nm and less. Generation of microfibrillated cellulose (MFC) from softwood
sulfite pulp has been reported through high-pressure homogenization action (Turbak et al.
1983, Herrick et al. 1983, Nakagaito and Yano 2004, 2005). The process led to opening up
and unraveling of the fibres to produce a mesh of smaller fibrils and microfibrils. It did not,
however, lead to the isolation of microfibrils as individual entities separate from the cell wall.
Fukuzumi et al. (2009) have used their cellulose-based nanofibres to fabricate transparent gels
and thin films that have remarkably high-oxygen barrier capability, high optical
transparency, high strength and a quite low coefficient of thermal expansivity, caused by
high crystallinity of native cellulose. The technique used by the scientists is based on the
selective oxidation of primary hydroxyl groups on the fibril surfaces to anionically-charged
carboxylate groups through 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO)-mediated
oxidation of native celluloses and the subsequent mild disintegration in water. The resulting
cellulose nanofibres maintained the crystallinity and crystal widths of the original wood
celluloses to be about 75% and 3-4 nm, respectively.
15
1.6.1 Refining
Refining is the most common method adopted for defibrillation of the fibre. The major
objective of refining is to change the fibre morphology to increase the bonding potential
among fibres. Refining is also used in mechanical pulping to beat the structure of wood and
increase the surface of the fibres. Two common approach in refining are
1.6.1.1 Disk refiner
Mechanical pulping is mainly brought about by absorption of energy by repeated
compression and decompression of the fibres (Pearson 1990). In the case of a disk refiner,
chips are fed in near the centre of rotation, and are transported through the action of
centrifugal force to the periphery where they are discharged. On the way, forces are
transferred to the chips, deforming and breaking them into progressively smaller pieces.
In a disk refiner, the fibre suspension to be treated is forced between rotor and stator disks. A
pictorial representation of the refiner plate is shown in Figure 10.1, Chapter 10. The fibre
suspension is forced into a confined zone whose thickness is called gap clearance. This
distance between the opposing bars ranges between 50 and 300 µm. During a bar crossing,
fibres are struck and strongly compressed, which contributes efficiently to the refining effect,
i.e., fibrillation, hydration and fibre breakage (Roux and Mayade 1999). The refining
mechanism and the refining model development are discussed in Chapter 10. Three most
commonly equipment used in the isolation of cellulose nanofibres are PFI mill, wet mill
grinder and high pressure homogenizer.
16
1.6.1.2 PFI mill
For beating under laboratory conditions, a PFI mill is commonly used (Figure 1.4). As
mentioned above, the starting material for the present research is bleached kraft pulp. The
PFI mill is more appropriate for this work, as this beater is operated with pulp as well, as
opposed to with chips in a disk refiner. The PFI mill was developed in 1948 by Stephansen at
the Paperindustriens Forkninginstitutt, Oslo, Norway. The PFI mill is primarily used to beat
kraft pulp fibres, increasing fibre flexibility and improving the properties of the resulting
paper. In the process, the refiner also produces fines and causes fibrillation leading to fibres
of smaller diameter.
The mill can be operated under a wide range of conditions as follows (Phillips et al. 1970):
(a) Mill variables – beating load, speeds of roll and housing, and beating with or without
a fixed minimum clearance
(b) Pulp variables – pulp charge and stock concentration
(c) Operating variables – temperature, conditioning of the beating surfaces and the rate of
application of the beating load
17
Figure 1.4 PFI mill & rotor
The action of any refiner is determined by shear and compression forces in the refining zone,
and by their distribution on single fibres. These forces are more evenly distributed in a
laboratory beater. In over-simplified terms, the beating action resembles a plunger moving
down into the pulp mass (Watson and Phillips 1964). These authors noted four primary
effects in the beating process that are related to the fibre properties and the fibre-water
system. These effects are:
(1) Intra-fibre bond breaking (internal fibrillation)
(2) External fibrillation
(3) Production of fines
(4) Fibre shortening
18
Nissan (1977) and Van den Akker (1957) have put forward several propositions on the
energy estimates in a refiner and also the various mechanism and their estimate energy
requirements while a fibre suspension is subjected to refining in a PFI refiner.
1.6.1.3 High pressure homogenizer
A description of the mode of operation of a high pressure homogenizer is provided in
Chapter 10.
1.6.1.4 Low consistency refining
Low consistency refining is a new approach being developed with the aim of minimizing
energy requirement for refining for mechanical pulp production (Heymer et al. 2005). Low
consistency refining research is being carried out at University of British Colombia in
collaboration with Aikawa, a subsidiary of Advanced Fibre Technologies
1.7 Research Significance
Enzymes have been widely used to modify cellulosic fibres for various applications,
especially in pulp and paper industry. Cellulolytic enzymes have been used as a pre-treatment
step in the isolation of cellulose nanofibres with the intention of minimizing the energy
requirement. This approach has partially achieved its objective with respect to energy
reduction, reducing the energy required for isolating cellulose nanofibres from over 50,000
kWh/T to less than 10,000 kWh/T. This approach utilizing cellulolytic enzymes tends to have
a negative impact on the reinforcing characteristics of the cellulose nanofibres due to the
random structural breakdown of the cellulose. Also, the use of purified enzymes for the pre-
treatment of cellulose has proved expensive negating the overall economics. The root cause
19
of the high energy requirement is the predominant hydrogen bonds existing between the
cellulose fibrils and there has not been any research to address this underlying chemistry.
The approach taken in this research project is to target and disrupt the predominant hydrogen
bonding network in the plant cell wall to bring bout internal defibrillation within the plant
cell wall. Specific characteristics of certain cell wall degrading enzymes, like their ability to
breakdown hydrogen bonds at microfibrillar level, are exploited here. An understanding of
the internal defibrillation mechanism at this molecular level and its exploitation to isolate
high strength micro and nano fibrils from plant cell wall in an economical manner will be a
huge step towards isolation of cellulose microfibrils and their commercial scale utilization in
various applications.
This project is directed at identifying a novel eco-friendly approach and explores the
underlying chemistry. This is the first ever scientific approach to use a hydrogen bond
specific enzyme from a cell wall degrading fungus to bring about internal defibrillation
within the plant cell wall to facilitate the isolation of cellulose nano-fibres. This approach, we
believe, will revolutionize the bio-composite and nano-bio-composite research and industry
and various other related industries identified in Section 1.4. Also, the scientific concept
presented here will inspire researchers in the field of bio-resources utilization to adopt and
expand on more environmentally friendly approaches developed and established here.
20
Chapter 2 Scientific Background
2.1 Structure of Cellulose
In this section, fundamental chemical and structural aspects of cellulose and the role of
hydrogen bonds in cellulose structure, cellulose / microfibril biosynthesis, and their structural
role in plant cell wall is discussed. The role of cell wall functional proteins and enzymes in
plant cell growth and their mechanism of action are discussed as this has a direct relevance to
the approach taken in this project.
2.1.1 Cellulose crystals and role of hydrogen bonding
Cellulose is the most important constituent of the cell wall, and forms a framework around
which all other cell wall components are deposited (Clowes and Juniper 1968). -Cellulose,
the fraction of the whole cell wall left after delignification and the extraction of
hemicelluloses and pectic substances, consists mostly of linear chains of 1,4 linked -
glucose. It differs from the structure of -glucose in the position of the OH group on carbon
1, as illustrated in Figure 2.1.
However, these -glucose molecules are not normally present in this native state. Glucose
mostly forms a ring structure in which an oxygen bridge links carbon atoms number 1 and 5
as shown in Fig 2.1. When two heterocyclic molecules condense with the loss of one
molecule of water, the disaccharide, cellobiose, is formed. The structure of cellobiose is
shown in Figure 2.2.
21
-Glucose β-Glucose
Figure 2.1 Structure of Glucose
Figure 2.2 Structure of Cellobiose
Figure 2.3 Structure of a cellulose molecule – (β -1, 4-linked-D-Glucose)
22
Further polymerization of glucose units results in long chain of molecules. The continuous
chain in a cellulose molecule is depicted in Figure 2.3. As the chemical structure shows, the
hydroxyl groups of C-atoms at 2, 3 and 6 in cellulose are free to form bonds with
neighboring chains. The hydroxyl groups of cellulose molecule tend to form hydrogen bonds
with hydroxyls of adjacent chains, giving the cellulose a superstructure of considerable
lateral order (Rydholm 1965). This relatively weaker but numerous hydrogen bonding among
cellulose chains imparts immense strength to the polymer structure.
(a)
(b)
23
(c)
Figure 2.4 Cellobiose molecules and hydrogen bonds. (a) View of the intra- and inter-chain
hydrogen bonding for (A) cellulose I and (B) cellulose II (Kontturi et al. 2006) (b) chain
length of each unit and distance between each molecule in a cellulose crystal (c) Crystal
lattice structure of cellulose (Clowes and Juniper 1968)
Depending on the sources of fibre, the average number of units in a cellulose molecule may
lie between 3,000 and 10,000, thus giving molecular lengths of between 1.5 and 5.0 microns
(Clowes and Juniper 1968). Rydholm (1965), for example, reported an average degree of
polymerization (DP) between 4,000 and 5,000 for cellulose in both softwood and hardwood.
Ohad and Mejzler (1965), however, mentioned that the average chain length might consist of
as low as 2,500 monomers.
24
The chain length of each cellobiose molecule is 1.03 nm, and one molecule is separated from
the other by a distance of 0.835 nm Figure 2.4 b. The widely accepted structure of an
elementary cell is a monoclinic one with the following dimension:
a = 0.83 nm b = 1.03 nm c = 0.79 nm β = 84o
The crystal lattice of cellulose is further shown in Figure 2.4 c. The hydroxyl oxygen’s of
two adjacent chains are at a distance of only 0.25 nm in the direction of the a-axis, allowing
complete hydrogen bonding (Rydholm 1965).
The structure cellulose fibres as detailed in Figure 2.4 is maintained by a combination of
intra-chain and inter-chain hydrogen bonds, and inter-sheet van der Waals forces (and
possibly inter-sheet hydrogen bonds) (O’Sullivan 1997, Kontturi et al. 2006, Walker 2006).
The intra-chain hydrogen bonds form between O3-H…O5 and O2-H…O6 give cellulose
their linear and rigid properties. The inter-chain hydrogen bonds between the O6-H…O3 and
O3…H-O6 form flat sheets of cellulose (Walker 2006). The sheets stack on top of one
another to form the characteristic three-dimensional structure. In this direction, only
hydrogen atoms project out axially, allowing the sheets to pack closely together so that van
der Waals forces stabilize the packing. There is also the possibility of an additional hydrogen
bond that is formed between the sheet layers that join 06-H…05. While hydrogen bonds are
relatively weak (with bond strengths of O-H…O, 10-40 kJ mol-1 and C-H…O, 2.5 kJ mol-1)
compared to covalent bonds (with bond strength of 200-800 kJ mol-1), they are formed along
the entire length of the chains (Walker 2006). This abundant hydrogen bonding network and
high level of organization give cellulose fibres exceptional strength.
25
2.1.2 Cellulose biosynthesis
Cellulose is biosynthesized in the plasma membrane and it is understood that an enzymatic
complex called Cellulose Synthase assists the process (Giddings et al. 1980) as detailed in
Figure 2.5. The hexagonal rosette is a complex of six subunits and each of these subunits
produces about six cellulose chains. Therefore, the 36-glucan chains produced by a terminal
complex can instantaneously associate by hydrogen bonding to form an elementary
microfibril. These elementary microfibrils can further associate with each other forming
bigger ones.
Figure 2.5 Cellulose biosynthesis: Rosette and a newly synthesized microfibril (Saxena and
Brown 2005)
2.1.3 Cellulose microfibril morphology within the cell wall
A number of structures for the microfibrils have been proposed. These models differ mainly
in the presentation of the less ordered regions. The most commonly considered microfibril
structure is that of a flat ribbon, having one dimension four times than the other (Preston and
CMF
synthase
26
Kuyper 1951). This structure, as illustrated in Figure 2.6, consists of several totally
crystalline micelles (M), between and around which lay the less regularly arranged glucose
chains, i.e., the paracrystalline region (P). This structure is widely considered as the basic
structural unit of cellulose. Linkages may also be made between the outside of the microfibril
and non-cellulose polysaccharides (N).
Figure 2.6 Microfibril structure (M: micelles; P: paracrystalline region; N: non-cellulose
polysaccharides (Preston and Kuyper 1951).
2.1.4 Organizationofhemicelluloseswithinplantcellwall The term hemicellulose was originally proposed to denote a substance somewhat similar in
character to cellulose and easily extractable, in comparison to cellulose, from higher plants
by aqueous alkaline solutions (O'Dwayer 1923, Aspinal 1959). Unlike cellulose,
hemicelluloses are branched heteropolysaccharides having β- (1→4)-linked backbones with
an equatorial configuration made up of pentoses such as xylose, and arabinose, hexoses such
as mannose, glucose, and galactose, and/ or sugar acids such as - D-glucuronic, - D- 4-O-
methylgalacturonic, and - D-galcturonic acids (Timmel 1964). Hemicelluloses are non-
27
crystalline, with a low molecular weight range of 10 - 26 kDa and a relatively low degree of
polymerization of 80 – 200 (Glaudemans and Timell 1958, Goring and Timell 1960,
Koshijima 1965). Typical structures and composition of hemicelluloses of soft wood are
shown in Figure 2.7 and Table 2.1 respectively.
Table 2.1 Hemicellulose characteristics in soft wood (Timmel and Syracuse 1967)
Wood type
Hemicellulose Percent of wood
Composition Parts Linkages Mol.wt. (Mn)
Mol.wt. (Mw)
Soft wood
Arabino-4-O-methylglucurono xylan Galacto glucomannan (water-soluble) Galacto glucomannan (alkali-soluble)
10-15 5-10 10-15
β-D-Xylp 4-O-Me-α-D-GlupA L-Araf β-D-Manp β-D-Glup α-D-Galp O-acetyl β-D-Manp β-D-Glup α-D-Galp O-acetyl
10 2 1.3 3 1 1 0.24 3 1 0.1 0.24
1-4 1-2 1-3 1-4 1-4 1-6 1-4 1-4 1-6
>120 >100 >100
>150 >150
28
Figure 2.7 Typical structures of hemicelluloses in wood: (a) 4-O-methy-D-Glucuronoxylan
(b) D-galacto-D-mannan; (c) D-gluco-D-mannan; (d) (L-arabino)-4-O-methyl-D-glucurono-
D-xylan.
(a)
(b)
(c)
(d)
29
2.1.5 Interaction of hemicellulose with cellulose
Five theories were reported to explain the network formation of hemicelluloses with cellulose
microfibrils (Cosgrove 2005)
(i) Hemicellulose spontaneously bind to the surfaces of cellulose microfibrils and
tether adjacent microfibrils together (Hayashi 1989, Fry 1989)
(ii) Polysaccharides (xyloglucan) might become entrapped during formation of the
ordered microfibril (b in the Figure 2.8) and the untrapped remainder of the
polysaccharides would be free to bind to other cellulose surfaces or to other
matrix polymers, thereby anchoring the microfibril firmly to its neighbors (Baba
et al. 1994, Hayashi et al. 1994, Brett and Walsron 1996).
(iii) Cellulose microfibrils might be simply coated with hemicelluloses (xyloglucan)
that adhere to other matrix polysaccharides, without direct linkage between
microfibrils, (Talbolt and Ray 1992)
(iv) Hemicelluloses (blue strands) might be covalently attached to pectin
polysaccharides (red strands), forming a macromolecule that anchors the
microfibrils by sticking of polysaccharides to cellulose surfaces (Thompson and
Fry 2000)
(v) Polysaccharides (arabinoxylans) might bind cellulose and be cross linked by
ferulic acid esters (A-F-F-A) (e in the Figure). This type of phenolic crosslink
might also crosslink other hemicelluloses and pectins, particularly in grass cell
walls (Zykwinska et al. 2005)
30
Figure 2.8 Polysaccharide network in lignocellulosic matrix (Cosgrove 2005)
2.1.6 Cellulose microfibrils and its structural role in plant cell wall
The formation of plasma membrane and middle lamella begins with cell division followed by
the deposition of the primary cell wall (Hodge and Wardrop 1950, Fengel 1984). As the cells
grow and expand, microfibrils synthesized by the plasma membrane are deposited onto the
primary cell wall. The microfibrils form the structural framework in to which all other
polysaccharides like hemicellulose, lignin, pectin, and cell wall proteins are deposited.
As the cells matures and stop growing, the secondary cell wall is deposited on to the inside of
the primary wall in a series of three layers – S1, S2 and S3 as depicted in Figure 2.9 (a). The
orientation of microfibrils deposited into these layers vary with S1 layer having a dispersed
mode, S2 layer having an alignment with high fibrillar angle and S2 having well aligned
microfibrils with a low fibrillar angle as shown in Figure 2.9 (b). There exists a good
31
correlation between the fibrillar angles in the S2 layer to the strength of the fibre (Barnett and
Bonham 2004).
(a) (b)
Figure 2.9 Plant Cell, (a) Structure of cell wall, (b) Orientation of microfibrils in cell wall, P
– primary cell wall, S1 and S2 – secondary cell wall (Barnett and Bonham 2004).
The primary cell wall, in addition to the presence of cellulose, hemicellulose and pectin, is
distinguished by the presence of cell wall proteins that have structural and functional role.
The cell wall proteins are particularly rich in amino acids hydroxyproline, proline, and
glycine.
2.1.7 Role of cell wall proteins in cell expansion and growth
The enlargement of plant cell wall is a key determinant of plant morphogenesis. Various cell
wall structure models have been put forward to explain how these structures accommodates
P
S2
S1
32
surface expansion and incorporates newly secreted polymers into the expanding load bearing
network – (a) sticky network model in which the cellulose microfibrils are directly tethered
to one another by xyloglucans that bind non-covalently to the surface of the cellulose (Fry
1989, Hayashi 1989 ), (b) the multi-coat model in which the cellulose is embedded in layers
of hemicelluloses and pectin fill the interstices (Talbott and Ray 1992), and (c) stratified
hybrid model, in which the pectin is largely restricted to the space between distinct layers or
lamellae of cellulose microfibrils which are connected laterally by xyloglucans as depicted in
Figure 2.10 (Ha et al. 1997). The concept of primary and secondary wall loosening agents
and the role of expasin, xyloglucan endotransglycosylase and endo-1,4--D-glucanase are
well established (Cosgrove 2000).
Figure 2.10 Hybrid model (Cosgrove 1997). Figure 2.11 Structure of Expansin
(Cosgrove 1997).
Cosgrove defines a primary wall loosening agent as a substance that induces stress relaxation
of the cell wall, resulting in secondary water absorption by the cell and expansion of the wall
(Cosgrove 1997). The substance could be an enzyme that acts to cleave a structural polymer
or to break apart associations between wall polysaccharides or it could be a wall polymer
33
with the ability to insert itself in the wall by competitive binding and exchange with load
bearing components of the wall. A secondary wall loosening agent does not itself cause the
crucial relaxation and expansion of the wall, but act as a synergist to make the wall more
sensitive to the action of primary agents. Hydrolytic enzymes, that can partially cleave the
matrix polysaccharides and thereby reducing the matrix viscosity, could achieve this. Of the
potential wall loosening agents, expansin has been found to be a primary wall loosening
agent where as other lytic wall enzyme might act a secondary wall loosening agent.
2.1.7.1 Expansin
These proteins bind strongly to cell walls, apparently to the crystalline regions of the
cellulose microfibrils (Mc Queen and Cosgrove 1995). They do not exhibit hydrolytic
activity against cell wall components. Expansin’s unique physical effects on plant cell walls
include rapid induction of wall extension and stimulation of stress relaxation (Mc Queen et
al. 1992). Expansin’s do not progressively weaken the cell wall, nor do they cause a lasting
change in wall structure (Fenwick and Cosgrove 1999), except that the wall is longer and
thinner after it extends. No ligands or cofactors have been identified as necessary for
expansin action, although thiol reductants help to maintain stable activity. The structure of
expansin is depicted in Figure 2.11.
Normally, expansin is a very minor component of the cell wall, for instance, in cucumber
seedlings expansin is found at roughly one part protein to 5,000 parts cell wall (on a dry mass
basis) and induces wall extension when added in amounts as low as 1:10,000. Binding and
activity both saturate at a protein-to-wall ratio of about 1:1,000 (Mc Queen and Cosgrove
1994). From the architecture of the plant cell wall, one might expect expansin’s loosening
34
action to result from hydrolysis of the matrix polymers that hold the cellulose microfibrils in
place. However, attempts to identify hydrolytic activity by expansin against the major wall
components have failed (McQueen and Cosgrove 1992, Mc Queen et al. 1995). Attempts to
mimic expansin’s effects with wall hydrolases have also failed (Cosgrove and Durachko
1998); such enzymes can reduce the mechanical strength of the cell wall, but do not induce
extension and stress relaxation, at least not until the wall is degraded to the point of
mechanical failure. Test based on a broad range of proteases (Cosgrove 1989) failed to detect
significant wall-extension activity. Thus, proteolysis does not appear to be a probable
mechanism of expansin action.
Figure 2.12 A model for expansin’s wall-loosening action. (Cosgrove 2000)
Cellulose microfibrils are connected to each other by glycans (thin yellow and red strands)
that can stick to the microfibril surface and to each other (Figure 2.12). The expansin protein
(blue) is hypothesized to disrupt the bonding of the glycans to the microfibril surface (a) or to
each other (b) Under the mechanical stress arising from turgor, expansin action results in a
displacement of the wall polymers (c) and slippage in the points of polymer adhesion
(compare b and c).
35
In contrast to these conventional enzymatic theories of wall loosening, an alternative
mechanism was proposed in which expansins weaken the non-covalent binding between wall
polysaccharides, thereby allowing turgor-driven polymer creep (McQueen and Cosgrove
1994). In this scheme, as shown in Figure 2.12, expansin would make use of the mechanical
strain energy in the wall to catalyze an inchworm-like movement, or repetition, of the wall
polymers. Expansin movement may be confined to lateral diffusion along the surface of the
cellulose microfibril, has been observed for other polysaccharide-binding proteins. Such
contained diffusion would enable expansin to search the microfibril surface, locally
loosening its attachment to the matrix, and allowing chain movement and stress relaxation.
This model of expansin action is consistent with the lack of progressive weakening of the cell
wall by expansin and also fits the biophysical characteristics of cell-wall extension, the rate
of which is dependent on the wall stress in excess of a minimum yield threshold (Cosgrove
1997). Other support for this mechanism comes from the finding that expansin weakens pure
cellulose paper, whose strength derives from non-covalent binding between cellulose fibres
(McQueen and Cosgrove 1995), and artificial composites composed of cellulose alone or
cellulose and xyloglucan (Whitney et al. 2000). Finally, although expansin lacks hydrolytic
activity by itself, it does enhance the hydrolysis of crystalline cellulose by cellulases
(Cosgrove and Durachko 1998). This synergistic action may indicate that expansin makes
glucans on the surface of the microfibril more accessible to enzymatic attack by cellulases.
2.1.7.2 Xyloglucan endotransglycosylase (XET) and endo-1,4--D-glucanase (EG)
Both of these enzymes have been proposed to act as cell wall loosening agents by cleaving
the xyloglucans that hypothetically knit cellulose microfibrils together. There is substantial
evidence that xyloglucan chains undergo substantial modification after their secretion into
36
the wall, and that these two classes of enzymes are likely to be of prime importance in these
modifications (Fry 1989, Hoson and Masuda 1995, Thomson and Fry 1997). XET is an
enzyme that cleaves xyloglucan mid-chain, forming a covalently bonded xyloglucan-enzyme
intermediate that can then transfer the xyloglucan to the non-reducing end of another
xyloglucan chain (Steel and Fry 1999, Sulova et al. 1998). The result of this molecular
grafting reaction is a new hybrid xyloglucan plus a fragment of the original chain. Like XET,
EG may cleave xyloglucans, but addition of water results in hydrolysis rather than
transglycosilation.
2.2 Biodegradation of Cellulosic Materials
In this section, a brief look at the role and recent developments in enzymes technology in
natural fibre processing, especially pulp and paper industry is presented followed by an
extensive overview of the structure and function of various cellulolytic enzymes and their
mode of action on cellulose.
Cellulose biodegradation involves principally hydrolytic depolymerization of cellulosic
materials to lower molecular weight compounds, finally yielding monomeric glucose units by
catalysis of cellulose enzyme systems of living organisms. Microorganisms cause major
deterioration of cellulose and wood based lignocellulosic materials. Fungi are well known for
their biocatalytic activity on cellulosic materials. Two major approaches are pursued in the
utilization of cellulosic materials (1) complete hydrolysis of cellulosic materials to their
monomeric glucose units and their subsequent utilization or modification – biofuels and
biopolymers, (2) Modification of cellulosic fibres – pulp and paper and biocomposite. The
major enzymes involved in these approaches are detailed in section 2.2.1 and 2.2.2 below.
37
2.2.1 Cellulosic fibre sector
The application of enzymes in fibre processing is mainly directed towards the degradation or
modification of hemicelluloses and lignin while retaining the cellulosic portion. The
enzymatic approach in the fibre processing sector has been based on the idea of selected
hydrolysis of certain components or limited hydrolysis of several components in the fibres.
Some of the important areas of applications are (1) Fibrillation, inter-fibre bonding and
strength enhancement (Bolaski et al. 1959, Yerkes 1968, Nomura 1985), (2) Drainage (
Fuentes and Robert 1988), (3) Modification of pulp properties (Uchimoto et al. 1988, Paice
and Jurasek 1984, Senior et al. 1988, Jurasek 1988), (4) Enzymatic pulping (Nazareth and
Mavinkurve 1987, Sharma 1987, Morvan et al. 1990), (5) Enzymatic pretreatments for
bleaching (Tolan and Canovas 1992, Scott et al. 1993, Viikari et al. 1990).
2.2.2 Enzymatic hydrolysis of cellulose
In wood, crystalline cellulose microfibrils are tightly packed in a complex network of
hemicellulose constituents and lignin. Most cellulolytic microorganisms produce, in addition
to cellulases that hydrolyze the -1,4-glucosidic bonds, a number of other cell-wall-
degrading enzymes, e.g. ligninases, xylanases, pectinases, etc. Only a few microorganisms
produce a complete set of enzymes capable of degrading native cellulose efficiently, e.g.
soft-rot fungi, such as Hypocrea jecorina (formerly known as Trichoderma reesei). Aerobic
and anaerobic microorganisms use different strategies to feed on cellulose. Whereas aerobes
generally secrete a set of individual cellulases, some anaerobes have evolved a multi-enzyme
complex- cellulosome, which is associated with the cell surface of the microorganism,
reviewed recently by Bayer et al. (2004).
38
The cellulolytic enzyme systems in fungi can be divided into two groups. The white-rot
fungi, such as Phanerochaete chrysosporium and soft-rot fungi, such as Hypocrea jecorina
and Penicillum pinophilum have complete cellulolytic enzyme systems capable of the
breakdown of crystalline cellulose to glucose. They consist of several secreted enzymes
acting at the ends (exoglucanases) or in the middle (endoglucanases) of the cellulose chains.
The released cellobiose is hydrolyzed to glucose by β-glucosidases. The second group of
fungi reportedly degrades cellulose by means of oxidative components together with
endoglucanases, but lack the strict cellobiohydrolases. A representative of this mechanism is
the cellulolytic system of the brown-rot fungus Postia placenta (Kleman-Leyer et al. 1992).
2.2.3 Functional classification
Cellulases can be classified by different means, according to their substrate specificities,
reaction mechanisms or structural similarities. Cellulases have traditionally been classified
into two distinct classes: cellobiohydrolase (1,4-β-D-glucan cellobiohydrolase, EC 3.2.1.91)
and endoglucanase (1,4-β-D-glucan glucanohydrolase, EC 3.2.1.4), based on their activity
toward a wide range of substrates. This is rather difficult, since the enzymes have
overlapping specificities toward substrates which themselves are poorly defined.
By definition, cellobiohydrolases release cellobiose from the non-reducing ends of the
cellulose chain, but the experimental evidence for this assumption is obscure. Enzyme
kinetics on soluble oligosaccharides and structural data on enzyme-oligosaccharide
complexes have showed that some cellobiohydrolases may have opposite chain-end
preferences (Barr et al. 1996, Divne et al. 1994, Koivula et al. 1998). Cellobiohydrolases are
thought to work processively, that is, one enzyme molecule can release several cellobiose
39
units from the cellulose chain without leaving the substrate. Cellobiohydrolases show little or
no activity on substituted celluloses, such as carboxy methyl cellulose (CMC), but
microcrystalline cellulose with relatively low DP is relatively rapidly degraded (Beguin and
Aubert 1994). Endoglucanases cut cellulose chains at random positions in less crystalline
regions, creating new chain ends. Extreme endoglucanases, often called CM-cellulases
(carboxymethyl-cellulases) have little activity towards crystalline cellulose, but hydrolyze
readily CMC, acid-swollen cellulose and even barley -glucan in a random fashion, resulting
in a rapid fall in the degree of polymerization (Kleman-Leyer et al. 1994).
The classification of cellulases as purely endoglucanases or exoglucanases is not absolute
and is an over-simplification, since several studies indicate that several cellobiohydrolases
can attack also the internal glucosidic bonds of the cellulose chain (Ståhlberg et al. 1993,
Armand et al., 1997, Boisset et al. 2000). Also, several endoglucanases have been shown to
hydrolyze cellulose processively, which is a common property of cellobiohydrolases
(Reverbel-Leroy et al. 1997, Gilad et al. 2003, Cohen et al. 2005, Zverlov et al. 2005). As a
result, cellulases seem to have a more or less continuous spectrum of properties ranging from
virtually random endoglucanases to highly processive strict cellobiohydrolases (Teeri 1997,
Hildén and Johansson 2004).
2.2.4 Hydrolytic mechanism
In glycosyl hydrolases, enzymatic hydrolysis of the glycosidic bond usually takes place via
general acid/base catalysis, which requires two critical residues: a proton donor (HA) and a
nucleophile/base (B-). Two aspartic or glutamic acid residues provide this catalytic activity.
40
Two different mechanisms can be distinguished- retaining and inverting mechanisms as
shown in Figure 2.13. In both cases the acid-base (HA) protonates, leaving glycosidic oxygen
with concomitant formation of a partial positive charge on the C1 carbon. In the inverting
mechanism, the base (B-) deprotonates a water molecule, which then attacks the C1 carbon
of the glucose ring in an SN2 type displacement reaction, resulting in inversion of the
configuration at the anomeric carbon C1. In the retaining mechanism, a glycosidic bond is
hydrolyzed via two single displacement steps. First, the nucleophile (B-) attacks directly the
C1 carbon, resulting in a covalent intermediate between the enzyme and the substrate and the
first product is released. In the second step, the acid-base activates a water molecule by
abstracting a proton from it, promoting an attack on the C1 carbon.
Recently, a fundamentally different glycosidase mechanism has been unveiled for NAD+ and
divalent metal ion-dependent GH4 glycosidases whereby hydride abstraction at C3 generates
a ketone, followed by deprotonation of C2 accompanied by acid-catalyzed elimination of the
glycosidic oxygen and formation of a 1,2-unsaturated intermediate. This 1, 2 unsaturated
species undergoes a base-catalyzed attack by water to generate a 3-keto derivative, which is
then reduced by NADH to complete the reaction cycle (Lodge et al. 2003, Rajan et al. 2004,
Varrot et al. 2005).
41
Figure 2.13 The two major mechanisms of enzymatic hydrolysis of the glycosidic bond.
(Koshland 1953). (A) The retaining mechanism. (B) The inverting mechanism.
2.2.5 Glycoside hydrolase families
The glycoside hydrolases can be classified into structurally related families based on
similarities in the distribution of hydrophobic amino acids in their sequences (Henrissat
1991). Up to date, 106 families have been distinguished and the continuously updated
information is available on Carbohydrate Active Enzymes Database server
(http://www.cazy.org/CAZY) (Coutinho and Henrissat 1999). Cellulolytic enzymes are
grouped into at least 14 families. The family classification reflects the structural features of
the enzymes and the evolution of glycoside hydrolases. Some families contain enzymes with
different substrate specificities.
42
2.2.6 Fungal cellulases
Cellulases face the difficult problem of working on a solid substrate. Most of the fungal
cellulases share a common molecular organization where a large catalytic domain (CD) is
connected by a highly glycosylated linker-peptide to a small carbohydrate-binding module
(CBM). Upon limited proteolysis with papain the enzymes can, in many cases, be cleaved
easily into the two functional domains (Tomme et al. 1988). The active site of a cellulase
consists of multiple binding sites for glucose units, which enhances the probability for the
enzyme to remain bound to the substrate after a catalytic cycle and thereby work processively
(Divne et al. 1994). The three hexagons in the CBM indicate the aromatic residues
responsible for interaction with the hydrophobic face of every second pyranose ring. The
gray area in the Figure 2.14 represents the loops covering the substrate-binding site. (Hildén
and Johansson 2004).
Generally, cellobiohydrolases have a tunnel-shaped active site, whereas the active site for
endoglucanases is more open, forming a cleft or groove, allowing the enzyme to bind to the
middle of the substrate chain and cleave it. Since some cellobiohydrolases also can perform
these internal cuts, the loops closing the tunnel must be flexible to allow a cellulose chain to
enter the active site. The CBD’s are believed to play an important role in cellulose
hydrolysis. Although these domains do not affect the activity of cellulases toward soluble and
amorphous substrates, they significantly enhance the capacity of the enzymes to hydrolyze
crystalline cellulose. The first structure of a fungal CBD was determined by nuclear magnetic
resonance (Kraulis et al. 1989). The CBD’s of the fungal cellulases have a wedge-shaped
fold containing a basic structure of a distorted β-sheet of three short antiparallel strands. One
face of the wedge is planar and contains three conserved aromatic amino acids separated by a
43
distance corresponding to the length of the repeating unit in cellulose, cellobiose (Tomme et
al. 1995). This interaction is often supplemented by polar residues forming hydrogen bonds
(Tormo et al. 1996). The other surface is rougher and less hydrophilic in character.
Figure 2.14 A sketch of fungal cellulase in action (Hildén and Johansson 2004).
2.2.7 Specific aspects of cellulase kinetics
The enzymatic degradation of solid cellulose is quite complicated, which takes place at a
solid-liquid phase boundary where the enzymes are the mobile components. Zhang and Lynd
(2004) investigated the various properties of the substrate that influence the kinetics of
enzymatic hydrolysis of cellulose and are:
(a) the crystallinity and the type of the cellulose crystals,
(b) the degree of polymerization,
(c) the molecular weight distribution,
44
(d) the accessible surface for the enzymes and the microstructure of the cellulose
surface
The enzymatic degradation of cellulose is characterized by a rapid initial phase followed by a
rather slow secondary phase that may last until all substrate is consumed. This has been
explained most often by the rapid hydrolysis of the readily accessible fraction of cellulose
followed by a strong product inhibition and slow inactivation of absorbed enzyme molecules
(Converse et al. 1988). The erosion of the cellulose surface by the cellulases has been
proposed as one of the rate retardation factors (Väljamäe et al. 1998). It has been shown that
the surface area of cellulose that is accessible to cellulase is the most important factor in
determining initial rates of hydrolysis (Thompson et al. 1992, Helle et al. 1993). The
efficiency of cellulose hydrolysis by an individual enzyme is dependent on the degree of
polymerization and crystallinity of the substrate. Generally, cellobiohydrolases are relatively
more active towards highly crystalline substrates with relatively low DP, endoglucanases
have only very limited action on crystalline substrates, but hydrolyze readily amorphous
cellulose (Henrissat et al. 1985).
2.2.8 Role of the cellulose binding domain (CBD)
The adsorption of cellulase enzyme on the surface of cellulose is a prerequisite step for
hydrolysis. The presence of the CBD enhances the overall binding efficiency of the cellulases
to the cellulose, and the activity is seen to correlate well with higher activity towards
insoluble cellulose (Tomme et al. 1988, Gilkes et al. 1988, Ståhlberg et al. 1993,
Reinikainen et al. 1995). At the same time, the strong binding via CBD to the cellulose
45
surface can lead to a population of nonproductively bound enzymes (Ståhlberg et al. 1991).
In addition to anchoring the enzyme molecules to the cellulose surfaces, the disruption of
cellulose microfibrils by family II CBD’s has been reported (Din et al. 1991). Many
cellulases, like other enzymes acting on polymeric substrates, have been thought to work
processively, i.e. they can perform several hydrolytic events without dissociating from the
substrate. In general, the kcat (reaction rate) value for oligosaccharide hydrolysis increases
with the DP of the substrate. Lee et al. (1983) found that the cellulase catalysis does not
significantly affect the DP of a solid cellulose substrate. These authors proposed that
cellulose chains are peeled off progressively from the fibrils by the cellulase enzymes, since
the DP remains constant during the course of hydrolysis. The thinning of cellulose
microfibrils by the action of cellobiohydrolases has been observed several times (Chanzy and
Henrissat 1985, Boisset et al. 2000, Lee et al. 2000). The processivity hypothesis is supported
by the structure of the CD of cellulases, which contain multiple binding sites for the glycosyl
units. The cellulose chains are held together in microcrystals by van der Waals interactions
and hydrogen bonds. In order to separate a cellulose chain from the cellulose crystal, a
cellulase molecule has to overcome an energy barrier in breaking the interaction between
cellulose chains, which may slow down the rate of hydrolysis (Skopec et al. 2003). Since the
active site of a cellobiohydrolase often is a long tunnel, the cellulose chain is enclosed and
held in the active site of the enzyme by numerous interactions, which makes the enzyme less
likely to dissociate after each hydrolytic step and thus compete more efficiently with the
interactions driving the cellulose chain back onto the cellulose crystal (von Ossowski et al.
2003).
46
The efficient hydrolysis of cellulose needs interplay between those two domains.
Experiments with H. jecorina Cel7A mutants with deletions in the hinge region connecting
the CD and CBD have shown that sufficient distance between CD and CBD is needed in the
cellulases for efficient hydrolysis of crystalline cellulose (Srisodsuk et al. 1993). Recently,
Mulakala and Reilly (2005) presented a model based on the interaction energies and forces
on cellooligosaccharides computationally docked to CD and CBD, where CBD wedges itself
under a free chain end on the crystalline cellulose surface and feeds it to the CD active site
tunnel. The energy for cellulose structure disruption comes ultimately from the chemical
energy of glycosidic bond breakage (Sinnott 1998).
2.2.9 Synergisms of cellulase systems
Native crystalline cellulose substrates are not so rapidly hydrolyzed by cellulases as the
amorphous, or water soluble, cellulose derivatives. So, truly cellulolytic fungi have long been
thought to have a specific factor or enzyme system affecting the supramolecular structure of
cellulose crystallite (Reese et al. 1950). Selby and Maitland (1967) and then Wood and
coworkers (1968, 1972) recognized the specific factor as one of the enzymes of the cellulase
system.
Effective degradation of crystalline cellulose requires cooperation between different types of
cellulases. This cooperation, resulting in higher total activity, is called synergism. Several
types of synergism between cellulases have been described: (a) the cooperation between
endoglucanases and cellobiohydrolases (endo-exo synergism), (b) between two
cellobiohydrolases (exo-exo synergism), (c) between endoglucanases, cellobiohydrolases and
47
-glucosidase, and (d) between enzymes from different sources. Generally, synergism
between endo and exoenzymes is highest on semicrystalline cellulose of high DP, lower on
amorphous cellulose and non-existent on soluble cellulose derivatives (Nidetzky et al. 1994,
Samejima et al. 1998).
The synergism has been found to be dependent on the relative proportions of the enzyme
components (Henrissat et al. 1985) and also on the degree of saturation of the substrate with
the enzymes, decreasing at higher enzyme concentration (Woodward et al. 1988). It is
generally assumed that the mechanism of endo-exo synergism can be discussed in terms of
sequential action where by the endoglucanase initiates random attack and the new chain ends
generated are then hydrolyzed by the endwise-acting cellobiohydrolase. Furthermore, -
glucosidase can work in synergy with cellulases by removing the cellobiose produced.
2.3 Research Rationale
The underlying principle of the approach adopted in this research is:
(1) Certain enzymes with similar sequence similarities with that of expansin as discussed in
Section 2.1.7, has the capacity to cleave the dominant non-covalent hydrogen bonding
between cellulose microfibrils and between microfibrils and hemicelluloses.
(2) Hydrogen bonding between the hemicelluloses and the cellulose microfibrils is the main
barrier in the isolation of cellulose microfibrils from delignified woody fibres.
48
It is envisioned that energy efficient isolation of cellulose nanofibres from cell wall by
loosening up the cell wall structure is achievable by one of the following mechanisms or a
combination of these in an environment catalyzed by extra-cellular enzymes.
a) Treatment of fibres with fungus that secrete enzymes that are typically known as cell
wall loosening agent (CWLA). These proteins bind strongly to cell walls, apparently
to the crystalline regions of the cellulose microfibrils (McQueen and Cosgrove 1995).
They do not exhibit hydrolytic activity against cell wall components. They weaken
the non-covalent binding between wall polysaccharides (McQueen and Cosgrove
1994). Certain enzymes like xyloglucan endotransglycosylase and endo-1,4--D-
glucanase have been proposed to act as cell wall loosening agents by cleaving the
xyloglucans that hypothetically knit cellulose microfibrils together. There is
substantial evidence that xyloglucan chains undergo substantial modification after
their secretion into the wall, and that these two classes of enzymes are likely to be of
prime importance in these modifications (Fry 1989, Hoson and Masuda 1995,
Thomson and Fry 1997). XET is an enzyme that cleaves xyloglucan mid-chain,
forming a covalently bonded xyloglucan-enzyme intermediate that can then transfer
the xyloglucan to the non-reducing end of another xyloglucan chain (Steel and Fry
1999, Sulova et al. 1998).
b) Treatment of fibres with fungus that secrets enzymes that has cellulolytic activity.
The cellulolytic activity of exoglucanase, endoglucanase and β-glucosidase secreted
by certain fungus can be utilized to partially loosen up the cellulose structure by their
hydrolytic activity on cellulose chain. This activity can also increase the accessibility
of CWLA to new cellulose chain and vice versa.
49
c) Treatment of fibres with xylanases that are produced by a wide variety of fungus, can
be a practical approach to loosen up the microfibril as it is believed that hemicellulose
tether the microfibrils and contributes to the rigidity of the wall. The hydrolysis of
the xylose-polymer backbone mainly involves two groups of enzymes, namely β-1,4-
xylanases and β-xylosidases (Wong et al. 1988).
d) Treatment of fibres involving an environment that contains a combination of enzymes
described in the above three approach namely CWLA, cellulolytic enzymes and
xylanases that can work synergistically to loosen the cellulose microfibrils can prove
valuable in the isolation of cellulose microfibrils. The selection of a microorganism
that can grow on fibrous cellulosic substrate and possessing the activities detailed
above would be a right candidate for the proposed research.
2.4 The Choice of Ophiostoma Ulmi
Ophiostoma Ulmi is a pathogenic causative agent of Dutch elm disease of native European
and North American Elm trees. In the opinion of some researchers, cell wall degrading
enzymes produced by Ophiostoma Ulmi are involved in the penetration of cell walls by
fungal hyphae. The cellulolytic enzymes that dissolve cellulose may be responsible for direct
penetration of the host cell walls by fungal hyphae. The cellulolytic activity (exoglucanase,
endoglucanase and β-glucosidase) of Ophiostoma Ulmi have been reported by various
researchers (Beckman 1956, Elgersma 1976, Przybyl et al. 2006).
Many observations indicate that extracellular xylanases might be involved in the interaction
between plants and pathogens. The hydrolysis of the xylose-polymer backbone mainly
involves two groups of enzymes, namely β-1,4-xylanases and β-xylosidases both of which
50
are secreted as extracellular enzymes by Ophiostoma Ulmi when grown in woody substrate
(Wong et al.1988, Binz and Canevascini 1996).
Certain enzymes like xyloglucan endotransglycosylase and endo-1,4--D-glucanase have
been proposed to act as cell wall loosening agents by cleaving the xyloglucans that
hypothetically knit cellulose microfibrils together. Xyloglucan endotransglycosylase and
endo-1,4--D-glucanase that are secreted by Ophiostoma Ulmi as part of their cellulase and
xylanase extracellular enzyme system has the capacity to cleave the non-covalent hydrogen
bonding that forms the major cohesive force between microfibrils and hemicelluloses.
The discussed above, growth and pathogenic propagation characteristic of Ophiostoma Ulmi
and the release of related extracellular enzymes can hypothetically provide an environment in
which the enzyme activities would assist in loosening the cellulosic structure and their
subsequent isolation.
51
Chapter 3 Research Hypothesis and Objectives
3.1 Hypothesis
Plant cell wall degrading enzymes produced by pathogenic fungus Ophiostoma Ulmi can
cleave the predominant non-covalent hydrogen bonding that exist between elementary
cellulose microfibrils and the hemicellulose in plant cell wall and substantially alleviate the
energy required to isolate them through subsequent mechanical defibrillation methods.
3.2 Research Objectives
a) Establish a fungal fibre treatment that will weaken or break the predominating
hydrogen bonds that exist between the cellulose microfibrils and the hemicellulose.
b) Investigate the effect of fungal treatment on fibres with respect to morphological,
physical and mechanical characteristics.
c) Scale-up and demonstrate the bio-treatment chemistry and the cellulose nano-fibre
isolation process.
d) Develop an energy consumption model and estimate the energy consumption in
isolation of cellulose nanofibres.
52
Chapter 4 Approach
The objective of this work is to first identify a fungal treatment that can produce exo-cellular
plant cell wall degrading enzyme and to investigate if a pre-treatment of this nature has any
impact on the internal structure of fibre cell wall.
The fibre source for this research is bleached kraft pulp with 14 - 15 % hemicellulose and
remaining cellulose. As described in the experimental section, 4.2.1, this research will
investigate the effect Ophiostoma Ulmi on fibres and its impact on the isolation of nanofibres
from the cell wall. The fungus Ophiostoma Ulmi, from previous studies (Wong et. al.1988,
Beckman 1956, Elgersma 1976, Przybyl et al. 2006, Binz and Canevascini 1996), has found
to have only a moderate effect on the cellulose part of the fibre and at the same time has
found to have a profound effect on the surface acid – base characteristics of the fibres. As a
first step, a thorough investigation on the effect of Ophiostoma Ulmi treatment of fibres on
the composition of the treated fibres like weight loss, cellulose and hemicellulose content and
the degree of polymerization of cellulose was performed.
Two different strategies were used for the bio-treatment of fibres - (a) the fibres were treated
with the Ophiostoma Ulmi in its original form without any refining; (b) the fungal treatment
was carried out after a light mechanical refining of the fibres to enhance the enzymatic
accessibility of the fibre.
Conventional PFI high shear refining and cryocrushing to isolate the cellulose microfibrils
from the fibres will follow the fungal treatment procedures discussed above. After the effect
53
of the fugal treatment is established, wet mill refining was investigated to develop a single
unit process for the isolation of cellulose nano-fibre following the bio-treatment. The effect
of fungal treatment on the fibres and its subsequent effect on the isolation of cellulose
microfibrils and their mechanical strength properties were investigated based on the
following methods:
a) The overall yield of cellulose nanofibres was compared for various treatments against
the yield from untreated fibres. The diameter distribution of nanofibres isolated was
also compared. Direct concentration measurement by drying, and optical techniques
like SEM and TEM was used to quantify the yield and distribution.
b) Weight loss of the fibre after enzyme treatment, fibre cellulose and hemicellulose
content, changes in the degree of polymerization, was determined using the
techniques described in experimental section, 5.2.1.
c) Effect of bio-treatment on the nature and density of Hydrogen bonding in the fibres
was investigated using FT-IR and 13C NMR Spectroscopy.
d) Effect of bio-treatment on the crystalline structure of the cellulose fibres was
investigated using FT-IR, X-Ray Crystallography and 13C NMR Spectroscopy.
e) Cellulose nanofibres isolated was investigated for their mechanical strength
properties.
f) Process scale-up demonstration of the wet mill refining unit operation and estimation
of the energy requirement for the isolation of nano-fibres was carried out.
g) An estimate of the actual energy requirement for isolating cellulose nano-fibres from
bio-treated fibre and untreated fibres was done to verify the impact of bio-treatment
on the energy consumption.
54
h) Rittinger’s constant (C’), which is a measure of the hydrogen bonding within the
fibre, for bio-treated fibres and untreated fibres was calculated using the energy
required for isolating cellulose nano-fibre and the diameter distribution.
55
Chapter 5 Experimental
5.1 Materials
5.1.1 Fibres
Bleached Kraft pulp fibres from black spruce were used as the starting material for the
isolation of microfibrils.
5.1.2 Enzymes
The fungus Ophiostoma ulmi sensu lata, obtained from Elm tree infected with Dutch elm
disease, was supplied by the Great Lakes Forest Center, Canada Forest Services, Sault Ste.
Marie, Ontario.
5.2 Methods
a) Biotreatment: Twenty-four grams of oven dry bleached kraft pulp fibre, based on the
capacity of the PFI mill, was soaked overnight and was thoroughly dispersed in 2
liters of water using a fibre disintegrator and autoclaved for 20 minutes. Disintegrator
is a fibre dispersion equipment with a pulp holder and a high speed mixing
mechanism that is typically used to make pulp suspension from fibre sheets. 100 ml
of Ophiostoma Ulmi fungal culture was added to this fibre suspension in a sterile
flask with 2 gL-1 sucrose and 1 gL-1 of yeast extract to support the fungal growth. The
fungus was left to act on the fibres at room temperature for four different time
duration, 24 hours, 48 hours, 72 hours and 96 hours with slow agitation. The fibres
56
b) were autoclaved (110 °C for 20 minutes) after their respective treatment time, washed
and made into sheets of 10% fibre consistency ready for the mechanical refining.
c) High Shear Refining: 24 g oven dry weight of this pulp was soaked in tap water for
30 minutes, and dispersed in a fibre disintegrator in 2 L of water for 15 min. The fibre
suspension was then filtered, made in to a thick sheet and water content adjusted to
obtain a consistency of 10%. The fibre was then sheared thoroughly in a laboratory
PFI mill for up to 125,000 revolutions. The refiner produced high shear capable of
forming individual microfibrils at the surface of the fibre bundles. PFI helps in
internal defibrillation.
PFI mill is commonly used for refining under laboratory conditions (Phillips et al.
1970). Four primary effects in the refining process that are related to the fibre
properties and the fibre-water system are (Watson and Phillips 1964), a) intra-fibre
bond breaking (internal fibrillation), b) external fibrillation, c) production of fines and
d) fibre shortening. Study of hand sheet properties and microscopic examination of
fibres have shown that all these effects take place in a PFI mill (Watson and Phillips
1964). During the operation of the mill, the head containing the bars are pushed at
one side of the casing as shown in Figure 5.1. As elaborated by Murphy (1962), the
stock in a PFI mill is centrifuged against the wall of the mill house. It is carried
around in a narrow band toward the beating gap where it converges with the moving
bars of the roll. The mill house at that point forms a smooth bedplate. In this work,
the aim of pre-refining of the fibres in a PFI mill is to bring about internal
defibrillation and also to increase the number of fibre ends which is achieved through
fibre shortening.
57
Figure 5.1 Working principle of PFI Mill (Chakraborty et al. 2005).
d) Cryocrushing: The refined fibres were then subjected to cryocrushing in which the
fibres were frozen using liquid nitrogen and high shear was applied using a mortar
and a pestle. This step is critical in liberating the microfibrils from the cell wall. The
cryocrushed fibres was then dispersed into water suspension using a disintegrator and
filtered through an 80-mesh filter (Chakraborty et al. 2005) to remove any un-
separated or large fibre remains. The filtrate, a dilute water suspension of microfibrils
was used for further investigation.
e) Homogenization: In order to understand the effect of pre-shearing of fibres before
bio-treatment on the fibre, a 0.5 to 1% suspension of the fibres with varying degree of
pre-shearing was prepared and passed through the high pressure homogenizer. The
resulting fibres were examined using Transmission Electron Microscope (TEM). A
high pressure homogenizer consists of a pressurizing chamber capable of pressurizing
the fibre suspension up to 3500 bar and an emulsifying cell. The fibre suspension was
58
introduced in the product inlet port which goes through a system of high pressure
cylinders and is pumped out as a high velocity jet through the orifice of the nozzle at
high pressure. This strong jet enters the absorption cell or the emulsifying cell where
forces of shear, cavitation, and impact break down and homogenize the feed, giving
out the product which is thoroughly mixed, uniform and has smaller particles.
f) Wet mill refining/grinding: The equipment used for this purpose is quite analogous
to the wet mills encountered in the food and agriculture industry, particularly for
crushing granular materials. It contains two disks with bars and grooves, one stator
disk and the other rotor. The fibre suspension, typically 2 – 3% was forced between
the rotor and the stator disk where the fibres are subjected to repeated cyclic stresses
during the refining process. The fibre suspension is forced in to confined zone whose
thickness is called the gap clearance. The distance between the opposing bars is
adjustable and ranges from 0.1 micron to 5 micron. During the bar crossing, the fibres
are compressed and sheared leading to internal and external defibrillation, hydration
and fibre breakage. The fibre suspension can be put through the refining process more
than once depending on the degree of refining required. One fibre suspension through
the unit is defined as one pass.
The grinder used in this research is a SUPER Masscolloider from Masuko Sangyo
Co. Ltd., Japan. The grinder disk, both stator and the rotor, used was specifically
made for pulverizing and emulsifying fibrous materials.
59
5.2.1 Characterization of fibres
a) Scanning electron microscopy (SEM) was used to understand the surface
morphology of the Ophiostoma Ulmi treated fibres. Hitachi S2500 model was used
for this purpose. A drop of a highly diluted suspension of the treated fibres was
placed on a SEM stub and allowed to dry under room conditions overnight. Three (3)
test stubs were prepared for each sample analyzed. The dried fibre samples were then
gold coated to provide a conductive surface for the SEM analysis. A voltage of 10 kV
was used to obtain the SEM image.
b) Transmission electron microscopy (TEM) was used to obtain images for
quantifying the distribution of nanofibre diameter. The instrument used was Philips
CM 201 model. A drop of highly diluted suspension of the nano-fibres was placed on
a pre-coated TEM grid and allowed to dry under room condition overnight. Three test
grids were prepared for each sample analyzed. The grid was then placed in the sample
chamber and images from several points of interest were taken for the analysis. The
diameter of the cellulose nano-fibres were found out using image processing program
called UTHSCA Image tool downloaded from
http://ddsdx.uthsca.edu/dig/itdesc.html. Minimum of fifty diameter values were
recorded for a single nano-fibre at various points along its length. The number
average diameter in a sample was calculated averaging at least five hundred average
diameter values of individual cellulose nanofibres.
60
c) Fibre composition: The Holocellulose (cellulose and hemicellulose) content of the
fibre after the bio-treatment was determined using the procedure adapted from Zobel
et al. (1966). The value reported is an average of three test results.
i. - Cellulose
- Cellulose was determined from holocellulose after hydrolyzing the
heterogeneous polysaccharides using 17.5% alkali. Holocellulose prepared for -
Cellulose determinations were not oven dried, but air dried for overnight in a
conditioning chamber.
ii. Hemicellulose
Hemicellulose was extracted from the Ophiostoma ulmi treated fibre using 4 wt%
NaOH solution. Wood was treated with 4% NaOH (solid to liquor ratio 1:10), at
a temperature of 70 °C for 2 hr. The reaction mixture was filtered and washed
with 50 mL of water to remove the solubles from the fibre. The filtrate and
washings were pooled together and neutralized to a pH of 4.6 using acetic acid.
The precipitated hemicellulose was allowed to settle down overnight and
centrifuged to separate from the liquid phase. The precipitate was then washed
with 95% ethanol and freeze dried.
d) Degree of polymerization of cellulose: ASTM D 1795-96 procedure was used to
estimate the average degree of polymerization of bleached kraft fibre. This test gives
the value of intrinsic viscosity, which can be converted to the average degree of
polymerization (DP) and polymer chain length.
61
14 mL of 0.5 M cupriethylene diamine (CED) solution was transferred in a 300 size
Ostwald viscometer placed in a water bath at 25C. After 5 minutes, when the
solution had reached the bath temperature, suction was applied, and the solution was
drawn past the upper mark of the viscometer. Time t0 required by the meniscus to
pass from this mark to the mark below the lower bulb was recorded.
Fibre samples were dissolved in CED in concentrations of 0.4 g dL-1, 0.2 g dL-1 and
0.1 g dL-1. Three samples were tested for each of these concentrations. 14 mL of the
solution was passed through the viscometer, and the time “t” for the solution to pass
between the two marks in the viscometer was noted. From these experiments, relative
viscosity (rel) for each sample at each of the three concentrations was obtained from
the ratios of outflow times as follows:
rel = t/t0
There are a number of different equations that are used to correlate the viscosity of a
non-Newtonian fluid with concentration. One equation in this regard is Martin's
equation (Utracki and Simha 1963, Bird et al. 1977), which is used in ASTM D 1795-
96 to relate the viscosity of the different cellulose solutions in CED with their
concentrations:
ln [(rel - 1)/c] = ln [] + k [] c
where k = 0.30, a constant, and c is the cellulose concentration (g dL-1) in the
solution. A plot of ln [(rel - 1)/c] was drawn against c, and the line was extrapolated
62
to the point c = 0. The intercept gives the value of intrinsic viscosity [],g dL-1. To
obtain the average degree of polymerization of the cellulose molecules, the value of
intrinsic viscosity was multiplied by 190 (ASTM D 1795). The length of each
cellobiose unit is 1.03 nm. A cellobiose molecule, in turn, is composed of two glucose
units. Therefore, the average cellulose chain length for the fibre was calculated by
multiplying the average degree of polymerization by 1.03/2.
e) Fibre length: Fibre length of the original fibres and the nanofibres were determined
in a Fibre Quality Analyzer (FQA). A very dilute suspension of the fibres to be
analyzed were prepared in a two littler flask and placed on the suction the chamber of
the Fibre Quality Analyzer. The accumulated results of the analysis were obtained in
a printed format with the following measurements –Weighted Fibre Mean Length,
Percentage fines, Mean Curl and Mean Kink. The Mean Length is the arithmetic
average contour length of all detected fibres in a given sample and the Weighted
Mean Length which is the length weighted average of all detected fibres was used
here to compare the samples.
5.2.2 Characterization of Ophiostoma Ulmi treated fibres
a) Weight loss: The weight loss of the bio-treated fibres was determined by simple
difference between the weight of fibres before and after treatment. The samples were
weighed to 0.01 mg scale. Six samples per treatment were used for weight loss
determination.
63
b) Surface morphology: High-resolution optical microscopy and SEM was used to
understand the surface characteristics of the biotreated fibres.
c) Fibre composition: The cellulose and hemicellulose content of the fibre after the bio-
treatment was determined using the procedure described above for the untreated fibre,
5.2.1.
d) Degree of polymerization of cellulose: The degree of polymerization of the bio-
treated fibre was determined as discussed for untreated fibre, section 5.2.1.
e) Hydrogen bond type and density: The chemical changes introduced by treatment on
fibres were investigated using FT-IR. Treated fibres saturated with deuterium (D2O)
were also analyzed using FTIR to confirm the hydrogen bonding associated with the
crystalline part of the cellulose. The detailed sample preparation and FITR spectra
determination procedure is provided in section 5.2.4.
f) Crystallinity determination by XRD and FT-IR: The details are provided in
section 5.2.5.
g) Mechanical strength: The mechanical strength properties of the cellulose
determined using micro tensile testing machine.
The cellulose nanofibre sheets for the mechanical test were prepared by vacuum
filtration. 0.5% nanofibre suspension was stirred for 45 minutes and the suspension
was then filtered on a Buchner funnel using 200 mesh filter screen. A basis weight of
65 gm-2 was targeted for the test papers. After filtration, the wet nanofibre sheets were
placed between filter papers and then between two metal plates and dried at 40 C for
about 24 hours. Three nanofibre sheets with basis weight 65±2 gm-2 were chosen as a
source for the test samples. The sheets were then cut to micro tensile test specimens
64
using standard ASTM die. Three to four test specimens per nanofibre sheet for a total
of 10 were tested on micro tensile testing machine.
5.2.3 Characterization cellulose nanofibres
a) Determination yield of cellulose nanofibres: The overall yield of cellulose
microfibrils from the two processes was calculated based on the solid content of the
microfibril suspension of specific amount of PFI refined fibres. Here, a specific
amount of PFI refined fibres were cryocrushed and dispersed in to a 2-liter water
suspension using a disintegrator. This suspension was then filtered through 60 meshes
and the solid content of this water suspension of nanofibres calculated as the overall
yield. The number average fibre diameter distribution was evaluated from TEM
images using image-processing software as described in section 5.2.1.
b) Scanning electron microscopy (SEM), and transmission electron microscopy
(TEM) were used characterize and understand the surface morphology of the treated
fibres and cellulose microfibrils isolated. The procedures were as described in section
5.2.1.
5.2.4 Hydrogen bond type and density
The hydrogen bond type and density associated with the treated and untreated fibre was
investigated using FT-IR. Bruker IFS 66 FT-IR spectrometer was used for all the
measurements. In order to understand some of the details of the band shapes and unresolved
band components Fourier self-deconvolution and Fourier derivation were applied using Opus
software supplied by Bruker.
65
5.2.4.1 FT-IR spectroscopy
There are many powerful tools which may be used to investigate cellulose chemistry and its
structure. X-ray, electron diffraction and microscopy, FTIR, FT – Raman and solid state
NMR are the major of which FT-IR is one of the most versatile techniques available to study
the hydrogen bond formation. Using FT-IR, Fengel (Fengel 1992, 1993) analyzed the
hydroxyl absorption bands by deconvoluting the spectra of cellulose. Michell (Michell 1988,
1990, 1993) used the 2nd derivative mode of the FT-IR spectra to improve its resolution
considerably. Sugiyama et al. (1991), and Michell (1993) used FT-IR spectral data to
establish and confirm the findings of Wiely and Atalla (1987), the crystalline dimorphism of
native cellulose i.e. the two phase, cellulose I and I β were considered to differ in their
hydrogen bonding rather than in their conformation. Tashiro and Kobayashi (1991) used IR
spectra to determine the O-H stretching frequencies due to intra- and intermolecular
hydrogen bonds in cellulose I and II. FTIR will be used here to study the hydrogen bond
density and their nature in the original fibre and the treated fibre.
5.2.4.2 Management of FT-IR spectra
All FT-IR spectra were base line corrected and compensated for CO2 and H2O before any
manipulation.
a) Fourier self-deconvolution: The aim of Fourier self-deconvolution is to enhance the
apparent resolution of a spectrum, or to decrease the line width. Fourier Self-Deconvolution
(FDS) assumes that the spectrum to be measured consist of well resolved lines which have
been convoluted by the same type of line broadening function (LBF). If the shape and width
of the LBF are known, its effects can be arithmetically excluded from the spectra. In general,
66
the deconvolution corresponds to a multiplication of the interferogram I (x) using the exp
(a*x) Deconvolution function for Lorentzian and exp (a*x*x) for Gaussian shapes. The
deconvolution factor is the maximum value of these functions at the end of the interferogram.
To avoid the increase of noise caused by the Deconvolution, a Blackman-Harris apodization
is simultaneously performed on the spectra.
b) Evaluation: This procedure primarily intends to calculate results from existing FT-
IR spectra.
i. Curve fit: Curve fitting procedure was used to calculate single components in a
system of overlapping bands. A model consisting of an estimated number of bands generated
is essential for the fitting calculation. The IR absorption bands for the OH stretching regions
were deconvoluted before the curve fitting procedure. To improve the calculation, the peaks
needs to be resolved and their number, position and areas were determined. The number of
peaks and their position was determined by the point at which the second derivative of the
spectrum contained peaks of interest. The position of the deconvoluted bands can give us
information about the nature and type of OH functionality.
ii. Region of interest, 3000 cm-1 - 3600 cm-1: An analysis of the various peaks in this
region should provide the modes of OH stretching vibrations. All the bands in this group are
due to OH stretching modes in alcoholic groups. A general property of hydrogen bonds,
including intramolecular hydrogen bonds, is that O-H bands which are due to hydrogen
bonded O-H groups are much more intense than the corresponding O-H bands with no
hydrogen bonds (Pimentel and McClellan1960). Another general property of hydrogen bonds
is that the stronger the hydrogen bond, the greater is the intensity of the corresponding O-H
band and the greater is the shift towards lower wavenumbers. All this implies that the bands
67
in the 3000 cm-1 - 3600 cm-1 region are mainly O-H bands. A small part at 3450 cm-1 or
higher wavenumbers can be assigned to O-H modes that are either free or weakly hydrogen
bonded.
The spectra of the hydroxyls in cellulose are relatively intense for any orientation of the fibre,
which is an indication that the intra- and intermolecular hydrogen bonds are well developed
in cellulose. A study by Suukhov (2003) has demonstrated that the stretching vibrations of
hydrogen bonded hydroxyl groups in the ordered regions of cellulose consist of five
components. Two of them have primarily parallel polarization, one has primarily
perpendicular polarization while the other two have mixed polarization, i.e. they appear in
both the spectra. The parallel polarized component has a maxima at 3370 cm-1 and 3275 cm-1
which corresponds to stretching vibrations of hydroxyl groups constituting intramolecular
hydrogen bonds and perpendicularly polarized component with maxima of 3410 cm-1 that
correspond to the band of stretching vibrations of hydroxyl groups forming intermolecular
hydrogen bonds. Mixed polarization usually has maxima at 3340 cm-1 and 3290 cm-1 and is
supposed to form multicentre bonds.
The stretching vibrational motions of OH groups are isolated from the other internal motion
of the cellulose molecule as in the case with CH motion. Hydroxyl stretching motion,
however, can couple with lattice modes due to their involvement intermolecular hydrogen
bonds. The relationship between band intensities and the orientation of the fibre axis relative
to polarization vector E, we should be able to suggest the orientation of the OH groups.
68
5.2.4.3 Sample preparation
Since handling individual fibre is not practically possible, thin sheets of treated and untreated
fibres with 15 – 20 gm-2 basis weight were prepared. This sample cut in to appropriate size
can be used with FT-IR spectroscopy. The sample cellulose fibre sheets were saturated with
heavy water (D2O) vapor to record the molecular spectra of the hydroxyl groups. The
hydroxyl groups in the unordered regions accessible to deuterium exchange were substituted
by OD groups, and the OH groups in the ordered regions which remained unsubstituted will
appear in the spectra in the 3000 cm-1 - 3600 cm-1 frequency region characteristic of their
stretching vibrations.
5.2.5 Crystallinity determination by FT-IR
The degree of crystallinity of the treated and untreated fibre samples was determined using
FT-IR. Dried cellulose samples were made into a pellet with KBr powder (1:5, cellulose:
KBr) and analyzed by FT-IR spectroscopy. The FT-IR spectra (256 scans, 4 cm−1) were
determined by the diffuse reflectance method (DRIFT) using a Bruker Tensor 27
spectrometer.
5.2.6 Crystallinity determination by X-ray diffraction
The bio-treated and untreated fibres were used as samples for the X-Ray Diffraction. X-ray
diffractometry curves were recorded in reflection mode by a Rigaku RU-200 BH with Ni-
filtered Cu-K radiation ( = 0.1542 nm) generated at 40 kV and 40 mA. Copper K was
eliminated by the use of nickel foil filters in the incident beam. The beam and detector slit
were set at 1° and 0.25° respectively. The scanning was made through 2 = 10° to 60°, and
intensity data was recorded with a digital recorder. Separation of peaks was done using a
69
least-squares profile fitting program, Peak fit, assuming Gaussian functions for each peak.
The diffraction angle was calibrated every time with the sodium fluoride diffraction line (d =
0.2319 nm).
5.2.7 Nuclear magnetic resonance spectroscopy (NMR)
One of the promising new experimental methods that can be applied to study the structure
and morphology of cellulose is solid state 13C nuclear magnetic resonance spectroscopy. 13C
spectra obtained with the combined techniques of proton-carbon cross polarization, high
power proton decoupling, and magic angle spinning can reveal unique information related to
various crystal forms in cellulose, identify different forms with in the same grouping such as
cellulose I. The distinction and identification arise due to either a morphological feature like
varying degree of disorderness and / or those associated with chains in the interior of the
crystallites.
The spectra were collected on a Bruker AVANCE 300MHz NMR Spectrometer using a 4
mm magic angle spinning (MAS) probe. The powder samples were spun at a rate of 5kHz at
ambient temperature (20 oC). The 13C-NMR spectra were acquired using cross-polarization
(CP) using a 3.5 ms contact time. The chemical shifts were reported with respect to
tetramethylsilane (TMS) as the shift reference.
70
Chapter 6 Results and Discussion
Bio-treatment of Natural Fibres: Effect on Fibres and Yield of Cellulose Nanofibres
6.1 Introduction
The results presented in this section focuses on the effect of Ophiostoma Ulmi treatment of
wood fibres on yield and diameter distribution of cellulose nanofibres obtained through
subsequent mechanical process as described in section 5.2. The action of fungal treatment on
the structural morphology and the capacity of the bio-treatment to facilitate the internal
defibrillation are extensively detailed here through scanning electron microscopy and
transmission electron microscopy. The results of the experiments to determine the impact of
bio-treatment and its extent of impact on the physical and chemical characteristics of the
fibre like weight loss, cellulose and hemicellulose content and the degree of polymerization
of cellulose in the treated fibres are also detailed in this chapter.
6.2 Bleached Kraft Fibre Characterization
The bleached Kraft pulp from northern black spruce was analyzed using procedure outlined
in Section 5.2.1 to obtain the Cellulose and hemicellulose content. The result is identified in
Table 6.1. The results are in line with typical analysis available for bleached Kraft pulp from
northern black spruce.
71
Table 6.1 Composition of bleached kraft wood pulp fibres.
Composition %
Cellulose 85 ± 1
Hemicellulose 14 ± 1
Others 1 ± 0.5
6.3 Effect of Ophiostoma Ulmi Bio-treatment of Fibres on Cellulose Nanofibres Yield
and Fibre Diameter Distribution
One of the major challenges impeding the isolation of cellulose nanofibres on a sizable scale
for any potential application is the high-energy requirement associated with neutralizing the
predominating hydrogen bonds between the cellulose microfibrils and also between
microfibrils and hemicellulose. Cellulose nanofibres were generated and isolated in
laboratory through a combination of high energy refining in a PFI mill, and subsequent
cryocrushing under the presence of liquid nitrogen. PFI refining of the fibres was carried out
here to cause internal defibrillation – a process where only a minor amount of the total
energy supplied to the PFI machine is utilized.
The interfibrillar hydrogen bonding energy has to be overcome in order to separate the
microfibres into individual entities. This association energy for cellulose ranges between 19
and 21 MJ/kg. mol, with 20 MJ/kg.mol being used as an average value in most cases (Nissan
et al. 1985). If this value is taken to be the intermolecular H-bond energy binding the fibres
72
together, then we should reasonably assume an expenditure of equal energy be supplied to
separate the microfibres into separate entities.
6.3.1 Yield of cellulose nanofibres
The yield of cellulose nanofibres determined by the procedure outlined in section 5.2 is
detailed in the Figure 6.1. The overall yield of cellulose nanofibres from Ophiostoma Ulmi
treated fibre decreased by an average of 3 to 4%. The decrease in yield of microfibrils is
noticeable only after a minimum of 3 days of treatment which indicates that the fungus needs
a minimum of 3 days to establish an active community and produce enzymes in an effective
quantity. The decrease in yield of nanofibres from treated fibres was found to be insignificant
compared to untreated fibres and this could be attributed to the loss of minor extractable
components from the fibre due to fungal treatment.
Figure 6.1 Yield of cellulose nanofibres with different Ophiostoma Ulmi treatment
conditions.
50
60
70
80
Untreated 3 days 5 days 8days
% N
an
ofi
bre
Yie
ld
Ophiostoma Ulmi Treatment
73
The yield of cellulose nanofibre is seen to even out after 5 days treatment period and there is
no noticeable decrease with any further time of treatment. This observation contradicts with
the earlier study of the effect of Ophiostoma Ulmi on hemp fibres, which showed a
significant activity of the fungus towards cellulose accompanied by a significant loss in the
fibre strength after 3 days of treatment (Gulati and Sain 2006). The hemp fibre for the study
was raw fibre and contained lignin and pectin and lacked a clear explanation on the effect on
Ophiostoma Ulmi treatment on cellulose content and the strength.
Figure 6.2 Weight loss of fibre with different Ophiostoma Ulmi treatment condition.
The observed low cellulolytic activity of Ophiostoma Ulmi is important in this project as the
objective of this work is to bring about an internal defibrillation in the fibre by minimizing
the hydrogen bonding and weak van der Waals forces that exist between the elementary
0
0.05
0.1
0.15
0.2
0.25
0 1 2 3 4 8 12
Wt.
los
s g
m /
2 g
m f
ibre
Ophiostoma Ulmi Treatment (Days)
74
fibrils while preserving the native characteristics of the cellulose chain. The low cellulolytic
activity of Ophiostoma Ulmi was further confirmed by a study of the weight loss, cellulose
content and the degree of polymerization of the cellulose of the treated fibres. The loss in
fibre weight as depicted in Figure 6.2 showed a gradual decrease up to a maximum 7.5 % of
original fibre weight for 4 days treatment and then the change tends to be insignificant with
increased time of treatment. A similar trend is seen with respect to the cellulose content of
the treated fibres where the cellulose content is seen to decrease with the extent of treatment
and the loss of cellulose is proportionate with the weight loss of the treated fibres. The
weight loss and a proportionate decrease in cellulose content of the treated fibres imply that
the action of fungal enzymes on the fibres is mostly limited to cellulose.
Figure 6.3 Cellulose content of fibres after different Ophiostoma Ulmi treatment condition.
70
75
80
85
90
0 1 2 3 4 8 12
Ophiostoma Treatment (Days)
% c
ellu
lose
co
nte
nt
75
As discussed in Chapter 2, Section 2.1.5, hemicellulose plays a bridging role between the
cellulose microfibrils providing the structural framework to the plant cell wall. The
hemicellulose content and the percentage extractable hemicellulose in the fungus treated
fibre were determined to understand if the fungal treatment had an effect on the structure of
hemicellulose with in the cell wall structure. The absence of any protein on the surface of the
treated fibre was verified before hemicellulose extraction.
Figure 6.4 Hemicellulose and the extractable hemicellulose content of fibres after different
Ophiostoma Ulmi treatment condition.
The hemicellulose content of the treated fibres showed a gradual decrease with a similar
trend as that of cellulose. The hemicellulose content of the treated fibre tends to level of after
4 days treatment. In order to understand the effect of treatment on the structure and their
interaction with cellulose, the extractable hemicellulose were determined using extraction
76
with 4% NaOH. The quantity of extractable hemicellulose showed a significant increase with
treatment time suggesting either a breakdown of hemicellulose chain or a reduction in the no-
covalent interaction between hemicellulose and cellulose chains or both.
The average degree of polymerization (DP) and the chain length of the raw fibre and the fibre
bio-treated for 4 days are shown on Table 6.2. The change in DP after 4 days of bio-
treatment does not indicate any significant level of cellulolytic activity by the exo-enzyme
secreted by the fungus. The observation is also in line with the cellulose content of the bio-
treated fibres as shown in Figure 6.3.
Table 6.2 Average degree of polymerization and chain length for biotreated and untreated
fibres
Fibres DP Average chain length, nm
Untreated 2650 ± 12 1365 ± 6
Bio-treated, 4 days 2480 ± 18 1277 ± 9
6.3.2 Cellulose nanofibre diameter distribution
Having understood the level of Ophiostoma Ulmi activity on cellulose, it is vital to
understand the effect of Ophiostoma Ulmi treatment on the internal defibrillation tendency of
the treated fibres during the subsequent mechanical defibrillation techniques such as PFI
refining or microfluidizer homogenization. This is the first step towards testing my
hypothesis which states that enzymes can help in internal defibrillation through either
77
weakening the hydrogen bonds that exist between microfibrils or loosening up the fibrils
through controlled hydrolytic activity and hydrogen bond cleavage.
Fibres treated with Ophiostoma Ulmi were PFI refined and the number average diameter
distribution of the cellulose nanofibres isolated from untreated fibres and fibres bio-treated
for a 4 days period is detailed in Figure 6.5. The number average diameter of the cellulose
nanofibres isolated from bio-treated fibres showed a significant shift towards the lower
diameter range with the maximum yield of fibres below 100 nm range while that for the
untreated fibre were between 100 – 150 nm range. The fibre diameter distribution did not
change in an appreciable manner with increase in treatment time longer than 4 days.
Figure 6.5 Effect of Ophiostoma Ulmi treatments on number average diameter distribution
of nanofibres after PFI refining for a 4 days treatment.
0
10
20
30
0-10 10-25.0 25-50 50-75 75-100 100-150 150-250
Fibre diameter nm
% Y
ield
OS Treated
Untreated
78
The shift in cellulose nano-fibre diameter distribution curve towards the lower diameter
range for bio-treated fibre after the PFI refining is of importance in this research as this
observed phenomenon can happen only if the bio-treatment had an effect on the internal
structure of the fibres that in turn assist the internal defibrillation in the fibre during PFI
refining.
Figure.6.6 SEM - Ophiostoma Ulmi treated fibre before PFI refining.
SEM photomicrograph of fungal treated fibres is shown in Figure 6.6 and it shows the fungal
growth on the fibres. The mechanism of enzyme action on fibre is not apparent yet, but a
good hypothesis is that the fungal enzymes interaction of enzyme with hydrogen bonding
between cellulose microfibrils and between the cellulose microfibrils and the hemicelluloses
is responsible for the internal defibrillation in the fibre cell wall. This observation is more
visible in the TEM images of the treated and untreated PFI refined fibres (Figure 6.7). The
20 µm
20 µm
20 µm
79
fibrillation of the treated fibres as seen in Figure 6.7 (b) is more pronounced after refining
compared to the untreated fibres. The actual separation of elementary fibres takes place to a
good extent with treated fibres while PFI refining seems to have a fewer fibril separation
effect on untreated fibres. This observation can explain the difference in fibre diameter
distribution associated with treated and untreated fibres.
(a) (b)
Figure 6.7 Effect of Ophiostoma Ulmi fibre treatments on internal defibrillation - (a) TEM of
untreated fibre after PFI refining, (b) TEM of treated fibre after PFI refining.
Cryocrushing is the final step that helps in the isolation of cellulose microfibrils into
individual entity from the defibrillated fibres. The noticeable difference in fibre diameter
distribution observed between treated and untreated fibres after PFI refining as detailed in
Figure 6.5 is no more evident in Figure 6.8 which details the fibre diameter distribution of
treated and untreated fibres after cryocrushing. The frozen PFI refined fibres are subjected to
high impact force during cryocrushing. This has a very destructive effect on the cell wall
32000x 500 nm
32000x 500 nm
80
structure and can release its structural content and the better defibrillation attained by
refining in treated fibres is subdued.
Figure 6.8 Effect of Ophiostoma Ulmi treatments of fibres on the yield and distribution of
cellulose nanofibres after refining and cryocrushing.
The fibre diameter distribution trend is similar for both treated and untreated fibres with
microfibrils from treated fibres showing a slight shift towards lower diameter range with
major fraction of fibres in the 0 – 50 nm range. The cellulose nanofibres isolated from treated
fibres after cryocrushing showed very clear and distinct separation as compared to cellulose
microfibrils isolated from untreated fibre as seen in Figure 6.9. This narrow shift in the fibre
diameter distribution as shown in Figure 6.8 stems from the fact that isolation of cellulose
-10
0
10
20
30
40
50
60
0-10 10.0-25 25-50 50-75 75-100 100-150 150-250
Fibre diameter nm
% y
ield
OS Treated
Untreated
81
microfibrils into distinct entity is not as good with untreated fibres as with treated fibres and
is evident from a closer look as shown in Figure 6.10.
Figure 6.9 TEM of cellulose microfibrils isolated from Ophiostoma Ulmi treated fibres
through PFI refining and cryocrushing.
Having demonstrated the encouraging effect of Ophiostoma Ulmi treatment on the
defibrillation of fibres during subsequent refining, it would be worthwhile adopting a one
step process like wet mill refining to authenticate the effect of fibre treatment and see if
fewer passes through the wet mill are enough to isolate cellulose microfibrils as compared to
the number of passes required for untreated fibres.
10000x 2 microns
2 micron
2 micron 10000x 2 micron 2 micron 10000x
82
Figure 6.10 TEM of cellulose microfibrils isolated from untreated fibres through PFI
refining and cryocrushing.
6.4 Conclusions
1. Ophiostoma Ulmi treatment of fibres was shown to produce internal defibrillation in
the fibres. This is a major breakthrough in the isolation of cellulose nano-fibres.
2. Ophiostoma Ulmi treatment of fibres has shown to have only a limited activity
towards cellulose which is of beneficial to this research as this minimizes the loss of
cellulose and the mechanical strength properties of the isolated cellulose nano-fibres.
This is evident from the degree of polymerization and the chain length of the
cellulose nanofibres isolated from treated and untreated fibres.
3. The increase in extractable hemicellulose content of the fungus treated fibres implies
a potential breakdown in the cellulose-hemicellulose interaction which is
predominantly non-covalent hydrogen bonding.
10000x 2 microns10000x 2 microns
10000x 2 microns
83
Chapter 7 Results and Discussion
Bio-Treatment of Natural Fibres: Impact of Pre-refining of Fibres on Bio-treatment
Efficiency and Nanofibre Yield
7.1 Introduction
The results presented in this chapter attempts to throw light on the impact of pre-shearing of
wood fibres would affect the bio-activity of the fungus and how such an approach would be
beneficial to the ongoing attempts to isolate cellulose nanofibres in an economical manner.
The effectiveness of pre-refining of fibres in enhancing the bio-activity of the fungus towards
fibre is evaluated here through (a) the weight loss of the bio-treated fibres, (b) cellulose
content of the bio-treated fibres and (c) the number of revolutions required in a high shear
refiner to affect appreciable nanofibre isolation. The yield of cellulose nano-fibres and their
number average diameter distribution was also determined and discussed.
7.2 Pre-Refining of Wood Fibres and Its Effect on Bio-treatment
The research in the direction of utilizing enzymatic pre-treatment of fibres as a means to
economically isolating cellulose nanofibres from plant fibre cell wall hit the crossroad when
we successfully used a fungus isolated from a fungus infected Dutch Elm tree for this
purpose. As mentioned earlier, we are exploring various methods to improve the
effectiveness of this bio-treatment by fibre modification before the bio-treatment step. The
impact of pre-refining of the fibres to be bio-treated is investigated here with respect to
overall cellulose nanofibre yield and cumulative diameter distribution of the nanofibres
84
obtained from the fibres affected with various levels of pre-refining and also the weight loss
and the cellulose content of these bio-treated fibres.
Figure 7.1 Effect of pre-refining of fibres on the effectiveness of bio-treatment – fibre
weight loss and cellulose content.
One of the major challenges impeding the isolation of cellulose nanofibres or microfibrils on
a sizable scale for any application is the high-energy requirement associated with neutralizing
the predominating hydrogen bonds between the cellulose microfibrils and also between
microfibrils and hemicellulose. The cellulose content and the weight loss of the bio-treated
fibres subjected to pre-shearing at 5000, 10000 and 15000 revolutions in a PFI mill is
identified in Figure 7.1. The cellulose content of the bio-treated fibres tend to decrease with
the degree of pre-shearing while the weight loss show a proportionate increase under same
75
77
79
81
83
85
87
89
Unrefined 5000 10000 15000
Pre-refining (PFI Revolution)
% C
ellu
los
e
0
0.02
0.04
0.06
0.08
0.1
We
igh
t lo
ss
g /
2 g
fib
re
85
conditions of pre-shearing. This should be attributed to the increased accessibility for the
enzymes secreted by Ophiostoma Ulmi to the internal structure of the fibre.
7.3 Cellulose Nanofibres Overall Yield and Diameter Distribution
The overall yield of cellulose nanofibres from bio-treated fibres did not show any significant
decrease (Chapter 6) while the % yield of cellulose nanofibres based on their diameter
distribution showed a substantial shift towards a lower diameter range. A good reason for this
effect may be attributed to the hydrogen bond specific activity of the plant cell wall loosing
enzymes (CWLE) secreted by the Ophiostoma Ulmi. The cell wall degrading enzymes like
Xyloglucan endotransglycosylase (XET) and endo-1-4-β-D glucanse (EG) are known to
neutralize the non-covalent hydrogen bonding mainly between the hemicellulose and the
cellulose microfibrils. Xylanases like β-1-4 Xylanases and β-Xylosidases which is secreted
by Ophiostoma Ulmi is known to hydrolyze the xylose polymer backbone. Ophiostoma Ulmi
cellulolytic enzymes like exoglucanase, endoglucanase and β-glycosidase which through
mild cellulolytic activity can provide better access to CWLE and the Xylanases. Pre-shearing
of the fibres before fungal treatment can further enhance the fibre internal accessibility to the
enzymes secreted by Ophiostoma Ulmi enhancing the internal defibrillation.
The versatility of cellulose nanofibres especially in nano bio-composites field is well founded
and has generated a lot of interest in the scientific community. The approach of utilizing
enzymes to disengage the hydrogen bonds between the microfibrils in an attempt to isolate
them in a sizable quantity had delivered interesting results.
86
Cumulative yield and diameter distribution of cellulose nano-fibres isolated from untreated
and bio-treated wood fibres pre-refined to various degrees is illustrated in Figure 7.2. The
yield of cellulose nanofibres isolated from the pre-refined fibres in the < 50 nm diameter
range has improved appreciably compared to the yield of cellulose nanofibre isolated from
unrefined wood fibres. Over 95% of the cellulose nano-fibres isolated from pre-sheared bio-
treated fibres had diameter < 50 nm compared to approximately 78% from fibres without any
pre-shearing before bio-treatment.
Figure 7.2 Effect of pre-refining of fibres on the effectiveness of bio-treatment.
The level of pre-refining of fibres above 5000 revolutions in a PFI mill does not seem to have
any visible improvement in the yield in the 0-50 nm range. The overall yield of cellulose
nanofibres from pre-refined wood fibres showed a marginal but obvious drop compared to
the yield of cellulose nanofibres from unrefined fibres which may be attributed to loss of
0
20
40
60
80
100
Microfibril diameter (nm)
Cu
mil
ati
ve
% Y
ield
Unrefined
5000 PFI revolution
10000 PFI revolution
15000 PFI revolution
0-10 10-25 25-50 50-75 75-100 100-150 150-2500
20
40
60
80
100
Microfibril diameter (nm)
Cu
mil
ati
ve
% Y
ield
Unrefined
5000 PFI revolution
10000 PFI revolution
15000 PFI revolution
0-10 10-25 25-50 50-75 75-100 100-150 150-250
87
cellulose and hemicellulose due to improved cellulolytic enzyme in pre-refined fibres. The
weight loss of fibres and the cellulose content of the fibres as depicted in Figure 7.1 clearly
support the above statement of increased enzymatic access due to pre-refining of the fibres.
Figure 7.3 TEM images of cellulose microfibrils isolated from cellulose fibres with various
levels of pre-refining followed by bio-treatment and 10 pass homogenization; (a) Unrefined
(b) Refined-5000 revolution (c) Refined–15000 revolutions
(a) (b)
(c)
88
As the extent of pre-refining increased there was a proportional increase in the weight loss
and a decrease in the cellulose content of the fibres. TEM images of cellulose nanofibres
isolated from these pre-refined and subsequently bio-treated fibres are shown in Figure 7. 3.
(a) Unrefined (b) Refined – 5000 PFI revolution
Figure 7.4 TEM images of cellulose fibres with various levels of pre-refining followed by
bio-treatment and 5 pass homogenization.
A certain level pre-refining of fibres to be bio-treated is believed to bring about two major
physical changes in the fibres that is believed to aid in the acceleration of the bio-activity of
the fungal enzymes towards the fibre. These two physical characteristic of the fibres are
a) A certain degree of internal defibrillation in the fibre would facilitate the easy
diffusive movement of enzymes secreted by the fungus and also increases surface
area for enzyme activity.
89
b) Increased number of fibre ends that can facilitate the easy separation of cellulose
microfibrils and also aid in the axial entry of enzymes in to the fibre structure which
is critical in a fibre with low level of internal defibrillation.
Figure 7.5 TEM images of pre-refined cellulose fibre end showing extensive defibrillation
after bio-treatment and 5 pass homogenization
Internal defibrillation of the fibres to be bio-treated is an important step in improving the
accessibility and action of the enzymes secreted by the fungus to the microfibril surface. A
careful observation has shown that, upon high shear refining, the separation of nano-sized
microfibrils begins to occur at the surface of the fibre in case of wood fibres that are neither
bio-treated nor pre-sheared; while for fibres that are not subjected to pre-shearing before bio-
treatment, a low level of internal nanofibre separation was also noticed. Fibres that are pre-
pre-sheared and bio-treated showed a substantial level of internal defibrillation and nanofibre
90
isolation up on subsequent high shear refining. These observations are detailed in Figure 7.4.
The effectiveness of bio-treatment and the defibrillation of fibres that helped the isolation of
nanofibres seem to improve with increase in fibre ends as depicted in Figure 7.5. These
physical changes to wood fibres can favor the bio-activity of the enzymes due to improved
fibre internal surface accessibility. Thus pre-refining of wood fibres in a PFI mill is an easy
and excellent technique to accelerate the bio-treatment and hence improve the separation of
cellulose nanofibres from plant cell wall.
7.4 Conclusions
The following conclusions are drawn from the above discussion
1. Pre-refining of wood fibres before the bio-treatment process has found to improve the
% yield of cellulose nanofibres in the less than 50 nm diameter range.
2. Pre-refining of wood fibres can loosen up the fibre cell wall structure and increase the
internal accessibility and also shorten the fibre leading to more fibre ends which in
turn has a favorable effect on the fungal treatment and the isolation of cellulose
nanofibres.
91
Chapter 8 Results and Discussion
Bio-treatment of Natural Fibres: Effect on Hydrogen Bonding Network
8.1 Introduction
This chapter focuses on understanding the effect of the bio-treatment on the hydrogen bond
density and their nature in the treated fibre. FT-IR is used extensively in exploring the OH
absorption bands in the 3000 to 3400 cm-1 region. FT-IR and Solid state 13C Nuclear
Magnetic Resonance spectroscopy is also used to understand the density of hydrogen
bonding in the treated and untreated cellulose and to confirm the structural disorder in the
crystalline region in the cellulose. In order to support the findings from FT-IR and Solid state
13C Nuclear Magnetic Resonance spectroscopy, this chapter also intend to introduce and
discuss some key findings of a parallel research (Draper 2010) that focused on isolation and
identification of the extracellular enzymes secreted by the fungus Ophiostoma Ulmi.
Some very specific properties of hydrogen bonds, including intramolecular hydrogen bonds
that are very beneficial in this investigation are
1. O-H... bands which are due to hydrogen bonded O-H groups are much more intense than
the corresponding O-H bands with no hydrogen bonds (Pimentel and Mc Clellan 1960,
Y. Marechal 1987).
2. Stronger the hydrogen bond, the greater the intensity of the corresponding O-H band and
the greater its shift towards lower wave numbers.
92
All this implies that the bands in the 3000 – 3600 cm-1 region are mainly O-H bands. The
small part that 3450 cm-1 or higher wavenumbers may be assigned to O-H modes that are
either free or weakly hydrogen bonded.
A study by Sukhov (2003) has demonstrated that the stretching vibrations of hydrogen
bonded hydroxyl groups in the ordered regions of cellulose consist of five components. Two
of them have primarily parallel polarization, one has primarily perpendicular polarization
while the other two have mixed polarization, i.e. they appear in both the spectra. The parallel
polarized component has maxima at 3370 cm-1 and 3275 cm-1 which corresponds to
stretching vibrations of hydroxyl groups constituting intramolecular hydrogen bonds and
perpendicularly polarized component with maxima of 3410 cm-1 that correspond to the band
of stretching vibrations of hydroxyl groups forming intermolecular hydrogen bonds. Mixed
polarization usually has maxima at 3340 cm-1 and 3290 cm-1 and is supposed to form
multicentre bonds.
8.2 Determination of Relative Intensity of Hydrogen Bonding by FT-IR
8.2.1 FT-IR spectra of cellulose fibres
Since the 1950’s infrared spectroscopy has been used in cellulose research. The introduction
of Fourier transform IR spectroscopy with special equipment such as diffuse reflectance
units, microscopes, and ATR units helped to understand the molecular structure and
interactions between the cellulose chains at molecular level. However, a major problem still
persisted in fully understanding the major hydroxyl functionality in cellulose and their
interactions with other hydroxyl groups within and between the same cellulose chains.
93
Figure 8.1 FT-IR spectra of black spruce cellulose fibres with various peaks selected in 2nd
derivative mode.
An FT-IR spectrum of cellulose normally contains these information in a broad band
between 3600 and 3200 cm-1 that is associated with the stretching vibration of the OH
functionality. This broad band hides several distinct bands and therefore a lot of information
about the interactions of OH group and hence hydrogen bonds and bond patterns (Tashiro
and Kobayashi 1991, Fengal 1993, O'Sullivan 1997). Research efforts to improve the
resolution of these overlapping bands by the use of techniques like dynamic FT-IR, polarized
light and mathematical processing of spectra has gained some attention now (Mann and
Marrinan 1956, 958, Tsuboi 1957, Liang and Marchessault 1959,1960, Siesler et al. 1975,
Michell, 1990, Fengel 1992, 1993).
500100015002000250030003500
Wavenumber cm-1
0.0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Abs
orba
nce
Uni
ts
3500 3000 20002500 1500 1000 500
Wavenumber cm -1
Ab
sorb
ance
Un
its
0.0
0.
1
0.2
0.3
0.
4
0.5
0.
6
0.7
OH band
CH stretchingvibration
Crystallinity sensitive
94
Table 8.1 Bands assignment of the OH band of cellulose from literature
Band position, cm-1 Assignment Literature
3230 – 3310 O(6)H…O(3), Intermolecular Liang & Marchessault 1959, Mann &
Marrinan 1956, Siesler et al. 1975, Ivanova et al. 1989
3240 Cellulose I Sugiyama et al. 1991
3270 Cellulose I β Sugiyama et al. 1991
3305 OH intermolecular H-bond in Liang & Marchessault 1959
101 plane
3309 Intermolecular H-bond Tashiro & Kobayashi 1991
3340-3375 O(3)H…O(5) intramolecular H-bond Liang & Marchessault 1959
Ivanova et al. 1989
3372 Stretching modes of intramolecular Tashiro & Kobayashi 1991
H-bonds
3405 OH intermolecular H-bonds in the Liang & Marchessault 1959
101 plane
3410-3460 O(2)H…O(6) intramolecular H-bonds Ivanova et al. 1989
3412 O-H stretching modes Tashiro & Kobayashi
1991
intramolecular H-bonds
3540-3570 Intermolecular H-bonds Bellamy, 1975
95
(general for polyalcohols)
3555 Free OH(6) Kondo 1997
3580 Free OH(2) Kondo 1997
Tashiro and Kobayashi (1991) were able to assign specific bands for intra- and inter
molecular hydrogen bonds based on values of the 3D elastic value constants obtained by
utilizing the symmetry properties of the cellulose crystal. Peaks corresponding to the two
crystalline allomorphs, I and I β in native cellulose determined by Sugiyama et al. (1991) by
following the conversion of I to I β by FT-IR spectroscopy. The free OH groups at the C-2
and C-6 position of cellulose were assigned at 3580 and 3555 cm-1 respectively by
methylation followed by curve fitting method (Kondo 1997). A typical FT-IR spectrum of
wood cellulose fibre is shown in Figure 8.1 and detailed description of the peaks associated
with the broad OH stretching band is given in Table 8.1.
8.3 Effect of Bio-treatment on the Hydroxyl Chemistry of Cellulose Fibres
In this research, the objective of enzymatic pre-treatment of wood fibres is to bring about
internal defibrillation that can facilitate the isolation of nanofibres by subsequent high shear
refining. Having established the fact that the enzymes bring about a certain degree of internal
defibrillation in the treated wood fibre, it is important to substantiate the validity of the
research hypothesis which identify the reason for internal defibrillation as the cleave of
hydrogen bonds between elementary cellulose fibrils.
96
Figure 8.2 Normalized FT-IR spectrum of cellulose wood fibres subjected to different levels
of treatment. S1 – 4 day OS treatment, S2 – 4 day treatment with OS extract, S3 –2 day OS
treated, S4 – untreated.
FT-IR spectrum of the untreated fibres and the enzyme treated fibres are FT- deconvoluted to
improve the resolution of the OH stretching band and evaluated by curve fitting to determine
the contributions of the integral intensities of each of the components to the total intensity of
the whole band. Normalized FT-IR spectrum of cellulose fibres subjected to various levels of
enzymatic treatment is shown in Figure 8.2. The spectrum was obtained with equal amount
500100015002000250030003500
Wavenumber cm-1
0.0
0.5
1.0
1.5
2.0
Abs
orba
nce
Uni
ts
S3
S4
S1
S22900
500100015002000250030003500
Wavenumber cm-1
0.0
0.5
1.0
1.5
2.0
Abs
orba
nce
Uni
ts
S3
S4
S1
S2
S3
S4
S1
S229002900CH stretchingVibration2900
OH bandAb
so
rba
nc
e U
nit
s0
.0
0.5
1
.0
1.5
2.0
Wavenumber cm -13500 3000 2500 2000 1500 1000 500
97
of cellulose fibres and KBr to contain relative quantitative information about the OH
functionality and their interaction in the sample.
As suggested by Tripp (1971), the band at 2900 cm-1 in Figure 8.1 and 8.2, which
corresponds to C – H stretching vibration, was relatively unaffected by the degree of
enzymatic treatment or other variables. Thus, the area under this 2900 cm-1 band was selected
to serve as an internal standard.
8.4 Decrease in the Relative Intensity of Hydrogen Bond Network
In cellulose, hydroxyl is the main functional group, having various hydrogen bonding
acceptors for the formation of hydrogen bonds. The comparison of peak areas as shown in
Table 8.2 of the absorption bands associated with OH stretching vibration in the cellulose
samples indicate a difference in their absorption level suggesting a difference in the relative
availability of OH in the samples analyzed. The untreated wood fibre sample, S4 shows 2.0
IR absorption units compared to the enzyme treated fibres S1 and S2 that show a value of 1.7
and 1.65 IR absorption units respectively. The difference in this IR absorption levels between
the enzyme treated and untreated cellulose can be attributed to the difference in the density of
hydrogen bonding in the fibre. Internal defibrillation that’s happening in the fibres due to
enzymatic treatment can be due to cleavage of accessible hydrogen bonding existing in the
fibre. In bleached Kraft fibre mainly contains cellulose and hemicellulose, the accessible
hydrogen boding are those associated with the amorphous region of the fibre and those that
are associated with the interface between the cellulose and hemicellulose.
98
To better understand the effect of treatment on the OH functionality and the hydrogen
bonding type and pattern in the cellulose fibre, the OH band associated with S1, S2, S3 and
S4 are deconvoluted and curve fitted to individual bands that have a major contribution to the
overall intensity of the OH band. To improve the calculation, the peaks needs to be resolved
and their number, positions accurately determined. The major the 2nd derivative peaks in the
region of interest (3600 to 3200 cm-1) associated with FT- Deconvoluted OH band was used
for this purpose.
Table 8.2 Change in IR index of cellulose fibres with treatment*
Enzyme OH peak area 2900 / cm * Hydrogen Bonds
Treatment 2997-3742 /cm Peak area
S1 624.958 69.92 8.94
S2 642.98 70.25 9.15
S3 647.366 70.63 9.16
S4 691.227 70.52 9.80
S1 – 4 day treatment, S2 – 4 day treatment with extract, S3 –2 day treated, S4 – untreated, *
Calculated by relative peak area with 2900 cm-1 as internal standard.
99
36
02
.85
35
81
.47
35
62
.50
35
39
.41
35
21
.08
35
02
.64
34
89
.26
34
76
.79
34
56
.86
34
47
.33
34
13
.46
33
90
.44
33
73
.34
33
46
.67
33
30
.37
32
87
.36
32
70
.25
32
53
.07
32
27
.85
32
11
.78
31
73
.88
31
59
.22
31
32
.11
31
16
.74
30
98
.70
310032003300340035003600
Wavenumber cm-1
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
Abs
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e U
nits
S1
36
02
.85
35
81
.47
35
62
.50
35
39
.41
35
21
.08
35
02
.64
34
89
.26
34
76
.79
34
56
.86
34
47
.33
34
13
.46
33
90
.44
33
73
.34
33
46
.67
33
30
.37
32
87
.36
32
70
.25
32
53
.07
32
27
.85
32
11
.78
31
73
.88
31
59
.22
31
32
.11
31
16
.74
30
98
.70
310032003300340035003600
Wavenumber cm-1
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
Abs
orb
anc
e U
nits
S1
3617
.86
3602
.12
3594
.89
3579
.68
3562
.37
3538
.49
3521
.54
3502
.06
3489
.41
3479
.18
3455
.74
3414
.23
3344
.67
3270
.50
3225
.33
3211
.56
3194
.73
3173
.12
3154
.43
3136
.57
3097
.82
310032003300340035003600
Wavenumber cm-1
0.6
0.8
1.0
1.2
1.4
1.6
1.8
Ab
sorb
anc
e U
nit
s
S2
3617
.86
3602
.12
3594
.89
3579
.68
3562
.37
3538
.49
3521
.54
3502
.06
3489
.41
3479
.18
3455
.74
3414
.23
3344
.67
3270
.50
3225
.33
3211
.56
3194
.73
3173
.12
3154
.43
3136
.57
3097
.82
310032003300340035003600
Wavenumber cm-1
0.6
0.8
1.0
1.2
1.4
1.6
1.8
Ab
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e U
nit
s
S2
100
3612
.30
3594
.46
3577
.10
3570
.28
3550
.16
3532
.85
3510
.94
3487
.05
3455
.93
3447
.03
3414
.04
3389
.11
3377
.34
3344
.43
3332
.55
3285
.74
3270
.12
3256
.07
3224
.07
3210
.91
3197
.57
3176
.22
3130
.80
3097
.52
310032003300340035003600
Wavenumber cm-1
0.6
0.8
1.0
1.2
1.4
1.6
1.8
Ab
sorb
anc
e U
nit
s
S3
3612
.30
3594
.46
3577
.10
3570
.28
3550
.16
3532
.85
3510
.94
3487
.05
3455
.93
3447
.03
3414
.04
3389
.11
3377
.34
3344
.43
3332
.55
3285
.74
3270
.12
3256
.07
3224
.07
3210
.91
3197
.57
3176
.22
3130
.80
3097
.52
310032003300340035003600
Wavenumber cm-1
0.6
0.8
1.0
1.2
1.4
1.6
1.8
Ab
sorb
anc
e U
nit
s
S3
3632
.02
3611
.42
3594
.25
3577
.30
3570
.01
3549
.48
3533
.62
3522
.14
3504
.81
3488
.88
3478
.75
3456
.09
3446
.50
3413
.44
3381
.87
3345
.28
3329
.99
3283
.06
3269
.45
3255
.73
3223
.93
3211
.03
3174
.04
3130
.93
3099
.50
310032003300340035003600
Wavenumber cm-1
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
Ab
sorb
anc
e U
nit
s
S4
3632
.02
3611
.42
3594
.25
3577
.30
3570
.01
3549
.48
3533
.62
3522
.14
3504
.81
3488
.88
3478
.75
3456
.09
3446
.50
3413
.44
3381
.87
3345
.28
3329
.99
3283
.06
3269
.45
3255
.73
3223
.93
3211
.03
3174
.04
3130
.93
3099
.50
310032003300340035003600
Wavenumber cm-1
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
Ab
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s
S4
101
Figure 8.3 FT- deconvoluted OH bands (3600 to 3200 cm-1) of S1, S2, S3 and S4.S1 – 4 day
treatment, S2 – 4 day treatment with fungal extract, S3 –2 day treated, S4 – untreated
cellulose fibre.
Major peaks associated with FT- Deconvoluted OH bands (3600 to 3200 cm-1) of S1, S2, S3
and S4 are pictured in Figure 8.3. The peaks of interest are picked from the FT-Deconvoluted
bands of S1, S2, S3 and S4 and a model is set up to run the curve fitting procedure.
Calculations were repeated until a best fit was obtained with rms error less than 0.01.
Investigators have suggested that the two bands between 3500 and 3400 cm-1 are derived
from the intramolecular hydrogen bonds between OH(3) and O(5), and that the bands
between 3400 and 3100 cm-1 are derived from intermolecular hydrogen bonds that are
310032003300340035003600
Wavenumber cm-1
0.5
1.0
1.5
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Abs
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S3
S4
S1
S2
310032003300340035003600
Wavenumber cm-1
0.5
1.0
1.5
2.0
Abs
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nits
S3
S4
S1
S2
S3
S4
S1
S2
102
between cellulose chains and cellulose microfibrils and the hemicellulose tethered to the
surface of the cellulose microfibrils.
Curve fitted OH bands, Figure 8.4, based on the peaks of interest picked was found to
provide more insight in to the individual peaks that contributed to the broad OH band. FT-IR
absorption characteristic bands associated with S4 - untreated fibre in the OH stretching
region demonstrated a marked difference with that of the peaks for S1 – 4 day treated fibres.
(a)
S1
FreeOH
Intra-H-bonds
Inter-H-bonds
FreeOH
Intra-H-bonds
Inter-H-bonds
3700 3600 3500 3400 3300 3200 3600
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Wavenumber, 1/cm
Abs
orba
nce
Uni
ts
FreeOH
Intra-H-bonds
Inter-H-bonds
FreeOH
Intra-H-bonds
Inter-H-bonds
3700 3600 3500 3400 3300 3200 3600
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Wavenumber, 1/cm
Abs
orba
nce
Uni
ts
3700 3600 3500 3400 3300 3200 36003700 3600 3500 3400 3300 3200 3600
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
0.0
0.2
0.4
0.6
0.8
1.0
1.2
1.4
Wavenumber, 1/cm
Abs
orba
nce
Uni
ts
103
(b)
Figure 8.4 Curve fitting and peak assignments for OH stretching regions in S1 and S4,
(a) S4 – untreated wood fibre, (b) S1 – 4 day treatment, rms error 0.01.
8.4.1 Intermolecular hydrogen bonds
A careful observation of the IR absorption characteristic curve (Figure 8.4) for treated and
untreated fibres in the 3400 to 3100 cm-1 region which is due to the intermolecular hydrogen
bonds that exist between the elementary cellulose fibres and between the hemicellulose and
the cellulose microfibrils, showed an apparent difference in the IR absorption intensity - 0.9
IR absorption units for untreated fibre compared to 0.48 IR absorption unit for enzyme
treated fibres. This observation can be considered as a direct validation of the research
hypothesis – Ophiostoma Ulmi treatment of wood fibres can facilitate the isolation of
104
nanofibres by reducing the intermolecular hydrogen bond density. As discussed in the
previous chapter, the increase in internal defibrillation of the treated fibres can be attributed
to the decrease in the intermolecular hydrogen bonds between the elementary cellulose
fibres.
8.4.2 Intramolecular hydrogen bonds
The characteristic band of intramolecular hydrogen bonds in a cellulosic fibre peaks in the
3500 to 3400 cm-1 region. The IR absorption intensity of the intramolecular hydrogen bond
for enzyme treated fibre showed a slight decrease compared to absorption intensity of the
band for untreated fibres – 0.71 IR absorption units for untreated fibres verses 0.65 IR
absorption units for enzyme treated fibres.
8.4.3 Free hydroxyl functionality
It is quite interesting to note that the peak at 3560 cm-1, characteristic of the free hydroxyl
groups in the cellulose fibres, showed a slight increase in the band intensity – 0.30 IR
absorption units for the untreated fibres compared to 0.35 IR absorption units for the enzyme
treated fibres. This increase may be as a result of the reduction in the intermolecular and the
intramolecular hydrogen bonds in the treated cellulose fibres. The increase in free OH groups
in cellulose fibres may not of benefit in the context of this research as the increased
hydrophilicity of these fibres can reduce their interfacial compatibility (fibre – matrix
interaction) in a hydrophobic polymer matrix in bio-composite application.
105
8.4.4 Hydrogen bonds and their association
The discussion above with respect to the effect of bio-treatment on hydrogen bond density in
the fibres has point to the fact that the extracellular enzyme produced by the fungus
Ophiostoma Ulmi has the ability to interact with the OH chemistry, indeed reducing the
hydrogen bonding in the fibre wall structure. The hydrogen bonds in the fibres may be either
associated with the amorphous region or the crystalline region. In order to differentiate and
understand the contribution of hydrogen bonds associated with the crystalline and the
amorphous regions to the IR absorption, the bio-treated fibres were saturated with Deuterium
(D2O) and the IR absorption spectra was taken and analyzed. When the fibres are placed in
an environment saturated with D2O, the OH group associated with amorphous region will be
preferentially replaced with the D2O. The OH associated with the crystalline region of the
fibres will remain unaffected due to very limited accessibility and the steric hindrance due to
the larger size of D2O.
Reduction in hydrogen bonding density in the treated fibres is due to the termination of the
OH group and is mainly from the most accessible part of the fibre structure. There is a
marginal decrease, approximately 4%, in hydrogen bond density of the treated and untreated
fibres that was saturated with D2O compared to the original treated and untreated fibres as
revealed from Table 8.2 and 8.3. This decrease in hydrogen bond density may be attributed to
any remaining easily accessible hydrogen bonds in the fibres that are found either in the
amorphous region of the fibre structure or the cellulose microfibril surface OH functionality
that form hydrogen bonds with hemicelluloses chains. D2O will not interfere with the interior
OH groups due to steric hindrance. Since the reduction in hydrogen bond density is quite
106
Figure 8.5 FT- deconvoluted OH bands (3700 to 3000 cm-1), S1 - -treated and S4 - Untreated
cellulose fibres.
Table 8.3 Change in IR index of D2O saturated cellulose fire with enzymatic treatment - OH
peak from 3000 to 3700 cm-1.
Bio- OH peak area 2900 / cm * Hydrogen Bonds
Treatment 3000-3700 /cm Peak area
S1 601.958 69.92 8.59
S4 652.227 70.52 9.24
S1 - treated fibre, S4 - untreated, *Calculated by relative peak area with 2900 cm-1 as internal
standard
3000310032003300340035003600
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an
ce
Un
its
0.2
0.4
0
.6
0
.8
1
.0
1
.2
3600 3500 3400 3300 3200 3100 3000
Wavenumber cm -1
S4
S1
107
small when the treated fibres were saturated with D2O, we can very safely assume the
reduction in hydrogen bond density due to Ophiostoma Ulmi treatment is mainly from the
breakdown of hydrogen bonds responsible for the cellulose microfibril – hemicelluloses
interface interaction.
8.5 Solid State 13C Nuclear Magnetic Resonance Spectroscopy
Solid state 13C Nuclear Magnetic Resonance Spectroscopy is currently being explored
extensively to better understand the structure and morphology of cellulose. 13C spectra
obtained with the combined techniques of proton-carbon cross polarization (CP)
(Hartmannand Hahn 1962), high power proton decoupling (Sarles and Cotts 1958), and
magic angle spinning (MAS) (Andrew 1959) can reveal unique information related to various
crystal forms in cellulose, identify different forms with in the same grouping such as
cellulose I. The distinction and identification arise due to either morphological feature like
varying degree of disorderness and / or those associated with chains in the interior of the
crystallites (Earl and VanderHart 1981, Atalla et al. 1980, Horii et al. 1982). As suggested by
these researchers, some of these later distinctions are due to morphological features, like the
varying degree of disorderness in the structure of cellulose. VanderHart and Atalla (1984)
have identified and assigned certain 13C resonance features associated with the chains in the
interior of the crystallites to morphologically distinct regions in cellulose I samples as shown
in Figure 8.6.
108
Figure 8.6. 13 C CP-MAS spectra of cotton linters. Horizontal bars represent spectral ranges
of the corresponding carbon sites in the anhydroglucose monomer unit of cellulose
(VanderHart and Atalla 1984)
The region from 60 to 70 ppm is assigned to C6, the cluster of resonance from 70 to 81 ppm
is assigned to C2, C3 and C5, the region from 81 to 93 is assigned to C4, and the region from
102 to 108 ppm (sometimes extending up to 96 ppm) is assigned to C1. C4 and C6 resonance
regions consists of sharper partially overlapping broader wings and studies by Earl (Earl,
1981) has assigned it to the crystallite surfaces. Also, two other points to be noted are the
Resonance from C atoms in the
interiors of crystallites
Resonance from C atoms in the
interiors of crystallites
109
sharper portions of the C4 resonance at 90 ppm and C6 resonance at 66 ppm are made of at
least two (2) and three (3) closely spaced components respectively. These multiplicities are
interpreted as arising from carbons on the chains in the interiors of the crystallites. The C1
resonance region between 102 and 108 ppm is also shows multiplicity and evidence of sharp
resonance. The difficulty in interpreting the C1 resonance, as suggested by Earl, arise from
the overlap of resonance arising from the interior crystallites with the C1 resonance arising
from the disordered surface as well as possible three dimensional disordered region with in
the cellulose.
The following elucidation by Vanderhart and Atalla of the 13C MNR spectra will form the
basis for the interpretation of the 13C MNR spectra of the bio- treated and untreated fibres.
- For a rather rigid, hydrogen bonded molecule like cellulose, in which all carbons
have directly bonded protons, the CP-MAS spectra are expected to exhibit
quantitative relative intensities, which means the intensities arising from each
carbon atoms are equal.
- Broad as opposed to sharp spectral features are associated with either disorder in
chain environment or significant molecular mobility and the broadness of the C4
and C6 wings of the spectra primarily reflects molecular packing disorder.
110
Figure 8.7 13C CP-MAS spectra of untreated fibres. Horizontal bars represent spectral ranges
of the corresponding carbon sites in the anhydroglucose monomer unit of cellulose fibres.
The spread of resonance frequencies in a disordered region can occur due to the following
reasons (VanderHart and Atalla 1981) – changes in the density of hydrogen bonding, changes
in bond geometries, conformational differences and due to the nonuniformities in the
neighboring chain environment.
C1
C2,3,5
C4 C6
ppm
C1
C2,3,5
C4 C6C1
C2,3,5
C4 C6
ppm
Sharp peeks Sharp peeks
111
Figure 8.8 13C CP-MAS spectra of biotreated cellulose fibres. Horizontal bars represent
spectral ranges of the corresponding carbon sites in the anhydroglucose monomer unit of
cellulose.
Figure 8.7 shows the 13C CP-MAS spectra of untreated cellulose fibres and is very much
similar to all the previously published cellulose rich natural fibres with respect to spectral
features, spectral relative intensity and positions (Vanderhart and Atalla 1980, 1984, Earl and
VanderHart 1981, Dudley et al. 1984, Horii et al. 1982).
Lower intensity
Lower intensity Broad peeks
112
The spectrum in Figure 8.8 shows the resonance peaks of the bio-treated wood fibres. These
spectrums are normalized to the same intensity and the first spinning side band of
tetramethylsilane (TMS) which is added as the chemical shift reference. The 13C CP-MAS
spectra of the untreated and bio-treated fibres has shown very similar spectral characteristics
and resonating positions with respect to the C1, C2,C3, and C5 carbon atoms. However, there
is a significant difference in the intensity and broadness of the resonance associated with both
C4 and C6 carbon atoms. This change in relative intensity and the broadness of the C4 and
C6 carbon atoms are due to the changes in the molecular packing in the crystallite region of
the cellulose and based on the previous studies referenced above this could be attributed to
the changes in the hydrogen bond density due to the bio-treatment of the cellulose fibres. The
decreased resonance intensity of the C4 and C6 resonance peaks also indicates a reduction in
the crystallinity of the bio-treated fibres which is being discussed in detail and supported by
FT-IR spectra analysis in the next chapter. As anticipated and established in chapter 6, the
bio-treatment of the fibres has resulted in decrease in the degree of polymerization (DP) of
the fibres compared to the un-treated fibres.
8.6 Isolation and Identification of Fungal Protein Secreted by Ophiostoma Ulmi
The function of cell wall loosening enzymes is to loosen the mechanical components of the
cell wall to allow it to undergo elongation during cell growth (Cosgrove 1998). The activity
of these enzymes was discovered when they were added to denatured cell walls and they
were able to restore the ability of the cell walls to extend, and hence were called expansins
(Li et al., 2003). In native cell walls, expansin acts at the interface between the microfibril
and the matrix by disrupting the hydrogen bonding between the cellulose and matrix
113
polysaccharides (Cosgrove et al. 2002). Fungi, including Trichoderma and Aspergillus
produce expansin-like proteins, swollenin, that induces a swelling effect on fibres. Swollenin
is a 75 kDa protein that contains an amino-terminal fungal type CBD that is connected by a
linker region to an expansin-like domain (Saloheimo et al. 2002). The authors postulated that
the swollenin opens the cross-linking of the fibres, which accounts for its overall weakening
effect. It acts as a swelling factor, a non-hydrolytic component to make a substrate more
accessible to the hydrolytic endo- and exo-acting enzymes and β-glucosidases that degrade
cellulose (Saloheimo et al. 2002).
The genome of Ophiostoma ulmi has not been completely sequenced yet, however there has
been efforts focused on identifying secreted proteins that interact with cellulose fibres
(Draper 2010). The addition of cellulose to fungal growth media induces the secretion of
cellulose related enzymes (English et al. 1971). This has been shown in several studies
where secretion of cellulases, swollenin and other cellulose modifying enzymes increases
with the addition of cellulose to the growth media (Acosta-Rodriguez et al. 2005, Saloheimo
et al. 2002). Draper (2010) examined the induced and uninduced conditions to compare
secretion profiles of Ophiostoma Ulmi in order to identify the proteins secreted in the
presence of fibres and thus identify potential candidate proteins involved in fibre
modification. It was found that the cellulose substrate induced culture had a lower number of
proteins compared to the uninduced culture.
To determine whether the proteins were interacting with the treated fibres, the fibres were
stained with Bradford Reagent. Bradford Reagent interacts with proteins and produces a
114
colorimetric response that reflects the amount of protein present in the sample (Kruger 1994).
The procedure is based on the formation of a complex between the dye, Coomassie Brilliant
Blue, and proteins in solution. When the dye forms a complex with a protein, it changes
from a red form of Coomassie into a stabilized blue form. Thus, the amount of protein-dye
complex present in the sample is a quantitative measure for the protein concentration (Kruger
1994). Intensely stained fibres would indicate that the proteins are bound to the fibres and
may account for the reduced number of proteins in the media. Upon the addition of the
indicating dye, the treated fibres turned an intense blue, while the untreated fibres had a very
low level of staining as depicted in Figure 8.9.
Figure 8.9 Protein concentration on fibres; (a) untreated fibres, (b) treated fibres (Draper
2010).
(a) (b)
115
Figure 8.10 Characterization of proteins isolated from treated fibres based on a biological
processes classification system, through GO Slim Mapper (Draper 2010).
116
The above observation suggests a strong interaction of some of the extracellular enzymes
secreted by Ophiostoma Ulmi with the fibres being treated. In order to throw light on the
extent of the adhesion of proteins to the fibre, the fibres were washed with Tris buffer (50
mM, pH 7.5) with moderate agitation to wash off the loosely bound proteins. To focus on
potential fibre modifying candidate proteins, the identified proteins were grouped into
molecular function and biological processing groups, using gene ontology (GO) Slim Mapper
(Figure 8.10).
Figure 8.11 SEM of treated and untreated fibres after sonication with glass beads. Images
are of (A) treated fibre ends, (B) untreated fibre ends, (C) treated fibre mid sections, and (D)
untreated fibre midsection. Arrows indicate areas of fibrillation (Draper 2010).
117
To further understand the effect of the enzymes on fibres, the activity of the fungal extracts
isolated from the induced fungal growth media was tested on the cellulose fibres (BKP). The
sonication of the treated fibres with glass beads showed fibrillation of the fibres as shown in
Figure 8.11.
The observations outlined above based on a parallel research at the centre (Draper, 2010) has
given us the evidence that proteins secreted by Ophiostoma Ulmi has strong interactions with
the fibres and as suggested by various researcher’s (Scheffer and Elgersma 1982, Beckman
1956, Przybyl et al. 2006) the Ophiostoma ulmi produces weak cellulolytic enzymes in the
presence of cell wall materials that facilitate its entry into the host. These enzymes do not
actively degrade the cell wall or other cellulosic materials. Therefore, they are described as
having a soft hydrolyzing effect. The effect of proteins like expansin and swollenin combined
with the soft hydrolyzing effect of the cellulases in the fungal exudate on the fibres is very
consistent with the observation on weight loss, degree of polymerization, cellulose content
and the crystallinity of the biotreated fibres. It also supports the inferences we had drawn
from the FT-IR and 13C NMR study of the hydrogen bonding network within the fibre
structure.
8.7 Conclusions
1. The results of investigation by Draper (2010) indicated that the cellulose fibres
induced protein secretion by the fungus. The research also demonstrated that the
proteins interact strongly with the fibres by binding, which was thought to facilitate
fibre modifications that are responsible for nanofibre formation. Also, our
118
investigation in to the structural characteristics of the biotreated has shown to
introduce internal defibrillation characteristics of the fibre.
2. FT-IR absorption characteristic of the enzyme treated fibres has shown a noticeable
decrease in the intermolecular hydrogen bonded OH functionality, while only a
marginal change in intramolecular hydrogen bonded OH group and free OH groups.
3. As inferred from 13 C CP-MAS spectra C4 and C6 resonance peak intensity and
broadness, suggest decrease crystallinity and the DP of the bio-treated fibres
supporting the fact that the bio-treatment can bring about internal defibrillation and
disorderness with in the crystalline structure of the fibres.
4. The fungus, during its growth on fibre substrate secretes enzymes capable of
interacting with the hydroxyl chemistry of the fibres thereby disrupting the cellulose -
hemicellulose network in the fibre cell wall.
119
Chapter 9 Results and Discussion
Bio-treatment of Natural Fibres: Effect on Cellulose Structure and Mechanical
Properties.
9.1 Introduction
In Chapter 8, the effect of the bio-treatment on the hydrogen bonding and their nature was
discussed. The detailed analysis of the FT-IR spectrum in the –OH region indicated that the
bio-treatment of the fibres decreased the hydrogen bonding density in the fibre. In this
chapter an attempt is made to validate the results of Chapter 8 and also to identify the
consequential effect of the decrease in hydrogen bonds on the structural attributes and the
mechanical properties of the nanofibres. Crystallinity of the treated fibres is investigated here
using FT-IR and X-Ray diffraction crystallography. Discussion on 13C NMR spectra from
Chapter 8 is also used here to support the findings from FT-IR and X-Ray Diffraction
Crystallography analysis.
9.2 FT-IR Bands in 1400 – 800 cm-1 Region
The enzyme treatment of fibres has shown to produce some marked difference in the IR
absorption characteristics in the 900 to 1100 cm-1 region. The IR absorption near the 900 to
1000 cm-1 region is found to be very sensitive to the amount of crystalline versus amorphous
structure of cellulose, i.e. broadening of the band reflects a higher degree of disordered
structure. Since the disorder of the cellulose structure is caused by the angle changes around
β-glycosidic linkage and rearrangement of hydrogen bonds (Blackwell 1977), examination of
120
the IR absorption characteristics of the enzyme treated fibres in this region showed a notable
broadening and a marked increase in intensity.
95010001050110011501200
Wavenumber cm-1
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
Abs
orba
nce
Uni
ts
S3
S4
S1
S2
95010001050110011501200
Wavenumber cm-1
0.4
0.6
0.8
1.0
1.2
1.4
1.6
1.8
2.0
Abs
orba
nce
Uni
ts
S3
S4
S1
S2
S3
S4
S1
S2
Ab
sorb
ance
Un
its
0.4
0
.6
0.8
1
.0
1.2
1
.4
1.6
1
.8
2
.0
1200 1150 1100 1050 1100 950
Wavenumber cm -1
Crystallinity sensitive
Figure 9.1 IR absorption characteristics of cellulose fibres in the 800 to 1200 cm-1 region
S1 – 4 day treatment, S2 – 4 day treatment with fungal extract, S3 –2 day treated, S4 –
untreated.
In the present context of this research, this observation is quite interesting for the fact that an
increase in disorderness in the cellulose structure can be viewed as an increase in the internal
defibrillation of the cellulose and also rearrangement of hydrogen bonds in the treated
cellulose structure supports the fact detailed in Figure 9.1. Although not conclusive, the
121
evidence presented so far support the fact that enzyme treatment of cellulose fibres bring
about structural disorderness in the cellulose and also rearrangement of hydrogen bonds in
the structure.
9.3 X-Ray Diffraction Crystallography
Cellulose is found in a number of structural variations – cellulose I, II, III and IV. Cellulose
I, also called the native cellulose is the most abundant in nature and has two polymorphs - I
and I. Cellulose II is the variety found in regenerated cellulose. The various celluloses have
distinct and well defined X-Ray diffraction patterns as a result of the crystalline nature of the
cellulose structure. The crystallinity, therefore, can provide an insight in to the macro-
structure of the cellulose. For a given cellulose source, the crystallinity of the cellulose fibres
has found to have a significant dependence on the treatment history. Physical treatments like
grinding, beating or bio-treatments that can interfere with the cellulose structure have found
to have a distinct effect on the X-Ray diffraction pattern (Ellefsen et. al. 1971, Blackwell
1971, Segal and Conrad 1957).
9.3.1 Cellulose crystallinity
There have been numerous attempts to examine and quantify the crystallinity of cellulose.
Although Acid hydrolysis approach (Philips et. al., 1947) and infrared measurements
(O’Connor et al. 1958, Basch et al. 1974) were used for qualitative crystallinity study, X-Ray
diffraction was extensively used for both the qualitative and qualitative estimate of cellulose
crystallinity.
122
In this study, a quantitative study of the cellulose crystallinity is done to understand the
effects of bio-treatment on the structure of the cellulose and also as tool to validate the FT-IR
spectral study done to correlate angle changes around -glycosidic linkage and
rearrangement of hydrogen bonds.
Figure 9.2 X-ray diffraction patterns of cellulose. S1: Treated cellulose fibres, S2: Untreated
cellulose fibres.
The crystallinity Index was used to quantify the crystallinity of the cellulose. The
crystallinity Index (CI) is defined as
CI = (I max - Imin) / Imax
0
100
200
300
400
500
600
700
0 10 20 30 40 50 60 70
2 Theta, (Deg)
Inte
nsit
y (
a.u
.)
S1
S4
2 = 19°
2 = 22.5°
123
where, Imax is the height of the peak at 2 = 22.5 and Imin is the height of the minimum at 2
= 19.
Table 9.1 Crystallinity index of biotreated and untreated Cellulose fibres.
Cellulose
Imax
Imin
CI
Untreated
576
182
0.69
Bio-treated
420
161
0.61
As can be seen from Figure 9.2, the X-Ray Diffraction pattern of untreated cellulose, S4
show a well-defined primary peak at diffraction angle 2 = 22.5 and two secondary peaks at
2 = 15 and 16.8. The X-Ray diffraction pattern of bio-treated cellulose show a marked
decrease in the intensity of the primary peak and also the secondary peaks at 2 = 15 and
16.8 tend to merge. This crystallographic pattern behavior in cellulose is only observed due
to degradation in the crystallite structure and hence decreased crystallinity.
Table 9.1 provides an estimate of the degree of reduction in the crystallinity index of the bio-
treated and untreated cellulose. The 8 – 10 % reduction in the crystallinity index, as
calculated from the X-Ray diffraction pattern is consistent with the FT-IR absorbance
intensity (A), (Auntreated- Atreated)/Atreated value calculated from Figure 9.1, at peak absorbance
intensity in the 800 to 1400 cm-1 region.
124
9.4 Fibre Length and Aspect Ratio of Cellulose Nanofibres
Aspect ratio which is the ratio of fibre length to their diameter is one of the most critical
parameter in evaluating the reinforcing potential of the fibre. Fibre length of the original
fibre and the length of the cellulose nanofibres isolated from both bio-treated and untreated
fibres were determined using a fibre quality analyzer as detailed in Section 5.2.1. Detailed
analysis results are provided in Appendix 1. Fibre diameters of the nano fibres were
calculated from the TEM photomicrophs. A typical nanofibre obtained after fungal treatment
is shown in Figure 9.3.
Figure 9.3 Transmission Electron Micrograph (TEM) of cellulose nanofibres obtained from
bio-treated fibres after single pass through the disk refiner.
125
Table 9.2 Diameter and aspect ratio of fibres and cellulose nano-fibres isolated from fibres.
Average Length*,
L, μm
Average
Diameter**, μm
Aspect ratio
L/D
Wood Fibre
2100 ± 40
23 ± 3.5
92
Nano-fibres isolated from
untreated fibres
300 ± 32
0.125 ± 0.05
2400
Nano-fibres isolated from
treated fibres
280 ± 28
0.065 ± 0.03
4300
* Weighted length from Fibre Quality Analyzer results, Appendix 1.
**Diameter of nano-fibres from diameter distribution, Figure 6.4, Chapter 6.
Aspect ratios of the cellulose nanofibres isolated from treated and treated fibres as identified
in Table 9.2, is a critical parameter with significance in reinforcing application.
9.5 Mechanical Strength Properties of Cellulose Nanofibres
As pointed out in the in the introduction, cellulose nanofibres are gaining much attention due
to their very high specific strength properties and reinforcing potential. The concept of
cellulose nanofibre reinforced bio polymers called nano-bio-composites is a key area of
research because of their huge potential especially in applications requiring strength and
100% biodegradability.
126
In this study, cellulose nanofibre films were prepared from their suspension via vacuum
filtration. During filtration the nanofibres were made to deposit on a 200 mesh filter. In
suspension, the interaction between the cellulose nanofibres is overcome by the fibre water
interaction, but when the fibres are deposited on the filter, the fibre-fibre interaction becomes
more prominent. As the nanofibre films dried, a strong nanofibre network is formed with
very high specific properties.
Figure 9.3 Stress-strain curves of cellulose nanofibre films prepared with nanofibres from S1
and S1-1: treated cellulose fibres and S4 and S4-1: untreated cellulose fibres.
The stress-strain behavior of the of the cellulose nanofibre films, made with cellulose
nanofibres isolated from original fibres and those isolated from bio-treated fibres are shown
in Figure 9.3. The curve exhibits two relatively linear regions, one up to a displacement of
127
0.5 mm and the other after the yield stress of approximately 0.012 kN. The films prepared
using nanofibres from original fibres and those isolated from treated fibres roughly followed
a similar stress-strain curve. The strain to failure ratio and hence the tensile strength of the
films from nanofibres isolated from treated fibres showed a higher valve compared to
nanofibres original fibres.
The strength properties of the cellulose nanofibre depend on three key parameters, namely
aspect ratio, degree of polymerization and the degree of Crystallinity. The discussion from
the previous chapters points us to the direction that bio-treatment of the fibres leads to a
decrease in the cellulose content, probably a decrease in the degree of polymerization and
also a decrease in the degree of crystallinity of the fibres. However, the cellulose nanofibres
isolated through bio-treatment had two distinct characteristics compared to the cellulose
nanofibres isolated through the conventional technique; (a) narrow diameter distribution and
hence high aspect ratio: (b) a more individualized and non-segregated fibres.
The average tensile strength of the nanofibre film made from nanofibre isolated through bio-
treatment showed a higher value compared to that isolated from the untreated fibre, 243 ± 8
MPa compared to 222 ± 10 MPa, respectively as shown in Table 9.2. This result is not
surprising based on the above point (a) and (b). It is self-evident that the nature and extent of
the surfaces of the fibres which make up a sheet have a profound effect on the properties of
the sheet.
128
Table 9.2 Average Mechanical properties of films prepared from cellulose nanofibres
isolated from bio-treated fibres and that isolated from untreated fibres.
Nanofibre film from Tensile Strength, MPa Modulus, GPa
Bio-treated fibres 244 ± 8 18 ± 2
Untreated fibres 222 ± 10 16 ± 2
9.6 Conclusions
1. Analysis of the FT-IR absorption characteristic spectra of the enzyme treated fibres
showed a marked decrease in the intermolecular hydrogen bonded OH functionality,
while only a marginal change was observed in intramolecular hydrogen bonded OH
groups and free OH groups.
2. FT-IR and X-Ray Crystallinity studies indicated a decrease in the crystallinity index
in the bio-treated fibres and an increase in the degree of disorderness in the macro-
molecular structure. This is an indication of the increased internal defibrillation
caused by bio-treatment.
3. The aspect ratio of the cellulose nanofibres isolated from bio-treated fibres was found
to be higher compared to those isolated from untreated fibres. This may be attributed
to the internal defibrillation caused by the bio-treatment.
129
4. The mechanical strength of the nanocellulose fibres isolated via bio-mechanical
process was marginally higher to that of those isolated via conventional mechanical
process. This is anticipated because the nanocellulose fibres isolated through fibre
bio-pretreatment have higher aspect ratio and are more distinct.
130
Chapter 10 Results and Discussion
Bio-treatment of Natural Fibres: Process Scale-up and Estimation of Energy
Requirement for the Isolation of Cellulose Nanofibres.
10.1 Introduction
This chapter briefly discusses the mechanism of fibre grinding in a wet mill disk refiner that
is used to scale up the isolation of cellulose nanofibres and attempts to estimate the actual
energy used to isolate cellulose nanofibres from bio-treated wood fibres. Mathematical
models to predict the kinetics of fibre breakage and its correlation to energy requirement per
unit mass of the fibre and the average impact intensity in the refiner is well established
(Smith 1923, Fox 1980, Goncharov 1971, Brecht 1967, Stephansen 1967, Mayade and Roux
1995).
10.2 Theory of Refining
10.2.1 Specific edge load theory
In a refiner, at the microscopic level, the refining effects are depended on two major factors –
the refining magnitude and the frequency of fibre deformation happening between the stator
and the rotor. In order to realize its relevance, the following assumptions are generally made:
(a) As the fibres are normally collected on the bar edges during refining, the greater the
number of bar edges available in the refining zone, the grater will be the number of fibres be
able to absorb a given refining load.
131
(b) Torque applied by a refiner motor is directly proportional to the normal force acting
to push a refiner rotor against a stator.
From the above two assumptions, it can be concluded that the average magnitude of fibre
deformation is directly related to the applied power divided by the product of rotating speed
and edge length. The concept of Specific Edge Load that was introduced in the late 60’s was
derived from these assumption identified above. The Specific Edge Load is also known as
refining intensity and is expressed in watt-second per meter (Ws/m).
In commercial operation of a refiner, there is always a significant power consumption
associated with hydraulic losses. This loss is typically in form of heat resulting from
acceleration and deceleration of the fluid in the refiner filling. This loss does not contribute to
any net refining effect on the fibres and is often referred as no load power. Therefore, the
Intensity of refining is calculated as follows:
I = (Applied Motor Power – No Load Power)/ [RPM x Bar Edge length x (min/60s)]
In order to define the refining process, it is also important to know the frequency or the
average number of deformation per unit mass of the fibres. The average deformation across
bar crossing per unit time in a refiner can be considered proportional to the rotating speed of
the rotor. Therefore, the number of deformation per unit mass (N) can be calculated as
follows:
132
N = (RPM x Bar Edge Length) / Tons per day
The amount of refining or the Specific Energy (P) is defined as
P = I x N
Where, I is the Intensity of refining and N is the number of deformation per unit
mass.
10.3 Overview of Fibre Refining Process for Cellulose Nano-Fibre Isolation
With the abundant potential application possibilities for cellulose nanofibres ranging from
paper additives to aerospace applications, the interest and attempts to isolate cellulose nano-
fibres from plant cell wall in an industrial scale has grown exponentially. Isolation of
cellulose nanofibres has been reported by researchers since 1996 either chemical or chemi-
mechanical process (Dufresne et al. 1997, Dinand et al. 1999). Cellulose nanofibres was
isolated from wood fibres by Taniguchi (1996) using a grinder type wet mill and the isolated
nanofibres were in the order of 100 nm or less in diameter.
The mechanism of fibre refining in any refiner is mainly attributed to the shear and
compression forces in the refining zone, and their distribution on single fibres. As identified
in Chapter 1, the four primary effects in refining a fibre suspension are:
a) Intra-fibre bond breaking (internal fibrillation)
b) External fibrillation
c) Production of fines
133
d) Fibre shortening
The effects stated above, while refining in a PFI mill were confirmed by various researchers
studying the handsheet properties (Watson and Phillips 1964).
In this approach, to isolate nanofibres from fibres to individualized nanofibres with high
aspect ratio, internal defibrillation is the focus effect during the refining. In a PFI mill
refining, the only way to affect internal defibrillation is to increase the number of revolution
and the expense of high energy. As discussed in Chapter 6, even at 125,000 revolutions, the
refining effect is predominantly external defibrillation with no actual useful scale of
nanofibre isolation from the fibres. Further processing of the refined fibres like high pressure
homogenization or cryo-crushing is required to affect the isolation of nano-fibres.
10.4 Disk Grinder
The theory of disk grinder is discussed here in detail. Based on the available literature and
the refining characteristics exhibited by these refiners, we consider it a right candidate for
any single step refining process that may follow the bio-treatment in the isolation of cellulose
nano-fibres.
The disk refiner contains two disks with bars and grooves, one stator disk and the other rotor,
as shown in Figure 10.1 and Figure 10.2. The fibre suspension, typically 1 – 2%, is forced
between the rotor and the stator disk where the fibres are subjected to repeated cyclic stresses
during the refining process. The fibre suspension is forced into confined zone whose
thickness is called the gap clearance.
134
Figure 10.1 Geometrical parameters of disk wet mill refiner, a - width of bars, b - depth of
grooves, () - sectorisation angle, ( ) - grinding angle, Roux (1999).
Figure 10.2 Stator and Rotor disk on a Disk wet mill Roux (1999).
The distance between the opposing bars is adjustable and ranges from 0.1 micron to 5
micron. During the bar crossing, the fibres are compressed and sheared leading to internal
135
and external defibrillation, hydration and fibre breakage. The fibre suspension can be put
through the refining process more than once depending on the degree of refining required.
One fibre suspension through the unit is defined as one pass. The defibrillation of the treated
and untreated fibres after refining is shown in Figures 10.3 to 10.5. It is clear from the
figures that nano fibres are formed after a single pass through the refiner, whereas in the case
of untreated fibres a minimum of 8 passes were required for the formation of nanofibres with
similar dimensions.
Figure 10.3 TEM of cellulose nanofibres obtained from treated fibres after single pass
through the disk refiner.
136
Figure 10.4 TEM of cellulose nanofibres obtained from un-treated fibres after single pass
through the disk refiner.
137
Figure 10.5 TEM of cellulose nanofibres obtained from un-treated fibres after eight pass
through the disk refiner.
10.5 Specific Edge Load
The typical mechanical action on fibres inside a refiner is shown in Figure 10.6. The fibres
are subjected to a combined effect of compression and shearing. In order to quantify the
impact intensity on fibres during refining, various concepts have been put forward by
researchers of which the most widely accepted one is the specific edge load for refining
138
machines. The role of bar length in fibre refining has been confirmed by observations in
modern refining machines, both in disk and conical refiners and has also established that the
maximum force occurs at the leading edge of the bars (Smith 1923, Fox 1980, Goncharov
1974).
Figure 10.6. Mechanical treatment of fibres between stator and rotor inside a refiner. (Roux
and Mayade 1999).
Smith (1964) has defined the cutting length of the bar per unit time as
.c s rL n n lN (1)
Where, ns and nr are the numbers of bars on the stator and rotor disk, respectively, l is the
average length of the bars and N is the frequency of rotation.
If a rotor bar is moving over sn stator bars, the cutting length per revolution is sn l . Therefore
for rn bars and N revolution per second, equation (1) provides the cutting length per unit
time.
139
Roux (1999) put forward a generalized equation to calculate the cutting length per unit time
and average impact intensity applied to the fibres during their residence in the refiner from
the engineering parameters of the stator and rotor disk.
. 2 3 3
3( )( )
4 ( )r r s s
c e i
a b a bPI P
L N
Where I is in J/m and quantifies the average impact intensity applied to the fibres during
their residence in the refiner.
Similarly, a comparison of energy requirements for production of increased external and
internal surfaces of pulp in the ball mill shows that such process is equivalent to the common
size reduction operations. The regular increase in both external and internal surface with time
indicates that Rittinger's Law for size reduction holds for the beating of pulp.
Rittinger's Law for size reduction assumes the mechanism of subdivision to be essentially
that of shearing and states that the energy consumed is proportional to the fresh surface
produced Brown et al. (1950). Since the mechanical characteristic of the ball mill and a disk
refiner is essentially that of constant power input, the energy expended on the pulp is a direct
function of time. However, the above models using comminution considered the cutting of
fibres along the fibre axis. Page et al. (1967) considered a process of delamination of the
fibres during fibre refining and Karnis (1994) went on to stress the forces acting on the fibres
during refining to be mainly along the fibre length.
140
In this research, although we have seen cutting effect along the length of the fibre, the
delamination of the fibres along the fibre length is considered significant in the process of
isolation of cellulose nano-fibres. Here the standard approach to theory of comminution
correlating the energy consumed to the new surface area produced is adopted.
Figure 10.7 Stress-strain curve of a hemp fibre (Chakraborty 2006).
Figure 10.7 illustrates the stress-strain behavior of a hemp fibre. The area under the stress-
strain curve provides the energy required to deform the fibre through its elastic and plastic
deformation zones. The energy is mainly expended is mainly for the plastic deformation of
the fibre until its breaking point. The fraction of energy expended for the elastic deformation
of the fibre is relatively negligible and is usually neglected for any modeling purpose.
141
10.6 Rittinger’s Law
Every fibre being refined is subjected to a degree of elastic and plastic deformation. In the
case of refining of bio-treated and the untreated fibres it is safe to assume that the
defibrillation of the fibres, both external and internal are due to the decomposition of the
molecular bond forces and the new surface thus generated is proportional to the energy
absorbed by the fibre in the plastic zone of deformation.
As per Rittinger’s Law, the energy consumed by any comminution operation is proportional
to the quantity of new surface are produced in the process. Applying this concept to the fibre
refining process in the wet mill
2 1'( )E C A A
E - Specific Energy or energy consumed per unit mass in JKg-1
'C - Rittinger’s constant, J.mKg-1
2A - Final specific surface or surface per unit volume, m-1
1A - Final specific surface or surface per unit volume, m-1
The value of the Rittinger’s constant 'C depends on the nature of the material subjected to
comminution and is a function of the hydrogen bond density in the cellulosic fibre being
refined.
142
10.7 Energy Estimation
The disk refiner motor used for the isolation of cellulose nanofibres had the following
electrical specification: Voltage: 200 V / Power: 11 kW
The bio-treated fibres and untreated fibres in suspension at 1.5 to 2% were passed through
the refiner. The refiner disk gap was adjusted to maintain motor amperage of 18 A
throughout the run. The samples took approximately 6 minutes to pass through the refiner.
P = V I watts = 200 V X 18A = 3600 watts.
Energy required for single pass through the refiner
E = P watts x t hour = 1 x 3600 x 6/60 = 360 watt hour or 0.36 kWh.
Weight of nanofibres produced from bio-treated fibres: 60 g
No of pass required: 1
Therefore, energy required for the isolation cellulose nano-fibres from bio-treated fibres is
E1 = 1 x 0.36 kWh x 106/60T = 6000 kWh/T
Weight of nanofibres produced from un-treated fibres = 60 g
No of pass required to achieve a comparable nanofibre diameter distribution = 8
Therefore, energy required for the isolation cellulose nano-fibres from untreated fibres is
E2 = 8 x 0.36 kWh x 106/60T = 48000 kWh/T
143
The energy requirement calculated above is the total energy consumed by the wet mill refiner
to produce a ton of dry nano-fibre and not the actual energy input on the fibres for the
isolation of cellulose nano-fibres.
The energy actually required to isolate the nano-fibres from the fibres or the specific energy
input on the feed fibre was calculated as follows.
Energy input on fibre = Total energy consumed by refiner for refining – energy consumed by
the refiner for dry run.
Dry run amperage draw = 12 A
Power expenditure for dry run = 200 V X 12 A = 2400 watts
Therefore, Energy consumed for dry run per pass = 2400 W x 6/60 h = 0.24 kWh
Actual energy input on fibres = 0.36 – 0.24 = 0.12 kWh.
This translates to net energy requirement of
E’1 = 1 x 0.12 kWh x 106/60T = 2000 kWh/T dry nano-fibre
Similarly, for untreated fibres, the net energy requirement is
E’2 = 8 x (0.36 – 0.24) kWh x 106/60T = 16,000 kWh/T
This assumes all heating losses during refining are negligible.
144
10.8 Rittinger’s Constant Calculation for Treated and Untreated Fibres
Net energy requirement for isolating cellulose nano-fibres from treated fibre was estimated at
2000 kWh/T which is equivalent to energy consumed per unit mass of fibre E = 7,200 kJ/Kg
= 7,200,000 JKg-1
Therefore, the Rittinger’s constant (C’) can be calculated as per Rittinger’s formula
2 1'( )E C A A
Figure 10.8 Diameter distributions of cellulose nanofibres from treated and untreated fibres
for single pass through disk refiner.
0.00
10.00
20.00
30.00
40.00
50.00
60.00
0-10 10.-25 25-50 50-75 75-100 100-150 150-250250-350 350-500
Untreated
Diameter distribution, nm
% Yield
Treated
145
The diameter distribution of cellulose nanofibres isolated from treated and untreated fibres
for a single pass through the disk grinder is depicted in Figure 10.8. Average value for
diameter at the diameter distribution peak is used for fibre specific area calculation. The
average diameter of cellulose nanofibres isolated from treated fibre is 40 nm and that of
untreated fibres is taken as 200 nm for further calculations.
Rittinger’s constant for bio-treated fibres:
For a fibre of length l m and diameter d m, the specific surface area has a value of
(πdl) / (πd2l/4) = 4/d
A2 for the cellulose nano-fibre which is equal to 4/Diameter
Average diameter of the cellulose nano-fibre = 40 nm
So, A2= 4/(40 x10-9) = 100 x 106 m-1
Similarly A1 for the fibre is equal to 4/Diameter
Average diameter of the fibre = 15 µm
So, A1= 4/(15 x 10-6) = 0.26 x 106 m-1
Thus, 7,200,000 JKg-1 = C’ (100 x 106 – 0.26 x 106) m-1
C1’ = 7,200,000/(100 x 106) = 0.7 x 10-1 JmKg-1
146
Rittinger’s constant for untreated fibres:
Net energy requirement for isolating cellulose nano-fibres from un-treated fibres was
estimated at 16,000 kWh/T which is equivalent to energy consumed per unit mass of fibre E
= 57,600 kJKg-1 = 57,600,000 JKg-1. However, the energy required for a single pass through
the disk refiner was maintained constant by manipulating the gap width between the disks.
Therefore, specific energy required for un-treated fibres for a single pass is 2,000 kWh/T
which is equivalent to energy consumed per unit mass of fibre E = 7,200 kJKg-1 = 7,200,000
JKg-1.
A2 for the cellulose nano-fibre which is equal to 4/Diameter
Average diameter of the cellulose nano-fibre = 200 nm
So, A2 = 4/(200 x 10-9) = 20 x 106 m-1
Similarly A1 for the fibre is equal to 4/Diameter
Average diameter of the fibre = 15 µm
So, A1 = 4/(15 x 10-6) = 0.26 x 106 1/m
Thus, 7,200,000 JKg-1 = C’ (20 x 106 – 0.26 x 106) m-1
C2’ = 7,200,000/(19.7 x 106) = 3.6 x 10-1 JmKg-1
147
As mentioned before, the C’ is an indication of the molecular hydrogen bonding existing in
the cellulose structure. A comparison of the C’ for the bio-treated fibres and untreated fibres
is also a direct indication of the hydrogen bond density in the fibres.
C2’/ C1’ = (3.6 x 10-1)/ (0.7 x 10-1) = 5.14
A comparison of the energy required to isolate cellulose nano-fibres from the bio-treated
fibres and the untreated fibres is given below.
E’2/ E’1 = 16,000 /2,000 = 8
Rittinger’s constant (C’) calculated based on the average diameter distribution of cellulose
nano-fibres obtained from bio-treated fibres and untreated fibres provides us with an
understanding on the degree of difficulty associated with isolation of cellulose nano-fibres
from the initial samples. For biotreated fibres, a single pass which is equivalent to a net
energy of 2,000 kWh/Ton as sufficient to provide > 90% yield of cellulose nanofibres with
diameter < 100 nm, whereas, it took almost close to 8 passes, which is equivalent of 16,000
kWh/Ton of energy, to achieve a yield > 90% of cellulose nanofibres with diameter < 100
nm.
148
Table 10.1 Rittinger’s constant for various passes of treated and untreated fibres.
No. of Pass
Diameter of untreated fibre, nm
Diameter of treated fibre, nm
Area, untreated fibre, m-1
Area, treated fibre, m-1
Rittinger's constant (Cut), untreated fibre, JmKg-1
Rittinger's constant, treated fibre (Ct), JmKg-1
Cut/Ct
1 200 40 2.0E+07 1.0E+08 3.6E-01 7.2E-02 5.05
2 160 30 2.5E+07 1.3E+08 5.8E-01 1.1E-01 5.38
3 120 22 3.3E+07 1.8E+08 6.5E-01 1.2E-01 5.49
4 90 18 4.4E+07 2.2E+08 6.5E-01 1.3E-01 5.02
As evidenced from Table 10.1, Rittinger’s constant calculated for untreated fibres at various
levels of refining showed a steep increase to a certain number of passes or energy input and
then tends to level out after the 3rd pass. The Rittinger’s constant calculated for the
biotreated fibres did not show a similar increase and leveling out after the 1st pass. The value
of Cut/Ct from Table 10.1 suggest that as the fibres are refined, the hydrogen bonding
energy that needs to be overcome in untreated fibres is at least 5 times that of the hydrogen
bonding energy that needs to be overcome in biotreated fibres. The ratio of the net energy
(E’2/ E’1) required to isolate cellulose nanofibres from treated and untreated fibres is 8, as
calculated above. The difference in the values of the ratios can be attributed to various other
deformational forces that need to be overcome during refining of the fibres. Also, the very
slow rise in the Rittinger’s constant for biotreated fibres with increasing specific energy can
be attributed to a high degree of internal defibrillation in the biotreated fibre and a
comparable level of energy input through refining can generate more new surface.
149
Figure 10.9 Variation in Rittinger’s constant with specific energy of refining.
10.9 Quantification of Energy Reduction
The two major binding forces that hold the cell wall structure in place are
a. The non-covalent intermolecular hydrogen bonding between the cellulose
chains forming the cellulose microfibrils.
b. The non-covalent hydrogen bonding between the cellulose and hemicellulose
chains which is mainly responsible for linking the cellulose microfibrils
forming the structural framework in the plant cell wall.
Cellulose and hemicellulose is the major constituent of the bleached kraft pulp used for the
research with less than 0.5% of lignin. It can be safely assumed that the major binding forces
y = ‐0.0011x2 + 0.0509x + 0.0567R² = 0.9951
y = ‐0.0001x2 + 0.0069x + 0.0299R² = 0.9829
0.0E+00
1.0E‐01
2.0E‐01
3.0E‐01
4.0E‐01
5.0E‐01
6.0E‐01
7.0E‐01
8.0E‐01
0 10 20 30 40
Untreated
Treated
Energy, MJ/kg
Rittinger’s constan
t, Jm/kg
150
holding the cellulose microfibrils together are the hemicellulose chains that are present to a
level of 14 to 15% in the fibre. The major non-covalent binding force within the cell wall
structure can be attributed to the hydrogen bonding between the cellulose chains forming the
cellulose microfibrils and a minor portion to those established by the hemicellulose chains
with the cellulose microfibrils.
Majority of the cellulose nanofibres isolated from Ophiostoma Ulmi fungus treated fibres
through wet mill grinding falls in the 20 to 25 nm ranges which suggest that the isolated
cellulose nano-fibres are mainly near individualized cellulose microfibrils. A small fraction
of the isolated cellulose nano-fibres also contained nano-fibres that were blow 5 nm in
diameter which suggest that a very minor portion of the isolated cellulose nano-fibres
originated from cellulose microfibrils. Therefore, the Ophiostoma Ulmi fungal treatment of
fibres mainly disrupts the non-covalent hydrogen bonding between the cellulose microfibril
and the hemicellulose to bring about the internal defibrillation in the fibre cell wall.
As discussed in Section 6.3.1, the hemicellulose content of the Ophiostoma Ulmi fungus
treated fibre showed a marginal decrease while the extractable hemicellulose in the treated
fibres increased significantly compared to untreated fibres. This observation indicates that the
cell wall structure has been modified by Ophiostoma Ulmi treatment with respect to
cellulose-hemicellulose interaction. The hydrolysis of the xylose-polymer backbone by
enzymes β-1,4-xylanases and β-xylosidases both of which are secreted as extracellular
enzymes by Ophiostoma Ulmi. Enzymes like Xyloglucan endotransglycosylase and endo-
1,4--D-glucanase have been proposed in earlier studies to act as cell wall loosening agents
151
by cleaving the xyloglucans that hypothetically knit cellulose microfibrils together.
Xyloglucan endotransglycosylase and endo-1,4--D-glucanase that are secreted by
Ophiostoma Ulmi as part of their cellulase and xylanase extracellular enzyme system has the
capacity to cleave the noncovalent hydrogen bonding that forms the major cohesive force
between microfibrils and hemicelluloses.
The study on hydrogen bond density in treated fibres showed an average decrease of 10%
compared to untreated fibres, as discussed in Chapter 9. The energy required to isolate
cellulose nano-fibres from the Ophiostoma Ulmi treated fibres through wet mill grinding was
found to be only one eighth that required for isolating cellulose nano-fibres from untreated
fibres. Therefore, the decrease in hydrogen bonding should arise mainly from the cellulose-
hemicellulose interaction and not from the hydrogen bonds within the core of the cellulose
microfibrils. The decrease in crystallinity of the treated fibres was also found to be
insignificant to assign it to the disruption of hydrogen bonding in the core of the cellulose
microfibrils.
10.10 Conclusions
1. Energy requirement for isolating cellulose nano-fibres from biotreated fibres was
estimated to be 6000 kWh/T.
2. Energy requirement for isolating cellulose nano-fibres from untreated fibres was
estimated to be 48,000 kWh/T.
152
3. The actual net energy requirement for isolating cellulose nano-fibres from bio-treated
fibres was estimated to be about 2000 kWh/T, considering the dry run energy
consumption of approximately equivalent to 4000 kWh/T.
4. The isolation of cellulose nanofibres from treated and untreated fibres by refining in a
disk refiner tends to obey the Rittinger’s law which states that the specific energy
requirement in refining is directly proportional to the new surface area generated.
5. In the process of cellulose nanofibre isolation, hydrogen bonding energy is the major
energy hill to overcome and a comparatively low value of the Rittinger’s constant for
biotreated fibre refining indicates a lower density of hydrogen bonding or a high
degree of internal defibrillation with in the fibres.
153
Chapter 11 Research Conclusions
The following conclusion can be drawn from the various discussions presented in this thesis.
a) The fungus Ophiostoma Ulmi, when allowed to grow in bleached Kraft pulp fibres
suspension secretes extracellular proteins with mild cellulolytic activity and also cell
wall degrading activity. The proteins secreted have a strong interaction with the
fibres.
b) Cellulose nanofibres isolated by high shear refining and cryocrushing of treated fibres
yielded very distinct microfibrils and a narrower microfibril diameter distribution
compared to that obtained for untreated fibres.
c) Pre-refining of natural fibres before the bio-treatment process has found to
substantially improve the percentage yield of cellulose nanofibres in the less than 50
nm diameter range. Pre-refining of wood fibres can loosen the cell wall structure of
the fibre and also bring about a degree of fibre shortening. These physical changes to
wood fibres can favor the bio-activity of the enzymes due to improved fibre internal
surface accessibility. Thus pre-refining of wood fibres in a PFI mill is an easy and
excellent technique to accelerate the bio-treatment and hence improve the separation
of cellulose nanofibres and microfibrils from plant cell wall.
d) Analysis of the FT-IR absorption characteristic spectra and 13C NMR spectra of the
enzyme treated fibres has shown a decrease in the intermolecular hydrogen bonded
154
e) OH functionality, while only a marginal change in intramolecular hydrogen bonded
OH group and free OH groups. Ophiostoma Ulmi treatment of fibres has shown to
reduce the hydrogen bond density in the crystalline structure of the cellulose fibre.
f) The decrease in hydrogen bonds within the cell wall structure was mainly from the
more accessible and amorphous regions which includes the interface non-covalent
hydrogen bonding between cellulose and hemicellulose.
g) FT-IR and X-Ray Crystallinity study had showed a decrease in the crystallinity index
in the bio-treated fibres and an increase in the degree of disorderness in the macro-
molecular structure. Ophiostoma Ulmi treatment of fibres has shown to increase the
internal defibrillation characteristics of the fibre.
h) The mechanical strength of the cellulose nano-fibres isolated via bio-mechanical
process was marginally higher to that of those isolated via conventional mechanical
process. This is anticipated because the cellulose nano-fibres isolated through fibre
bio-pretreatment have higher aspect ratio and are more distinct.
i) Wet mill refining was established as a suitable one step unit operation for the
isolation of cellulose nanofibres from natural fibres. Also, scaled-up and continuous
flow through refining to isolate cellulose nanofibres from fibres was demonstrated.
j) In the Wet Mill refiner, the net energy required to isolate cellulose nanofibres from
treated fibres was found to be 2000 kWh/T while the net energy required to isolate
155
cellulose nanofibres from untreated fibres was estimated to be 16000 kWh/T. This
amounts to a substantial cost reduction in the isolation of cellulose nano-fibres. The
action of enzymes secreted by the Ophiostoma Ulmi discussed in Chapters 6, 8, 9 and
10 can therefore be summarized and firmly understood to have created a structural
disorder in the crystalline region of the cellulose by interrupting the hydrogen bonds
that holds the structure together. This internal defibrillation created with the fibre is
crucial in the isolation of cellulose nano-fibre from natural fibre.
k) The Rittinger’s constant (C’) calculated for treated and untreated fibres, which is an
indication of the hydrogen bond density in the fibres being refined, suggest that the
major energy barrier that needs to be overcome in the isolation of cellulose nanofibre
is the hydrogen bonding in the fibre. The existence of a very good correlation
between the specific energy for refining, the new surface area generated and the
Rittinger’s constant is also established.
l) The close correlation between the energy requirement for isolation of cellulose
nanofibres from bio-treated fibres and untreated fibres and the calculated Rittinger’s
constant C’ confirms the validity of application of theory of comminution and hence
Rittinger’s law in the refining of fibres.
156
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13. Appendix
See attached.
