ionic liquid pretreatment and fractionation of sugarcane...
TRANSCRIPT
Ionic liquid pretreatment and
fractionation of sugarcane bagasse for
the production of bioethanol
By
Sergios K. Karatzos
B.Sc. (Hons), M.Sc.
A THESIS SUBMITTED FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
Faculty of Science and Technology
Queensland University of Technology
2011
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IMPORTANT NOTICE
The information in this thesis is confidential and should not be disclosed for any
reason nor relied on for a particular use or application. Any invention or other
intellectual property described in this document remains the property of
Queensland University of Technology.
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© Copyright 2011
By Sergios K. Karatzos
Queensland University of Technology
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Keywords
Sugarcane; bagasse; lignocellulosics; lignin; cellulose; ionic liquids;
pretreatment; decrystallisation; fractionation; aqueous biphasic systems;
saccharification; ethanol; biofuel.
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Abstract
Pretretament is an essential and expensive processing step for the
manufacturing of ethanol from lignocellulosic raw materials. Ionic liquids are a new
class of solvents that have the potential to be used as pretreatment agents. The
attractive characteristics of ionic liquid pretreatment of lignocellulosics such as
thermal stability, dissolution properties, fractionation potential, cellulose
decrystallisation capacity and saccharification impact are investigated in this thesis.
Dissolution of bagasse with 1-butyl-3-methylimidazolium chloride
([C4mim]Cl) at high temperatures (110 °C to 160 °C) is investigated as a
pretreatment process. Material balances are reported and used along with
enzymatic saccharification data to identify optimum pretreatment conditions (150
°C for 90 min). At these conditions, the dissolved and reprecipitated material is
enriched in cellulose, has a low crystallinity and the cellulose component is
efficiently hydrolysed (93 %, 3 h, 15 FPU). At pretreatment temperatures < 150 °C,
the undissolved material has only slightly lower crystallinity than the starting. At
pretreatment temperatures ≥ 150 °C, the undissolved material has low crystallinity
and when combined with the dissolved material has a saccharification rate and
extent similar to completely dissolved material (100 %, 3h, 15 FPU). Complete
dissolution is not necessary to maximize saccharification efficiency at temperatures
≥ 150 °C.
Fermentation of [C4mim]Cl-pretreated, enzyme-saccharified bagasse to
ethanol is successfully conducted (85 % molar glucose-to-ethanol conversion
efficiency). As compared to standard dilute acid pretreatment, the optimised
[C4mim]Cl pretreatment achieves substantially higher ethanol yields (79 % cf. 52 %)
in less than half the processing time (pretreatment, saccharification, fermentation).
Fractionation of bagasse partially dissolved in [C4mim]Cl to a polysaccharide
rich and a lignin rich fraction is attempted using aqueous biphasic systems (ABSs)
and single phase systems with preferential precipitation. ABSs of ILs and
concentrated aqueous inorganic salt solutions are achievable (e.g. [C4mim]Cl with
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200 g L-1 NaOH), albeit they exhibit a number of technical problems including phase
convergence (which increases with increasing biomass loading) and deprotonation
of imidazolium ILs (5 % - 8 % mol). Single phase fractionation systems comprising
lignin solvents / cellulose antisolvents, viz. NaOH (2M) and acetone in water (1:1,
volume basis), afford solids with, respectively, 40 % mass and 29 % mass less lignin
than water precipitated solids. However, this delignification imparts little increase
in saccharification rates and extents of these solids.
An alternative single phase fractionation system is achieved simply by using
water as an antisolvent. Regulating the water : IL ratio results in a solution that
precipitates cellulose and maintains lignin in solution (0.5 water : IL mass ratio) in
both [C4mim]Cl and 1-ethyl-3-methylimidazolium acetate ([C2mim]OAc)). This
water based fractionation is applied in three IL pretreatments on bagasse
([C4mim]Cl, 1-ethyl-3-methyl imidazolium chloride ([C2mim]Cl) and [C2mim]OAc).
Lignin removal of 10 %, 50 % and 60 % mass respectively is achieved although only
0.3 %, 1.5 % and 11.7 % is recoverable even after ample water addition (3.5 water :
IL mass ratio) and acidification (pH ≤ 1). In addition the recovered lignin fraction
contains 70 % mass hemicelluloses. The delignified, cellulose-rich bagasse
recovered from these three ILs is exposed to enzyme saccharification. The
saccharification (24 h, 15 FPU) of the cellulose mass in starting bagasse, achieved by
these pretreatments rank as: [C2mim]OAc (83 %)>>[C2mim]Cl (53
%)=[C4mim]Cl(53%). Mass balance determinations accounted for 97 % of starting
bagasse mass for the [C4mim]Cl pretreatment , 81 % for [C2mim]Cl and 79 %for
[C2mim]OAc. For all three IL treatments, the remaining bagasse mass (not
accounted for by mass balance determinations) is mainly (more than half) lignin
that is not recoverable from the liquid fraction. After pretreatment, 100 % mass of
both ions of all three ILs were recovered in the liquid fraction.
Compositional characteristics of [C2mim]OAc treated solids such as low
lignin, low acetyl group content and preservation of arabinosyl groups are opposite
to those of chloride IL treated solids. The former biomass characteristics resemble
those imparted by aqueous alkali pretreatment while the latter resemble those of
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aqueous acid pretreatments. The 100 % mass recovery of cellulose in [C2mim]OAc
as opposed to 53 % mass recovery in [C2mim]Cl further demonstrates this since the
cellulose glycosidic bonds are protected under alkali conditions. The alkyl chain
length decrease in the imidazolium cation of these ILs imparts higher rates of
dissolution and losses, and increases the severity of the treatment without changing
the chemistry involved.
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List of publications
Poster presentations
• Karatzos, S. K., Edye L.A. and Doherty W.O.S., 2010, Optimisation of
lignocellulose dissolution in ionic liquids as a pretreatment strategy for
ethanol production. 32nd Symposium on Biotechnology for Fuels and Chemicals: Tampa, FL, USA.
• Karatzos, S. K., Edye L.A. and Doherty W.O.S., 2009, Evaluation of
lignocellulose dissolution in ionic liquids as a pretreatment strategy for
ethanol and lignin production. 31st Symposium on Biotechnology for Fuels and Chemicals: San Francisco, CA, USA.
• Doherty W.O.S, Edye, L.A., O’Hara, I., Nanayakkara, B., Rainey, T., Tan, S., Cronin, D., and Karatzos, S. K., 2008, Comparative study of effects of sugarcane biomass
fractionation strategies for production of chemicals and biofuels. The International Conference on Biorefinery: Beijing, China.
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Acknowledgements
I thank my supervisors for invaluable help particularly with the last weeks of
writing, my lab colleagues for hints and chats, my flatmates and friends for fun and
food, and my family and girlfriend for all the love and support.
Funding was generously provided by the Greek State Scholarship Foundation
(IKY), the Queensland Government, and Queensland University of Technology. The
Joint BioEnergy Institute (Emeryville, CA, USA) kindly provided funding and facilities
from January to April 2010 during my research project at their laboratories.
Supervisory team
Dr. Leslie A. Edye, QUT
Dr. William O.S. Doherty, QUT
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The work contained in this thesis has not been previously submitted to meet
requirements for an award at this or any other higher education institution. To the
best of my knowledge and belief, the thesis contains no materials previously
published or written by another person except where due reference is made
Signature...........................
Date..................................
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Contents
Abstract ................................................................................................................... iv
Abbreviations and Nomenclature ........................................................................... xx
CHAPTER 1 INTRODUCTION ................................................................................. 1
1.1 Background ................................................................................................ 1
1.1.1 Renewable liquid fuels and chemicals from lignocellulosic biomass .. 1
1.1.2 Sugarcane bagasse ............................................................................ 2
1.1.3 The importance of pretreatment and fractionation ........................... 3
1.1 Research aim .............................................................................................. 4
1.2 Objectives .................................................................................................. 5
1.3 Novelty ....................................................................................................... 5
1.4 Summary of chapters ................................................................................. 6
CHAPTER 2 LITERATURE REVIEW ......................................................................... 8
2.1 Overview .................................................................................................... 8
2.2 Lignocellulosic biomass: chemical and structural characteristics ................ 9
2.2.1 Cellulose ............................................................................................ 9
2.2.2 Hemicelluloses ................................................................................ 12
2.2.3 Lignin .............................................................................................. 13
2.2.4 Lignin-carbohydrate bonds .............................................................. 17
2.2.5 Cellulose microfibrils: The foundation units of the cell wall construct .
........................................................................................................ 19
2.2.6 The cell wall layers .......................................................................... 21
2.2.7 Mechanism of cell wall swelling....................................................... 22
2.3 Pretreatment ........................................................................................... 23
2.3.1 Overview of the conversion of biomass to ethanol fuel ................... 23
xi
2.3.2 Goals of pretreatment ..................................................................... 27
2.3.3 Pretreatment technologies .............................................................. 30
2.3.4 Conventional cellulose solvents ....................................................... 34
2.3.5 Enzyme saccharification of cellulosics after pretreatments .............. 38
2.4 Ionic liquid based pretreatment technologies ........................................... 40
2.4.1 Ionic liquids: properties and history ................................................. 40
2.4.2 Cellulose dissolution using ionic liquids ............................................ 43
2.4.3 Lignin dissolution in ionic liquids ...................................................... 49
2.4.4 Biomass dissolution and pretreatment in ionic liquids ..................... 50
2.5 Rationale .................................................................................................. 53
CHAPTER 3 METHODOLOGY ............................................................................... 55
3.1 Bagasse ..................................................................................................... 55
3.2 Chemicals ................................................................................................. 55
3.3 Uncertainty (or error) analysis of quantitative measurements .................. 56
3.4 Mass values .............................................................................................. 56
3.5 Karl Fischer titration ................................................................................. 57
3.6 Determination of IL dissolution extent and losses ..................................... 57
3.6.1 Dissolution ....................................................................................... 57
3.6.2 Recovery of undissolved solids (UND) and dissolved-then-
precipitated solids (DS) .................................................................................... 57
3.6.3 Gravimetric determination of percent mass dissolution ................... 58
3.6.4 Gravimetric determination of percent mass losses .......................... 59
3.7 Bagasse soda lignin preparation................................................................ 60
3.8 Real time FTIR and reaction calorimetry ................................................... 60
3.9 Differential Scanning Calorimetry ............................................................. 61
3.10 Thermogravimetric analysis ...................................................................... 61
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3.11 Cellobiose hydrolysis kinetics ................................................................... 62
3.12 Compositional analysis of solid fractions .................................................. 62
3.13 Preparation of IL pretreated samples for enzyme saccharification............ 63
3.14 Preparation of dilute acid pretreated samples .......................................... 63
3.15 Enzymatic saccharification ....................................................................... 64
3.16 XRD cellulose crystallinity measurement .................................................. 65
3.17 Saccharification and fermentation ............................................................ 66
3.18 ATR-FTIR ................................................................................................... 67
3.19 Aqueous biphasic systems ........................................................................ 67
3.19.1 Preparation of ABSs ......................................................................... 67
3.19.2 Cloud point titrations ...................................................................... 68
3.19.3 Ion concentration determination (for ABS distribution ratios) ......... 68
3.20 Quantification of [C4mim]Cl deprotonation using an acid titration ........... 69
3.21 Mass balance determinations for three IL treatments .............................. 70
3.21.1 Compositional analysis of “solid fraction 1” ..................................... 72
3.21.2 Compositional analysis of monosaccharides in liquid fraction 1 ....... 72
3.21.3 Compositional analysis of oligosaccharides in liquid fraction 1 ........ 73
3.21.4 Acetyl bromide for lignin quantification in solid fractions 2 and 3 ... 73
3.21.5 Recovery of IL .................................................................................. 74
3.21.6 Enzymatic saccharification of solids from 3 IL treatments ................ 74
CHAPTER 4 RESULTS – PRETREATMENT ............................................................. 76
4.1 Biomass dissolution in IL and recovery by addition of water ..................... 76
4.1.1 Ionic liquids used ............................................................................. 77
4.1.2 Factors affecting biomass dissolution .............................................. 78
4.1.3 Thermal stability of bagasse components in [C4mim]Cl ................... 88
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4.1.4 Ionic liquid pretreatment comparison with dilute acid pretreatment ..
........................................................................................................ 95
4.1.5 Summary ....................................................................................... 102
4.2 Role of non-dissolution pretreatment effects on enzyme saccharification ...
............................................................................................................... 104
4.2.1 Compositional analysis .................................................................. 104
4.2.2 Enzyme saccharification ................................................................. 107
4.2.3 X-Ray diffractometry (XRD) of bagasse ........................................... 110
4.2.4 “High temperature phase” of crystalline cellulose ......................... 111
4.2.5 ATR-FTIR analysis of undissolved fractions ..................................... 113
4.2.6 Summary ....................................................................................... 116
CHAPTER 5 RESULTS - FRACTIONATION ............................................................ 117
5.1 Aqueous biphasic systems ...................................................................... 117
5.1.1 Choice of kosmotropic salts for aqueous biphasic systems ............ 122
5.1.2 Evaluation of ABS stability with coexistence curves ....................... 124
5.1.3 Evaluation of the phase divergence of ABS using distribution ratios ...
...................................................................................................... 129
5.1.4 Effect of biomass loading on distribution ratios of ABSs ................. 132
5.1.5 Chemical instability of imidazolium ILs in alkaline ABSs .................. 133
5.1.6 Summary ....................................................................................... 136
5.2 Aqueous single phase fractionation systems ........................................... 136
5.2.1 Summary ....................................................................................... 141
5.3 Preferential precipitation by incremental additions of water .................. 141
5.4 Comparison of three IL pretreatment and fractionation systems ............ 144
5.4.1 Compositional analysis .................................................................. 147
5.4.2 Structural analysis by ATR-FTIR ...................................................... 149
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5.4.3 Enzyme saccharification ................................................................ 153
5.4.4 Precipitation of solid fraction 2 and 3 ............................................ 156
5.4.5 Mass recovery of bagasse components after pretreatment ........... 161
5.4.6 Mass recovery of the ionic liquid solvent after pretreatment ........ 167
5.4.7 Effect of IL anion and cation on pretreatment ............................... 168
5.4.8 Summary ....................................................................................... 168
CHAPTER 6 CONCLUSIONS ............................................................................... 171
6.1 Findings .................................................................................................. 172
6.1.1 Chapter 4: Pretreatment ............................................................... 172
6.1.2 Chapter 5: Fractionation ................................................................ 174
6.2 Future work ............................................................................................ 178
Appendix I ............................................................................................................ 181
Appendix II ........................................................................................................... 183
Appendix III .......................................................................................................... 184
References ........................................................................................................... 185
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LIST of FIGURES
Figure 2.2.1: Molecular structure of cellulose .......................................................... 9
Figure 2.2.2: Most probable hydrogen bond patterns of cellulose allomorphs ........ 11
Figure 2.2.3: Molecular structure of glucuronoarabinoxylan ................................... 13
Figure 2.2.4: Lignin monomer units ......................................................................... 14
Figure 2.2.5: The most common linkages between lignin phenylpropane units ....... 15
Figure 2.2.6: Partial structure of a hypothetical lignin molecule from European
beech (Fagus sylvatica) ........................................................................................... 16
Figure 2.2.7: Commonly occurring covalent linkages between GAX and lignin in
grasses .................................................................................................................... 18
Figure 2.2.8: Possible covalent cross-links between polysaccharides and lignin in cell
walls ....................................................................................................................... 19
Figure 2.2.9: Detailed structure of cell walls ............................................................ 20
Figure 2.2.10: Cell wall layers and organisation of the cellulose microfibrils............ 21
Figure 2.2.11: Light microscope image showing ballooning of a sulphate pulp fibre
(Pinus silvestris) ...................................................................................................... 23
Figure 2.3.1: Gross representation of the main steps in a biomass to ethanol process
............................................................................................................................... 24
Figure 2.3.2: Consolidation of bioprocessing in cellulosic ethanol production ......... 26
Figure 2.3.3: The effects of lignin, acetyl groups, and crystallinity on enzyme
adsorption and enzymatic hydrolysis of biomass .................................................... 29
Figure 2.3.4: The participating inputs and outputs in a pretreatment process ......... 31
Figure 2.3.5: Conventional cellulose solvents .......................................................... 36
Figure 2.3.6: Gross schematic of the hydrogen bonding formed between NMMO and
cellulose hydroxyls upon dissolution ....................................................................... 37
Figure 2.3.7: EDA interactions between cellulose and a non-derivatising solvent (e.g.
NMMO) .................................................................................................................. 38
Figure 2.4.1: Common ions in ionic liquids .............................................................. 41
Figure 2.4.2: Structure proposed for a covalent binding of [C2mim]OAc to a
cellooligomer (DP 6-10) .......................................................................................... 45
Figure 2.4.3: Proposed dissolution mechanism of cellulose in [C4mim]Cl ................ 46
xvi
Figure 3.6.1: Process for recovering undissolved and dissolved-then-precipitated
solids. ..................................................................................................................... 58
Figure 3.8.1: The Mettler-Toledo RC1e reaction calorimeter and ReactIR FTIR probe
............................................................................................................................... 61
Figure 3.16.1: Diffractogram of bagasse ................................................................. 65
Figure 3.20.1: Linear relationship of refractive index to [C4mim]Cl concentration in
water ...................................................................................................................... 69
Figure 3.21.1: Flow chart of the fractionation process used in mass balance
experiments ........................................................................................................... 71
Figure 4.1.1: ILs used in this study .......................................................................... 77
Figure 4.1.2: Effect of temperature on bagasse dissolution in [C4mim]Cl for 90 min
............................................................................................................................... 81
Figure 4.1.3: Effect of residence time on bagasse dissolution in [C4mim]Cl (150 °C)
............................................................................................................................... 82
Figure 4.1.4: Effect of bagasse moisture content on bagasse dissolution in
[C4mim]Cl .............................................................................................................. 83
Figure 4.1.5: Effect of ionic liquid choice on bagasse dissolution ............................ 85
Figure 4.1.6: Real time FTIR of bagasse polysaccharides upon dissolution in
[C4mim]Cl .............................................................................................................. 87
Figure 4.1.7: Differential scanning calorimetry profiles ........................................... 89
Figure 4.1.8: First derivative of thermogravimetric analysis curves ......................... 91
Figure 4.1.9: Cellobiose hydrolysis and glucose accumulation in [C4mim]Cl ........... 93
Figure 4.1.10: Hydrolysis of cellobiose in the absence of water .............................. 93
Figure 4.1.11: Enzyme saccharification of bagasse pretreated with [C4mim]Cl and
dilute acid............................................................................................................... 97
Figure 4.1.12: Images of [C4mim]Cl-pretreated bagasse at 140 °C and 150 °C ........ 98
Figure 4.1.13: Initial rates of enzyme saccharification and XRD crystallinity indices
for IL- and dilute acid-pretreated bagasse (TRS) ..................................................... 99
Figure 4.1.14: Glucan and xylan saccharification extent after 121 h for IL- and dilute
acid- pretreated bagasse (TRS) ............................................................................. 100
Figure 4.1.15: Fermentation kinetics of [C4mim]Cl-treated bagasse after enzyme
saccharification .................................................................................................... 101
xvii
Figure 4.2.1: Saccharification of the undissolved bagasse after [C4mim]Cl
pretreatment at different conditions .................................................................... 107
Figure 4.2.2: Initial rates of enzyme saccharification and XRD crystallinity indices for
[C4mim]Cl-pretreated bagasse fractions ............................................................... 108
Figure 4.2.3: Glucan and xylan saccharification extent after 121 h for [C4mim]Cl-
pretreated bagasse fractions ................................................................................ 109
Figure 4.2.4: Diffractograms of undissolved bagasse after [C4mim]Cl pretreatment
............................................................................................................................. 110
Figure 4.2.5: Optical microscopy images showing swelling of miscanthus grass
particles in [C2mim]Cl ........................................................................................... 112
Figure 4.2.6: FTIR spectra of IL- and dilute acid-pretreated bagasse fractions ....... 114
Figure 5.1.1: A NaOH / [C4mim]Cl ABS with 1% mass bagasse load ....................... 119
Figure 5.1.2: FTIR spectra of each phase of two NaOH / [C4mim]Cl ABSs .............. 120
Figure 5.1.3: FTIR spectra of each phase of a NaOH / [C4mim]Cl ABS loaded with 15
% soda lignin ......................................................................................................... 121
Figure 5.1.4: The Hofmeister series (ions relevant to this study in bold) ................ 123
Figure 5.1.5: Coexistence curves of [C4mim]Cl with selected kosmotropic salts .... 125
Figure 5.1.6: Phase diagrams of [C4mim]Cl with various salts ............................... 127
Figure 5.1.7: Activity coefficients of NaOH and KOH at different molarities .......... 128
Figure 5.1.8: Distribution ratios of ions in ABSs and their molal composition ........ 130
Figure 5.1.9: Ion migration diagrams based on distribution ratios ......................... 131
Figure 5.1.10: The effect of bagasse loading on the ion distribution ratios in ABSs 133
Figure 5.1.11 : Carbene formation from imidazolium-based ILs ............................ 133
Figure 5.1.12: HCl titration of the IL phase of a [C4mim]Cl / NaOH ABS ................. 134
Figure 5.2.1 : Enzyme saccharification of total recovered solids (TRS) from partial
bagasse dissolution in [C4mim]Cl using different antisolvents .............................. 138
Figure 5.2.2 Enzyme saccharification of completely dissolved bagasse (DS)
precipitated from [C4mim]Cl using different antisolvents ..................................... 140
Figure 5.3.1: pH of [C2mim]OAc and [C4mim]Cl aqueous solutions at different water
: IL mass ratios ...................................................................................................... 142
Figure 5.3.2: Lignin and cellulose precipitation observed at different water : IL mass
ratios of [C2mim]OAc and [C4mim]Cl aqueous solutions ...................................... 143
xviii
Figure 5.4.1: Process flow chart of a fractional precipitation separation of IL treated
bagasse using incremental additions of water ...................................................... 145
Figure 5.4.2: FTIR spectra of bagasse treated with different ILs ............................ 148
Figure 5.4.3: FTIR spectra of DS and UND bagasse treated with different ILs ........ 152
Figure 5.4.4: Glucan saccharification of extracted bagasse treated with 3 ILs ....... 153
Figure 5.4.5: Xylan saccharification of extracted bagasse treated with 3 ILs ......... 154
Figure 5.4.6: FTIR spectra of precipitate recovered after precipitation in 3.5 water :
IL mass ratio (acidified to pH < 1) in three ILs........................................................ 158
Figure 5.4.7: Mass distribution of bagasse components in [C4mim]Cl pretreatment
fractions ............................................................................................................... 162
Figure 5.4.8: Mass distribution of bagasse components in [C2mim]Cl pretreatment
fractions ............................................................................................................... 163
Figure 5.4.9: Mass distribution of bagasse components in [C2mim]OAc
pretreatment fractions ......................................................................................... 165
Figure 5.4.10: Fraction of original bagasse polysaccharides saccharified in 24 h (15
FPU g-1 glucan) after pretreatment in three ILs ..................................................... 167
LIST OF TABLES
Table 2.3.1: Enzymatic saccharification from selected pretreatment systems ......... 39
Table 4.1.1: Compositional analysis of bagasse pretreated with [C4mim]Cl and dilute
acid ........................................................................................................................ 94
Table 4.1.2: Comparison of ethanol yields from IL and from dilute acid pretreatment
............................................................................................................................. 102
Table 4.2.1: Compositional analysis of dissolved-then-precipitated solids (DS) and
undissolved solids (UND) from [C4mim]Cl pretreatment of bagasse ..................... 105
Table 4.2.2: Effect of residence time on the composition of undissolved bagasse
after [C4mim]Cl pretreatment at 150°C ................................................................ 105
Table 4.2.3 : Assignments of FTIR-ATR absorption bands for bagasse ................... 115
Table 4.2.4: Ratios of FTIR absorbances attributed to ester bonds and the aromatic
ring of lignin. ........................................................................................................ 115
xix
Table 5.1.1: Gibbs free energies of hydration (∆Ghyd) of selected ions ................... 123
Table 5.1.2: Water solubilities of selected inorganic salts ..................................... 123
Table 5.1.3: Deprotonation of imidazolium IL in top phase of ABSs ....................... 135
Table 5.2.1: Compositional analysis of total recovered solids (TRS) from partial
bagasse dissolution in [C4mim]Cl using different antisolvents .............................. 137
Table 5.2.2 : Compositional analysis of completely dissolved bagasse (DS)
precipitated from [C4mim]Cl using different antisolvents ..................................... 139
Table 5.4.1: Compositional analysis of SF1 solids from pretreatment of ethanol-
extracted bagasse with three different ILs. ........................................................... 146
Table 5.4.2: FTIR crystallinity indices of IL-pretreated solids .................................. 150
Table 5.4.3: Mass recovery, delignification and enzyme saccharification resulting
from treatment with different ILs ......................................................................... 155
Table 5.4.4: Mass recovery and lignin content of solids recovered from the liquid
fraction after treatment with three ILs .................................................................. 157
Table 5.4.5: Mass balance of bulk biomass and of biomass components from three
treatments with different ILs ................................................................................ 160
Table 5.4.6: Mass recovery of ionic liquid ions after use ....................................... 168
xx
Abbreviations and Nomenclature
[Allylmim]Cl: 1-allyl-3-methylimidazolium chloride
[C1mim] MeSO4: 1-methyl-3-methylimidazolium methyl sulphonate
[C2mim]Cl: 1-ethyl-3-methylimidazolium chloride
[C2mim]OAc: 1-ethyl-3-methylimidazolium acetate
[C4mim]BF4: 1-butyl-3-methylimidazolium tetrafluoroborate
[C4mim]CF3SO3: 1-butyl-3-methylimidazolium trifluoromethanesulphonate
[C4mim]Cl: 1-butyl-3-methylimidazolium chloride
[C4mim]PF6: 1-butyl-3-methylimidazolium hexafluorophosphate
[C4mmim]Cl: 1-butyl-2,3-dimethylimidazolium chloride
ABS: aqueous biphasic system
AFEX: ammonia fibre explosion
AIL: acid insoluble lignin
ARP: ammonia recycle percolation
ASL: acid soluble lignin
ATR: attenuated total reflectance
BASF: BASF, the chemical manufacturing corporation
b.p.: boiling point
CBP: consolidated bioprocessing
df: degrees of freedom
DMA / LiCl: dimethylacetamide / lithium chloride
DMA: dimethylacetamide
DMSO: dimethylsulphoxide
DP: degree of polymerisation
DS: dissolved-then- precipitated fraction
DSC: differential scanning calorimetry
EDA: electron donor-acceptor
EOL: ethanol organosolv lignin
FPU: filter paper units
FTIR: Fourier transform infrared spectroscopy
xxi
HMF: hydroxymethylfurfural
HPLC: high-pressure liquid chromatography
IC: ion chromatography
IL: ionic liquid
LCB: lignocellulosic biomass
LF: liquid fraction
m.p.: melting point
n/a: not applicable
n/d: not determined
NMMO: N-Methylmorpholine-N-oxide
NMR: nuclear magnetic resonance
NREL: National Renewable Energy Laboratory (Golden, CO, USA)
PEG: polyethylene glycol
rpm: revolutions per minute
SF: solid fraction
SHF: separate hydrolysis and fermentation
SRS: sugar recovery standard
SSCF: simultaneous saccharification and co fermentation
SSF: simultaneous saccharification and fermentation
STEX: steam explosion
TGA: thermogravimetric analysis
TRS: total recovered solids (sum of undissolved and precipitated solids)
UND: undissolved fraction
XRD: X-ray diffractometry
YPD: solution containing yeast extract, peptone and dextrose
1
CHAPTER 1 INTRODUCTION
1.1 Background
1.1.1 Renewable liquid fuels and chemicals from lignocellulosic biomass
Lignocellulosics, whether in the form of dedicated energy crops such as
sorghum, switchgrass and cardoon, agricultural residues such as sugarcane bagasse
and corn stover, or from forestry residues, present a renewable resource with an
energy value of approximately 300 x 1018 J worldwide [1]. With world energy
demand predicted to increase in the near future [2], and fossil fuel reserves being
depleted and non-renewable, biomass resources have drawn much attention as
renewable feedstocks for alternative fuels and chemicals. This attention is further
driven by issues such as the need to reduce CO2 emissions, the need to rely on local,
renewable and sustainable fuel sources (e.g. biofuel from crops farmed on marginal
land) and the need to reduce dependence on remote and unstable fuel sources (e.g.
petroleum imports). Among the strategies for biomass valorisation is hydrolysis and
fermentation. Ethanol fuel and other products of fermentation can be
manufactured from sugars, and high value polymers can be synthesized from lignin.
When these are derived from lignocellulosic biomass (LCB), a non-food renewable
resource, they present a promising sustainable alternative to petroleum based fuels
and chemicals.
This thesis investigates the conversion of sugarcane bagasse to ethanol fuel
using ionic liquid pretreatment. The initial focus of this study was optimisation of
pretreatment of sugarcane bagasse by dissolution in ionic liquid and fractionation
using aqueous salt biphasic systems [3]. Problems with this pretreatment process
(specifically with increasing convergence of biphases as biomass loading increases)
and, at the time, lack of published works on biomass – ionic liquid interactions, led
to a broader study of bagasse-imidazolium ionic liquid (IL) interactions and impacts
of these interactions on enzymatic saccharification of the polysaccharide
component of treated bagasse.
2
This chapter presents the characteristics of bagasse (Section 1.1.2), the
importance of pretreatment and the benefits and challenges of ionic liquids (Section
1.1.3). The aim and objectives of this study are described in Sections 1.1 and 1.2,
the novelty of the work in Section 1.3 and finally the thesis Chapter layout is
provided in Section 1.4.
1.1.2 Sugarcane bagasse
Sugarcane (Saccharum officinarum) is a sugar crop that thrives in tropical
climates and produces biomass prolifically; it is a member of the grass family
(Poaceae), has a high photosynthetic efficiency and produces a total biomass yield
of between 20 t ha-1 yr-1 to 30 t ha-1 yr-1 on a dry basis [4]. Accordingly, high CO2
sequestration capacity is an inherent advantage of this crop, with a reported CO2
fixation rate of 49 t ha-1 yr-1 for south Texas USA, more than three times that for
mid-latitude temperate forests [5].
The extraction of sucrose from sugarcane stems produces a biomass residue
known as bagasse. Bagasse, containing ca. 40 % - 45 % cellulose, 25 % - 30 %
hemicellulose and 25 % - 30 % lignin, is an ideal candidate for the production of
ethanol and biopolymers [4]. Infrastructure for the processing of sugarcane is
already well established, and the feedstock is already delivered at central locations
as part of the sugar manufacturing process. The energy available in harvested cane
biomass is well in excess of that required to process cane to raw sugar. As a result
most Australian raw sugar factories are configured to operate at low
thermodynamic efficiencies to dispose of the surplus fibre and avoid an unwanted
accumulation of bagasse at the end of crushing season. With only modest
improvements to boiler and process steam efficiencies, the energy required for
sugar processing could still be met whilst 35 % of the bagasse produced at the
factory made available for ethanol or power production. Based on a 34 million
tonne cane harvest for the Australian industry, this 35% bagasse surplus
corresponds to approximately 1.7 million tonnes of available dry fibre [6, 7].
3
1.1.3 The importance of pretreatment and fractionation
Ethanol is produced by hydrolysis and fermentation of the polysaccharides
in LCB. However these processes are inhibited by the complex structure of LCB.
Therefore pretreatment of LCB is necessary prior to hydrolysis and fermentation.
LCB, found in the structural tissue of plants, is a complex material designed
by nature to resist physical, chemical and biological (e.g., microbial and enzymatic)
attack. It is predominantly comprised of three biopolymers viz. cellulose,
hemicelluloses and lignin. It also contains small amounts of extractives (soluble non-
structural materials) and ash. This complex material is recalcitrant to the chemical
and/or biological processing involved in the production of cellulosic ethanol and
needs to be pretreated.
The conversion of lignocellulose into ethanol involves three main processing
steps, viz.:
• Pretreatment (opening up of the complex lignocellulosic structure to
increase surface area and improve further processing by enzymes)
• Saccharification (hydrolysis of the holocellulose or polysaccharide fraction of
lignocellulosics to monosaccharides)
• Fermentation (conversion of the monosaccharide source to ethanol using
yeast or other organisms)
This study investigates ionic liquids as agents for the first two steps:
pretreatment and fractionation. Pretreatment is an indispensible and expensive
processing step in the conversion of lignocellulosic biomass into fermentable sugars
[1, 8, 9]. Fractionation is an optional step that follows pretreatment and makes use
of the chemical properties of the pretreated/accessible biopolymers in order to
isolate them in a pure or partially purified form. Lignin is a known inhibitor of
enzyme hydrolysis [10, 11] while it is also potentially a high value feedstock for the
polymer industry. Separating the lignin from the polysaccharide fraction enhances
saccharification yields and may add a high value lignin product to the process.
4
Fractionation can also remove fermentation inhibitors such as sugar degradation
products (e.g. hydroxymethylfurfural).
Most pretreatment technologies are either physical (e.g., size comminution,
steam explosion and hydro-thermolysis) or chemical, utilizing organic solvents, acids
or alkalis [12-14]. These chemical pretreatment technologies occur by either acid or
alkali mechanisms at high temperatures and pressures (often at extreme pH values)
and produce products that may be inhibitory to enzymatic saccharification or
fermentation. Harsh pretreatment renders lignin in a condensed, non-reactive form
and reduces its potential value for functionalisation and polymer manufacturing
[15].
Recently, ionic liquids (ILs) have drawn a great deal of attention as “green”
solvents for processing of lignocellulosics. ILs are a class of organic salts that are
liquid at temperatures below 100 °C. Many ILs are non-volatile, non-explosive,
stable at a wide range of temperatures and reaction severities and compatible with
a wide array of organic and inorganic functional chemicals and solvents. ILs have
unique solubilisation characteristics compared to conventional molecular solvents
and some are known to achieve solvation of the whole lignocellulosic structure.
Formation of homogeneous solutions of lignocellulose in IL is a property responsible
for a number of beneficial pretreatment and fractionation characteristics. Such
characteristics include the dissolution-then-precipitation of a disordered
(decrystallised) cellulose and the potential for clean fractionation of cellulose, lignin
and hemicelluloses [3, 16-18].
1.1 Research aim
The overarching objective of this study is to investigate and optimise the
performance of imidazolium ionic liquids as a pretreatment and fractionation
strategy for bagasse.
5
1.2 Objectives
The objectives of this work are to:
• Investigate factors affecting dissolution of biomass in IL 1-butyl-3-
methylimidazolium chloride ([C4mim]Cl) and improve current understanding
of this pretreatment process
• Assess the performance of optimised IL ([C4mim]Cl) pretreatments and
compare with dilute acid pretreatment
• Investigate the lignin-polysaccharide fractionation efficiency of single and
biphase aqueous systems after IL ([C4mim]Cl) pretreatment of bagasse.
• Compare bagasse treatment in three imidazolium ILs ([C4mim]Cl, 1-ethyl-3-
methylimidazolium chloride or [C2mim]Cl, 1-ethyl-3-methylimidazolium
acetate or [C2mim]OAc) and understand effects of anion and cation
variation on saccharification yields, lignin fractionation efficiency, and total
mass balances.
1.3 Novelty
While ionic liquids have been extensively studied as solvents for cellulose over
the last decade, there are few accounts in the literature of whole biomass
dissolution and the effect of this dissolution on saccharification kinetics. Despite the
recent increase in research reports on IL pretreatment of biomass (which is cited in
the results and discussion of this thesis), this work remains novel and contributes
new knowledge. The compositional and structural analysis of dissolved and
undissolved fractions has the potential of improving the understanding of ionic
liquid pretreatment and no such detailed analysis is available at present. The direct
comparison of saccharification kinetics between different ionic liquids and dilute
acid pretreatment is also a novelty. The high viscosity of ionic liquids in combination
with their interference and occasional incompatibility with analytical
instrumentation (e.g. chromatography and spectroscopy) has discouraged the
scientific community from reporting extensively on full mass balance closures of
such processes. In this work mass balance closures are presented for pretreatment
processes using three different ionic liquids.
6
1.4 Summary of chapters
This chapter introduces the background, the objectives and the novelty of
this work.
Chapter 2 reviews the relevant literature that motivated this study while it
provides a background for the discussion of the emerging results. It covers the
structure of lignocellulosics, the characteristics of pretreatment technologies and
the properties of ionic liquids in the context of cellulose and biomass dissolution.
Chapter 3 describes the methodology and instrumentation used to produce
the results. It specifies the pretreatment and enzyme saccharification reaction
conditions and it details: a) protocols for the quantification of dissolution rates and
associated losses b) standardised wet chemistry methods for the compositional
analysis of pretreated bagasse and the associated liquid effluents (e.g. acid
hydrolysis and acetyl bromide digestion), c) spectroscopic instrumentation for the
structural analysis of bagasse (e.g. infrared spectroscopy and X-ray diffraction, XRD)
and d) methods for assessing the stability of biphasic systems (e.g. cloud point
titrations and calculation of phase divergence coefficients). It also provides the
protocol by which the challenging task of monitoring mass balances of ionic liquid
pretreatment processes was carried out.
Chapters 4 and 5 present and discuss the results emerging from the
experimentation of this project. Chapter 4 reports the results on a simple IL
([C4mim]Cl) pretreatment based on partial dissolution and precipitation using
water. The extent of dissolution and associated losses at different conditions are
examined. The saccharification performance and compositional/structural
characteristics of IL treated bagasse are discussed and compared to untreated and
dilute acid treated bagasse. Chapter 5 reports on experimentation with
fractionation systems. The stability and divergence of biphasic systems and the
associated fractionation difficulties are revealed. Single phase fractionation systems
employing solutions that are lignin solvents / cellulose antisolvents are also
investigated. The use of incremental additions of water in IL / bagasse partial
dissolutions to precipitate cellulose and keep lignin in solution is examined as a
7
fractionation strategy. Finally mass balances are determined for three IL
pretreatments ([C4mim]Cl, [C2mim]Cl and [C2mim]OAc). All results are compared
to those of previous works and their impact on current knowledge emphasised.
Chapter 6 summarises the findings and draws the conclusions from this
study.
Appendices present extra experimentation and data to which the main text
occasionally refers.
8
CHAPTER 2 LITERATURE REVIEW
2.1 Overview
This chapter covers the literature relevant to ionic liquid pretreatment of
lignocellulosics for the purpose of enzymatic hydrolysis of polysaccharides to
fermentable sugars (saccharification). The literature post 2008 is reviewed in
comparison to the results of this work.
The description of lignocellulosic biomass, beginning from component
molecules (e.g. cellulose) and extending to the structural characteristics of the
whole plant tissue (e.g. cell wall layers), is covered in Section 2.2. In this section the
swelling of cell wall layers is emphasized as it is an important precursor to other
events such as dissolution and saccharification. Section 2.3 covers pretreatment as
a first step in the process of producing fermentable sugars. It describes the
structural changes contributing to ease of LCB saccharification. Finally it reviews
some representative pretreatment technologies and how they effect these changes.
Ionic liquids as solvents for cellulose and LCB are reviewed in the final section
(Section 2.4). In this section, the characteristics of ionic liquids as pretreatment and
clean fractionation agents are emphasized.
9
2.2 Lignocellulosic biomass: chemical and structural characteristics
Lignocellulosic biomass (LCB, the mass of mature terrestrial plants) primarily
comprises woody (lignified) fibre in the cell walls of dead (no longer metabolically
active) tissues that provide mechanical support to the plant. For example
sclerenchyma tissue found in stems, trunks and branches is rich in LCB. As the term
LCB suggests, it is predominantly comprised of the lignin (a phenolic polymer) and
polysaccharides (namely cellulose and hemicelluloses). In a simplified depiction,
cellulose can be seen as the skeleton of the cell wall which is surrounded by
hemicelluloses as a filling matrix and lignin as an encrusting material [19]. In reality,
its structure is complex and varies among plant genotypes and even among
phenotypes. However, the general cell wall characteristics discussed here are
common to most terrestrial plant species that yield LCB.
2.2.1 Cellulose
Cellulose comprises 40 % to 45 % of the dry mass of LCB and it is located
predominantly in the secondary wall. It is an unbranched homopolysaccharide that
consists of β-(1→4) linked D-glucopyranosyl units. Each glucose unit is rotated 180o
with respect to its neighbour, so that the structure repeats itself every cellobiose
(glucose dimer) unit (see Figure 2.2.1). The three hydroxyl groups at C-2, C-3 and C-
6 positions of the glucopyranosyl units are involved in the hydrogen bonding in
cellulose crystal structures. An aldehyde group in a hemiacetal structure is found at
the C-1 end of the cellulose chain and a hydroxyl group at the C-4 end. The C-1 end
has reducing properties while the C-4 end is non-reducing. Finally, the conformation
of the glucopyranosyl unit is a 4C1 chair [20].
Figure 2.2.1: Molecular structure of cellulose
10
The stereochemical conformation of cellulose favours regular tight packing
of its long chains (degree of polymerisation (DP) ≥ 10000) resulting in crystalline
regions in native cellulose. These crystalline regions provide for a dense network of
intramolecular and intermolecular hydrogen bonds and make cellulose a high
tensile strength, water insoluble polymer. Other glucose polymers (glucans) with
different stereochemical conformation have very different physical and chemical
behaviour to cellulose. For example starch, which is a mixture of linear and highly
branched α-anomeric glucans, has very low tensile strength and dissolves readily in
water [19, 21].
The solubility of the homologous series of β-(1→4) linked D-glucopyranosyl
oligosaccharides in water decreases as the DP increases. Glucose is soluble in water
(54.6 g (100 mL)-1 at 30 °C [22]), cellohexose (cellulose oligomer of DP 6) is less
soluble and a cellulose oligomer of DP 30 is completely insoluble [20].
Cellulose is capable of forming a number of crystal structures, or allomorphs,
which differ in conformation and packing arrangement. The allomorph of native
cellulose is known as cellulose I, whereas the allomorph found in crystalline regions
of swollen or dissolved cellulose is known as cellulose II.
The unit cell of the cellulose I crystal allomorph is composed of four glucose
moieties in two parallel (i.e. reducing end at same end of adjacent cellulose chains)
cellulose chains (see Figure 2.2.2). This conformation provides for two types of
intramolecular hydrogen bonds, namely, from O(6) in one glucose residue to O(2)H
in the adjacent glucose and also from the ring oxygen (O(5)) to O(3)H. The chains
are then held together by hydrogen bonds from O(3) in one chain to O(6)H in the
other.
Cellulose II is formed by swelling of cellulose fibres containing regions of the
cellulose I allomorph with chemical agents such as strong alkali and subsequent
addition of water. Since the strongly hydrogen bonded cellulose II is
thermodynamically more favoured than cellulose I, it cannot be reconverted to
cellulose I. Unlike cellulose I, cellulose II is composed of chains which run
11
antiparallel (i.e. reducing ends at opposite ends to adjacent chains). The structure of
cellulose II (see Figure 2.2.2) results in less intramolecular and more intermolecular
hydrogen bonding as compared to cellulose I. The O(3)H to O(5) bond is maintained
as the only intramolecular hydrogen bond in cellulose II while the O(6) to O(2)H in
020 plane and the O(2)H to O(2) to the chain along the diagonal in the 110 plane
(not shown, into and out of the page), account for the intermolecular bonding [20,
21].
Figure 2.2.2: Most probable hydrogen bond patterns of cellulose allomorphs
(from Kroon-Batenburg [23])
Weimer et. al. [24] studied the digestibility of different cellulose allomorphs
by ruminal cellulolytic bacteria and concluded that cellulose I is more digestible
12
than cellulose II. Wada et al. [25] reported that the hydrated form of cellulose II is
more amenable to enzyme saccharification than cellulose I [25]. They also reported
the saccharification rate for an anhydrous cellulose II sample to be higher than that
of a cellulose I sample. However, this is not an effect of the allomorph transition
since upon conversion of cellulose I to cellulose II, the crystallinity index of the latter
was also reduced. This indicates that the enhanced saccharification of the
anhydrous cellulose II substrate is due to the reduction in crystallinity rather than
due to the change in cellulose allomorph. It can be thus concluded that the order of
saccharification efficiency of the macromolecular structures of cellulose is:
amorphous cellulose > hydrated cellulose II > cellulose I > anhydrous cellulose II.
The relative proportions of these cellulose structures in pretreated biomass solids
will play a role in their enzyme saccharification performance.
2.2.2 Hemicelluloses
Hemicelluloses comprise 20 % to 30 % of the dry mass of LCB. As opposed to
cellulose, they are a collection of branched heteropolysaccharides with shorter
chain lengths (maximum DP of about 200) and no crystalline structures. They
consist of hexoses (e.g. D-glucose) and pentoses (e.g. D-xylose and L-arabinose) in
addition to uronic acids and acetyl groups with the exact composition depending on
the type of hemicellulose. The composition and structure of hemicelluloses differ
characteristically between plant types (especially between hardwoods, softwoods
and grasses) and tissue types [26].
Glucuronoarabinoxylan (GAX, Figure 2.2.3) is the predominant type of
hemicellulose found in the grass family [26, 27]. It consists of a β-(1→4)-D-
xylanopyranosyl backbone which is partially substituted at C-2 with 4-O-methyl-α-
D-glucuronic acid (GlcA) (at ca. 2 xylose units out of every 10) and acetyl groups (at
ca. 1.2 xylose units out of every 10) and at C-2 or C-3 with α-L-arabinofuranose units
(at ca. 1.3 xylose units out of every 10). The glycosidic bonds of the xylose backbone
and the arabinose side chains are easily hydrolysed by acids but resistant to alkali,
whereas the uronic acid (GlcA) linkages with xylan are alkali labile and relatively
resistant to acids [19, 26, 28]. The bonds with acetyl groups can be easily cleaved by
13
alkali treatment [11, 19]. Acetyl groups are more abundant in softwoods and
hardwoods than they are in grasses.
Figure 2.2.3: Molecular structure of glucuronoarabinoxylan
2.2.3 Lignin
Lignin accounts for 20 % to 30 % of LCBs dry mass. It is a hydrophobic
‘cementing’ and ‘insulating’ agent of the plant cell wall and it is deposited mainly in
cell walls of supporting and water-conducting tissues. It is a phenolic polymer
formed from the polymerisation of three monomer units, p-coumaryl, coniferyl and
sinapyl alcohols (Figure 2.2.4). Lignins made up of these three monomers are called
p-hydroxyphenyl (H), guaiacyl (G) and syringyl (S) lignins respectively [21, 29].
14
OH
OOCH3H3C
OH
OH
OCH3
OH
6
54
3
21
α β
γ
OH
OH
p-coumaryl alcohol coniferyl alcohol sinapyl alcohol
Figure 2.2.4: Lignin monomer units
The lignin monomers form macromolecular structures via ether bonds (ca.
2/3 of monomer linkages) and carbon-carbon bonds [19]. The most common
linkages and dimer structures in lignin are shown in Figure 2.2.5 and a
macromolecular structure of a hypothetical lignin of European beech is presented in
Figure 2.2.6. A study on the structure of sugarcane bagasse by Sun et al. [30]
reports presence of all three types of phenylpropanoid units (H,G,S) linked to each
other mainly via β-O-4 ether bonds, and carbon-carbon bonds such as β-β, 5-5’ and
β-5.
The relative proportions of the individual phenylpropanoids contained in
lignin (H,G,S) vary between plant species. Dorrestijn et al. [31] report that pyrolysis
of grass lignins results in 45 % H, 39 % G and 16 % S phenylpropane units. However,
Meier et al. [32] determined that for sugar cane bagasse derived pyrolysis oil the
proportions of phenylpropanoids derivatives were 61 % H, 28 % G and 11 % S.
Ruggiero et al. [33] showed that unbleached acidolysis bagasse lignin had
proportions of phenylpropanoids of 56 % H, 37 % G and 7 % S whilst bleached
acidolysis lignin had 50 % H, 44 % G and 6 % S. It would appear that even for LCB of
the same species H:G:S ratios can vary, but for grasses in general the predominant
monomer is H followed by G, while S is substantially lower.
15
Figure 2.2.5: The most common linkages between lignin phenylpropane units
(from Sjostrom [19])
16
Lignin contains phenolic hydroxyl, benzylic hydroxyl and carbonyl groups.
Their frequency varies and from a processing point of view the relative frequency of
these groups in extracted lignin, determines its potential for processing towards
value-added products.
Figure 2.2.6: Partial structure of a hypothetical lignin molecule from European
beech (Fagus sylvatica)
(from Nimz [34])
17
2.2.4 Lignin-carbohydrate bonds
Hemicelluloses bind covalently to lignin but not to cellulose. However,
sufficient adhesion between cellulose and hemicelluloses is provided by hydrogen
bonds and van der Waals forces [19].
The covalent bonds between hemicelluloses and lignin are reported to
involve ester and ether bonds and they influence the reactivity of biomass when
exposed to chemical processing. For example, ferulic (or coniferic) acid esters are
known to make grass cell walls recalcitrant to enzymatic saccharification prior to
fermentation to biofuels [35].
Some common types of lignin-hemicellulose linkages found in the cell walls
of grasses are (depicted in Figure 2.2.7):
• Direct ester (e.g. uronic acid ester bonds formed by the attachment
of the carboxyl group of the hemicellulose GlcA branching unit to
phenolic hydroxyl sites in lignin [36].)
• Direct ether (e.g. benzyl-α-ether bonds formed between lignin and
the O5 position of arabinofuranose in hemicelluloses [36-38].)
• Hydroxycinnamic acid ester (e.g. ester bonds formed by the
attachment of carboxyl groups from lignin hydroxycinnamic acid to
the primary alcohol hydroxyls of the arabinofuranose unit of
hemicelluloses [27, 36].)
The most common hydroxycinnamic acids encountered in lignin of grasses
are ferulic acid and p-coumaric acid. Both acids participate in covalent linkages
between lignin and hemicelluloses. p-Coumaric acid is only known to form ester
bonds, while ferulic acid forms both ester and ether bonds. In addition, ferulic acid
can form dimeric (dehydrodiferulic) bridges between lignin and polysaccharides and
between different polysaccharide chains. These varying bonding possibilities of
hydroxycinnamic acids along with some direct ester and ether linkages are shown in
Figure 2.2.8.
18
Figure 2.2.7: Commonly occurring covalent linkages between GAX and lignin in
grasses
It is worth mentioning that the exact in situ bonding of lignin to
polysaccharides is not yet fully elucidated [36, 38] and that structures presented
here correspond to representations based mainly on ex situ characterisations of
lignin. In addition, plant cell walls may vary in chemical structure depending on
which tissue of the plant they pertain to (e.g. leaves, stems, young or old tissue),
and on the environmental stress experienced by the plant during growth (e.g.
drought, disease, mechanical stress by wind). Sun et al. [30] reported that the
lignin-carbohydrate bonds in bagasse consist mainly of coumaric acid esters and
ferulic acid ethers.
19
Figure 2.2.8: Possible covalent cross-links between polysaccharides and lignin in
cell walls
(from Iiyama et al. [36], www.plantphysiol.org Copyright American Society of
Plant Biologists)
2.2.5 Cellulose microfibrils: The foundation units of the cell wall construct
Cellulose microfibrils are the rod-like foundation units of the cell wall
structure. Native cellulose is a long polymer whose molecule stretches to a length of
at least 10000 glucose units. Parallel cellulose molecules, held together by hydrogen
bonds, form the smallest building element of the cellulose skeleton known as the
microfibril [19]. The microfibrils wind together to form threads that coil around
each other, like strands in a cable. Each ‘cable’ forms the next size building unit
20
called a macrofibril (Figure 2.2.9). Cellulose molecules wound in this fashion have a
tensile strength approaching that of steel (50-160 kg mm-2) [29].
Cellulose has crystalline properties resulting from the orderly arrangement
of cellulose molecules in microfibrils. This crystalline arrangement is restricted to
parts of the microfibril known as micelles (Figure 2.2.9).
Amorphous and disordered cellulose as well as hemicelluloses and lignin are
located in the spaces between the microfibrils. Hemicelluloses are considered
amorphous and lignin is both amorphous and isotropic [19].
Figure 2.2.9: Detailed structure of cell walls
(from Evert et al. [29]).
A, strand of fibre cells. B, transverse section of fibre cells showing layering: a layer of primary and three layers of secondary wall. C, fragment from the middle layer of the secondary wall showing macrofibrils (white) and interfibrilar spaces (black). D, fragment of a macrofibril showing microfibrils. E, structure of microfibrils showing the long cellulose molecule which in some parts forms orderly micelles. F, fragment of a micelle, G, two glucose residues forming the repeating unit (cellobiose) of the cellulose polymer
21
2.2.6 The cell wall layers
The mature, lignified cell wall is organised in layers, namely middle lamella
(ML), primary wall (P), outer layer of secondary wall (S1), middle layer of secondary
wall (S2) and inner layer of the secondary wall (S3) (Figure 2.2.10). These layers
have distinct structure and composition. The microfibrils wind around the cell axis
in different directions, either to the right (Z helix) or to the left (S helix) [19] in the
direction of growth.
Figure 2.2.10: Cell wall layers and organisation of the cellulose microfibrils
(adapted from Raven et al. [39]).
The middle lamella fills the intercellular spaces and binds the cells to each
other. At maturity of the cell, this layer is predominantly composed of lignin.
The primary wall is a thin layer consisting of cellulose, hemicelluloses, pectin
and protein completely embedded in lignin. In the outer portion of this layer, the
cellulose microfibrils form an irregular network, while in the interior, they are
oriented nearly perpendicular to the cell axis.
ML
S3
S2
S1
P
22
The secondary cell wall represents most of the cell mass, especially its thick
middle layer (S2). The three layers of the secondary wall are built of near-parallel
microfibrils of cellulose between which lignin and hemicelluloses are intertwined.
The orientation and angle of the helices formed by the microfibrils vary among the
three layers. In the outer secondary wall (S1) the orientation of the helices is both Z
and S and at angles to the long cell wall axis that are large and sometimes
perpendicular. In the mid-layer (S2), the angle is small and the slope of the helix
steep (close to vertical to the long axis). In the inner layer (S3), the microfibrils are
deposited as in S1, at large angle to the long axis [29, 39].
2.2.7 Mechanism of cell wall swelling
Swelling is a natural phenomenon of cell walls which allows space for the
lateral deposition of newly-formed fibre. In presence of solvents, swelling is the first
step in the process of dissolution of cell walls and also occurs without dissolution.
Swelling of biomass in ionic liquids without dissolution is covered in this thesis and
therefore it is worth reviewing here.
In the presence of reagents that induce swelling, the thick S2 layer swells
laterally (unidirectional microfibril helices at steep angles) while the primary wall
gets peeled off as the S1 layer around the fibre expands [19]. This combination of
events results in the formation of balloon structures seen in Figure 2.2.11.
The complex structure of LCB is comprised of polysaccharides and lignin that
are intricately intertwined inside the cell wall layers. The abundance of LCB and its
rich polysaccharide content make this material an attractive carbon resource for
manufacturing renewable fuels. However, its complex structure poses challenges to
fuel manufacturing based on fermentation of the monosaccharide products of
saccharification. Pretreatment is the chemical, mechanical and/or biological process
by which the recalcitrant and inaccessible LCB structure becomes available for
fractionation and further processing towards fuel and other added value products.
Pretreatment is reviewed in the following section.
23
Figure 2.2.11: Light microscope image showing ballooning of a sulphate pulp fibre
(Pinus silvestris)
(from Illvessalo-Pfaffli [40])
2.3 Pretreatment
2.3.1 Overview of the conversion of biomass to ethanol fuel
The conversion of the LCB polysaccharides to ethanol fuel and/or other
products of fermentation (e.g. butanol) involves hydrolysis, fermentation and
distillation. However, these polysaccharide molecules are not readily hydrolysed
since they are contained in the chemically recalcitrant and structurally robust
lignocellulosic matrix described in Section 2.2. Therefore, a pretreatment step is
added to the process prior to hydrolysis in order to improve saccharification of the
polysaccharides. The process steps involved in the conversion of LCB to ethanol are
shown in Figure 2.3.1.
Pretreatment is the first step in this process and its goal is to ‘open up’ the
LCB structure. This entails structural modification of the material (e.g. reduce
cellulose crystallinity and increase surface area) and chemical modification (e.g.
minimise lignin content). This step is the subject of this thesis and will be reviewed
in more detail in this section.
The ribbon like, unrolled primary wall (P) surrounds the swollen secondary wall. S1 is the swollen outer layer of the secondary wall, under which the microfibrils of the middle layer, nearly parallel to fibre axis, are dimly visible. S3 is the inner
24
Since pretreatment is the first step in the process, it has to be designed to
minimise inhibition towards downstream process steps, namely hydrolysis and
fermentation.
Figure 2.3.1: Gross representation of the main steps in a biomass to ethanol
process
Hydrolysis can be catalysed by mineral acids or enzymes derived from
cellulolytic fungi such as Trichoderma reesei. Although concentrated mineral acid
hydrolysis is more technologically mature than enzymatic hydrolysis, the enzymatic
processes are expected to have cost advantages as cellulase research advances.
Moreover, acids are associated with greater environmental liabilities and the
formation of fermentation inhibiting products such as furfurals [8]. With the
exception of comminution prior to direct hydrolysis of LCB in concentrated acid,
pretreatment is tailored to optimise the LCB substrate for enzyme rather than acid
hydrolysis.
lignocellulosic biomass (LCB)
'opened up' LCB accessible polysaccharides
monosaccharides
ethanol
PRETREATMENT
HYDROLYSIS
FERMENTATION
25
The saccharification of LCB after a number of pretreatments are compared
in Section 2.3.5 (after all these pretreatments have been discussed)
Fermentation to ethanol is carried out by yeasts or bacteria. Yeast
(Saccharomyces cerevisiae), has been traditionally used in the brewery industry to
convert hexoses to ethanol. However, a substantial proportion of the sugars in most
LCB substrates are pentoses (20 % to 30 %) and there is a need for a microorganism
that can ferment these too. Microbiologists are still improving yeast and bacterial
strains in order to achieve fermentation of both hexoses and pentoses to ethanol or
butanol [8].
Both enzymatic hydrolysis and fermentation are biologically catalysed
reactions and they are collectively known as the ‘bioprocessing’ steps of the
cellulosic ethanol process. Ideally, all bioprocessing steps would be carried out by a
single system of organisms that would simultaneously exhibit the following
properties: (a) synthesis of an active cellulose enzyme system at high levels, (b)
fermentation and growth on sugars arising from both cellulose and hemicellulose,
and (c) production of ethanol at high selectivity and high concentration.
Unfortunately all compatible combinations of known microorganisms fall short of
this ideal, on account of two main limitations: (a) an inability to utilize the range of
carbohydrates present in biomass (e.g. cellulose, hemicellulose) while also
producing ethanol at high yield or (b) differing requirements for oxygen for various
functions essential to the process. For example S. cerevisiae, is unable to ferment
pentose and cannot coexist with T. reesei because the latter requires oxygen for
growth while the former requires low oxygen conditions for fermentation. Two
approaches have been employed to overcome this incompatibility; viz. create
recombinant organisms that are compatible, or carry out each bioprocess in
separate reactors. These approaches including their intermediate forms are listed in
Figure 2.3.2. When all bioprocessing takes place in a single reactor, the process is
referred to as consolidated bioprocessing (CBP), when the enzyme is produced in a
separate incubator, the process is referred to as simultaneous saccharification and
co fermentation (SSCF), when all four bioprocesses take place in different reactors,
26
the process is referred to as separate hydrolysis and fermentation (SHF) [8].
Although CBP is the most favoured approach in terms of less infrastructure
requirements (single reactor), the biotechnology of compatible microorganisms is
still at low maturity and less consolidated forms of bioprocessing are currently the
most commonly employed options.
The ethanol fuel is recovered from the fermentation broth by distillation.
Generally, the unfermented solids (lignin, unreacted polysaccharide fractions and
enzymes) accumulate at the bottom of the distillation column. These solids are
dried and combusted for generation of thermal energy [8]. The type of
pretreatment and extent of bioprocessing consolidation influence the composition
of these unfermented solids. In biorefinery processes based on clean fractionation,
lignin and hemicelluloses may be separated from the cellulose prior to
saccharification, and the residue after distillation may be suitable for purposes
other than combustion (e.g. animal feed).
Processing strategy (each box represents a bioreactor not to scale)
Biologically
mediated event
SHF
(separate
hydrolysis and
fermentation)
SSF
(simultaneous
saccharification
and fermentation)
SSCF
(simultaneous
saccharification
and co
fermentation)
CBP
(consolidated
bioprocessing)
Cellulose
production
Cellulose
hydrolysis
Fermentation of
C6 sugars
Fermentation of
C5 sugars
Figure 2.3.2: Consolidation of bioprocessing in cellulosic ethanol production
(from Lynd [8])
27
2.3.2 Goals of pretreatment
The benefit of pretreating biomass prior to enzyme saccharification has long
been recognised [14]. Unless a very large excess of enzyme is used (with
consequent higher processing costs), the final cellulose conversion in native
biomass is very low (< 20 % of theoretical), whereas with appropriate pretreatment,
it can often reach 100 % of theoretical mass [13]. Optimising performance and
reducing cost of pretreatment is a current research activity aimed at enhancing the
commercialisation potential of cellulosic ethanol.
The goal of pretreatment is to disrupt certain structural and chemical
characteristics of native biomass that are thought to be responsible for its low
enzyme saccharification rates. These include, the crystallinity of cellulose, the
presence of lignin, the protection of cellulose by lignin and hemicellulose, and the
small surface to volume ratio (porosity) of the material.
Lignin in biomass undoubtedly interferes with enzymatic hydrolysis of
glycosidic linkages. The phenolic polymer restricts physical access of the enzymes to
cellulose [41] while it also provides sites to which cellulase enzymes adsorb
unproductively and irreversibly [42]. Research shows that rates and extent of
biomass saccharification increase with increasing lignin removal [11].
Hemicellulose removal is also reported to enhance enzyme saccharification
of cellulose [43]. Hemicellulose removal increases internal surface area, provides
more immediate access of enzymes to cellulose, and reduces unproductive binding
of cellulases on hemicellulose sugars [10, 43, 44]. Acetyl ester linkages are
detrimental to enzyme accessibility and the selective removal of acetyl groups has a
positive effect on saccharification of LCB [10, 11, 43]. For example, Grohmann et al.
[43] reported saccharification rate increase of 5-7 times for hemicellulose and 2-3
times for cellulose after selective removal of 75 % of acetyl groups of wheat straw
following de-esterification with hydroxylamine solutions. Acetyl groups are not
frequent on xylans of grasses and are therefore not of major influence to the
reactions in this study.
28
Highly crystalline cellulose is less accessible to enzyme attack than
amorphous cellulose [10, 11, 44]. Experiments on pure microcrystalline cellulose
(e.g. Avicel) provide strong evidence that cellulose decrystallisation improves
enzyme saccharification rate [44, 45]. Dadi et al. [45] produced amorphous cellulose
using dissolution in ionic liquid and reported a 50-fold higher saccharification rate
compared to crystalline cellulose. Jeoh et al. [44] decrystallised cellulose using a
DMSO-paraformaldehyde technique that had been demonstrated to produce
amorphous cellulose without affecting its DP. Their results showed a 15-fold
enhancement of cellulase enzyme accessibility compared to crystalline cellulose.
Holtzapple and co-workers [10, 11], have also demonstrated the positive effect of
decrystallisation on the saccharification of LCB cellulose. “Selective”
decrystallisation of LCB cellulose in these studies was achieved by ball milling. Work
by Gharpuray et al. [46] demonstrated that ball milling reduced particle size and
increased surface area and in the process produced less crystalline biomass.
Therefore the effect of ball milling on saccharification rate was not due to
decrystallisation alone. However, Holtzapple and co-workers supported the
selectivity of their methodology by citing a preceding publication where it was
demonstrated that further reduction of biomass particle size below 40-mesh (the
authors’ starting material) did not enhance the saccharification rate [10, 11].
Lignin-hemicellulose covalent bonds are another source of recalcitrance to
hydrolysis. For example, expression of ferulic esterase enzymes in tall fescue grass,
improved enzyme saccharification of its cell walls by rumen fluid inocula by 10 % -
14 % compared to the control [35].
All aforementioned factors play a role in the enzyme saccharification of LCB.
Their relative importance is hard to define due to the fact that ‘selective’ chemical
removal of one barrier usually affects the state of at least one other barrier. Chang
and Holtzapple [11] appear to have successfully isolated the effects of three of
these factors (namely delignification, deacetylation and decrystallisation of poplar
wood) and reported their relative importance both on cellulose and hemicellulose
saccharification. Their results indicated that among the three structural features,
29
lignin content and crystallinity had the greatest effects on saccharification of total
polysaccharides. As compared to cellulose, hemicellulose saccharification was less
affected by decrystallisation (since it is not crystalline) and more affected by
delignification and deacetylation (since it is covalently linked to both lignin and
acetyl groups). Zhu et al. [10] took this work further and investigated interrelations
and relative importance of each characteristic at different stages of the
saccharification reaction (1 h, 6 h and 72 h). As shown in Figure 2.3.3, this work
demonstrated a causal relationship between enzymatic saccharification and enzyme
adsorption extent (amount) and effectiveness, which are in turn related to the
structural features of the LCB substrate. The amount of enzyme that binds to
polysaccharides is reflected in the extent of saccharification whilst the enzyme’s
effectiveness plays a role at the initial saccharification rates. The amount of enzyme
that binds was increased with increasing lignin content and to a lesser extent with
increasing acetyl content, while the effectiveness was correlated with the cellulose
crystallinity of biomass. These conclusions can aid the interpretation of enzyme
hydrolysis results from various pretreatment-substrate combinations.
Figure 2.3.3: The effects of lignin, acetyl groups, and crystallinity on enzyme
adsorption and enzymatic hydrolysis of biomass
(reproduced from Zhu et al.[10])
CRYSTALLINITY EFFECTIVENESS 1-h Saccharification
extent
ACETYL
AMOUNT
LIGNIN
6-h Saccharification
extent
72-h
Saccharification
extent
Enzyme adsorption Structural features
of LCB
Enzyme
saccharification
extent
Thicker arrows indicate a more significant effect
30
Apart from the aforementioned pretreatment aim of increasing
saccharification, an efficient pretreatment technology has to take into consideration
a number of variables related to the processes’ general viability. These include:
• the potential for added value co-products (e.g. added value lignin co-
product)
• release of inhibitory by-products to downstream processing
• losses of carbohydrate raw material
• energy usage (heat, mechanical)
• resource usage (water)
• use of reagents that are expensive or toxic to humans and the
environment
• the cost of biomass and its appropriateness for the pretreatment
• overall capital and operation costs
• the contribution to life cycle impact factors
These criteria as well as the saccharification rates are the basis on which
different pretreatment technologies should be compared to each other. Figure 2.3.4
schematically represents the relation of such criteria to the inputs and outputs of a
conventional pretreatment process that does not result in clean fractionation of
biomass components.
2.3.3 Pretreatment technologies
Pretreatment is usually physical or chemical or a combination of the two. At
least to date, biological pretreatments using microorganisms (e.g. white rot fungi)
have been too slow (order of weeks) and therefore not favoured [14, 47].
Physical pretreatments involve the application of mechanical force and hot
water or steam. They exclude the use of any additive chemicals. These treatments
increase the surface area of the substrate while they can also solubilise part of the
non-cellulosic fraction of the biomass structure.
31
Figure 2.3.4: The participating inputs and outputs in a pretreatment process
Mechanical comminution reduces particle size, increases surface area and in
the case of ball milling also disrupts cellulose crystallinity. However the energy
usage of comminution is high and increases exponentially with decreasing particle
size. Therefore comminution is usually limited to ‘coarse milling’ before it starts
becoming prohibitively energy-intensive. This coarse milling is sometimes needed to
reduce the size of material that is destined for chemical pretreatment [13].
Steam explosion (STEX) is a physical treatment very commonly used for the
pretreatment of biomass. In this method, biomass is impregnated with high
pressure saturated steam (160 °C to 260 °C for 1 min to 10 min) and then released
to explosively decompress to atmospheric pressure. It removes some
hemicelluloses and lignin while it increases the surface area of the STEX solid LCB
substrate. Since water acts as an acid at high temperatures, some of the
characteristics of STEX pretreated biomass resemble those of acid treated. In fact,
P
R
E
T
R
E
A
T
M
E
N
T
Pretreatment Additives
Minimise, recycle and avoid toxicity
Biomass
Low cost + fit for pretreatment
Liquid stream
Minimise inhibitors and carbohydrate losses
Pretreated solid
Maximise
saccharification
Vapour stream
Minimise vapour + avoid loss of carbohydrate or reagent
Energy (heat, mechanical)
Minimise
INPUTS OUTPUTS
Lignin fraction
Added value co-product
Combustion of residue
Heat and power
White: Inputs and outputs. Grey: the corresponding criteria/targets associated with each input and output. Light grey: enzyme saccharification as the central criterion
32
addition of acid in steam explosion increases this resemblance; nearly all
hemicellulose is removed and more sugar degradation is incurred [13, 14].
Chemical treatments are reactions that use aqueous acid or alkali solutions
at elevated pressures and temperatures. It is generally understood that acid
pretreatments remove hemicelluloses, alkali pretreatments remove lignin and both
increase the internal surface area of the substrate.
Dilute acid hydrolysis is the most widely studied pretreatment. Dilute H2SO4
has been used to commercially produce furfural from cellulosic materials [48].
Dilute acid treatment at concentrations of H2SO4 below 4 % and at high
temperature (160 C° to 190 C°) and pressure for about 10 min appear very effective
for quantitative removal of hemicelluloses and render the rest of the LCB more
digestible for enzymes [13, 14]. Fermentation inhibitors such as furfurals are a
byproduct of dilute acid pretreatment and should preferably be extracted from the
liquid stream destined for fermentation [49, 50].
Alkaline pretreatment processes utilize lower temperatures and pressures
compared to other pretreatment technologies. Lime (calcium hydroxide)
pretreatment is carried out at low temperatures (100 °C to 150 °C) but depending
on temperature may require a long residence time (typically 2 h to 12 h but up to a
number of days) [11, 14, 51, 52]. Kim et al. [52] demonstrated that lime treatment
deacetylates and delignifies the LCB substrate. Deacetylation of 90 % was achieved
regardless of temperature or reaction conditions. Delignification extent increased
with increasing temperature and in the presence of oxygen [52].
Ammonia fibre explosion (AFEX) is similar to STEX although the chemistry
involved differs. In a typical treatment, equal masses of liquid ammonia and dry
biomass are heated under pressure to 90 °C for 5 min to 30 min and then the
pressure is suddenly released [14, 53]. AFEX increases the surface area and
interferes with internal bonding of the LCB without removing substantial mass from
the substrate [53]. According to Teymouri et al. [53] AFEX can also reduce cellulose
crystallinity although they provide no direct evidence or measurement of
33
decrystallisation. Kumar et al. [54] provide evidence that AFEX can effect a slight
decrystallisation on corn stover although not on poplar. A comparative
disadvantage of AFEX is reported to be the slow saccharification kinetics in
substrates of high lignin content (e.g. aspen wood chips with 25 % lignin, only
reached 50 % saccharification extent after AFEX) [14].
The ‘organosolv’ processes are based on pulping technologies that use
combinations of solvents to remove lignin. For example, in an organosolv process
using an organic solvent (e.g. ethanol) mixed with an aqueous solution of dilute acid
(e.g. H2SO4) at high temperature and pressure (180 °C for 60 min), the organic
solvent acts as a delignifying agent while the acid aids in the removal of
hemicelluloses. The resulting solid material is a soft cellulose pulp of low lignin and
hemicelluloses content. Pan et al. [55] applied the organosolvation process to the
conversion of poplar to ethanol. The process resulted in the fractionation of poplar
chips into a cellulose-rich solids fraction; an ethanol organosolv lignin (EOL) fraction;
and a water soluble fraction containing hemicellulosic sugars, sugar breakdown
products, degraded lignin and other components.
The above examples cover most chemical pretreatments proposed to date.
The underlying chemical mechanisms are predominantly governed by the pH of the
medium. Even in organosolv or hot water treatments with no added acid, organic
acids (principally acetic acid) released from LCB upon treatment result in acid like
processes (known as autohydrolysis). However there are numerous pretreatment
variations based on choice of equipment. One variation is the use of continuous
percolation reactors (e.g. ‘flow-through’ reactor), which press the solvent through a
biomass ‘cake’ at high temperatures (usually around 160 °C) and recycle it back in
the process in a continuous mode. Examples include ammonia recycle percolation
(ARP), hot water and dilute acid percolation, where biomass is processed with liquid
ammonia, water or dilute acid, respectively [13].
Most pretreatment technologies require expensive, pressure-rated
equipment, have high energy requirements and use corrosive or volatile chemicals
[13]. Acids or bases and organic solvents at high temperatures and pressures create
34
conditions that are corrosive to common stainless steel industrial equipment. Many
of these processes have associated workplace health and safety issues which also
may increase costs. In addition, acidic or alkali output streams need to be
neutralized prior to enzyme saccharification, producing mineral salts that are
difficult to recycle. Notwithstanding these problems, none of the above
pretreatments discussed here demonstrate much ability to disrupt the crystallinity
of cellulose [54]. While the crystallinity is somewhat reduced (e.g. AFEX or ball
milling), these outcomes are secondary to the intended primary effect.
Pretreatments that result in solid materials with retained cellulose crystallinity
require high enzyme loads and long saccharification times to effect complete
saccharification [10]. Such processes have capital and operating costs that are still
too high for lignocellulosic ethanol to be competitive with petroleum [56].
2.3.4 Conventional cellulose solvents
The use of cellulose solvents is another possible pretreatment. Among the
cellulose solvents are concentrated mineral acids which depolymerise cellulose and
those solvents which dissolve cellulose in polymeric form. Dissolving the LCB in a
polymeric form provides the opportunity for clean fractionation of the dissolved
biomass molecules and the precipitation of cellulose in a decrystallised form.
Cellulose solvents disrupt the crystalline order of native cellulose [20, 44,
45]. Concentrated acid is a long known means of chemically decrystallising cellulose
[58]. Concentrated acid treatment of cellulosic materials has been practiced in
various forms during times of fuel shortages [59] and more recently is the subject of
a patent by DuPont & Co [60] and other patents [61, 62]. However, large scale
industrial applications are still limited by the corrosivity, safety risk, high water
usage and disposal problems associated with concentrated acid. Combinations of
aprotic solvents (e.g. dimethylsulphide or dimethylacetamide) and metal salts (e.g.
LiCl or FeCl3) are cellulose solvating systems often used for laboratory scale
preparation of amorphous cellulose [20, 44]. Generally their industrial applications
are limited to products of higher value than fuels due to the volatility, toxicity and
cost of these solvents.
35
In fact, cellulose is a versatile starting material for chemical conversion to
renewable/biocompatible films, fibres and packaging materials. Dissolution of
cellulose in polymeric form is an essential step prior to such conversion. Since its
crystalline structure does not facilitate chemical interaction with solvents,
dissolution has been a challenging and long standing goal in research for cellulose-
based artificial polymers.
Until about 1950, only cuprammonium was well-known and widely used as a
solvent for cellulose. Ten years later, the discovery of solvents based on transition
metals broadened the spectrum of cellulose solvents. Since then a large number of
organic solvents have been added to this list [20]. Figure 2.3.5 gives an overview of
the conventional cellulose dissolving systems. Depending on the interaction of the
solvent with polysaccharide these solvents are classified as derivatising and non-
derivatising. Derivatising solvents covalently interact with cellulose to form unstable
intermediates such as cellulose esters, ethers or acetates. Non-derivatising solvents
have Coulombic interactions only with the substrate and therefore there is no
formation of intermediates and probably limited covalent bond cleavage.
Non-derivatising solvents are more versatile in terms of further processing
of the dissolved cellulose and are more relevant to pretreatment applications.
These solvents are systematically divided into a number of subcategories
comprising varying combinations of polar organic solvents, inorganic salts,
transition metals and amino groups. The most relevant to practical uses is the
subcategory that takes advantage of the strong intermolecular interaction between
the polymer and some dipolar aprotic organic compounds with N-O or C=O dipoles.
These solvents can be subdivided into the two groups, viz. salt-free and salt-
containing systems. N-Methylmorpholine-N-oxide (NMMO) is representative of a
salt-free solvent and dimethylacetamide / LiCl (DMA / LiCl) is a commonly used
example of a salt containing system.
36
O
N
O
O S O
O
N
NMMO (N-Methylmorpholine-N-oxide)
Li
Cl
DMA / LiCl (Dimethylacetamide / LiCl)
H H
O
O
mineral acids (sulfuric acid)
ONH
NN O
O
O
O
N2O4/DMF (dinitrogen tetroxide/dimethylformamide)
OH Na
Sodium hydroxide (NaOH)
N
S
O
F
DMSO / TBAF (dimethylsulfoxide / tetrabutylammonium fluoride)
LiClNH
HN
O
Dimethylimidazolone / LiCl
ClO4
SCN Li
Cl ClZn
Li
molten salt hydrates
Figure 2.3.5: Conventional cellulose solvents
(reproduced from Pinkert et al. [57])
Solvent systems with molecular interactions or bonds of high dipole moment
(of near ionic character) and strongly electronegative anions (i.e. Cl- and F-) seem to
be common characteristics among the solvents listed in Figure 2.3.5. In that regard,
Spange et al. [63] have demonstrated that the chloride ion contributes about 80 %
of the dipole-dipole interactions between DMA and cellulose in DMA/LiCl solvation
systems and is primarily responsible for hydrogen bond disruption in cellulose and
consequent dissolution.
NMMO and its monohydrate form is the solvent of choice in the process
used for manufacturing the cellulosic apparel fibre known as Lyocell on a technical
scale of about 100,000 tonne per annum [64]. The solvation power of NMMO is due
37
to its ability to disrupt hydrogen bonds. Dissolution is facilitated by acid-base
(donor-acceptor) interactions resulting in disruption and restructuring of the
hydrogen bond network of native cellulose. NMMO is a weak base (pKB = 9.25) and
its most prominent feature is the highly polar N-O group with a dipole moment of
4.38 D [65]. This dipole is symbolized either as ionic (with positive charge on the
nitrogen and negative on the oxygen) or as donative with an arrow pointing at the
oxygen (see Figure 2.3.6). NMMO was originally introduced as a cheaper, faster and
more environmentally benign alternative to the Viscose process. However the
NMMO treatment causes severe fibrillation of fibres, while the Viscose process
based on NaOH and carbon disulphide, produces fibres with properties similar to
cotton. Moreover, the use of NMMO – a thermally unstable solvent – also requires
a major investment in safety technology. Consequently the Viscose process is still
used in the manufacture of ca. 95 % of modified cellulose fibre [66].
ONOHOCellulose
Figure 2.3.6: Gross schematic of the hydrogen bonding formed between NMMO
and cellulose hydroxyls upon dissolution
In the case of salt-containing systems, a direct complexation between the
cation and the cellulosic hydroxyl group is assumed. This interaction is facilitated by
the participation of the polar organic medium in which these solvations take place.
DMA / LiCl is the most widely used system for the dissolution of cellulose for
analytical purposes. After preactivation, even high molecular weight cellulose can
be dissolved without residue and detectable chain degradation [20].
Cellulose dissolution without covalent derivatisation can be generally viewed
as an electron donor-acceptor (EDA) interaction where the amphoteric cellulose
takes the role of either donor or acceptor or both, depending on the solvent
structure in hand [20]. This generic model is presented schematically in Figure 2.3.7.
38
Figure 2.3.7: EDA interactions between cellulose and a non-derivatising solvent
(e.g. NMMO)
According to Cuissinat and Navard [67], dissolution of cellulose microfibrils,
either as cotton or wood fibres, follows specific patterns upon dissolution. These
patterns are governed by the efficiency of the solvent and the orientation of
cellulose chains in fibres. Highly efficient solvents disrupt the hydrogen bonding
network as fast as they penetrate the fibre (fast dissolution by disintegration of rod-
like fragments). Less efficient ones penetrate faster than they dissolve, leading to
the formation of swollen balloon structures that eventually burst and dissolve. Poor
solvents penetrate only without disrupting any of the H-bond network (swelling
with no dissolution). Finally non-solvents are unable to cause either swelling or
dissolution. These dissolution patterns are relevant to pretreatment since both
swelling and dissolution enhance enzyme access to crystalline cellulose ([68] and
own data).
2.3.5 Enzyme saccharification of cellulosics after pretreatments
The enzyme saccharification yields following various pretreatments are hard
to compare directly since the substrates, the conditions and the enzyme properties
vary among studies in the literature. The Biomass Refining Consortium for Applied
39
Fundamentals and Innovation (CAFI) [69] was the first attempt to compare sugar
recovery data from different biomass pretreatments. The laboratories that
participated assessed enzyme saccharification and total sugar recovery for a
number of pretreatments using identical protocols. Although this initiative provided
a useful single source of comparison for pretreatment technologies, the reaction
conditions among the compared pretreatments still varied considerably. For
example pretreatment residence times varied from 5 min for AFEX to 4 weeks for
lime treatment. Some pretreatments perform better at initial saccharification rates
while others may be slower but achieve a higher final saccharification extent. In
Table 2.3.1, a selection of studied pretreatments is compared for saccharification
yields both at the early stages (24 h) and at end of reaction (≥48 h).
Table 2.3.1: Enzymatic saccharification from selected pretreatment systems
Pretreatment
24h
Saccharification
(% cellulose1)
Final extent of
saccharification
(≥48 h)
(% cellulose1)
Substrate Enzyme
loading
(FPU/g
cellulose1)
Reference
AFEX 66 96 Corn stover 15 Teymouri et al. [70]
Dilute acid 80 80 Idem Idem Lloyd et
al. [71] Hot water
(flow
through)
ND 96 Idem Idem Kim et al. [72]
Lime 80 100 Idem Idem Kim et al. [73]
Organosolv 92 98 Hybrid poplar
20 Pan et al. [74]
Phosphoric
acid (84%)
97 97 Corn stover 15 Zhu et al. [75]
Ionic liquid
[C2mim]OAc
96 99 Switchgrass 50 mg protein /g cellulose
Li et al. [76]
Conventional cellulose solvents (e.g. phosphoric acid) seem to yield near 100
% cellulose conversion at 24 h of exposure to enzymes. This is largely attributed to
the complete decrystallisation of cellulose. Dilute acid achieves about 80 % at 24 h,
1 cellulose = mass of cellulose recovered after pretreatment
40
but the highly crystalline cellulose and high content of lignin do not permit higher
yields. Lime on the other hand, which removes lignin, reaches near 100 % final
cellulose conversion. Organosolv in presence of acid achieves both delignification
and hemicelluloses-acetyl removal and approaches the performance of cellulose
solvents.
Ionic liquids are a new class of solvents that can be used for LCB
pretreatment. Preliminary reports on IL pretreatment show both high rates and
extents of saccharification (see Table 2.3.1) while the ability of the IL properties to
be tuned offers the potential of optimising dissolution performance and minimising
the aforementioned solvent-related problems.
LCB pretreatments based on ionic liquids are the subject of investigation in
this thesis and current knowledge of this is reviewed in the following section.
2.4 Ionic liquid based pretreatment technologies
2.4.1 Ionic liquids: properties and history
Ionic liquids are low-melting salts (< 100 °C), which form liquids that consist
of cations and anions only. Characteristics that contribute to the low melting points
are large ions with low symmetry and delocalized charge [77]. Ions can be inorganic
or organic ions, often featuring an aromatic or cyclic structure and long alkyl chains.
Many ILs have negligible vapour pressure, are non-flammable and can be designed
to have high thermal stability [78, 79] and low toxicity [80, 81].
The first reporting of an IL dates back to the mid 19th century. Chemists
performing an AlCl3-catalysed Friedel-Crafts alkylation observed the formation of a
‘red oil’. With the advent of nuclear magnetic resonance (NMR) techniques, this ‘oil’
was later identified as a stable intermediate comprised of a carbocation and a
tetrachloroaluminate anion [82].
41
Interest in major practical applications of ILs began in the 1960’s, when the
US Air Force Academy studied low-melting salts as alternative electrolytes for
thermal batteries. These salts were binary mixtures of 1-butylpyridinium chlorides
and aluminium chlorides. ‘First generation’ ILs suffered from easy electrochemical
reduction and air sensitivity. In the 1990’s, ‘second generation’ ILs were designed to
overcome these problems. For example, in 1992, Wilkes and Zaworotko [83]
prepared and characterised a series of air and water stable low melting salts based
upon the 1-ethyl-3-methylimidazolium cation. These stable salts sparked new
interest in IL research and since then the number of ILs prepared and the related
publications and applications have grown considerably. Examples of common ions
that form stable ILs are listed in Figure 2.4.1.
Figure 2.4.1: Common ions in ionic liquids
One of the main reasons for rapidly growing research interest in ILs is the
ability of their properties to be tuned. To the best estimate of Holbrey and Seddon
[84] the number of accessible IL anion and cation combinations equate to about
one trillion. Properties such as melting temperature, conductivity, refractive index,
42
thermal stability, acid-base character, toxicity, hydrophilicity, polarity, density and
viscosity can be tailored to a certain degree [57, 85]. This ability of ILs to be tuned
makes them attractive in applications beyond the electrolytes that spurred their
discovery (e.g. as solvents in industrial applications).
Aside from tunability, ILs offer a variety of physical properties that make
them attractive alternatives to other solvents. Ionic liquids involving fully
quaternised nitrogen cations are non-flammable and have very low or negligible
vapour pressure. This means reduced risk of explosion or fire accompanied by
reduced need for respiratory protection and exhaust systems. ILs of the
imidazolium cation type have demonstrated ability to dissolve a wide range of
organic and inorganic compounds including cellulose and biomass [57, 82, 85]. This
facilitates the formation of homogeneous solutions of disparate reagents, reactants
and products [85]. The large liquidus range of ILs is another attractive characteristic
when compared to conventional solvents. ILs maintain fluidity and volume in a
range as great as 300 °C [85]. Most conventional solvents would freeze or boil
across such a large temperature range. Due to the wide thermal stability, many
more chemical processes may be undertaken in these solvents. The thermal
stability also affords strategies for recovery of the solvents.
Replacing conventional solvents with ILs has the potential to enable safer,
more stable and more efficient chemical processes. This potential has gained ILs a
central place in a new field of chemistry known as “green chemistry”. Green
chemistry is the term coined to describe the recent international efforts in science
and policy to prioritise sustainability in chemical processes. The Montreal Protocol
which aims at phasing out the use of ozone-depleting substances is an example of
the policy aspect of such international efforts [84]. The BASIL™ (Biphasic acid
scavenging using ionic liquids) process, established by BASF in 2002 is based on IL,
and forms an example of a greener industrial chemical process [86].
Among the many areas where ionic liquids are investigated as replacements
for solvent applications is the dissolution of cellulose [57]. Interest in the dissolution
of cellulose was initially aimed at improving chemical functionalisation and fibre
43
spinning for the polymer and textile industry [20]. Recently ILs have also attracted
attention as a pretreatment solvent for lignocellulosic ethanol. This is due to their
ability to solvate cellulose and LCB, their favourable physical characteristics, their
facile recyclability and their tunability [85, 87, 88].
2.4.2 Cellulose dissolution using ionic liquids
Molten salts have been known to dissolve cellulose since the 1930’s.
However, it wasn’t until the turn of the century that cellulose dissolution attracted
extensive research interest. Graenacher [89] filed a patent in 1934 claiming that
molten salts (e.g. benzylpyridinium chloride) can readily dissolve cellulose. In 2002
(after the discovery of stable and low-melting ILs [82, 83]) Rogers and co-workers
[90, 91] reported using melt salts as non-derivatising solvents for cellulose and
demonstrated that 10 % to 25 % mass cellulose in IL solutions were achievable by
heating at 100 °C or by short pulses of microwave heating. This result, only
attainable with [C4mim]Cl, was attributed to high chloride content of the solvent.
They speculated that chlorides interacted readily with the cellulose hydroxyls via
hydrogen bonding and this would be similar to the mechanism and role of Cl- in
DMA / LiCl. The list of cation-anion combinations examined in this first study was
limited. Especially in terms of cations, only alkylimidazolium salts were used. Since
then, but predominantly in the last three years, more than 40 ILs [87] have been
tested on cellulose and biomass.
2.4.2.a IL structure and cellulose dissolution
Hydrogen bond basicity (β, a Kamler-Taft solvation parameter) and strong
dipole moments of the IL have been reported as pivotal for the performance of ILs
as both cellulose and biomass solvents [87, 92, 93]. Conventional non-aqueous
cellulose solvent systems such as DMA / LiCl exhibit notably high hydrogen bond
basicity and high polarity. Not surprisingly the same is observed in cellulose
solvating ionic liquids. [C4mim]Cl, the most cited IL for cellulose dissolution, is a
highly polar IL with a high β value [94] and [Allylmim]formate solvated higher
amounts of cellulose than [Allylmim]Cl due to the hydrogen bond basicity of the
former being 1.2-fold that of the latter [87, 93].
44
The effect of dialkyl imidazolium cation structure on cellulose solubility has
been systematically studied. Generally, cellulose was found to be soluble in chloride
salts of imidazolium with ethyl butyl and allyl side chains with decreasing solubility
as alkyl chain length increases. It was also observed that even numbers of carbon
atoms show higher cellulose dissolution in the series C2 to C20 as compared to odd
numbers [95]. This unexpected and unexplained outcome was later corroborated by
Vitz et al. [96]. The same authors also demonstrated that this pattern was no longer
observed when the chloride anions were replaced with bromides. A clear and
viscous solution of 14.5 % cellulose in [Allylmim]Cl was achieved at 80 °C in a little
more than 30 min by Zhang et al. [93]. Generally [Allylmim]Cl outperformed
[C4mim]Cl in cellulose dissolution experiments [97]. This could be due to the low
viscosity of [Allylmim]Cl, attributed to the double bond on its side chain. Low
viscosity allows IL ion mobility and thus increases cellulose swelling rate and
enhances dissolution.
As alternatives to the corrosive and viscosity-inducing halides, halide-free ILs
with high hydrogen bond basicity have been successfully employed. For example,
Fukaya et al. [98] found that the low viscosity [Allylmim]formate dissolves ca. 20 %
mass of microcrystalline cellulose (DP ca. 250) at 80 °C, whereas [Allylmim]Cl
dissolved only ca. 2 % under the same conditions. The same team continued the
investigation for halide-free ILs by synthesizing and testing some [C2mim]+ ILs with
varying phosphonate anions [99]. Their results indicated that these anions where
exceptionally good cellulose solvents at very mild temperatures (i.e. 45 °C). All ILs
tested had high dipolarity, high hydrogen bond basicity and low viscosity to which
their success was attributed. [C2mim]methylphosphonate dissolved cellulose at
high concentrations (10 % mass) at 45 °C and 30 min and could also dissolve lower
concentrations (2 % to 4 % mass) at room temperature. The closely analogous
[C2mim]diethylphosphate was reported as a good solvent since cellulose
degradation during dissolution was low [96].
Recently, 1-ethyl-3-methylimidazolium acetate has received considerable
attention as a solvent for both cellulose and lignocelluloses [66, 100-102]. Its low
45
toxicity, relative compatibility with cellulose enzymes and high solvating capacity
are its most prominent attributes. However, it has been demonstrated that
[C2mim]OAc does not act solely as a solvent but also covalently interacts with the
reducing ends of cellulose chains. Kohler et al. [103] investigated the interactions of
IL cations using model cellulose oligomers. On the basis of 13C NMR studies he
reported that [C2mim]OAc forms a carbon-carbon bond between C-1 of glucose and
C-2 of the imidazolium ring (see Figure 2.4.2). This chemistry was further confirmed
by means of 13C-isotopic labelling experiments carried out by Ebner et al. [104].
Surprisingly these imidazole glycosides do not form with [C2mim]Cl [103], which
leads to speculation that either the glycoside formation is catalysed by the basicity
of the acetate anion, or the stronger ion pairing network that chlorides form with
the imidazole ring prevents covalent bond formation [103].
O
HO
OH
O
OH
O
HO
OH
O
OH
OH
HO
OHOH
OH
H
O CH3
O
N
NH3C
CH3
Figure 2.4.2: Structure proposed for a covalent binding of [C2mim]OAc to a
cellooligomer (DP 6-10)
(From Kohler et al. [103])
It would appear from the literature that IL characteristics favourable for LCB
and cellulose dissolution include low viscosity, small polarising ions and high
hydrogen bond basicity. In an industrial setting the characteristics of low toxicity,
corrosivity and cost should be added. One of the advantages of ILs as solvents is
that anions and cations can be chosen and modified (i.e. by changing alkyl side
chain lengths and ring structures) to obtain these characteristics. This tuning of IL
structure and function is the subject of current research activity. Extensive research
is under way to identify the characteristics responsible for dissolution of cellulose
46
and for enhanced biomass dissolution in ILs [87]. These characteristics will aid
synthetic chemistry in tuning ILs.
2.4.2.b Mechanism
The mechanism of cellulose dissolution in ILs is not fully understood, but
there is evidence to suggest that both the cation and the anion take part in the
hydrogen bond disruption of the cellulose chains. 13C NMR and 35/37Cl NMR
relaxation studies indicated that there is a 1:1 stoichiometric interaction between
the chloride ions in [C4mim]Cl and the hydroxyl groups in cellulose [105]. Electron-
donor-acceptor (EDA) interactions between the IL anion and the cellulose hydroxyl
hydrogens and between the IL cation and cellulose hydroxyl oxygens have been
proposed [93, 106, 107] (see Figure 2.4.3 with [C4mim]Cl as an example).
Figure 2.4.3: Proposed dissolution mechanism of cellulose in [C4mim]Cl
This model is based on the generic model of polar cellulose solvents as seen
in Figure 2.3.7. It has been shown that the hydroxyls participating in this interaction
are primarily the C-6 and C-3 [108] (viz. the same hydroxyls that are responsible for
the interchain bonding in the cellulose I allomorph. Zhang et al. [107] have
employed 13C NMR to probe further into these EDA interactions for [C2mim]OAc
and cellulose. They suggested that the acetate anion favours the formation of
hydrogen bonds with hydrogen atoms of hydroxyls, and the aromatic protons in the
bulky imidazolium cation especially the most acidic proton at the C-2 position,
prefer to associate with the oxygen atoms of hydroxyls with less steric hindrance.
Furthermore, Zhang et al. [107] estimated the stoichiometric ratio of [C2mim]OAc :
hydroxyl groups to be between 3:4 and 1:1 in the primary solvation shell, suggesting
[C4mim]+ Cl- [C4mim]+
+[C4mim]
47
that, at least for [C2mim]OAc, there is a possibility that the imidazolium cation
forms some hydrogen bonds with the saccharides. Regardless of stoichiometry of
these interactions, it would appear that the solvents interact with the hydroxyl
groups that are involved in hydrogen bonding in crystal structures.
The acetate anion of [C2mim]OAc has also been observed to participate in
covalent bonding with cellulose. Kohler et al. [109] have observed that while
attempting to form feruoyl and triphenyl ethers of cellulose in [C2mim]OAc
solution, unexpected acetylation was taking place instead. At the same time some
conversion of [C2mim]OAc to [C2mim]Cl was reported. These outcomes suggest
that the [C2mim]OAc did not act purely as a solvent since its acetate ion was being
consumed. [C2mim]OAc has been used in numerous experiments for cellulose and
LCB dissolution [68, 76, 101, 110] but there are no reports on whether or to what
extent the solvent is being consumed.
2.4.2.c Effect of reaction conditions
Apart from the IL properties, a number of reaction conditions have been
identified which influence the rate of cellulose dissolution in ILs. Microwave
irradiation has been shown to substantially accelerate the dissolution rates [91, 96,
111-113]. This is not surprising since ILs are polar molecules which absorb
microwave energy directly and this internal heating is more effective than heat
transfer based heating. However, care must be taken not to overheat and pyrolyse
the cellulose [106]. Mikkola et al. [97] reported that upon use of high-power
ultrasound, the dissolution rate increased and complete dissolution was achieved in
a matter of few minutes in [C4mim]Cl and especially in [Allylmim]Cl [97]. However,
Rogers and co-workers [91] reported no significant benefit from utilising sonication.
Elevated pressures between 0.2 and 0.9 MPa can assist dissolution [111]. It has also
been reported that the use of polar co-solvents may limit the solubility of cellulose.
For example, use of DMSO in [C4mim]Cl (3 : 1 mixture of [C4mim]Cl : DMSO) slowed
down the dissolution rate when compared to pure [C4mim]Cl by decreasing the
ionic strength of the solvent system [114].
48
Water is known to affect the physicochemical properties of ILs [115] and in
the case of cellulose dissolution it plays a dual role. At low concentrations it can
prevent formation of degradation products (e.g. furfurals) and at high
concentrations it competes with the IL for hydrogen bond sites resulting in
decreased solvation which may completely prevent dissolution. In the case where
cellulose is dissolved in dry IL, the addition of water precipitates the cellulose. The
general convention is that ILs are sensitive to low concentrations of water [96, 116].
According to Rogers and co-workers [91], 1 % mass water in IL is enough to limit the
solubility of cellulose in [C4mim]Cl at 100 °C. However, [C2mim]Cl / cellulose / acid
and [C2mim]Cl / LCB / acid solutions studied by Vanoye et al. [117] and by Binder
and Raines [118] respectively were found to tolerate 5 % water without
compromising dissolution. The higher effective concentration of chloride ions in
[C2mim]Cl (cf. [C4mim]Cl) and the presence of acid in the latter examples may be
the reason for their higher water tolerance.
2.4.2.d Properties of cellulose precipitated from IL solutions
Cellulose dissolved in ILs can be precipitated out of the solution with the use
of an antisolvent such as ethanol, methanol or water [45, 119]. This precipitated
cellulose generally exhibits a lower degree of crystallinity than native cellulose
[101]. Similar impact on crystallinity is observed for conventional cellulose solvents
[20]. The precipitated celluloses from ionic liquid dissolution generally retained 25
% – 42 % of native cellulose crystallinity [87]. Decrystallisation is known to greatly
enhance cellulose saccharification by cellulase enzymes[10, 11, 25]. Dadi et al. [45],
have reported 50-fold enhancement of initial saccharification rates after treatment
of Avicel cellulose with [C4mim]Cl. Liu et al. [120] reported the effect of ionic liquid
treatment on enzymatic saccharification of whole biomass. Wheat straw treated
with microwave heated [C4mim]Cl, rendered the straw more digestible to enzymes.
This effect of ILs on biomass created a new niche for IL applications as biomass
pretreatment systems for the manufacturing of fermentable sugars.
The above review of cellulose dissolution in ILs demonstrates the influence
of the ionic liquid characteristics on cellulose solubility and the importance of
49
carefully selecting the structure of the anion, cation and reaction conditions in
order to achieve efficient cellulose dissolution. It also explains the role of ILs in
pretreatment research since cellulose precipitated from IL solutions is expected to
exhibit high enzyme saccharification rates.
2.4.3 Lignin dissolution in ionic liquids
About 26 million tonnes of lignin are manufactured annually as a by-product
of the environmentally harsh Kraft pulping process. This lignin is thiolated and
mainly used as combustion fuel. Since it has a relatively high initial water content,
its fuel value is low, producing less than ¼ as much energy for an equivalent mass as
middle distillate (diesel, jet and boiler) fuels [121]. Moreover, it has been
demonstrated that native lignin (i.e. lignin that is not thiolated or sulphonated) can
be converted to value-added products such as adhesives, coatings, polymer blends,
and carbon fibre composites [121-124]. In this study, lignin is viewed as a valuable
product stream. If lignin is to be functionalised, blended, polymerised, or sold for its
antioxidant properties, a high reactivity and low degree of condensation are the
sought-after characteristics [124, 125]. Capturing these high value product streams
is the intention of the lignin fractionation experiments reported in this thesis. The
potential of IL treatment to extract and fractionate lignin with preservation of
native structure is explored here.
A number of ILs have been tested for their ability to dissolve lignin:
imidazolium salts containing methyl, ethyl, allyl, butyl, hexyl and benzyl groups in
the imidazolium ring and with a number of common anions, such as chloride,
bromide, tetrafluoroborate, acetate, trifluoromethanesulfonate and methylsulfate
[68, 126]. Although it is challenging to compare results for lignin solubility across
studies, some general observations can be made. Bearing in mind that only
imidazolium cations were screened, the anions appear to influence lignin solubility
the most. In imidazolium salt based ILs, large non-coordinating anions, such as BF4-
and PF6- as well as bromide were not good lignin dissolving solvents. In order of
preference, the anions methylsulfate, acetate and chloride imparted good solubility.
The effect of cation is not insignificant, for example, [Allylmim]Cl outperforms
50
[C4mim]Cl. This is possibly due to the π electrons of the allyl group interacting with
the phenolic π electrons of lignin [17, 68].
Lee et al. [68] compared a few combinations of ions for their ability to
dissolve isolated lignin and wood flour. The highest lignin solubility was obtained
using [C1mim] MeSO4 and [C4mim]CF3SO3; solvents that do not result in
appreciable solubility of wood flour. At the other end of the spectrum, chloride
anions enabled relatively high wood flour solubility (10 g kg-1 to 30 g kg-1) while
retaining > 100 g kg-1 lignin solubility. [C4mim][BF4] and [C4mim][PF6] were not
effective at dissolving either lignin or wood flour.
Separation of lignin from LCB via an ionic liquid dissolution process prior to
saccharification has a dual benefit. Delignification is known to enhance enzyme
saccharification [11] and a lignin co-product can improve the economic viability of
the overall process [121].
2.4.4 Biomass dissolution and pretreatment in ionic liquids
Biomass is a very variable substrate and its dissolution in ILs depends on the
plant genotype, phenotype and degree of processing prior to dissolution. The
obvious choices for biomass dissolution are ILs that are good in dissolving both
lignin and cellulose. Examples of such ILs include [C4mim]Cl, [C2mim]Cl,
[Allylmim]Cl, [C2mim]OAc and some dialkyl phosphate ILs. However the rate of
whole biomass dissolution is expected to be slower than that of isolated cellulose or
lignin. Plant architecture, hemicelluloses-lignin bonding and other polymer
interactions in biomass would be expected to impart recalcitrance to dissolution.
In 2005, Myllymaki and Aksela [111] filed a patent on dissolution of whole
biomass using ILs. Their examples included use of microwave- and pressure-assisted
dissolution of straw, softwood chips and sawdust in [C4mim]Cl. In 2007, Rogers and
co-workers [16] published the first comprehensive peer-reviewed article on whole
biomass dissolution in ionic liquids wherein partial dissolution of both hardwoods
(oak, eucalyptus, poplar) and softwoods (pine) in [C4mim]Cl were achieved.
Precipitation of decrystallised cellulose by the addition of water was demonstrated
51
and a lignin-containing liquid effluent was reported. However the dissolution of 5 %
mass solutions of biomass in IL was still not complete after 24 h at 100 °C. When
comparing this reaction time with that for the dissolution of ex situ pure cellulose (a
few minutes, as shown earlier), it becomes apparent that cellulose in LCB is
recalcitrant to dissolution in ILs. This recalcitrance is due to the complex bonding of
the surrounding cell wall matrix. However, these dissolutions were carried out in
mixtures of IL and deuterated DMSO (15 % mass DMSO-d6) as opposed to pure IL
(for the purpose of 13C NMR analysis) and, as already noted (p. 46), Vitz et al. [114]
reported that 25 % mass DMSO in [C4mim]Cl slowed down the cellulose dissolution
when compared to pure [C4mim]Cl. Rogers and co-workers supported their
methodology by citing previous work [127] where it was observed that 15 % mass
DMSO addition in [C4mim]Cl did not reduce the solubility of cellooligomers. The
cellooligomers investigated in the work cited were of DP ≤ 6 and thus do not exhibit
the crystallinity that full cellulose molecules do (DP > 30) or, more importantly,
native in situ cellulose. Thus DMSO may well be contributing to a slow dissolution in
these first experiments.
Kilpellainen, Argyropoulos and co-workers [17, 128] showed that pine
dissolution rate was fastest (8 % mass in 8 h at 80 °C) when [Allylmim]Cl (as
opposed to [C4mim]Cl), small particle size (e.g. ball-milled wood powder) and
higher dissolution temperatures (up to 130 °C) were used. They also showed that
spruce dissolved in [AllylMim]Cl and precipitated with water imparted enhanced
cellulose saccharification and decrystallised cellulose. The slower dissolution rates
previously reported by Rogers were attributed by Kilpellainen and co-workers to
insufficient biomass drying.
As discussed earlier, the ILs with an acetate anion have a number of
favourable characteristics when compared to those with chloride (viz. high
hydrogen bond basicity, low corrosivity, low toxicity, low melting point and
biocompatibility). BASF, one of the leaders in industrial applications of ILs, has
recently used [C2mim]OAc for dissolution of cellulose [101]. Interestingly, Kamiya et
al. [129] reported that the rate of cellulose enzyme saccharification conducted in an
52
aqueous solution of 20 % volume [C2mim]OAc was double that conducted in pure
water. This indicates an advantage of [C2mim]OAc over other imidazolium ILs which
have been reported to irreversibly unfold and inactivate cellulase enzymes. For
example, cellulase activity in [C4mim]Cl aqueous solutions as dilute as 22 mM was
diminished [130]. However it should be noted that enzyme-friendly ILs have
industrial utility only in a process where fermentation can be performed in IL /
water / LCB mixtures and where product and solvent can be recovered post-
fermentation. If extra steps have to be employed to recover the sugars from the IL
solutions prior to fermentation, there is no obvious benefit from using such ILs. In
addition, ILs are expensive solvents and their recovery/reusability may be reduced
by the contaminants introduced by in situ saccharification and fermentation.
In the period from ca. 2008 to 2010, a number of publications have
appeared in the literature. Lee et al. [68] have reported efficient lignin extraction
and enhanced enzyme hydrolysis by treating maple wood flour with [C2mim]OAc.
Rogers and co-workers [101] compared dissolutions of southern yellow pine and
red oak in [C4mim]Cl, [C2mim]Cl and [C2mim]OAc and reported on the
delignification imparted when acetone in water is used for precipitating the
dissolved lignocellulose. Singh et al. [131] have employed confocal microscopy on
switchgrass cross sections exposed to [C2mim]OAc and heat and identified swelling
patterns and a preferential dissolution of lignin. Li et al. [88] demonstrated that
[C2mim]diethylphosphate is outperforming most commonly used ILs at low
temperatures (100 °C) due to lower viscosity. Zavrel et al. [132], have used a light-
scattering technique to screen ILs for their ability to dissolve Avicel cellulose. Arora
et al. [102] have carried out substrate mass balance and temperature/time
optimisation studies for [C2mim]OAc treatment of switchgrass. Li et al. [76]
presented a direct comparison of [C2mim]OAc pretreatment of switchgrass with
dilute acid pretreatment. These publications are discussed in more detail in the
results and discussion chapters of this thesis.
53
2.5 Rationale
The theory and experimental work reviewed here serves as a reference for
the discussion and comparison of the results presented in Chapters 4 and 5.
While the chemistry of lignocellulosics is relatively well understood and
several biomass pretreatments for saccharification and fermentation have been
described (and in many cases demonstrated at pilot scale), substantially the utility
of ILs in biomass processing has not been explored. Descriptions of IL-based LCB
pretreatment processes are vague and limited to a few research accounts of
imidazolium salts’ swelling and dissolution properties. The dissolution mechanisms
are not well understood and there are few reported studies on process
optimisation. Data on the effect of fractionation strategy and process conditions on
saccharification efficiency and on mass balances are scant. This thesis investigates
the potential of ILs for LCB processing.
A number of process strategies can be envisioned. Edye et al. [3, 133, 134]
have listed examples of such process strategies, viz.:
• Dissolution of lignin in IL (with or without cosolvent)
(delignification/pulping)
• Complete or partial dissolution of biomass in IL (with or without
cosolvent) with water precipitation (single liquid phase)
• Complete or partial dissolution of biomass in IL (with or without
cosolvent) with dilute aqueous salt / base (e.g. NaOH) precipitation (single liquid
phase)
• Complete dissolution of biomass in IL (with or without cosolvent) with
aqueous salt / base (e.g. NaOH) precipitation (biphasic liquid)
• Complete dissolution in IL (with or without cosolvent) and in
situ saccharification (i.e. acid catalysed hydrolysis)
54
One of the first described processes is found in the patent claims of Edye
and Doherty in 2007 [3, 134] wherein the IL [C4mim]Cl was used to dissolve
biomass at high temperatures (130 °C – 190 °C) and fractionation of dissolved
components achieved by forming an aqueous biphasic system (ABS) with
concentrated alkali (e.g. NaOH). The examples of this patent showed no
optimisation work on dissolution, no yields or solid to liquid ratios and limited
performance metrics on the ABSs. These knowledge gaps constitute the starting
point for the experiments in this study.
In this work, numerous aspects of this IL strategy are thoroughly
investigated and alternative IL technologies proposed and tested. First, the
dissolution reaction is optimised taking into consideration dissolution extent,
material losses and saccharification kinetics. Second, a selection of fractionation
systems is assessed starting with the NaOH ABS described in the patent and
expanding to other ABSs and single phase fractionation systems using preferential
precipitation. Finally the mass balance closures and saccharification kinetics of three
processes based on three different ILs are reported and discussed.
55
CHAPTER 3 METHODOLOGY
3.1 Bagasse
Sugarcane bagasse (Rocky Point sugar mill, Pimpama, Queensland)
comprising long cuticle fibres and core pith particles (mostly ca. 5 mm to 50 mm)
was air dried for a week on metal trays then size reduced (to < 10 mm) with a knife
mill before being mixed and subsampled by the cone and split method and stored at
4 °C. Before use the bagasse was ground (to < 2 mm) using an electric lab mill
(Retsch SM100, Haan, Germany). The material was ground for ca. 1 min per batch to
avoid excess heating, placed on top of 2 brass sieves (0.5 mm and 0.25 mm) and in a
sieve shaker for 20 min, and the fraction collected between the two sieves was used
as the starting material. Moisture content was measured gravimetrically
(convection oven, 105 °C, overnight) before every use and was 10 ± 1 % mass
except where otherwise indicated.
3.2 Chemicals
The ionic liquids (1-butyl-3-methylimidazolium chloride [C4mim]Cl (≥ 95 %)
melting point (m.p.) 73 °C, 1-butyl-2,3-dimethylimidazolium chloride [C4mmim]Cl (≥
97 %) m.p. 96 °C - 99 °C, 1-ethyl-3-methylimidazolium chloride [C2mim]Cl (≥ 95 %)
m.p. 80 °C, and 1-ethyl-3-methylimidazolium acetate [C2mim]OAc (≥ 90 %) m.p. –20
°C, Sigma-Aldrich, NSW) were all dried in a vacuum oven (at 80 °C – 90 °C, ca. 4 mm
Hg, > 12 h) prior to use. Initial moisture content (at the time of weighing the IL for
each use) was typically ca. 2 % of total mass for [C4mmim]Cl and 1 % for [C2mim]Cl
and [C2mmim]OAc as measured by Karl Fischer titration. At this point it is worth
noting that although the m.p. of neat [C4mim]Cl is 73 °C, its 2 % moisture content
was sufficient to maintain it in liquid phase at room temperature. Cellulose (Avicel
PH-101), dimethyl sulphoxide (DMSO) (99.9 %) and Karl Fischer HYDRANAL titrant
2E and solvent E were purchased from Sigma-Aldrich (Sydney, NSW). Cellulase / β-
glucosidase mixture (Accelerase 1000) was purchased from Genencor (Danisco A/S,
56
Denmark). Water was Millipore-filtered and deionised (Milli-Q-plus) to a specific
resistivity of 18.2 µS at 25 °C. All other solvents and chemicals were analytical
grade.
3.3 Uncertainty (or error) analysis of quantitative measurements
The data values reported in this thesis represent either single measurements
or the mean of duplicate measurements. The precision of the measurement
techniques used to produce these quantitative data was calculated as the estimate
of standard deviation of duplicate measurements of similar samples analysed
identically. The higher the number of sets of duplicate measurements (degrees of
freedom or df), the less uncertainty (higher confidence) can be placed on the
precision of the technique. The estimate of standard deviation was calculated using
Equation 1 as seen in Taylor [135]. The resulting standard deviation along with the
degrees of freedom form part of the description of each technique and provide an
estimate of its precision.
� = �∑��2�
Equation 1
Where: s Estimate of standard deviation d Difference between duplicate measurements
k Number of sets of duplicate measurements ν = k degrees of freedom
3.4 Mass values
All mass or percent-mass values reported in this thesis are on a dry basis
except where otherwise noted.
57
3.5 Karl Fischer titration
A Karl Fischer automated titrator (Radiometer Copenhagen TIM 900) with
ethanol based HYDRANAL reagents was used to measure moisture content of ILs
after drying and prior to use.
3.6 Determination of IL dissolution extent and losses
3.6.1 Dissolution
In a typical dissolution or pretreatment reaction, IL (5 g on a dry basis) was
placed in a 50 mL beaker in an oil bath (clear silicon oil), heated by a hot plate (RET
Basic IKA Laboritechnik) with magnetic stirring (300 rpm), in the open atmosphere.
The selected temperature for dissolution was controlled by a thermocouple (IKA
ETS-D5) immersed in the oil. Bagasse (0.250 g) was added after 60 min, to allow for
temperature stabilisation before the start of the reaction. Dissolutions of bagasse
were carried out in [C4mim]Cl at varying temperatures (110 °C to 160 °C), times (30
min to 180 min) and bagasse moisture contents (1 % to 49 %). Bagasse dissolution
in different ILs ([C2mim]Cl and [C2mim]OAc) was also conducted.
3.6.2 Recovery of undissolved solids (UND) and dissolved-then-precipitated
solids (DS)
To determine the dry mass % of bagasse that dissolved at each trial a
variation of the gravimetric method as described by Sun et al. [101] was used (see
Figure 3.6.1). After bagasse / IL mixtures were subjected to dissolution conditions,
DMSO (ca. 5 mL) was added to reduce the viscosity. The mixture was stirred (300
rpm, 10 min without heating) and then filtered through a pre-weighed nylon filter
(20 μm porosity, 90 mm diameter, Millipore) using a Buchner filtration system. The
residue was washed with additional DMSO (30 mL) and then with deionised water
(100 mL) to remove all residual DMSO on the fibre. This fraction was dried to a
constant mass (convection oven, 105 °C, overnight) and weighed for gravimetric
determination of the undissolved fiber mass (UND). Water washings were added to
the original filtrate and then stirred (300 rpm, 20 min) to precipitate the dissolved
58
material (IL soluble but water / IL insoluble). The precipitate was collected by
filtration (Whatman 54 paper followed by a 0.2 µm Sartorius membrane filtration
with a glass fibre prefilter, all filters preweighed), dried (105 °C, overnight) and
weighed to determine IL / water insoluble dissolved material mass (DS).
Figure 3.6.1: Process for recovering undissolved and dissolved-then-precipitated
solids.
3.6.3 Gravimetric determination of percent mass dissolution
The percent of bagasse dissolved was calculated according to Equation 2
��. = 100 ∗ �1 � ������� �
Equation 2
Where: Diss. Dissolution (% mass of original bagasse added)
mOB Mass of original bagasse added (g)
mUND Mass of undissolved residue recovered (g)
OB:
original (starting) bagasse mass
UND:
undissolved solids mass
dissolved mass = OB - UND
DS: mass dissovled and recovered as a
solid precipitate after addition of water
Losses =
OB - UND - DS
DISSOLUTION FILTRATION PRECIPITATION
WITH WATER
59
The estimate of standard deviation (absolute) of this technique is 6 % mass
of bagasse and is based on 5 df only, due to its cumbersome nature and time
constraints. Note that duplicate experiments for the critical conditions 140 °C and
150 °C for 90 min were included in the calculation of this standard deviation
estimate.
3.6.4 Gravimetric determination of percent mass losses
The percent of bagasse losses to components non-recovered in solid form
was calculated by difference, according to Equation 3. These losses are assumed to
be, for the major part, components which are soluble in the IL / water liquid
fraction. However, they may also represent small amounts of volatile losses from
biomass degradation products (such as acetic acid (b.p. 118.1 °C) or furfural (b.p.
161.7 °C)), since these dissolutions were conducted “in the open atmosphere”.
���� = 100 ∗ (1 −��� +����
���
)
Equation 3
Where: Loss Losses (% mass of original bagasse added) mOB Mass of original bagasse added (g)
mUND Mass of undissolved residue recovered (g) MDS Mass of precipitated-dissolved residue recovered (g)
The estimate of standard deviation of this technique is 3 % mass of bagasse
(i.e. absolute) and is based on 5 df only, due to its cumbersome nature and time
constraints.
60
3.7 Bagasse soda lignin preparation
Bagasse soda lignin was prepared by soda pulping of bagasse (175 °C, 2 h,
bagasse 10 % mass, NaOH 10 % mass) and precipitating the resulting black liquor
with acid (2 M H2SO4) add to reduce the pH to 3.0. The precipitate was then
redissolved in aqueous NaOH (10% mass) and reprecipitated by addition of acid to
reduce the pH to 3.0. The recovered lignin solids were washed and dried (40 °C,
vacuum oven).
3.8 Real time FTIR and reaction calorimetry
Real time FTIR (Fourier transform infrared) spectroscopy of bagasse
dissolution in IL was carried out in a Mettler-Toledo RC1e Reaction Calorimeter (for
accurate temperature control) equipped with a ReactIR FTIR probe (see Figure
3.8.1). The 1 L reaction glass vessel was heated by a silicon oil jacket and the
temperature controlled with temperature probes in the jacket and in the reaction
mass. An ATR (Attenuated total reflectance) -FTIR probe was immersed in the
reaction mass and 64 scan spectra were recorded every 60 s. The height of the
absorption bands at 1070 cm-1, 1510 cm -1 and 1560 cm-1 were monitored during
the course of the reaction. The ‘valley to valley’ method was used to determine the
baseline for the calculation of each band height. [C4mim]Cl (700 g) was added to
the reaction vessel and the temperature stabilised at 70 °C (IL liquid due to 2 %
moisture). The heat capacity of the IL was measured via the response of the
reaction mass temperature to fluctuations of the jacket temperature. Bagasse (35 g)
was added and the same procedure repeated for calculating the heat capacity of
the IL / bagasse mixture. The reaction mass temperature was accurately controlled
and monitored and FTIR spectra were acquired in real-time and band heights
plotted over time. Heat flow of the reaction mass was also monitored with the
calorimeter but no thermal events of interest were detected and the results are
presented in Appendix III.
61
Figure 3.8.1: The Mettler-Toledo RC1e reaction calorimeter and ReactIR FTIR
probe
3.9 Differential Scanning Calorimetry
Differential Scanning Calorimetry (DSC) was performed on samples (ca. 3 mg
in sealed aluminium pans) using a Q100 TA (TA Instruments, New Castle, DE, USA)
by a heat/cool/heat cycle starting at 25 °C and reaching 180 °C at 30 °C min-1 under
nitrogen sweeping gas. The first heat ramps were used to delete thermal history
and the second heat ramps are reported.
3.10 Thermogravimetric analysis
Samples (5 mg to 20 mg, depending on specific gravity) of solids or ILs were
placed in platinum crucible for thermogravimetric analysis (Setaram TGA-DTA/DSC
LabSys, Caluire, France) and heated starting from 25 °C and increasing to 600 °C at 5
°C min-1 to 30 °C min-1. The first derivative of the mass loss was plotted against
temperature to indicate the onset temperature of thermal decomposition.
62
3.11 Cellobiose hydrolysis kinetics
[C4mim]Cl (3 g) in 20 mL test tubes (2) were heated in an oil bath until
desired temperature (130 °C and 150 °C) was reached. D-Cellobiose (150 mg) was
added to each tube and samples (ca. 50 mg) were removed periodically and placed
in pre-weighed Eppendorf tubes. Water (1 mL) was added, the tubes were agitated
vigorously until a homogeneous solution was observed and then injected (20 μL to
50 μL) into the high-pressure liquid chromatograph (HPLC) for sugars analysis (see
Section 3.12).
3.12 Compositional analysis of solid fractions
Compositional analysis was carried out using the standard NREL procedure
for determination of structural carbohydrates and lignin in biomass [58]. All samples
were freeze dried overnight prior to analysis. Each sample (250 mg) was treated
with H2SO4 (72 % mass) at 30 °C for 1 h. These samples and a sugar recovery
standard (SRS, containing known concentrations of glucose, xylose and arabinose)
were then exposed to dilute H2SO4 (4 %) at 121 °C for 1 h. The hydrolysis products
were determined by HPLC (Waters) equipped with a RI detector (Waters 410) and a
Bio-Rad HPX-87H column operated at 85 °C. The mobile phase consisted of 5 mM
H2SO4 with a flow rate of 0.6 mL min-1. The glucose, xylose and arabinose results
were corrected for acid decomposition using the % mass recovery from the SRS. The
polysaccharide and acetyl mass content were calculated by conversion of the
monosaccharide and acetic acid results with appropriate multiplication factors (0.90
for glucose, 0.88 for xylose and arabinose, 0.683 for acetic acid). The acid- insoluble
lignin (AIL) after acid hydrolysis was measured as the mass loss of insoluble residue
at 575 °C. The acid-soluble lignin (ASL) was measured by UV-Vis spectrophotometer
(Cintra 40) at 240 nm with an extinction coefficient value of 25 L g-1 cm-1 [58]. Ash
was determined by placing separate sample fractions at 575 °C.
The estimates of standard deviation (absolute) of this technique for each
component (as % dry mass of bagasse) are: 0.4 for ash, 0.4 for AIL, 0.2 for ASL, 0.4
63
for total lignin (AIL + ASL) ii, 1 for glucan, 0.4 for xylan, 0.04 for arabinan and 0.04 for
acetyl. These estimates are based on 15 df.
This technique was used for all compositional analysis results shown in this
thesis, except where otherwise noted.
3.13 Preparation of IL pretreated samples for enzyme saccharification
IL pretreated bagasse samples destined for enzymatic saccharification trials
were prepared at a larger scale and without drying. Oven or air-drying can
irreversibly collapse the pore structure of biomass and affect enzyme
saccharification kinetics. Bagasse (2.2 g) in IL (40 g) mixtures were placed in 150 mL
beakers and subjected to pretreatment conditions in the manner described in
Section 3.6.1. The UND was filtered and washed as described in Section 3.6.2 (40 mL
DMSO and 400 mL of water) and weighed without significant air-drying. The DS was
collected by centrifugation in preweighed 250 mL tubes (Beckman J2 MC, JLA-
10.500 rotor, 14300 x g, for 10 min), the supernatant was decanted and the
sediment was resuspended and recentrifuged with 3 x 200 mL deionised water. In
cases where UND and DS were not separated, a total solid residue (TSR) was
obtained by adding water to the pretreated IL bagasse mixtures, the precipitate was
collected by centrifugation and washed by centrifugation (3 x 200 mL). All solid
fractions were transferred to capped glass vials, which were stored moist at 4 °C till
used for enzyme saccharification trials.
3.14 Preparation of dilute acid pretreated samples
Dilute acid pretreatment was carried out according to the LAP-007 NREL
protocol for the preparation of dilute acid pretreated biomass [136]. Bagasse (20.77
g at 3.73 % moisture) and deionised water (167.75 mL) were stirred (175 rpm) and
heated in a Parr reactor (0.5 L SS316) to 160 °C for approximately 10 min, acid
ii Estimate of standard deviation for total lignin equals to the square root of the sum of squared estimates of standard deviation for AIL + ASL.
64
(16.22 mL of 9 % mass H2SO4) and then water (15.25 mL) were injected with a high
pressure feeding pump (Prominent Beta / 4) over 165 s and the temperature was
maintained at 160 °C for 10 min. The reactor was then cooled by placing the
reactor in an ice bath and running tap water through the internal cooling coil. The
pretreated solids were recovered by filtration (Whatman 5) and washed with
distilled water until pH of the washate was > 5.0. The washed and moist solids were
stored at 4 °C till use.
3.15 Enzymatic saccharification
Cellulose contents of bagasse and pretreated bagasse were determined by
compositional analysis prior to enzymatic saccharification (Section 3.12). Moisture
content was determined prior to calculating sample weights required (estimated
moisture) and also at the time of actually weighing the samples (actual moisture).
The former was used to determine enzyme loadings (by oven drying to constant
mass overnight at 150 °C) and the latter was used for final cellulose and xylan
concentration calculations.
Bagasse and pretreated bagasse samples (100 mg cellulose equivalents)
were suspended in citrate buffer (10 mL, 50 mM, pH 4.7) and equilibrated on a
temperature controlled rotary shaker (150 rpm, 50 °C). Accelerase 1000 (Genencor)
was added to achieve an enzyme activity of 15 FPU g-1 (50 µL of Accelerase as
received). Samples (0.5 mL) were removed periodically placed in ice, centrifuged at
4 °C and then frozen. After thawing of the samples at room temperature, cellobiose,
glucose and xylose concentrations were measured by HPLC (HPLC system as in
section 3.10). The glucose and cellobiose results were converted to glucan mass
equivalents and xylose was converted to xylan mass equivalents using appropriate
multiplication factors.
The estimates of standard deviation (absolute) of this technique are 2 %
mass of glucan and 2 % mass of xylan and are based on 15 df.
65
This technique was used for all enzyme saccharification monitoring results
shown in this thesis, except where otherwise noted.
3.16 XRD cellulose crystallinity measurement
Pretreated and the untreated bagasse samples were scanned on a
diffractometer (PANalytical X’Pert MPD, Cuα (1.5418 Å) radiation) with a scan speed
of 0.18° min-1 and a step size of 0.018° (see Figure 3.16.1 for example).
untreated
0 10 20 30 40 50 60 70 800
50000
100000
150000
Figure 3.16.1: Diffractogram of bagasse
The cellulose crystallinity index (CrI) was determined using Equation 4 as
reported by Thygesen et al. [137] .
IAM ITOT
2θ[°]
cou
nts
66
��� = � � � �!"� �
Equation 4
Where: CrI Crystallinity index ITOT Intensity at about 2ϑ = 22° (represents the crystalline and amorphous material)
IAM Intensity at the “valley” between the two peaks at about 2ϑ = 18°. (represents the amorphous material)
There is controversy as to which baseline should be used for the
measurement of the intensity values in Equation 4 [137]. The straight baseline used
in this study was drawn by baseline normalisation (“valley to valley”). This approach
may lead to an overestimation of crystallinity (since there is no account of
background scatter). However, this overestimation will be of similar magnitude for
all samples examined and thus should not affect the overall reliability and
consistency of conclusions. The estimate of standard deviation (absolute) of this
technique is 0.01 and is based on 3 df only due to limited time of instrument
availability.
This technique was used for all cellulose crystallinity index results shown in
this thesis, except where otherwise noted.
3.17 Saccharification and fermentation
Bagasse (35 g) in [C4mim]Cl (464 g) was reacted (150 °C for ca. 1 h) in the
RC1 reactor (same reactor setup as in Section 3.8), cooled to 70 °C and precipitated
with water (ca. 300 mL). The recovered solids were centrifuged and washed as
described in Section 3.13 and stored moist at 4 °C.
Enzymatic hydrolysis reactions were performed under sterile conditions for
3 days in 100-mL Erlenmeyer flasks (equipped with water traps) on a rotary shaker
(150 rpm, 50 °C) in volumes of 40 mL with a biomass load of 2 g cellulose equivalent
and Accelerase 1000 (Genencor) activity of 5 FPU g-1 in 50 mM citrate buffer (pH
4.7) containing 4 mL of a YP solution (100 g L-1 yeast extract and 200 g L-1 peptone).
67
At the end of the 3 days, 0.5 mL of yeast cell suspension (see preparation below)
was added to achieve a final optical density (O.D.) of 0.5 and incubated under
sterile conditions (32 °C, 130 rpm, 72 h); samples were removed at regular intervals
and after appropriate dilutions, injected onto the HPLC (as in section 3.10) for
quantification of glucose and ethanol concentrations.
Yeast cells (Saccharomyces cerevisiae) were prepared as described in the
relevant protocol by the National Renewable Energy Laboratory (NREL) [138]. A
frozen stock culture was suspended in a sterilised flask with YPD (10 g L-1 yeast
extract, 20 g L-1 peptone, 50 g L-1 dextrose) medium and incubated overnight at 32
°C with orbital agitation. Cells were then harvested by centrifugation and washed
with sterile water (3 x 38 mL, 5 min, 4500 rpm). The resulting solids were suspended
in 2.5 mL sterile water and optical density measured with a spectrophotometer at
600 nm.
3.18 ATR-FTIR
For liquid samples, a drop was placed on the diamond probe of a Thermo
Nicolet 870 FTIR (software: OMNIC 7.3). For solid samples, a small amount of freeze
dried fibre, enough to cover the surface of the probe, was used. The sample was
pressed with an anvil to increase the surface contacting the probe. Sixty-four scans
were acquired for each spectrum and the two replicate spectra for each sample
were overlayed. No differences in the replicate spectra of this study were observed
and thus only the first spectrum of each sample was used for analysis.
3.19 Aqueous biphasic systems
3.19.1 Preparation of ABSs
ABSs were prepared using the proportions described in the patent of Edye
and Doherty [3, 134] by mixing IL (6.3 g) with 20 % NaOH (8.4 mL) in a 25 mL volume
68
graduated cylinder, agitating and allowing to stand overnight except where
otherwise indicated.
3.19.2 Cloud point titrations
The coexistence curves were determined using cloud point titration at
ambient temperature in a similar manner as reported by Bridges et al. [139]. The
titration started with a solution of known and high concentration of ionic liquid in
water. Dropwise addition of kosmotropic salt solution (of known concentration) to a
monophasic (clear) IL solution was followed by vigorous vortexing, and time to
settle. Upon settling, if a cloudy solution formed (which would yield a biphasic
solution if allowed to separate completely), the cloud point was deemed reached
and the mass of titrant added was recorded. The same was then repeated with the
water titrant until the solution became clear again and the water mass added
recorded. This was continued until enough points were measured for an accurate
coexistence curve. The mass additions and the concentrations of the starting
solution and the titrant were used to determine the IL molality and the kosmotropic
salt molality at each cloud point observed.
3.19.3 Ion concentration determination (for ABS distribution ratios)
The phases of biphasic systems comprising an IL top phase and an aqueous
salt bottom phase were sampled separately. Sampling of the bottom phase was
carried out with care not to contaminate it with top phase solution. A Pasteur
pipette was passed through the top phase with an air bubble maintained at its tip.
After sampling the bottom layer and upon exit, small amounts of bottom phase
solution were being expelled while the pipette tip was crossing the top phase. Upon
exit the exterior of the pipette tip was wiped with a tissue.
The samples were diluted with deionised water and ion concentrations
determined by ion chromatography (Metrohm 761 with a conductivity detector).
For cation analysis, samples were acidified (to pH 3.5, 2M HNO3, ca. 1 µL mL-1) and
injected onto a Metrosep C 2 150 (150 mm x 4 mm) column with an aqueous mobile
phase (25 % volume acetone, 6 mM tartaric acid and 0.75 mM dipicolinic acid) at 1
mL min-1. For anion analysis samples were injected onto a Metrosep ASupp5 (150
69
mm x 4 mm) column with an aqueous mobile phase (1 mM NaHCO3 and 3.2 mM
Na2CO3) at 0.7 mL min-1 and suppression by post-column addition of H2SO4 (50 mM).
The estimate of relative standard deviation (RSD) of this technique is 0.5 % for
chloride ions and is based on 9 df.
3.20 Quantification of [C4mim]Cl deprotonation using an acid titration
HCl (0.2 M) was used as a titrant on an aqueous solution (50 mL) containing
ca. 1.5 g of the IL phase sample. The [C4mim]Cl content (% mass) of this IL phase
sample was determined using refractive index (Bellingham Stanley RFM320
refractometer). The linear relationship of refractive index to [C4mim]Cl
concentration (% mass) in aqueous [C4mim]Cl solutions was determined using 7
standards as shown in Figure 3.20.1. This relationship is in close agreement with Liu
et al. [140].
Figure 3.20.1: Linear relationship of refractive index to [C4mim]Cl concentration in
water
y = 0.0019x + 1.3319R² = 0.9982
1.32
1.34
1.36
1.38
1.40
1.42
1.44
1.46
0 10 20 30 40 50 60
Re
fra
ctiv
e i
nd
ex
[C4mim]Cl concentration (% mass)
70
In the case of the [C4mim]Cl / Na2CO3 and trisubstituted [C4mmim]Cl /
NaOH ABSs, the IL content of their IL phases was determined via the more
cumbersome ion chromatography technique (see Section 3.19.3) due to
unavailability of the refractometer.
3.21 Mass balance determinations for three IL treatments
Mass closure experiments for three ionic liquid treatments of bagasse were
carried out in sealed tubes to avoid volatile losses such as acetic acid (b.p. 118.1 °C)
or furfural (b.p. 161.7 °C) or from the degradation of xylose. The fractionation
process was designed by the author and is shown in Figure 3.21.1. The amounts of
water used for each of the three preferential precipitations are based on the results
of Section 5.3. The mass of each of the original reactants was recovered in either
one of the liquid or solid fractions depending on their solubility in the three
different concentrations of water in IL used (0.5, 2.0 and 3.5 (+acidification) of
water : IL mass ratio).
Bagasse (0.25 mm – 0.5 mm) was extracted with ethanol and water using a
Sohxlet device according to the NREL protocol for biomass extractives [141]. ILs (ca.
30 g of either [C4mim]Cl or [C2mim]Cl or [C2mim]OAc in duplicate) were weighed in
sealable pressure glass tubes (ACE glass 50 mL). At this point, IL (ca. 0.5 g) was
weighed and set aside for IL recovery analysis (see section 3.19.5). Extracted
bagasse (3.5 % moisture) (ca. 1.5 g for [C4mim]Cl and [C2mim]Cl and 0.75 g for
[C2mim]OAc) was added to each pressure tube, sealed with Teflon stoppers and
placed in an oil bath which was stabilised at 150 °C with magnetic stirring at 200
rpm. The tubes were left in the oil bath for 60 min (25 min of which at temperature
ramp and 35 min at 150 °C) and, upon removal, placed in an ice, bath with magnetic
stirring to quench the reaction. After 2 min, the tubes were removed from the ice
bath and water was added equal to 0.5 mass fraction of the originally added IL. The
tube was sealed again and agitated vigorously until a homogenous solution
between water and IL appeared to form. The contents of each tube were
quantitatively transferred into a preweighed polypropylene centrifuge tube and
centrifuged at 10000 x g for 20 min. The liquid contents of the centrifuge tube were
71
decanted to a new preweighed polypropylene centrifuge tube and weighed (liquid
fraction 1). The pellet (solid fraction 1) was centrifuge washed with distilled water (5
x 30 mL at 10000 x g and 5 min - 10 min cycles), freeze dried overnight (-85 °C, 80
mT) and weighed. Liquid fraction 1 was precipitated with additional water resulting
to a water : IL mass ratio of 2.0. Precipitation and coagulation of solids was aided by
storing at 4 °C overnight followed by shaker incubating at 55 °C - 70 °C for 60 min.
The resulting lignin rich precipitate (solid fraction 2) was centrifuge washed, freeze
dried and weighed.
Figure 3.21.1: Flow chart of the fractionation process used in mass balance
experiments
biomass dissolution
precipitation in water : IL mass
ratio = 0.5
LIQUID FRACTION 1
precipitation in water : IL mass
ratio = 2
LIQUID FRACTION 2
Precipitation in water : IL mass ratio = 3.5
+ acidification to pH <1.0
LIQUID FRACTION 3 SOLID FRACTION 3
SOLID FRACTION 2
SOLID FRACTION 1
72
Losses of liquid components to washings of pellets were accounted for by
weighing pellets prior to washing and after drying (it is assumed that the
composition of these lost liquid components is the same as the bulk liquid).
Similarly, subsampling for analysis was accounted for by careful attention to mass
changes.
Solid fraction 1 and the starting biomass were characterised using the NREL
acid hydrolysis protocol (see Section 3.12). Solid fractions 2 and 3 were
characterised for lignin content with the acetyl bromide protocol described by
Iiyama and Wallis [142]. The sample of liquid fraction 1 was directly injected onto
the HPLC and the Ion Chromatograph (IC) for quantification of monosaccharides
and IL ions respectively while the soluble oligosaccharides were determined by acid
hydrolysis. All methods are described in detail in the following sections. The
distribution of cellulose, hemicellulose and lignin between solid fractions, liquid
fraction monosaccharides and liquid fraction oligosaccharides was reported as a
percent mass of the components in the starting material.
3.21.1 Compositional analysis of “solid fraction 1”
The “solid fraction 1” of each reaction was characterised according to the
NREL protocol described in Section 3.12.
The estimates of standard deviation (absolute) of this analysis (on the basis
of duplicate IL pretreatments, 3 degrees of freedom) for each component (as % dry
mass of starting bagasse) are: 2 for glucan, 2 for xylan, 3 for arabinan, 1 for acetyl
and 2 for lignin.
3.21.2 Compositional analysis of monosaccharides in liquid fraction 1
Each sample of “liquid fraction 1” (0.5 mL) was weighed in 1.5 mL Eppendorf
tubes and diluted with water (0.5 mL). The contents were vortexed thoroughly,
filtered through a 0.45 µm nylon filter and injected to a Waters HPLC as described in
Section 3.12. Glucose, xylose, arabinose and acetic acid masses were converted to
glucan, xylan, arabinan and acetate masses using appropriate multiplication factors
(see Section 3.12). In addition, it was assumed that the detected
73
hydroxymethylfurfural (HMF) and furfural were products of cellulose and xylan
degradation respectively. Therefore, HMF and furfural masses were converted to
cellulose and xylan mass equivalents using multiplication factors of 1.28 and 1.38
respectively.
The estimates of standard deviation (absolute) of this analysis (on the basis
of duplicate IL pretreatments, 3 df) for each component (as % dry mass of starting
bagasse) are: 0.2 for glucan, 0.2 for xylan, 20 for arabinan and 0.7 for acetyl. The
unacceptably high standard deviation for arabinan is attributed to its very low
concentrations in this analysis.
3.21.3 Compositional analysis of oligosaccharides in liquid fraction 1
Each sample of “liquid fraction 1” (0.5 mL) and SRS solution (0.5 mL) were
weighed in 2 mL twist-top Eppendorf tubes, diluted with water (1 mL) and acidified
(with 72 % mass H2SO4) to a pH of 0.3. The contents were vortexed thoroughly and
autoclaved (121 °C for 60 min; autoclaving did not affect mass). After cooling to
room temperature, the autoclaved tube contents were filtered through a 0.45 µm
nylon filter and injected onto the same HPLC system as in section 3.12. After SRS
correction for acid decomposition of sugars (see Section 3.12) and subtraction of
the monosaccharide composition results (Section 3.21.2), the difference was
converted to polysaccharide mass equivalents (using appropriate multiplication
factors as in Section 3.12) in order to arrive at the composition of the soluble
oligosaccharides in liquid fraction 1.
The estimates of standard deviation (absolute) of this analysis (on the basis
of duplicate IL pretreatments, 3df) for each component (as % dry mass of starting
bagasse) are: 1 for glucan, 2 for xylan, 20 for arabinan and 3 for acetyl. The
unacceptably high standard deviation for arabinan is attributed to its very low
concentrations in this analysis.
3.21.4 Acetyl bromide for lignin quantification in solid fractions 2 and 3
The acetyl bromide method as described by Iiyama and Wallis [142] was
used to determine the % mass lignin content of solid fractions 2 and 3. Freeze dried
74
solids (ca. 10 mg) were weighed in glass tubes and acetyl bromide in acetic acid (25
% mass, 10 mL) and then perchloric acid (70 % mass, 0.1 mL) were added. The tubes
were sealed with Teflon screw caps and placed in temperature controlled rotary
shaker (70 °C and 100 rpm for 30 min). After cooling to room temperature the tubes
were opened and 2 M NaOH (10 mL) and then glacial acetic acid (25 mL) were
added. After agitation, absorbance (280 nm, quartz cuvettes, Cintra UV
spectrometer) was measured against glacial acetic acid. The resulting solution was
analysed with a Cintra-40 UV spectrometer and the absorbance was referenced to a
cuvette with glacial acetic acid. Dilutions with glacial acetic acid were necessary for
some samples so that the absorbance was < 1.0. The absorbance was converted to
percent mass concentration of lignin using an extinction coefficient of 25 L·g-1·cm-1.
The extinction coefficient was determined with the use of a calibration curve based
on bagasse of known lignin content. The estimate of standard deviation (absolute)
of this technique (duplicate samples of untreated bagasse and soda lignin, 4 df) (as
% dry mass of solid analysed) is 3.
3.21.5 Recovery of IL
IL set aside at the start of mass balance experiments (starting IL) was
brought to a volume of 50 mL with deionised water. Similarly, known masses of
liquid fraction 1 were diluted with deionised water and injected onto the ion
chromatograph as described in Section 3.19.3. IL mass balance was determined
from the results of these analyses. The estimate of standard deviation (absolute) of
this technique (as % mass of ions in starting IL) for both cations and anions and for
all 3 ILs is 2 (based on duplicate IL pretreatments, 6 df).
3.21.6 Enzymatic saccharification of solids from 3 IL treatments
Enzymatic hydrolysis reactions were performed in 20 mL scintillation vials on
a rotary shaker (150 rpm, 50 °C) in volumes of 5 mL with a biomass load of 50 mg
cellulose equivalent and Accelerase 1000 (Genencor) activity of 15 FPU g-1 (25 µL of
Accelerase as received) in 50 mM citrate buffer (pH 4.7). Samples (0.2 mL) were
periodically removed, placed in ice, then in boiling water (2 min) and centrifuged.
The sugars analysis and conversion to glucan an xylan masses was done as in
75
Section 3.12 except a Shodex SPO-810 HPLC column at 85 °C with a mobile phase of
ultrapure water at 0.6 L min-1 were used.
The estimates of standard deviation of this analysis (based on duplicate IL
pretreatments’ saccharification extents at different time points, 18 df) are 2 % mass
of glucan (i.e. absolute) and 0.9 % mass of xylan.
76
CHAPTER 4 RESULTS – PRETREATMENT
Pretreatment is the process by which the LCB structure is ‘opened up’ to
facilitate enzyme saccharification of the polysaccharide fraction. In this chapter a
simple ionic liquid pretreatment system is investigated. Bagasse is partially
dissolved in IL and the pretreated solids are recovered with the addition of water.
Section 4.1 investigates the pretreatment performance of this system and compares
it with dilute acid pretreatment. Section 4.2 studies the characteristics of the
undissolved bagasse in [C4mim]Cl and draws conclusions on the non-dissolution
effects of IL pretreatment, such as structural (viz. fibre swelling and cellulose
decrystallisation) and compositional changes (viz. preferential dissolution patterns)
of the undissolved fraction.
4.1 Biomass dissolution in IL and recovery by addition of water
The utility of ILs in biomass pretreatment is attributed to their ability to be
tuned and be compatible with a wide array of processes entailing a biomass
dissolution step as listed in Section 2.5. The first undertaking of this study is the
exploration of a simple system entailing partial dissolution of bagasse with
[C4mim]Cl and precipitation with water.
The extent of dissolution and material losses (unrecovered portion of
biomass lost to soluble components, e.g. monosaccharides, lignin monomers,
degradation products) incurred when bagasse is reacted in ionic liquids at
temperatures between 110 °C and 160 °C are examined in this section. Most ionic
liquid work so far quotes long biomass dissolution times (in the order of days) at
temperatures ≤ 110 °C [16, 91, 101]. The rationale for using relatively low
temperatures is (albeit with little evidence) that polysaccharide degradation is
assumed to be high at higher temperatures. The reaction parameters that
77
determine the extent of dissolution and degradation of bagasse in [C4mim]Cl are
also investigated in this section in order to optimise this simple system at high
temperatures.
The optimised [C4mim]Cl system is then compared to dilute acid
pretreatment in terms of saccharification and fermentation yields and processing
time.
4.1.1 Ionic liquids used
Three most cited ionic liquids for cellulose and biomass dissolution are
[C4mim]Cl, [C2mim]Cl and [C2mim]OAc (see Figure 4.1.1). [C2mim]Cl is a difficult IL
to handle since it is solid up to about 70 °C and mixtures of bagasse in [C2mim]OAc
are difficult to stir when biomass loading in IL exceeds 2.5 % mass. Consequently
[C4mim]Cl was chosen for investigating the parameters affecting high temperature
dissolutions. The dissolution extents in all three ionic liquids are also compared in
this section.
2
N34
5
N 1
CH3
CH3
Cl
N
N
CH3
CH3
ClO CH3
ON
N
CH3
CH3
1-butyl-3-methylimidazolium chloride
1-ethyl-3-methylimidazolium chloride
1-ethyl-3-methylimidazolium acetate
Figure 4.1.1: ILs used in this study
78
4.1.2 Factors affecting biomass dissolution
4.1.2.a Background
Dissolution is the process wherein substances (solids, liquids or gases)
disperse in a liquid (solvent) to form a solution. In the cases of dissolution of solids
and liquids, the solution is formed by dissociation of solute material (e.g.
breakdown of crystal lattice) and association or solvation of individual solute
molecules with solvent molecules. Dissolution rate is dependent on properties of
the solvent and the solute and the conditions under which they interact. These
variables are expressed in Equation 5.
���# = $� ��% � �&�
Equation 5
Where: m Amount of dissolved material (kg)
t Time (s)
A Surface area of the solid material (m2)
D Diffusion coefficient (m2 s-1)
d Thickness of the boundary layer of the solvent at the surface of the dissolving substance (m)
Cs Concentration of the substance in the boundary layer (kg m-3)
Cb
Concentration of the substance in the bulk of the solvent (kg m-3)
The relation of diffusion to temperature is defined by the Arrhenius
equation (Equation 6)
= '()*+,
Equation 6
Where: D Diffusion coefficient (m2 s-1)
D0 Maximum diffusion coefficient (at infinite temperature) Ea Activation energy for diffusion (kJ mol-1)
T Temperature (°K) R The universal gas constant (J K
-1 mol
-1)
These equations describe the behaviour of pure solids dissolving in pure
solvents and assume that the surface of the solids is chemically homogeneous.
79
Biomass, as described earlier, is a complex structure and is not expected to exhibit a
chemically homogeneous surface. Furthermore as the biomass dissolves the
composition of its surface changes (i.e. the proportion of the more soluble
components reduces). However, the equations indicate the parameters that
accelerate biomass dissolution and their relative importance. In this work, the initial
surface area and concentration of biomass solids are held constant by using
consistent particle size, agitation conditions and low biomass loading (5% mass)
across experiments. The parameters varied are temperature, residence time,
bagasse moisture and ionic liquid type.
According to Equation 6, temperature and activation energy of diffusion are
the main variables influencing the diffusion coefficient. The diffusion coefficient,
and thereby the dissolution rate, will increase exponentially with increasing
temperature and/or reducing activation energy. The apparent activation energy for
biomass diffusion would be influenced by the activation energies of diffusion of its
individual components.
4.1.2.b Effect of temperature
Bagasse (0.250 g) dissolution in [C4mim]Cl (5 g) for 90 min was conducted at
different temperatures (110 °C to 160 °C) and the extent of dissolution along with
the biomass losses (biomass ending up in liquid fraction) after recovery by addition
of water was measured for each temperature (results shown in Figure 4.1.2).
Dissolution extent was determined by weighing the undissolved material recovered
as the filtration residue after diluting the reaction mass with DMSO. Losses were
determined by the difference of the mass dissolved and the mass of the recoverable
filtrate after precipitating the dissolved mass with water (see Section 3.6).
At this point, it is important to clarify that DMSO dilution as described above
does not cause further dissolution of bagasse or precipitation of dissolved
components. Although DMSO is a solvent for carbohydrate-free lignin, it has been
reported by Rogers and coworkers [101] that it does not interfere with native lignin
or carbohydrates and thus does not influence dissolution results. It must be noted
however, that the author has observed gel formation upon addition of DMSO in the
80
[C2mim]OAc / bagasse solutions. It is also important to clarify that DMSO dilution as
described above may cause slightly different components of bagasse to form part of
the losses as compared to IL / water liquid fractions. However if such bias is taking
place it is internally consistent for all samples analysed and compared.
Significant dissolution was observed at temperatures above 130 °C, where
the extent of dissolution appears to more than double with every 10 °C
temperature increment (Figure 4.1.2). This is only an empirical observation since, as
discussed in Section 4.1.2.a, the Arrhenius kinetics equation cannot be directly
applied to the continuously altering surface of biomass in dissolution. At 160 °C, the
dissolution rate and extent are high but the losses are also high. At 150 °C and
below the mass of losses are fairly consistently 1/3 of mass dissolved. It is therefore
concluded that temperature should be maintained at or below 150 °C to avoid
excessive losses.
At 160 °C the dissolution approaches its practical end point (dissolution = 92
% mass), only the more recalcitrant material remains and consequently the
dissolution rate slows. However depolymerisation and degradation reactions of the
solvated material continue resulting in excessive losses (53 % mass, cf. 17 % mass at
150 °C). These findings were recently corroborated by Rogers and co workers [101]
who demonstrated that the last 10 % mass of the starting pine or oak, in a range of
ILs, required as much (or more) time to dissolve as the first 90 %. The same workers
reported losses of 40 % mass for near complete dissolution of pine in [C2mim]OAc
(110 °C for 16 h). This ratio of losses to dissolution is close to the 1:3 found here
although slightly higher possibly due to the fact that the dissolution extent for
Rogers was closer to 100 %.
The increased losses are due to depolymerisation of solvated biomass
leading to formation of molecules soluble in the water / IL mixture. While it is
possible or even likely that dissolution involves breaking of intramolecular C-C and
C-O bonds, it is not possible from these results to infer much about the molecular
masses (or DP) of solutes. However, it is certain that depolymerisation does occur
since the lost material is of low enough molecular weight (or DP) to be soluble in
81
the water / IL mixtures. Therefore, depolymerisation may be a consequence of the
dissolution process and it is reasonable to expect more depolymerisation after
dissolution (i.e. in the solvated state).
Figure 4.1.2: Effect of temperature on bagasse dissolution in [C4mim]Cl for 90 min
4.1.2.c Effect of time
To investigate the effect of reaction time on dissolution, bagasse (0.250 g)
dissolution in [C4mim]Cl (5 g) at 150 °C was conducted for five different residence
times (30 min to 180 min) and the extent of dissolution along with the associated
biomass losses was measured for each time. These time series results are presented
in Figure 4.1.3 and they confirm the dissolution pattern observed in the
temperature series experiments. Generally, in cases where ca. 75 % or less of the
material is dissolved, the ratio of losses to dissolution extent is about 1:3. The
dissolution appears to be ca. three times faster than the combined rates of
depolymerisation and degradation (which lead to losses).
0
10
20
30
40
50
60
70
80
90
100
110 130 140 150 160
ba
ga
sse
(%
ma
ss)
Temperature (°C)
dissolution losses
82
Figure 4.1.3: Effect of residence time on bagasse dissolution in [C4mim]Cl (150 °C)
At least for these biomass : IL ratios, optimum conditions appear to be 150
°C and 90 min. At lower temperatures and times the extent of biomass dissolution is
low and at higher temperatures and times depolymerisation and degradation
reactions lower recovery. Recently, Varanasi et al. [119] reported using 150 °C and
90 min for pretreatment of corn stover in [C4mim]Cl and [C2mim]OAc. Fu et al.
[143] reported using the same conditions for triticale straw in [C2mim]OAc.
Furthermore, these authors suggested these conditions to be optimum for high
saccharification rates of the IL treated biomass.
4.1.2.d Effect of bagasse moisture
Moisture is an inherent component of biomass and it is an important factor
in its dissolution due its dual role as a reactant in the hydrolysis of glycosidic bonds
and an antisolvent of cellulose and lignin. Bagasse at the sugar mill gate is received
0
10
20
30
40
50
60
70
80
90
100
30 60 90 120 180
ba
ga
sse
(%
ma
ss)
Time (min)
dissolution losses
83
at ca. 50 % moisture. Air-dried bagasse contains about 10 % moisture. Bagasse
samples (0.225 g each, on a dry basis) of different moisture contents (viz. 48.5 %,
10.5 % and 1.1 % moisture) were reacted in 5 g of [C4mim]Cl (90 min at 150 °C) and
the dissolution and losses of all three samples is shown in Figure 4.1.4. There is little
difference between air-dried (10.5 % moisture) and oven-dried (1.1 % moisture)
bagasse both in terms of extent of dissolution and losses (Figure 4.1.4). This
outcome could be related to the high reaction temperature (150 °C) where most
water is evaporated quickly. It is not possible to distinguish between a simple water
concentration effect and a more complex competition between water and IL in
occupying pore and fibre structures. However, the solubility of bagasse at 48.5 %
moisture content seems to be significantly diminished and it is possible that when
bagasse moisture is high enough and water is inside the biomass structure, it is slow
to be displaced by ILs. The ratios of losses to dissolution extent appear to remain
around 1:3 at all moisture contents tested.
Figure 4.1.4: Effect of bagasse moisture content on bagasse dissolution in
[C4mim]Cl
0
10
20
30
40
50
60
70
80
1.1 10.3 48.5
ba
ga
sse
(%
ma
ss)
moisture (%)
dissolution losses
84
4.1.2.e Loading
In preliminary dissolution experiments, it was observed that an initial
loading of ca. 9 % mass bagasse loading in [C4mim]Cl (150 °C) rendered the mixture
very viscous and impossible to stir with magnetic stirring. However, when the
reaction was mechanically stirred in the RC1 reactor (setup described in Section 3.8)
and the loading was incrementally dosed, much higher loadings were achieved.
Bagasse loading started with a ca. 8.6 % mass (33.7 g) in [C4mim]Cl (356 g , 150 °C)
and subsequently bagasse was added in increments of 10 g when the viscosity of
the reaction mass appeared to drop. The loading achieved was 15.3 % (64.4 g)
within the first 2 h and 20.6 % (94.4 g) in 5 h. The fact that polysaccharides were still
dissolving at these loadings was confirmed with monitoring of the 1070 cm-1 FTIR
absorption band as described in Section 4.1.2.g.
4.1.2.f Effect of Ionic liquid choice
The dissolution of bagasse in a choice of three ILs under identical conditions
(150 °C, 90 min and 5 % mass bagasse in IL) was investigated and the results are
shown in Figure 4.1.5. It has to be noted that the [C2mim]OAc dissolution at this
loading (5 % mass) was very viscous and hard to stir.
One of the most attractive characteristics of ILs is that the vast range of ion
combinations which allow for great ability of their physicochemical properties to be
tuned. The variation in IL ions has a major influence on dissolution extent and ratio
of losses. The effect of cation size is exhibited when comparing [C4mim]Cl with
[C2mim]Cl. In agreement with the literature [57], the smaller [C2mim] cation
imparts higher dissolution and this may be due to the enhanced penetration of the
smaller solvent molecule resulting in higher dissolution. However, the mass losses
are nearing the mass dissolved which indicates increased depolymerisation of
solutes. The effect of anion is studied by comparing [C2mim]Cl to [C2mim]OAc. The
acetate anion seems to favour dissolution as opposed to losses. Moreover, losses
may be exacerbated due to the fact that dissolution (96 %) is well into the last
recalcitrant bagasse fraction. It is probable that by reducing the severity of the
reaction conditions, the ratio of dissolution to losses will be improved. Acetate has a
85
higher hydrogen bond basicity than chloride [92] and thus its ability to disrupt
hydrogen bonds and dissolve cellulose is higher. Out of the ILs tested, [C2mim]OAc
appears the most favourable.
Figure 4.1.5: Effect of ionic liquid choice on bagasse dissolution
The superiority of [C2mim]OAc to [C4mim]Cl as a solvent for biomass has
been reported in the literature recently. Rogers and co-workers [101] measured
93.5 % dissolution extent of southern yellow pine in [C2mim]OAc and only 26 % in
[C4mim]Cl under the same conditions (particle size 0.25 – 0.50 mm, 5 % mass
loading, 110 °C for 16 h). However [C2mim]OAc is not extensively used in this
research due to the high viscosity of [C2mim]OAc / bagasse solutions. Preliminary
experiments with bagasse loading as low as 3 % mass in [C2mim]OAc resulted in
reactions that were difficult to stir with magnetic stirring. The higher biomass
0
10
20
30
40
50
60
70
80
90
100
[C4mim]Cl [C2mim]Cl [C2mim]OAc
ba
ga
sse
(%
ma
ss)
Ionic liquid
dissolution losses
86
loadings reported in the literature are attributed to the different substrate used
(viz. pine as opposed to bagasse).
Comparatively, Zavrel et al. [132] reported in 2009, the use of a light
scattering technique to screen ILs for their ability to dissolve Avicel cellulose. The
scattered light beam extinction after passing through a dissolution reaction is
positively related to the amount and size of suspended undissolved solids. With this
technique the solubility was ranked as follows
[C2mim]OAc>[C2mim]Cl>[AllylMim]Cl>[C4mim]Cl. However, when these ILs were
tested for dissolution of wood chips (both hardwood and softwood), [Allylmim]Cl
was found better than the [C2mim]+ ILs.
Lee et al. [68] conducted incremental additions of maple wood flour in a
number of ILs (80 °C for 24 h). Conversely, they report a wood solubility of > 30 g kg-
1 for [C4mim]Cl and < 5 g kg-1 for [C2mim]OAc. Their methodology, based on
qualitative visual observation of dissolution, may be introducing bias to these
results as compared to the methodology used by all above authors and the one
used in this research.
4.1.2.g Monitoring dissolution kinetics using real time FTIR - ATR
The FTIR spectra of bagasse soda lignin (prepared according to Section 3.7),
glucose and cellulose (each dissolved in [C4mim]Cl) were obtained and analysed.
Lignin concentration was found to be linearly related to absorbance at 1510 cm-1.
Likewise, glucose and cellulose concentrations were linearly related to absorbances
at 1050 cm-1 and 1070 cm-1, respectively. These wavenumbers were used to
monitor biomass dissolution by ATR-FTIR. The nature of these absorbances and the
quantification method are provided in Appendix I.
FTIR spectra from a dissolution reaction with 5 % mass bagasse in [C4mim]Cl
were acquired in real time (see Section 3.8 for details). The 1070 cm-1 band was
attributed to the sum of all polysaccharides that were dissolved in the IL. The 1510
cm-1 band was not detectable, possibly due to the low concentration of lignin in the
biomass / IL solution. The absorbance at 1570 cm-1 was attributed to the
87
imidazolium ring of [C4mim]Cl. Figure 4.1.6 shows the extent of polysaccharide
solvation during the temperature ramp by plotting the trend of the ratio of the
polysaccharide absorbance (1070 cm-1) to that of the background [C4mim]Cl
absorbance (1570 cm-1). Polysaccharide solvation appears stagnant to very slow at
70 °C, even after 120 min and it starts accelerating significantly when the
temperature is increased. Dissolution appears to accelerate at temperatures above
150 °C. Certainly, dissolution rate has significantly increased as the temperature
approaches and exceeds 160 °C, but then slows down again when the temperature
is returned to 150 °C. This outcome suggests that high temperatures are indeed
associated with accelerated dissolution and requires further investigation.
Figure 4.1.6: Real time FTIR of bagasse polysaccharides upon dissolution in
[C4mim]Cl
0
0.1
0.2
0.3
0.4
0
20
40
60
80
100
120
140
160
180
0 50 100 150 200
FT
IR p
ea
k r
ati
o
Te
mp
era
ture
( °
C)
Time (min)
reaction temperature (°C )
polysaccharide (FTIR peak ratio)
88
4.1.3 Thermal stability of bagasse components in [C4mim]Cl
In order to investigate the thermal stability of bagasse components in
[C4mim]Cl, DSC and TGA analysis was used and the kinetics of glycosidic bond
cleavage of cellobiose in [C4mim]Cl were studied.
4.1.3.a Differential scanning calorimetry of bagasse and bagasse lignin
The dissolution at high temperatures can be accelerated by biomass
softening phenomena triggered at certain temperatures. For example, biomass-
softening phenomena occurring at high temperatures at and above the glass
transition of lignin effect disentanglement and fibre swelling which increases
surface area. Lignin, hemicelluloses and the amorphous component of bagasse
cellulose are all viscoelastic materials and can be expected to exhibit glass transition
temperatures [144]. Dissolution of biomass close to the glass transition
temperature of lignin is thought to influence dissolution and pretreatment effects
[102, 131, 145]. In this study, differential scanning calorimetry (DSC) was used to
identify such possible material softening phenomena in biomass at high
temperatures. Glass transition represents the change from a glassy state of an
amorphous polymer to its rubbery state. When this change occurs it is associated
with a sudden change in heat capacity at that temperature which is detectable as an
endothermic transformation in a DSC thermogram.
DSC profiles of bagasse, NaOH extracted bagasse lignin (i.e. soda lignin), and
bagasse in [C4mim]Cl and [C2mim]OAc were acquired according to Section 3.9 and
are shown in Figure 4.1.7. The arrows are showing points of maximum endothermic
transitions, only in the temperature range of 110 °C to 160 °C (temperatures used in
this work), as calculated by the thermal analysis software. The transition in the
lignin is clear and characteristic and thus can be attributed to a glass transition
temperature at 122 °C. This figure is in close agreement to the glass transition
temperature for corn stem rind lignin reported by Donohoe et al. [145] at 120 °C
and it is also close to the glass transition determined for bagasse soda lignin by
Moussaviun and Doherty [146] at 130 °C. Glass transition may vary for bagasse
lignins extracted under different conditions and processes. The DSC curve for
89
bagasse indicates a transition at 140 °C which is less sudden and occurs over a wide
temperature range. This is not surprising since the amorphous component of
bagasse is more complex than extracted lignin and the heat capacity change may be
a result of a number of transitions resulting in a summative broad transition. The
transitions observed in the bagasse DSC curves are relatively subtle and broad, and
thus a sharp glass transition temperature cannot be identified. Nevertheless, when
bagasse is reacted in ionic liquids, the transitions seem to occur at lower
temperatures with [C2mim]OAc having a greater effect on this thermal transition
than [C4mim]Cl. The transition temperatures can be considered broad indicators of
slight softening of bagasse at these temperature ranges, namely between 130 °C
and 145 °C.
bagasse
lignin (NaOH extracted)
30 % bagasse in [C4mim]Cl
30 % bagasse in [C2mim]OAc
0 20 40 60 80 100 120 140 160
-1.5
-1
-0.5
0
Figure 4.1.7: Differential scanning calorimetry profiles
Temperature range of interest
122 °C
140 °C
137 °C
129 °C
Temperature (°C)
He
at
flo
w (
m W
-1)
90
4.1.3.b Thermogravimetric analysis (TGA) of bagasse
It has been demonstrated by DSC analysis in Section 4.1.3.a, that bagasse
undergoes structural changes when reacted in [C4mim]Cl at temperatures between
130 °C and 145 °C. It has also been demonstrated in Section 4.1.2 that some of the
starting bagasse is lost post [C4mim]Cl dissolution and precipitation with water. The
amount of these losses has been extensively investigated, however their
composition needs to be understood. TGA (as described in Section 3.10) was used
to determine whether any mass of reactants is lost to volatile molecules at the
targeted temperature range. The peaks of the first derivative curve of TGA curves
are an indication of the temperature at which thermal decomposition is fastest. In
Figure 4.1.8 the peak at ca. 100 °C is a result of moisture loss. The first bagasse
component to degrade is the hemicellulose as it is the most thermolabile of the
biomass components [147]. Both hemicellulose and bagasse degrade at lower
temperatures in the presence of [C4mim]Cl. [C4mim]Cl is more thermally stable in
IL / bagasse mixtures than by itself. Accordingly, Wendler et al. [148] have
demonstrated that [C2mim]OAc is more stable in IL / cellulose mixtures than by
itself. The thermal decomposition temperatures for both bagasse and bagasse in
[C4mim]Cl lie significantly above the targeted temperature range of 110 °C to 160
°C used in the pretreatment experiments of this study. Therefore, bagasse losses
measured in Section 4.1.2 must be predominantly due to biomass depolymerisation
towards IL / water soluble molecules as opposed to losses of volatile molecules.
91
Figure 4.1.8: First derivative of thermogravimetric analysis curves
4.1.3.c Cellobiose hydrolysis in [C4mim]Cl
The hydrolysis of glycosidic bonds is likely to be one of the main reasons for
losses incurred upon biomass dissolution in ILs and the rate of this hydrolysis needs
to be understood.
The rate of cellobiose hydrolysis to glucose and subsequent glucose
degradation in [C4mim]Cl (as described in Section 3.11) at two different
temperatures are plotted in Figure 4.1.9. The hydrolysis of the cellobiose glycosidic
linkage is considered to be a model for the hydrolysis of glycosidic linkages in fully
dissolved cellulose.
-10
-9
-8
-7
-6
-5
-4
-3
-2
-1
0
0 100 200 300 400 500 600
Δw
ΔT
-1/
mg
°C
-1
Temperature (°C)
bagasse
bagasse hemicellulose
30 % bagasse in[C4mim]Cl
3 % hemicellulose in[C4mim]Cl
[C4mim]Cl
92
When cellulose or biomass is dissolved in a chloride imidazolium ionic liquid,
the glycosidic bond hydrolysis has been shown to occur at random points across the
cellulose chain [117]. This random chain scission is not expected to result in
significant glucose formation. Nevertheless, the stability of glucose monomers
under these reaction conditions is also shown in this experiment. Both cellobiose
hydrolysis rate and glucose degradation rate increase with increasing temperature.
Although it is expected that glucose would be less stable at 150 °C than at 130 °C,
glucose initially accumulates at 150 °C, but does not accumulate at 130 °C. This
initial accumulation may be attributed to the mechanisms of cellobiose hydrolysis
and glucose decomposition. Glucose decomposition proceeds either by Lobry de
Bruyn - van Ekenstein rearrangement to fructose (which is less stable than glucose)
and subsequent decomposition, or via ring opening and retro-aldol condensation
(e.g. initially to glyceraldehydes and dihydroxyacetone). In the absence of water,
cellobiose hydrolysis proceeds by electrophilic attack of hydrogen ions on the
glycosidic oxygen lone pair of electrons and initially results in the formation of
glucose and a glucose carbocation which then reacts with water to form glucose
and regenerates the hydrogen ion. The carbocation product formed under
anhydrous conditions (1,6-anhydro-β-D-glucopyranose - see Figure 4.1.10) is more
thermally stable than glucose [149]. Consequently, at 130 °C cellobiose in the
presence of small amounts of water initially forms glucose which rapidly
decomposes. Later in the reaction, after the water has been consumed or lost, 1,6-
anhydro-β-D-glucopyranose accumulates. At 150 °C and with little or no water
present, 1,6-anhydro-β-D-glucopyranose accumulates early in the reaction.
While it has been established that glycosidic bond cleavage is rapid for
saccharides in solution at high temperatures, the extent to which this bond cleavage
contributes to cellulose losses depends on the DP of the cellooligomers resulting
from the aforementioned random chain scission (low DP cellooligomers are water
soluble and are expected to be lost as they will not precipitate on addition of water
antisolvent).
93
Figure 4.1.9: Cellobiose hydrolysis and glucose accumulation in [C4mim]Cl
Figure 4.1.10: Hydrolysis of cellobiose in the absence of water
-60
-40
-20
0
20
40
60
0 50 100 150 200 250
con
cen
tra
tio
n in
[C
4 m
im]C
l (
mg
g-1
)
Time (min)
Cellbiose 130 °CCellbiose 150 °CGlucose 130 °CGlucose 150 °CGlucose loss 130 °CGlucose loss 150 °C
94
Table 4.1.1: Compositional analysis of bagasse pretreated with [C4mim]Cl and dilute acid
% dry mass ratios
sample Mass
recovery
Ash
AIL
ASL
Total
lignin
Glucan
Xylan
Arabinan
Acetyl
Arab/
xylan
Acetyl/
xylan
Untreated 100 3.4 21.2 5.0 26.2 41 22.7 1.84 2.48 0.08 0.11
DIL ACID 66 2.8 28.3 2.3 30.6 62 4.7 0.42 0.36 0.09 0.08
IL 140 °C 93 2.5 20.4 4.7 25.0 41 21.3 1.31 2.49 0.06 0.12
IL 150 °C 83 3.8 23.2 3.7 26.9 48 15.8 0.82 1.96 0.05 0.12
95
4.1.4 Ionic liquid pretreatment comparison with dilute acid pretreatment
The total recovered solids (TRS, described in Section 3.13) from water
precipitation of [C4mim]Cl-treated bagasse (at the optimised dissolution conditions
determined in Section 4.1.2) and dilute acid-pretreated bagasse (prepared
according to the standard NREL process described in Section 3.14) were analysed
compositionally and compared for saccharification performance.
4.1.4.a Compositional analysis
The composition of untreated bagasse, the TRS of bagasse treated with
[C4mim]Cl (for 90 min at 140 °C and 150 °C) and bagasse treated with dilute acid
was analysed according to Section 3.12 and the results are shown in Table 4.1.1.
The composition of the untreated bagasse indicates that the substitution of GAX
hemicellulose is 0.8 arabinosyl and 1.1 acetyl groups for every 10 xylose units
(glucuronyl groups were not measured). This is in near agreement with the GAX
hemicellulose substitution reported in the literature (1.3 arabinosyl and 1.2 acetyl
groups for every 10 xylose units as seen in Section 2.2.2) and confirms that the GAX
structure depicted in Figure 2.2.3 is representative of the GAX structure found in
the starting bagasse of this study. The dilute acid-treated solids have low
arabinoxylan and acetyl content and are enriched in lignin and cellulose (glucan). It
is known that dilute acid pretreatment of biomass removes hemicelluloses and this
dissolution may be accompanied by hydrolysis of ester bonds between
hemicelluloses and lignin. Consequently, low xylan and arabinan content can be
expected. Ionic liquid treatment at 140 °C imparts low losses (7 % mass),
consequently few compositional changes are measurable and the recovered solids
appear compositionally almost identical to the untreated bagasse. In IL treatment at
150 °C the arabinoxylan is removed similarly to the dilute acid treatment. However
[C4mim]Cl appears to be less effective at removing acetyl groups. It is interesting
that arabinan seems to be selectively removed by the ionic liquid (cf. dilute acid
pretreatment).
Lee et al. [68] and Fu et al. [143], have investigated the effect of
[C2mim]OAc pretreatment on wood flour and wheat straw, respectively. The
96
compositional changes of pretreated solids with increasing temperatures up to 150
°C were reported at 90 min of reaction time in both works. The most pronounced
difference in their results, when compared with those reported here, is that the
lignin of their solids diminishes with increasing temperature. Although these studies
are all on different plant substrates, it is probable that lignin is more soluble in
water / [C2mim]OAc mixtures than in water / [C4mim]Cl mixtures and consequently
there is less lignin precipitation upon addition of water. In fact, the pH of 0.5 water :
IL mass ratio for [C4mim]Cl is ca. 6.6 and for [C2mim]OAc is ca. 7.9 (see Figure
5.3.1). This pH difference may explain variable lignin recovery (i.e. lignin is generally
alkali soluble) and will be investigated in more detail in Section 5.3.
4.1.4.b Enzyme saccharification
The enzyme saccharification of cellulose in the untreated bagasse, the TRS of
bagasse treated with [C4mim]Cl (for 90 min at 140 °C and 150 °C), the DS (dissolved-
then-precipitated fraction only, excludes the undissolved fraction) of bagasse
treated with [C4mim]Cl (for 90 min at 150 °C) and bagasse treated with dilute acid
was monitored according to Section 3.15 and the results are shown in Figure 4.1.11.
Both ionic liquid and dilute acid pretreatments imparted high cellulose
saccharification rates and extents as compared to those of untreated bagasse. The
enzyme saccharification rate and extent of the TRS from ionic liquid treatment at
150 °C is similar to that of the dissolved bagasse fraction only (DS) from ionic liquid
treatment at 150 °C. Its saccharification reaches a practical endpoint in less than 3 h
at which point its saccharification extent (93 % mass) is more than twice as high as
that of TRS from ionic liquid treatment at 140 °C (42 %) and dilute acid treatment
(31 %). At 24 h the [C4mim]Cl treatment at 150 °C still imparts close to two times
more cellulose saccharification than dilute acid treatment (viz. 96 % cf. 55 %).
Comparatively, in 2010, Li et al. [76] reported 24 h cellulose saccharification of
[C2mim]OAc treated switchgrass to be ca. two times higher than dilute acid
treatment (viz. 96 % cf. 48 %). It is noteworthy that the IL pretreatment reported by
Li et al., using a higher cellulase enzyme loading than used here, imparts a practical
saccharification end-point only after 24 h (cf. 3 h in Figure 4.1.11).
97
It is here indicated that Ionic liquids can outperform dilute acid as a
pretreatment for bagasse while temperature plays a pivotal role in the performance
of ionic liquid treatment. It may also be deduced from the data in Figure 4.1.11 that
complete dissolution is not necessary to maximise saccharification efficiency.
[C4mim]Cl treatment of bagasse at 150 °C for 90 min imparts 52 % mass dissolution
(viz. Section 4.1.2.b.), yet the bagasse partially dissolved under these conditions
(TRS) exhibits a similar saccharification profile to that of completely solubilised
bagasse (DS in Figure 4.1.11). The sudden large increase in enzyme saccharification
rate, between 140 °C and 150 °C, has recently (2010) been reported by Arora et al.
[102] for switchgrass treated with [C2mim]OAc. These authors measured two times
the initial rate of total reducing sugars released at 150 °C than at 140 °C and
attributed this phenomenon to the glass transition temperature of lignin, without
providing direct evidence.
Figure 4.1.11: Enzyme saccharification of bagasse pretreated with [C4mim]Cl and
dilute acid
0
20
40
60
80
100
0 4 8 12 16 20 24
glu
can
in
pre
tre
ate
d s
oil
ds
( %
ma
ss)
Time (h)
150 °C DS
IL 150 °C
IL 140 °C
Dil Acid
Untreated
98
The effect of the temperature increment from 140 °C to 150 °C on the
structure of IL-treated bagasse can be seen in Figure 4.1.12. At 140 °C and 90 min in
[C4mim]Cl (5 % mass bagasse in IL), the structure of the fibre is still discernible
while at 150 °C, and otherwise identical reaction conditions, the pretreated bagasse
looks more like a paste.
Figure 4.1.12: Images of [C4mim]Cl-pretreated bagasse at 140 °C and 150 °C
In Figure 4.1.13, the initial saccharification rates of both cellulose and
hemicellulose (as xylan) and the XRD-derived crystallinity indices (described in
Section 3.16) of solids recovered from IL pretreatment (TRS at 140 °C and 150 °C for
90 min) are compared to those of untreated and dilute acid treated bagasse.
Hemicellulose saccharification is less rapid than that of cellulose for all
solids. This may be a result of hemicellulose being covalently linked to lignin and
thus forming part of the enzyme-recalcitrant lignin-hemicellulose fraction of
bagasse. It may also be related to the fact that hemicellulose saccharification is very
slow due to the low concentration of xylanases in the “Accelerase 1000” enzyme
cocktail used here. As expected, the crystallinity index seems to be inversely related
to the initial saccharification rates of all solids. Interestingly, the crystallinity index
Pretreated bagasse at 140 °C
(ca. 50 % moisture)
Pretreated bagasse at 150°C
(ca. 70 % moisture)
99
of IL treatment at 140 °C is three times higher than that at 150 °C, despite the fact
that the extent of dissolution at 150 °C is only ca. twice that at 140 °C (as reported
in Section 4.1.2.b). Preferential dissolution of the non-crystalline component of
cellulose is not surprising, but it would appear that temperatures higher than 140 °C
are required to either dissolve or effect decrystallisation of the crystalline
component. It is notable that although only ca. 50 % of the bagasse dissolved in the
150 °C pretreatment, the crystallinity of the recovered material is less than ¼ of the
original bagasse. At 150 °C in [C4mim]Cl, bagasse need not be dissolved to disrupt
the crystal regions of the bagasse. This is a key finding.
Figure 4.1.13: Initial rates of enzyme saccharification and XRD crystallinity indices
for IL- and dilute acid-pretreated bagasse (TRS)
In Figure 4.1.14, the final saccharification yields after 121 h of incubation
with enzymes are plotted. At 121 h, the saccharification is considered complete (i.e.
no further saccharification is expected beyond this point). While 98 % mass
cellulose saccharification is reached by the ionic liquid treatment at 150 °C, the
extents of saccharification of the rest of the pretreatments are still much lower. For
example, dilute acid reaches a maximum of only 72 % cellulose conversion. Final
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0.80
0
10
20
30
40
50
60
70
80
90
100
Untreated DIL ACID IL 140 °C IL 150 °C
CrI
init
ial
sacc
ha
rifi
cati
on
ra
te (
% m
ass
h-1
)
glucan xylan CrI
100
hemicellulose saccharification is consistently lower than that of cellulose and this is
particularly pronounced for the IL pretreatment at 150 °C. As compared to
untreated bagasse, the IL treated solids at 150 °C have a 30 % lower xylan to lignin
content ratio (see Table 4.1.1) and it was thus deduced that the lignin bound
hemicellulose is more recalcitrant to the IL. The higher content of lignin-bound
hemicellulose in the IL treated solids at 150 °C as compared to the other solids may
be responsible for its less complete saccharification, particularly since lignin is a
known inhibitor of enzyme saccharification.
Figure 4.1.14: Glucan and xylan saccharification extent after 121 h for IL- and
dilute acid- pretreated bagasse (TRS)
4.1.4.c Saccharification and fermentation of IL treated bagasse
The purpose of this experiment was simply proof of concept that these
materials can be fermented and no attempt to optimize conditions was made.
Nevertheless, some ethanol yield comparisons to dilute acid pretreatment can be
made. Bagasse (35 g) in [C4mim]Cl (464 g) was reacted (150 °C for 2 h) in the RC1
reactor (described in Section 3.8) cooled to 70 °C and precipitated with water (ca.
0
10
20
30
40
50
60
70
80
90
100
Untreated DIL ACID IL 140 °C IL 150 °C
sacc
ha
rifi
cati
on
ex
ten
t (
% m
ass
)
glucan xylan
101
300 mL). The recovered solids were saccharified with enzymes (3 days, 5 FPU) and
subsequently fermented with yeast (0.5 O.D.), as described in Section 3.17, and the
kinetics are shown in Figure 4.1.15. The highly saccharified cellulose of [C4mim]Cl-
treated bagasse (91 % mass of theoretical on the basis of pretreated solids) yielded
ethanol equivalent to 76 % of theoretical in < 24 h and possibly actually achieved
this in < 16 h (based on the initial fermentation rate and the fermentation kinetics
presented in other studies under the same conditions). The 76 % ethanol yield for
this experiment is equivalent to a yield of 0.43 g g-1
of glucose or 85 % mass of
theoretical yield on the basis of glucose fed to fermentation. The equivalent
glucose-to-ethanol conversion efficiency for dilute-acid-pretreatment-derived
glucose is 95 % of theoretical yield according to the latest NREL report [150]. These
glucose-to-ethanol efficiencies together with the cellulose-to-glucose efficiencies
from Figure 4.1.11 and cellulose recoveries from Table 4.1.1 were used for
calculating potential ethanol production from IL and dilute acid-pretreated biomass
shown in Table 4.1.2.
Figure 4.1.15: Fermentation kinetics of [C4mim]Cl-treated bagasse after enzyme
saccharification
0
10
20
30
40
50
60
70
80
90
100
0 10 20 30 40 50 60 70 80
% m
ass
of
the
ore
tica
l
(on
th
e b
asi
s o
f p
retr
ea
ted
so
lid
s)
Time (h)
Glucose
Ethanol
102
Table 4.1.2: Comparison of ethanol yields from IL and from dilute acid
pretreatment
Pretreatment Dilute Acid Ionic Liquid
([C4mim]Cl)
Temp (°C) 160 150 time (h)
Pretreatment 0.17 1.50 Saccharification 24.0 3.0 Fermentation 16.0 16.0
Total processing 36.2 16.5
Mass (% of theoretical yield)
Cellulose recovery 100 100 Cellulose saccharification 55 93
Glucose to ethanol 95 85 Total ethanol yield 52 79
From the comparison in Table 4.1.2, IL treatment appears superior to dilute
acid both in terms of processing time (16.5 h cf. 36.2 h) and ethanol yield (79 % cf.
52 % mass of theoretical based on starting biomass). The performance of ILs is
attributed to the ability to cleave covalent bonds while also decrystallising cellulose
and dilute acid is only capable of the former.
4.1.5 Summary
In this section, it was established that at the targeted high temperatures of
110 °C to 160 °C, dissolution of bagasse in [C4mim]Cl increases with both time and
temperature while the decomposition temperature of the reactants is not
exceeded. At the early stages of dissolution (e.g. < 75 % mass dissolution), the
losses are proportionately low and generally account for about 1/3 of the dissolved
material when the temperature is kept at ≤ 150 °C and time ≤ 120 min. At 150 °C
and 90 min, cellulose is fully recovered and the losses comprise primarily
hemicellulose components (viz. xylan and arabinan). As the dissolution nears 100 %
mass (e.g. 160 °C, 90 min), the recalcitrance of the undissolved material increases
and the dissolution rate slows while the losses continue to rise. Bagasse at 50 %
moisture dissolves slower in [C4mim]Cl than air-dried bagasse, while no difference
is observed between the dissolution rates of air-dried (10 % moisture) and oven-
dried bagasse (1 % moisture). Using incremental bagasse dosing, loadings of up to
103
20.6 % mass were achieved in [C4mim]Cl. Dissolution of bagasse in different ILs was
conducted and the effect of the IL ion variation on dissolution and losses were
discussed. The acetate IL anions, as compared to chloride anions, appear to afford
an increased dissolution to losses ratio while the shorter alkyl chains of imidazolium
cations seem to accelerate both dissolution and losses.
Cellobiose monomerisation under anhydrous conditions in [C4mim]Cl
appeared to induce the accumulation of 1,6-anhydro-β-D-glucopyranose which is
more thermally stable than glucose.
Fermentation of [C4mim]Cl-pretreated bagasse was successfully conducted
and the ethanol yield measured. [C4mim]Cl pretreatment (with partial dissolution
at 150 °C, 90 min) afforded a much higher ethanol yield than standard dilute acid
pretreatment (79 % cf. 52 % mass theoretical – on the basis of cellulose in starting
biomass) in less than half the processing time (pretreatment + saccharification +
fermentation = 16.5 h cf. 36.2 h). This is mainly due to the persistence of crystalline
cellulose in dilute acid pretreated solids which limits initial saccharification rates.
The saccharification extent at practical saccharification endpoint (reached at
≤ 3 h for all materials) achieved from partial dissolution of bagasse in [C4mim]Cl at
140 °C and 90 min were slightly higher than those of dilute acid. However, this
saccharification extent was more than doubled when the temperature of [C4mim]Cl
treatment was increased by 10 °C (to 150 °C) and it equalled those of bagasse
completely dissolved in [C4mim]Cl. Temperature plays a pivotal role in this IL
pretreatment, while bagasse need not be completely dissolved to impart high
saccharification rates and extents. This, together with the fact that the crystallinity
of cellulose (in the IL pretreated bagasse) at 150 °C, was markedly lower than that
at 140 °C, are key outcomes.
104
4.2 Role of non-dissolution pretreatment effects on enzyme
saccharification
In section 4.1.4.b, (see Figure 4.1.11) it was observed that biomass treated in
[C4mim]Cl at 150 °C, but incompletely dissolved, imparted a similar saccharification
profile to completely dissolved material. It appears likely that structural changes of
the undissolved fraction contribute significantly to saccharification rate and extent.
This section describes the outcomes of experiments where the undissolved material
was separated from the dissolved material prior to precipitation (see Section 3.6.2)
4.2.1 Compositional analysis
In a typical dissolution, bagasse (2.5 g) was reacted with [C4mim]Cl (50 g) for
90 min at 140 °C and 150 °C. The resulting mass was diluted with 50 mL DMSO and
filtered. The residue retained on the filter was recovered and analysed as the
undissolved fraction. The filtrate (which contained the dissolved fraction) was
precipitated with 100 mL water and centrifuged. The precipitated solid was
recovered and analysed as the dissolved solids (DS or dissolved-then-precipitated
fraction).
As shown in Table 4.2.1, the composition of the undissolved fraction at 140
°C does not differ markedly from that of the untreated bagasse, indicating little if
any preferential dissolution or recovery of any particular component. The
undissolved fraction at 150 °C however, differs significantly. It contains half of the
original glucose, indicating that at around 150 °C preferential cellulose dissolution
starts occurring. The acetyl content is high in the undissolved fraction at both
temperatures showing the recalcitrance of acetyl groups to [C4mim]Cl.
105
Table 4.2.1: Compositional analysis of dissolved-then-precipitated solids (DS) and undissolved solids (UND) from [C4mim]Cl pretreatment of
bagasse
Mass (g) % dry mass ratios
Sample Sample
dry mass Ash AIL ASL
Total lignin
Glucan Xylan Arabinan Acetyl Arab/ xylan
Acetyl/ xylan
Untreated 2.50 3.4 21.2 5.0 26.2 41 22.7 1.84 2.48 0.08 0.11
140°C UND ca. 1.95* 2.1 21.3 6.1 27.4 39 25.2 1.50 2.86 0.06 0.11
140°C DS ca. 0.37* 3.0 12.2 3.5 15.7 77 9.2 0.52 0.88 0.06 0.10
150°C UND ca. 1.20*
2.0 32.2 5.2 37.4 22 28.1 1.65 3.47 0.06 0.12
150°C DS ca. 0.88* 1.9 8.0 2.4 10.4 82 7.16 0.44 0.67 0.06 0.09
Table 4.2.2: Effect of residence time on the composition of undissolved bagasse after [C4mim]Cl pretreatment at 150°C
Mass (g) % dry mass ratios
Sample Sample
dry mass Ash AIL ASL
Total lignin
Glucan Xylan Arabinan Acetyl Arab/ xylan
Acetyl/ xylan
Untreated 6.00 3.4 21.2 5.0 26.2 41 22.7 1.84 2.48 0.08 0.11 30 min UND 4.93 1.4 23.5 6.5 30.0 41 21.7 1.49 3.00 0.07 0.14 60 min UND n/d 2.2 28.1 7.2 35.4 32 18.8 1.28 3.20 0.07 0.17 90 min UND 3.36 2.0 32.2 5.2 37.4 22 28.1 1.65 3.47 0.06 0.12
* Masses do not correspond to the same experiment but have been estimated from previous dissolution experiments which used the same conditions and the same solids recovery method (see Section 4.1.2.b)
106
The composition of the dissolved-then-precipitated fractions (DS in Table
4.2.1) is notably rich in cellulose (77 % at 140 °C and 82 % at 150 °C). Lignin and
hemicellulose are also present in the dissolved fractions (albeit at a low
percentages), but decrease as temperature increases (i.e. with increasing
temperature lignin and hemicellulose content in the DS is reduced). While this is
partly due to preferential dissolution of cellulose, it is likely also due to increased
degradation of hemicelluloses and lignin in solution to material that is soluble in IL /
water (i.e. lower molecular mass products of bond cleavages).
Arabinosyl glycosidic bonds are generally the most labile of glycosidic
linkages in the lignocellulosic matrix (certainly this is the case in an acidic aqueous
environment and also under pyrolysis conditions). It is evident from Table 4.2.1 that
cellulose is preferentially dissolved by [C4mim]Cl. [C4mim]Cl also dissolves lignin
and hemicellulose but to lesser extents.
Preferential dissolution patterns were also monitored over time. Bagasse (6
g) was reacted in [C4mim]Cl (120 g) for 30 min, 60 min and 90 min and the
undissolved solids separated and characterised (Table 4.2.2). There seems to be
little change in composition after 30 min. Over time, the lignin, xylan and acetyl
content increase in the undissolved fraction while the cellulose gets preferentially
removed (solubilised).The same dissolution behaviour is evident, i.e. initially all
components dissolve at the same rate, and then cellulose is preferentially dissolved.
Either arabinose is cleaved from the hemicellulose solids or arabinose is cleaved
from dissolved hemicellulose. It seems likely from the composition of the dissolved
material that both may happen. Cleavage of arabinosyl glycosidic linkages leads to
arabinose in solution, unless the arabinose is covalently linked to lignin.
Lignin and hemicellulose are more recalcitrant and cellulose is preferentially
dissolved. IL treatment results in changes to undissolved hemicellulose, viz.
cleavage of arabinosyl glycosidic bonds and enrichment in lignin, xylan and acetyl
content.
107
4.2.2 Enzyme saccharification
The enzyme saccharification profiles of UND fractions, at different IL
pretreatment temperatures and times, were exposed to enzyme saccharification
and the profiles are shown in Figure 4.2.1. It appears that increasing pretreatment
conditions severity, in terms of both temperature and time, increases the enzyme
accessibility of the undissolved fractions. This result confirms that changes in the
undissolved fraction (e.g. swelling) contribute to higher saccharification rates and
extent. Starting with the effect of time, at 150 °C the difference in saccharification
extent at 24 h of the 90 min sample to the 60 min one is as large as the difference of
the latter to the 30 min sample. At 90 min and 150 °C, the effect of temperature is
sudden since the difference of the 24h-saccharification extent of the 150 °C sample
to the 140 °C sample is as large as the difference of the latter to the untreated
bagasse.
Figure 4.2.1: Saccharification of the undissolved bagasse after [C4mim]Cl
pretreatment at different conditions
0
10
20
30
40
50
60
0 10 20 30 40 50
glu
can
in
un
dis
solv
ed
so
lid
s (
% m
ass
)
Time (h)
150 °C, 90min
150 °C, 60min
140 °C, 90min
150 °C, 30min
Untreated
108
The initial rates of saccharification and XRD crystallinity indices for the
undissolved and dissolved fractions from [C4mim]Cl treatment at 140 °C and 150 °C
are presented in Figure 4.2.2 and final saccharification yields in Figure 4.2.3. The
crystallinity indices of all dissolved fractions are lower than untreated material.
Interestingly, while the crystallinity index of the undissolved fraction at 140 °C is
higher than that of the dissolved material, the crystallinity of the undissolved and
dissolved fraction at 150 °C are similar. This explains the higher initial cellulose
saccharification rates of the 150 °C undissolved fraction at 26.4 % h-1 as compared
to 11.6 % h-1 for 140 °C. Initial hemicellulose saccharification rates are very slow (< 5
% h-1) for all solids, except the ones treated at 150 °C (both DS and UND). For the
UND fraction, increased perturbation/swelling of the LCB structure at 150 °C allows
more rapid hemicellulose saccharification. For the DS fractions, the persistence of
lignin-hemicellulose bonds results in low hemicellulose saccharification rates.
Figure 4.2.2: Initial rates of enzyme saccharification and XRD crystallinity indices
for [C4mim]Cl-pretreated bagasse fractions
0.00
0.10
0.20
0.30
0.40
0.50
0.60
0.70
0
20
40
60
80
100
120
Untreated 140 °C UND 140 °C DS 150 °C UND 150 °C DS
CrI
init
ial
rate
of
sacc
ha
rifc
ati
on
(%
ma
ss h
-1)
glucan xylan CrI
109
While the initial cellulose saccharification rate of the undissolved fraction at
150 °C is remarkably high, the final saccharification yield after 121 h (the effective
endpoint) is below 60 % mass. Given that this fraction has low crystallinity but high
lignin content, its saccharification pattern is in agreement with the analysis of
Holtzapple and co-workers [10, 11], who suggest that crystallinity plays a role in the
initial saccharification rates while lignin content limits mainly the final
saccharification yield. It is also interesting that while the dissolved fractions at both
temperatures reach full saccharification of cellulose (100 % mass) they only reach
about 70 % hemicellulose saccharification. The xylan content of these fractions is
only 7 % to 9 % mass and it is possible that this remaining xylan fraction is close to
lignin-hemicellulose bonds, which inhibit the activity of xylanase enzymes; this
would confirm that some of the hemicellulose has been solubilised with its lignin
bonds intact. However, this is only a speculation as it is not possible to determine
from these data which bonds are preserved upon dissolution.
Figure 4.2.3: Glucan and xylan saccharification extent after 121 h for [C4mim]Cl-
pretreated bagasse fractions
0
10
20
30
40
50
60
70
80
90
100
Untreated 140 °C UND 140 °C DS 150 °C UND 150 °C DS
sacc
ha
rifi
cati
on
ex
ten
t (%
ma
ss)
glucan
xylan
110
4.2.3 X-Ray diffractometry (XRD) of bagasse
The cellulose crystal structures of the undissolved solids at 140 °C and 150 °C
were analysed using XRD diffractograms (acquired according to Section 3.16) shown
in Figure 4.2.4. In the comparison of 140 °C to 150 °C pretreated material, there is
evidence of decrystallisation as indicated by the absence of the peak at 16° in the
2θ range. There is also evidence of a transition of cellulose to its “high temperature
phase” as indicated by the shift of the peak at 22° towards lower 2θ angles (20.5°)
(see arrow on Figure 4.2.4). Hori et al. [151] attribute this shift to the lateral thermal
expansion of cellulose crystals, characteristic of its transition to its “high
temperature phase”.
untreated
140 °C UND
150 °C UND
0 10 20 30 40 50 60 70 800
50000
100000
150000
Figure 4.2.4: Diffractograms of undissolved bagasse after [C4mim]Cl pretreatment
2θ[°]
cou
nts
111
4.2.4 “High temperature phase” of crystalline cellulose
Structural studies of naturally occurring crystalline cellulose (cellulose I)
using XRD and dynamic molecular modelling have reported a change in its crystal
phase at high temperatures described as “high temperature phase” [151, 152]. This
change occurs suddenly at a specific temperature and takes the form of an
anisotropic thermal expansion of the unit cell of cellulose I. This thermal expansion
increases the volume of the unit cell and results in a sudden swelling of the whole
cellulose crystal structure [151]. Dynamic molecular modelling shows that the
hydrogen bonding of the high temperature phase results in a less crystalline
cellulose structure with fewer and weaker inter-chain hydrogen bonds [152]. For
isolated cellulose I, XRD studies report the high temperature phase transition to
occur at 180 °C [151] while dynamic molecular modelling places it at around 176 °C
[152]. This transition may occur at lower temperatures in ionic liquid reacted
cellulose due to the capacity of Cl- to disrupt hydrogen bonds. These interactions
may also be responsible for reducing the thermal decomposition temperature of
bagasse hemicellulose by about 50 °C when reacted in IL as seen in the TGA shown
in Figure 4.1.8.
Transition to the high temperature phase of cellulose is likely to be a critical
first step in rapid dissolution of the crystalline regions in native cellulose. The
increased swelling and reduced crystallinity of the cellulose structure should
facilitate the diffusion of the ionic liquid and also enhance the enzyme accessibility
of cellulose chains remaining in the swollen undissolved biomass fractions.
The cellulose decrystallisation measured in the undissolved fraction is most
likely a consequence of extensive swelling that bagasse cellulose undergoes prior to
dissolution. This mechanism of extensive swelling corresponds to the cellulose
dissolution mode 2 as described by Cuissinat et al. [67] for cellulose microfibrils in
NMMO (viz. “Large swelling by ballooning followed by dissolution”). This ballooning
pattern has also been observed in the swelling of pine sulphate pulp fibre and
swelling was primarily taking place in the secondary cell wall (see Figure 2.2.11).
112
At this point it has to be noted that swelling of cellulose or LCB without
phase transition of the cellulose crystal structure is also possible and it may take
place with protracted exposure to chemical swelling agents at low temperatures.
However, at the high temperatures used in this study, swelling and phase transition
of cellulose mostly happen concomitantly and the phenomena are difficult to
distinguish from each other.
Recently (2009) other studies have been published that support this swelling
pattern upon IL pretreatment. Singh et al. [131] have shown fluorescence
microscopy images of switchgrass cross sections exposed to [C2mim]OAc and heat;
they observed swelling primarily in the secondary cell walls prior to dissolution.
According to Lee et al. [68], [C2mim]OAc pretreated maple wood flour resulted in
comparatively little dissolution but effected high enzyme saccharification rates. This
was attributed to the extensive delignification, swelling and decrystallisation of the
undissolved wood flour. Vanoye et al. [117] have shown swelling of miscanthus
grass in optical microscopy images (see Figure 4.2.5).
Figure 4.2.5: Optical microscopy images showing swelling of miscanthus grass
particles in [C2mim]Cl
(from Vanoye et al. [117])
t0 t = 2h t = 20h
100 °C, 0.5 mm mesh (white arrow is 0.34 mm)
113
4.2.5 ATR-FTIR analysis of undissolved fractions
A selection of [C4mim]Cl-treated bagasse fractions were analysed using
infrared spectroscopy (as described in Section 3.18). Figure 4.2.6 shows FTIR spectra
of the IL undissolved fractions at 140 °C and 150 °C and the dilute acid pretreated
bagasse.
Table 4.2.3 lists the assignments of the absorption bands of interest. The
1236 cm-1 absorbance is generally attributed to syringyl ring and C-O stretching
vibration in lignin, xylan and ester groups [153]. However, the syringyl lignin content
of bagasse is very low so, in this study, this band is going to be associated mostly
with the C-O stretching vibration of ester groups.
The acetyl, lignin and C-O ester bands (1730 cm-1, 1510 cm-1 and 1236 cm-1
respectively) are more intense for the undissolved fraction at 150 °C than for the
untreated bagasse. This indicates that the IL recalcitrant fraction is rich in lignin,
acetyl groups and ester groups/linkages. In agreement with the compositional
analysis above (Table 4.2.1), the FTIR analysis shows that [C4mim]Cl dissolves
cellulose leaving the UND fraction rich in lignin and hemicellulose. A similar
conclusion is presented by Sun et al. [101] in a recent publication of FTIR analysis on
undissolved fractions of wood (oak, pine) in [C2mim]OAc, which states that lignin-
bound carbohydrates are more recalcitrant to IL dissolution. The spectra in Figure
4.2.6 also show that dilute acid treatment removes more acetyl groups and
hemicellulose-lignin ester bonds than IL treatments. In addition the “OH” band at
1100 cm-1 is more pronounced in dilute acid indicating higher cellulose crystallinity
[76]. This interpretation of the FTIR spectrum and of dilute acid treated bagasse is in
agreement with the analysis on dilute acid treated biomass by Kumar et al. [54].
114
Figure 4.2.6: FTIR spectra of IL- and dilute acid-pretreated bagasse fractions
(absorbance – common scale)
The relative abundance of ester bonds compared to lignin content can be
indicated by comparing the relative FTIR absorbances at 1236 cm-1 (for ester
bonding) and 1514 cm -1 (for lignin content). Table 4.2.4 shows these ratios for the
spectra in Figure 4.2.6. Dilute acid pretreatment affords a solid that is rich in lignin
and cellulose and with hemicellulose largely removed. The FTIR band ratio is low
indicating that ester bonds have been broken in the pretreatment process.
Untreated bagasse, IL UND and IL DS have the same FTIR band ratios indicating that
ester bonds between lignin and hemicellulose are preserved in the IL pretreatment.
Dilute acid treated bagasse [C4mim]Cl undissolved fraction (UND) at 150 °C [C4mim]Cl dissolved reprecipitated (DS) fraction at 150 °C Untreated bagasse
POLYSACCHARIDES
LIGNIN
Wavenumbers (cm-1
)
115
Table 4.2.3 : Assignments of FTIR-ATR absorption bands for bagasse
(from [153-156])
Band position
(cm-1
)
Assignment
1730 C=O stretching vibration in acetyl groups of hemicelluloses
1600 C=C stretching vibration in aromatic ring of lignin
1510 C=C stretching vibration in aromatic ring of lignin
1421 CH2 scissoring at C(6) in cellulose
1368 Symmetric C–H bending in cellulose
1236 C-O stretching vibration in ester groups
1100 O-H association band in cellulose and hemicelluloses (associated with crystalline cellulose)
1030 C-O stretching vibration in cellulose and hemicelluloses
974 C-O stretching vibration in arabinosyl side chains in hemicellulose
895 Glucose ring stretch, C1-H deformation
Table 4.2.4: Ratios of FTIR absorbances attributed to ester bonds and the aromatic
ring of lignin.
Band heights Ester bonds Lignin
aromatic ring
Height ratio
1236 cm -1
/ 1514 cm -1
baseline (cm-1
) 1294-1190 1539-1483
band (cm-1
) 1236 1514
Untreated 0.017 0.008 2.2
Dilute acid 0.013 0.170 0.1
150 UND 0.025 0.013 2.0
150 DS 0.015 0.007 2.1
FTIR spectra confirm previous observations that lignin and lignin-bound
hemicellulose are recalcitrant to IL dissolution. If selective removal of arabinan in
the IL is associated with concomitant reduction in ferulate cross-linking,
saccharification of IL-treated solids would be enhanced over and above the
decrystallisation effect. Ferulic acid esters are common in grass hemicelluloses (viz.
GAXs) and are known to be interlinked to the O-5 position of the arabinofuranosyl
branch residues in GAXs. These ferulate esters oxidatively cross-link GAXs and form
116
bonds with lignin. These cross-links are assumed to be recalcitrant to herbivore
digestion and more generally to enzyme saccharification [26]. However, the
arabinan removed by the IL may be ferulic acid ester-linked in which case the
removal would mean cleavage only of the ether bond to the xylan backbone. With
the data available in this thesis, it is not possible to conclude which of the two
arabinan dissolution mechanisms is taking place in the IL.
4.2.6 Summary
Preferential dissolution patterns have been identified which are consistent
across different temperatures and times and more pronounced with increasing
reaction severity. Cellulose and arabinose are preferentially dissolved by [C4mim]Cl,
as opposed to lignin, xylan and acetyl groups. It is also proposed that lignin dissolves
in [C4mim]Cl while preserving covalent bonding to hemicellulose. Preliminary FTIR
analysis suggests that these bonds may be predominantly ester bonds. This may be
the reason for the incomplete final saccharification of hemicellulose in the
dissolved-the-precipitated solids (DS).
It is clear from the data presented here and elsewhere that in the
undissolved bagasse in [C4mim]Cl at 150 °C there is evidence of a transition of
crystalline cellulose I to its “high temperature phase” which reduces its crystallinity.
This is not the case at 140 °C and the transition appears sudden and temperature
dependent.
While the IL undissolved bagasse (UND) at 150 °C is decrystallised to the
same extent as the completely dissolved material (DS), the enzyme saccharification
extent of the former is still significantly lower than that of the latter. The
preferential dissolution imparted by the IL results in a preserved covalent lignin-
hemicellulose structure that renders the cellulose difficult to access by enzymes
regardless of its being decrystallised. It is thus concluded, that while
decrystallisation alone accelerates enzyme saccharification of pretreated solids,
high yields of monosaccharides (high extents of saccharification) require dissolution
of components of the biomass and perturbation of lignin-hemicellulose
interactions.
117
CHAPTER 5 RESULTS - FRACTIONATION
Pretreatment is the process used for ‘opening up’ the lignocellulosic
structure prior to enzyme saccharification. It has to be inexpensive and ideally
should enhance both rate and extent of enzyme saccharification. Fractionation
implies separation into component parts, i.e. separate lignin, hemicellulose and
cellulose. It provides the opportunity to treat three parts separately and differently
and therefore has greater value-creating opportunities. Fractionation is a central
process when envisioning multiple products from biomass processing in the context
of biorefineries.
The compatibility of ionic liquids with other solvents and the opportunities
for liquid–liquid separations of IL solutes make them attractive for fractionation
operations. In this chapter, the use of aqueous biphasic systems (ABSs) and
preferential cellulose precipitation with antisolvents are investigated as tools for
efficient separation of biomass into lignin-rich and polysaccharide-rich fractions.
Finally mass balances are determined for three partial dissolutions of bagasse in IL
([C4mim]Cl, [C2mim]Cl, [C2mim]OAc) fractionated with incremental additions of
water as an antisolvent.
5.1 Aqueous biphasic systems
Ionic liquids form ABSs with aqueous salt solutions and this property has
some potential for industrial separations. Ionic liquids are chaotropic or water
structure disrupting salts and can be salted out by kosmotropic or water structuring
salts such as K3PO4 and Na2SO4 [139, 157-159]. This property has the potential of
concentrating IL / water mixtures while at the same time fractionating biomass
polymers according to their preference for chaotropic or kosmotropic solutions. For
example it has been reported that in polyethylene glycol (PEG) / salt ABSs, lignin has
118
a preference for the polymer-rich (chaotropic) phase as opposed to the inorganic
salt-rich (kosmotropic) phase [160]. Moreover, from an industrial point of view,
ABSs are attractive because potentially they provide a low energy separation
system [158].
One of the objectives of this project is to use ABSs for the separation of the
biomass into its component parts. A claim in the patent by Edye and Doherty [3,
134] formed the starting point for ABS experiments in this thesis. It was claimed
that addition of 200 g L-1 NaOH in a solution of bagasse in [C4mim]Cl yields a
biphasic system comprising a top phase where the IL and water report and a
bottom phase where the aqueous NaOH, the lignin in solution and the precipitated
cellulose (in suspension) report.
In initial experiments in this research, where ca. 1 % mass bagasse (0.166 g)
appeared to completely dissolve in [C4mim]Cl (16.627 g) in 60 min at 150 °C,
subsequent addition of NaOH (200 g L-1, 20 mL), vigorous shaking and let settling for
two days, produced an ABS with clear phase separation and a sharp boundary (see
Figure 5.1.1). The top phase is IL-rich, chaotropic and dark coloured and the bottom
phase is NaOH-rich, kosmotropic and light coloured. The volume of the IL phase
(top) is larger than the volume of the original IL due to water migration from the
NaOH (bottom) phase. A polysaccharide-resembling white fluffy solid accumulated
at the top of the NaOH phase while the dark colour of the top phase suggests a
lignin rich solution. At a 5 % mass bagasse loading (0.330 g) in [C4mim]Cl (6.3 g, at
150 °C for 170 min), the addition of aqueous NaOH (8.4 mL) in the same proportion
did not result in an ABS. Twice as much aqueous NaOH (16.8 mL) was needed to
separate the phases. It is likely that more water was needed to satisfy the hydration
requirement of the biomass solutes and effect phase separation.
FTIR spectra of upper and lower phase of the 5 % bagasse and a biomass-
free NaOH / [C4mim]Cl ABS were acquired (as described in Section 3.18) and are
shown in Figure 5.1.2. It is apparent that the biomass-free ABS phases are clearly of
different composition whereas spectra of the ABS with 5 % bagasse have similar
features, indicating more mixing of the phases. Therefore the formation of ABS in
119
the absence of biomass was investigated (see Sections 5.1.1 to 5.1.3).
Notwithstanding this problem, the literature reports deprotonation of dialkyl
imidazolium ions under alkali conditions to form neutral carbenes. This reaction
may lead to loss of ILs and consequently is also investigated here (see Section 5.1.5)
Figure 5.1.1: A NaOH / [C4mim]Cl ABS with 1% mass bagasse load
In order to examine the phase preference of lignin in the [C4mim]Cl / NaOH
ABS, 1.4 g of soda lignin were dissolved in 9.7 g of [C4mim]Cl (170 °C for 22 min).
Aqueous NaOH solution (20 g L-1, 13 mL) was added and the reaction shacked
vigorously and left for a week to phase separate. Each layer was sampled and their
infrared spectra acquired (see Figure 5.1.3). The characteristic lignin band
absorption around 1510 cm-1 has shifted to 1496 cm-1 and it is only discernible at
the spectrum of the [C4mim]Cl phase of the ABS. This indicates that the lignin has a
preference for the IL phase of this system. The 1510 cm-1 band is clearly visible in
the reference spectrum of 15 % lignin in [C4mim]Cl while it is absent from the neat
[C4mim]Cl background.
IL phase (containing lignin?)
Cellulose-resembling fluffy solid
NaOH phase
120
Figure 5.1.2: FTIR spectra of each phase of two NaOH / [C4mim]Cl ABSs
Regarding the distribution of lignocellulosics within such ABSs, the original
claims in the patent by Edye and Doherty [3, 134, 161] were that cellulose
precipitates and the lignin remains in solution in the inorganic salt layer. Visual
observations of the ABS (Figure 5.1.1) and FTIR analysis (Figure 5.1.3) suggest that
the cellulose precipitates at the top of the inorganic salt phase and lignin remains in
solution in the [C4mim]Cl phase. It’s also worth stressing that Rogers and co-
workers have reported the separation of lignin from cellulose in a PEG / NaOH ABS
[160]. In this system, the cellulose is indeed precipitating in the NaOH phase of the
ABS and lignin remains in the chaotropic PEG phase. Willauer et al. [162] have
established that cellulose demonstrates a clear preference for the NaOH aqueous
phase while the three types of lignin studied (Indulin AT, Indulin C, and Reax 85A)
show a preference for the PEG phase. These authors report that the partitioning of
the three lignins investigated is affected by the free energy of hydration of the salt
forming the ABS. They also stress that both cellulosic samples used (fibrous
[C4mim]Cl phase of biomass-free ABS [C4mim]Cl phase of 5% bagasse loaded ABS NaOH phase of 5% bagasse loaded ABS NaOH phase of biomass-free ABS
Wavenumbers (cm-1
)
121
cellulose and diethylaminoethyl cellulose) are of hydrophilic nature and they do not
dissolve, but rather report to the salt-rich phase of an ABS [162].
Figure 5.1.3: FTIR spectra of each phase of a NaOH / [C4mim]Cl ABS loaded with
15 % soda lignin
The poor phase separation as shown by the resemblance of the FTIR spectra
of the phases in the ABS is an undesirable outcome. Further investigation and use of
alternative ABS compositions (e.g. alternative kosmotropic salts) were employed in
order to improve phase separation. It was considered practical to start this
investigation with “biomass free” ABSs. The coexistence of biomass, ILs, inorganic
salts and their by-products in a biphasic system would render analytical data
complex and difficult to unravel. Therefore the ABSs that seemed prima facie to
have an application in the fractionation of IL-biomass solutions were initially
analysed excluding biomass.
[C4mim]Cl phase of 15 % soda lignin ABS NaOH phase of 15 % soda lignin ABS [C4mim]Cl 20 % soda lignin in [C4mim]Cl
1496 cm-1
Wavenumbers (cm-1
)
122
5.1.1 Choice of kosmotropic salts for aqueous biphasic systems
The stability and purity of each of the two phases in an ABS depend on the
choice of kosmotropic salt for a given IL. The salting-out strength of the
kosmotropic salts follows the well-established Hofmeister series, as observed in
polymer–salt ABSs, and can be directly related to the ions’ Gibbs free energies of
hydration (∆Ghyd) [139, 157]. The strong dependence of ABS formation on ∆.hyd of
each of the inorganic salt ions was also demonstrated by Najdanovic-Visac et al. and
Trinidade et al. [158, 159].
The Hofmeister Series is a classification of ions from kosmotropic to
chaotropic (water structuring and water structure-disrupting respectively). Figure
5.1.4 demonstrates this classification and the properties associated with each ion.
This classification becomes relevant when selecting salts for salting out ILs.
Out of all the kosmotropic salts that are expected to form ABS with
[C4mim]Cl, the following salts were selected for investigation in this study.
• NaOH, since it has been claimed in Edye and Doherty’s patent [3,
134] to form a stable ABS with [C4mim]Cl in presence of biomass.
NaOH has also been reported by Rogers and co-workers [160] to
contain cellulose in PEG / NaOH systems.
• Na2CO3, since it was observed that as biomass loads increase it is
increasingly more difficult to form an ABS in aqueous NaOH.
Consequently, a salt with higher ∆Ghyd was required.
• KOH and K2CO3, for the purpose of comparing method outcomes
with the literature (viz. Bridges et al. [139]).
The ions of these salts are found towards the left side of the Hofmeister
series (Figure 5.1.4) denoting a tendency to form water structuring solutions and a
high ∆Ghyd . The main parameters that affect salting out strength of these salts are
the ∆Ghyd of their ions (presented in Table 5.1.1) and the solubility of the salts in
water (Table 5.1.2).
123
Figure 5.1.4: The Hofmeister series (ions relevant to this study in bold)
(reproduced from Jakubowski [163])
Table 5.1.1: Gibbs free energies of hydration (∆Ghyd) of selected ions
(from Bridges et al. [139])
Cation ∆Ghyd /kcal mol-1
Anion ∆Ghyd /kcal mol-1
H+ -252 OH
- -104
Na+ -89.6 CO3
2- -353
K+ -72.7
Table 5.1.2: Water solubilities of selected inorganic salts
(from Lide [22])
Inorganic salt Solubility (g / 100 g H2O @ 25 oC)
NaOH 100 Na2CO3 30.7
KOH 121 K2CO3 111
NaCl 36.0 KCl 36.0
THE HOFMEISTER SERIES
↓Surface tension Easier to make cavity ↑Solubility in hydrocarbons Salt in (solubilise) Less negative ∆Ghyd Less kosmotropic
NH4+ K+
Na+ Li+ Mg2+ Ca2+ guanidinium+
CATIONS
ANIONS
CO32-
SO42-
Acetate F-
OH-
Cl- NO3
- ClO3
- I
- SCN
-
↑ Surface tension Harder to make cavity ↓Solubility in hydrocarbons Salt out (aggregate) Very negative ∆Ghyd
More kosmotropic
124
5.1.2 Evaluation of ABS stability with coexistence curves
Biphasic systems that maintain phase separation over a wide range of
concentrations of both IL and kosmotropic salts are considered more stable and less
sensitive to concentration changes (e.g. changes of water availability in the system).
Coexistence curves drawn by the cloud point method (described in Section 3.19.2)
are used for evaluating the stability of ABSs formed between [C4mim]Cl and the
selected kosmotropic salts. The curves delineate the concentration threshold
between single and two phase systems (viz. aqueous mineral salt solutions and IL
water mixtures) and determine their sensitivity to concentration changes. The area
under the curve shows concentration ranges for single phase systems. The area
above the curve shows concentrations of salts and the ILs that will lead to formation
of ABS (notwithstanding other considerations, e.g. the solubility of the mineral salt
in water). Accordingly, the larger the area above the curve, the more stable is the
ABS.
The coexistence curves in Figure 5.1.5 represent the author’s cloud point
titrations for the aforementioned selected salts which formed ABSs with [C4mim]Cl.
It is evident (and in agreement with reports of others [139, 158]) that the energy of
hydration of the ions comprising these salts determines, to a large extent, the
distance from the origin of these curves, i.e. as the Gibbs free energy of hydration of
the kosmotropic aqueous solutions becomes more negative, the biphasic systems
become more stable and preferred over a greater molality range
.
125
Figure 5.1.5: Coexistence curves of [C4mim]Cl with selected kosmotropic salts
0
1
2
3
4
5
6
7
8
9
0 1 2 3 4 5 6 7 8 9
NaOH
Na2CO3
KOH
K2CO3
[C4
mim
]Cl
mo
lali
ty
salt molality
B
A
Exponential trend lines have been drawn as visual aids. The horizontal lines (A and B) mark the points where
rapid crystal formation and collapse of the ABS occurred.
Na2CO3
K2CO3
KOH
NaOH
126
The difference in energy of hydration between the anions (OH- and CO3 2-) is
in the order of 150 Kcal mol-1, whereas for the cations (Na+ and K+) it is in the order
of 17 kcal mol-1. This pattern is depicted in the positioning of the corresponding
coexistence curves in Figure 5.1.5. The Na2CO3 and K2CO3 curves have a smaller
single phase region (positioned closer to the origin) with the K2CO3 curve slightly
closer to the origin. The NaOH and KOH curves on the other hand have a
significantly larger single phase region (positioned far from the origin) with the
NaOH curve positioned slightly more towards the origin.
Bridges et al. [139] report coexistence curves for [C4mim]Cl ABSs with K2CO3
and KOH (see Figure 5.1.6). These curves agree with those reported here. However,
Bridges et al. do not report on other possible events, such as salt crystal
precipitations, that can occur when the two phases are competing for water.
Work by Trindade and co-workers [158, 159] has reported the otherwise
overlooked effect of inorganic salt precipitation in ABSs of ILs (viz. [C4mim]Cl,
[C4mim]BF4 and other) with inorganic salts (viz. K3PO4, NaCl, Na2SO4, and Na3PO4). In
these systems, increasing concentrations of IL reduced the solubility of the
kosmotropic salts as compared to their solubility in pure water and led to their
precipitation out of the ABS.
The horizontal lines in Figure 5.1.5 (marked A and B) represent the last cloud
point sample before precipitation of the kosmotropic salt and collapse of the ABS
occurred. In the case of KOH / [C4mim]Cl ABS, the crystal precipitate was harvested,
washed with acetone and analysed with ion chromatography (described in Section
3.19.3). The analysis indicated that the precipitate was quantitatively pure KCl salt.
A metathesis reaction occurs between KOH and [C4mim]Cl to form KCl. Since KCl
has a much lower water solubility than the other salt combinations in the mixture
(see Table 5.1.2), once the solubility is exceeded KCl crystallises and drives the
metathesis reaction. The metathesis reaction is initiated by high concentrations of
[C4mim]Cl, rather than high concentrations of KOH. In the cloud point
determination, KCl precipitates from a cloudy solution (i.e. a solution on the verge
of forming a two phase system). In biphasic systems, where the water solubility of
127
KCl is exceeded, it is likely that, under these conditions, Cl- ions migrate to the KOH
phase easier than K+ ions to the imidazolium phase because the imidazolium cations
strongly partition to the imidazolium phase (as shown in Section 5.1.3 below).
Figure 5.1.6: Phase diagrams of [C4mim]Cl with various salts
(from Bridges et al. [139])
Although the solubilities of NaCl and KCl in water are the same (36 g / 100 g
H2O at 25oC, see Table 5.1.2) a similar metathesis reaction in the NaOH / [C4mim]Cl
system does not occur. This can be explained by comparing the effect of molality on
the activity coefficients of KOH and NaOH (see Figure 5.1.7). Although KOH and
NaOH have the same ionic activity at low concentrations, at high concentrations,
the KOH activity is comparatively much higher. This phenomenon of higher ion
activity of K+ at high concentrations is further accentuated given that the KOH
aqueous solution originally added, in the ABS gets further concentrated by the
migration of water towards the [C4mim]Cl phase (in order to satisfy the IL’s
hydration requirement).
( ) K3PO4, ( ) K2HPO4, ( ) K2CO3, (◊) KOH
Salt molality
[C4
mim
]Cl
mo
lali
ty
128
.
Figure 5.1.7: Activity coefficients of NaOH and KOH at different molarities
(values from Lide [22])
In the Na2CO3 / [C4mim]Cl ABS on the other hand, precipitation of Na2CO3
occurs at high concentrations of Na2CO3. Yet this is not the case in the “counterpart
cation” system with K2CO3 ABS since there is no precipitation observed at any point
during the cloud point titration. This is simply attributed to the fact that the
maximum solubility of K2CO3 (111 g / 100 g H2O) is much higher than that of Na2CO3
(30.7 g / 100 g H2O) (see Table 5.1.2). Aside from salt crystal precipitation, some gas
bubble formation was observed upon mixing of [C4mim]Cl with the Na2CO3
solution. This gas is CO2. Deprotonation of the IL imidazolium ions in presence of
concentrated Na2CO3 aqueous solution (as detected in [C4mim]Cl / Na2CO3 ABSs in
Section 5.1.5 below) produces carbenes and carbonic acid which in turn produces
CO2 and water (hydration equilibrium constant Kh = [H2CO3]/[CO2] = 1.7 * 10-3). The
reaction is further driven towards carbene and CO2 formation by the escape of CO2
gas from the reaction mixture.
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0 1 2 3 4 5
Act
ivit
y c
oe
ffic
ien
t
Molarity
KOH
NaOH
129
Na2CO3 + 2 [C4mim]Cl → 2 NaCl + CO2 + 2 carbene + H2O
That this reaction did not completely consume all reactants may be
attributed to slow carbene formation or to the phase barrier. The consequences of
excessive mixing or contact time on these reactions are not known.
5.1.3 Evaluation of the phase divergence of ABS using distribution ratios
Phase divergence is the difference in concentration between phases of
components in a biphasic system i.e. as phase divergence increases, there is less
miscibility of a solvent and its solutes in the solvent of the other phase and
consequently the ABS is more stable. Distribution ratios are used to quantify the
phase divergence of ABSs. The calculation of these ratios is explained in Equation 7.
The ion concentrations of each ABS phase, required for the equation, were
measured according to Section 3.19.3.
= /0/1
Equation 7
Where: D Ion distribution ratio between phases
cH Ion concentration in phase with higher apparent concentration cL Ion concentration in phase with lower apparent concentration
The distribution ratios of the ions participating in a [C4mim]Cl / NaOH ABS
are compared to the distribution ratios of the ions participating in a [C4mim]Cl /
Na2CO3 and a [C4mim]Cl / KOH ABS (Figure 5.1.8). In agreement with the
coexistence curves in Figure 5.1.5, the distribution ratios express the extent of
mutual exclusion of each phase among the three ABSs. The distribution ratios for all
ions are high for the Na2CO3 system, lower for NaOH and very low for KOH. Na2CO3
forms a stable ABS since it exhibits a large region of two phases at lower
concentrations as shown by the coexistence curves. Even though the molality of the
Na2CO3 ABS in Figure 5.1.8 is significantly lower than the KOH and NaOH ABSs, the
Na2CO3 partitions better than the hydroxides. However, at high concentrations of
Na2CO3, the salt’s solubility is exceeded resulting in its precipitation. Carbon dioxide
production was also observed in the Na2CO3 / [C4mim]Cl ABS, but this reaction
130
required vigorous mixing to occur to any great extent in laboratory experiments
because the [C4mim]+ has strong preference for the top phase. However it might be
difficult to control (and limit) in an industrial setting. The phase divergence of the
KOH ABS, is the lowest out of the three, reflecting the extensive miscibility and low
competition for water between the two phases. Precipitation of KCl crystals is
observed at high concentrations of [C4mim]Cl in the KOH ABS. This is a result of the
migration of Cl- ions into the KOH phase resulting in the formation of KCl whose
solubility in water is much lower than that of KOH.
ABS [C4mim]Cl / Na2CO3 [C4mim]Cl / NaOH [C4mim]Cl / KOH
Salt molality 1.8 5.5 5.5 IL molality 4.3 4.8 4.8
Figure 5.1.8: Distribution ratios of ions in ABSs and their molal composition
Unlike other ions in the ABS, [C4mim]+ is not miscible to any extent in the
aqueous salt phase. The Cl- ion is able to migrate to the aqueous salt phase and the
Na+ ion is able to migrate to the imidazolium phase (which contains a significant
amount of water).
0
50
100
150
200
250
300
350
[C4mim]Cl / Na2CO3 [C4mim]Cl / NaOH [C4mim]Cl / KOH
D (
Dis
trib
uti
on
Ra
tio
)
D Cl-
D Na+
D [C4mim]+
D Cl-
D Na+
D [C4mim]+
/ Na2CO3
131
As shown in the ion migration diagrams in Figure 5.1.9, the salt precipitation
observed in the two aforementioned ABSs is driven by different mechanisms. In the
stable ABS with Na2CO3, the precipitation is driven by competition for water. Water
migrates to the IL phase and thus the Na2CO3 solubility is exceeded. For KOH the
two phases are more miscible and the competition for water is low. The driver for
KCl salt precipitation here is the high migration of Cl- ions to the KOH phase, causing
the formation of KCl which is far less soluble than KOH in water.
Na2CO3
KOH
Figure 5.1.9: Ion migration diagrams based on distribution ratios
Cl- [C4mim]+
K+ [C4mim]Cl phase
KOH phase
KCl precipitation ↓
H2O
Cl- Na+
[C4mim]Cl phase
Na2CO3 phase
[C4mim]+
Na2CO3 precipitation ↓
H2O
CO
2
The thickness of the arrows reflects the relative distribution coefficients of ions - not in scale
132
5.1.4 Effect of biomass loading on distribution ratios of ABSs
The effect of biomass loading on the distribution ratios of aqueous NaOH
ABSs with [C4mim]Cl and [C4mmim]Cl are shown in Figure 5.1.10. The [C4mim]Cl
system was loaded with 5 % bagasse while the [C4mmim]Cl with ca. 1 % only (0.052
g bagasse in 5.200 g [C4mmim]Cl at 150 °C for 60 min, precipitated with 5.2 mL of
200 g L-1 NaOH). Bagasse loading leads to a significant lowering of phase separation
capacity (distribution ratio) for all ions. While the imidazolium ions are different for
the 1 % mass and 5 % mass biomass loadings, the results still show increasing
miscibility (or decreasing ABS phase divergence) as biomass load increases. The
difference in distribution ratios between the dialkyl and trialkyl substituted
imidazolium ions may be related to their tendency to form carbenes.
It was observed that the addition of biomass in model ABSs affected
negatively their distribution ratios (see Equation 7). Thus the enhanced separation
of ABSs with higher biomass loads required the use of ions with higher ∆GHyd and
the availability of more free water to satisfy the hydration requirements of the
lignocellulosic biomass (mainly the polysaccharide fraction since it is highly
hydrophilic).
All the above leads to the conclusion that the initial proposal for lignin to
cellulose separation in an ABS has a real potential, however the distribution of the
lignin is probably reverse to what was claimed in the patent by Edye and Doherty [3,
134]. The results from ABS experiments in combination with previously discussed
literature [162], indicate that ions which rank higher in the Hofmeister series
(higher ∆GHyd), such as the [CO3]-2 ion, could contribute towards improved phase
divergence on biomass-loaded ABSs. However, as demonstrated earlier, K2CO3
would be better than Na2CO3, since Na2CO3 has a low solubility in H2O and can
precipitate and collapse the ABS as water migrates to the IL phase.
133
Figure 5.1.10: The effect of bagasse loading on the ion distribution ratios in ABSs
5.1.5 Chemical instability of imidazolium ILs in alkaline ABSs
An issue of significant concern that emerged while experimenting with ABSs
where the imidazolium-based [C4mim]Cl mixed with alkali salts is the phenomenon
of deprotonation of the imidazolium ring to form carbenes. This reaction is depicted
in Figure 5.1.11, where a base attacks the acidic proton on the imidazolium ring to
form a neutral carbene [164]. Deprotonation is an undesirable phenomenon
because it leads to loss of IL to carbenes or necessitates the use of acid to
reprotonate the carbene and recycle the IL.
CH
N
CHHC
NCH3R
C
N
CHHC
NCH3R
alkali
Imidazolium cation carbene
Figure 5.1.11 : Carbene formation from imidazolium-based ILs
(reproduced from BASF [86])
0
10
20
30
40
50
60
70
80
[C4mim]Cl / NaOH [C4mim]Cl / NaOH+ 5 % bagasse
[C4mmim]Cl /NaOH
[C4mmim]Cl /NaOH + 1 %
bagasse
D (
Dis
trib
uti
on
ra
tio
)
D Cl
D Na
D [C4mim] or[C4mmim]
D Cl-
D Na
+
D [C4mim]+
or [C4mmim]+
134
Since alkaline salts are used in the ABSs investigated in this project it
became necessary to develop a method for determining the extent of imidazolium
IL cation deprotonation in alkaline biphasic systems. A titration method was
devised.
The extent of deprotonation was determined by titration with HCl titrant
and pH monitoring. The resulting inflection points are indicators of the number of
HCl mmol required to reprotonate the same number of carbene mmol in a 1:1
stoichiometry (see Equation 8 and Figure 5.1.12).
�(2��# = 100 ∗ �0� ��0��31
Equation 8
Where: deprot Deprotonation of IL in IL layer (% mol)
mH1 Total HCl mol till 1st inflection point
mH2 Total HCl mol till 2nd
inflection point mIL Total IL mol originally added in titration solution
Figure 5.1.12: HCl titration of the IL phase of a [C4mim]Cl / NaOH ABS
-100
-90
-80
-70
-60
-50
-40
-30
-20
-10
0
0
2
4
6
8
10
12
14
0 0.5 1 1.5 2
∆p
H Δ
mm
ol -1
(pH
HC
l mm
ol -1)
pH
HCl (mmol)
pH
∆ pH
Inflection point 2: reprotonation of
carbenes
Inflection point 1: neutralization of base
(OH-)
pH
∆pH Δmmol-1
135
The presence of two inflection points in the acid titration of the IL phase of
the [C4mim]Cl / NaOH ABS indicates that there are two species reacted. After
titration of a neat NaOH aqueous solution, the first inflection point was attributed
to the neutralisation of OH- ions present in the top phase and the second to the
reprotonation of carbenes back to [C4mim]+ ions. Imidazolium deprotonation for
the selected ABSs was measured by this method and listed in Table 5.1.3.
Table 5.1.3: Deprotonation of imidazolium IL in top phase of ABSs
Salt % imidazolium moles
reprotonated by HCl
[C4mim]Cl / NaOH 8
[C4mim]Cl / KOH 8
[C4mim]Cl / Na2CO3 7
[C4mmim]Cl / NaOH 5
Deprotonation of [C4mim]Cl is significant and does not seem to vary greatly
with salt used. Surprisingly, Na2CO3 is inducing almost as much deprotonation of the
imidazolium ring as NaOH. Given that Na2CO3 is less alkaline than NaOH, this
deprotonation was attributed to the loss of CO2 as described in section 5.1.2. The
deprotonation extents measured also indicate that the imidazolium ions in the top
phase are not quantitatively deprotonated.
One way to reduce deprotonation was thought to be the substitution of the
acidic proton of the [C4mim]Cl imidazolium ring with a methyl group as found in the
ionic liquid 1-butyl-2,3-dimethylimidazolium chloride or [C4mmim]Cl. Surprisingly,
the HCl titration of the IL phase of the trisubstituted [C4mimm]Cl / NaOH ABS also
exhibited a second inflection point. This indicates that the imidazolium ion still
reacted with HCl (5 % mol). Whether this means that the alkaline solution
demethylated the imidazolium ion in the C-2 position creating a nucleophilic site or
that some transalkylation resulted in a similar effect is not deducible from this data.
136
5.1.6 Summary
ABSs present an opportunity for the clean fractionation of lignocellulosics.
Salts with high ∆GHyd form [C4mim]Cl ABSs with high degrees of phase divergence.
However issues such as deprotonation of [C4mim]Cl in alkaline solutions and the
fact that higher biomass loads lead to phase convergence may limit the utility of IL
ABS in biomass processing. In an industrial setting, these issues represent less clean
fractionation but also difficulty in recycling the IL. Deprotonation can lead to IL mass
losses while with increasing miscibility of the two phases, the IL is present in both
phases and therefore the cost and energy needed to recover the IL are higher. Due
to these technical impediments, experiments with ABSs were not extended to
monitoring the enzyme kinetics of the resulting bagasse solids. Single phase
separation systems formed the basis of the rest of the experimentation on
fractionation systems (viz. section 5.2).
Other useful observations attained from this section include the mechanism
of chloride salt precipitation in inorganic salt / [C4mim]Cl ABSs affecting phase
separation (viz. metathesis reactions taking place between the IL anion and the
cation of the kosmotropic salt in ABSs with K2CO3). As far as the separation of lignin
and cellulose is concerned, the hypothesis of separation is still valid with the only
difference that the partitioning of the lignin in each phase of the ABS seems to be
opposite to what was originally claimed. Based on the data produced in this project
so far, identification of a “preferred composition ABS” should be possible.
5.2 Aqueous single phase fractionation systems
Edye and Doherty’s [3, 134] choice of NaOH was based on the belief that
since lignin is soluble in aqueous NaOH, it would remain in solution in a single
aqueous phase [161]. Cloud point plots show that there are aqueous conditions
wherein NaOH and [C4mim]Cl will coexist as a single phase. Therefore it seemed
that the use of aqueous NaOH as an antisolvent but at concentrations producing a
single phase would be worthy of consideration.
137
The lignin solvents that are also polysaccharide precipitation agents were
dilute NaOH and aqueous acetone and are compared with water. The
deprotonation of the imidazolium IL by dilute NaOH (0.2 M) was tested on biomass-
free systems (7.8 g of 0.2 M NaOH mixed with 7.5 g of [C4mim]Cl for 5 min) prior to
experimentation, using the HCl reprotonation titration method (titrant 0.05 M HCl)
used in Section 5.1.5. The resulting titration curve indicated only 0.3 % mol
deprotonation. Acetone in water (1:1 volume basis) is miscible with [C4mim]Cl and
it has been used by Sun et al. [101] to dissolve lignin and precipitate
polysaccharides from a pine wood / [C2mim]Cl dissolution system.
Three partial dissolution reactions were carried out at identical conditions (1
g of bagasse in 20 g of [C4mim]Cl at 150 °C for 90 min in the setup described in
Section 3.6.1) and were precipitated with 20 mL of the three selected antisolvents.
The total recovered solids (TRS) were characterised and compared to each other in
Table 5.2.1.
Table 5.2.1: Compositional analysis of total recovered solids (TRS) from partial
bagasse dissolution in [C4mim]Cl using different antisolvents
% dry mass
Dry mass (%) Ash AIL ASL Total lignin
Glucan Xylan Arabinan Acetyl
Untreated 3.4 21.2 5.0 26.2 41 22.7 1.84 2.48
Total recovered solids
(TRS)
Dilute NaOH
(0.2 M) 3.1 18.5 6.0 24.4 44 20.4 0.60 n/d
Acetone in water
(1:1 v/v) 2.4 18.5 5.4 23.9 39 19.9 0.55 n/d
Water 3.1 20.0 6.2 26.2 44 20.3 0.54 n/d
Compositionally there is no apparent significant difference between the
solids recovered (TRS) with the different antisolvents.
Enzyme saccharification of these solids is shown in Figure 5.2.1. No
differences in initial saccharification rate and little difference in saccharification
138
extent were imparted by the antisolvents used. Shown differences in extent may be
due to differences in hemicellulose bonding to lignin.
Although the rate and extent of saccharification of water-precipitated
biomass in Figure 5.2.1 appear to be different to previous experiments with the
same pretreatment conditions (cf. Figure 4.1.11), the conclusions from the results in
Figure 5.2.1 are still reliable as the data is internally consistent. The reason for this
discrepancy could be a fault in the heating element of the oil bath resulting in large
temperature fluctuations or insufficient heating.
Figure 5.2.1 : Enzyme saccharification of total recovered solids (TRS) from partial
bagasse dissolution in [C4mim]Cl using different antisolvents
0
10
20
30
40
50
60
70
80
90
0 10 20 30 40 50
glu
can
in
pre
tre
ate
d s
oli
ds
(% m
ass
)
Time (h)
dilute NaOH
water
Acetone : water
Untreated
dilute NaOH
water
acetone: water
untreated
139
In order to more thoroughly examine the fractionation efficiency of these
antisolvents they were also tested on bagasse completely solubilised in [C4mim]Cl.
The dissolved solids only (DS) from a reaction of bagasse (7.5 g) in [C4mim]Cl (150 g,
150 °C, 3 h) were isolated with DMSO (150 mL) and filtration, split in three equal
mass portions and each portion precipitated with one of the selected antisolvents
(ca. 100 mL). The composition of the precipitated solids from each solvent is
presented in Table 5.2.2.
Table 5.2.2 : Compositional analysis of completely dissolved bagasse (DS)
precipitated from [C4mim]Cl using different antisolvents
% dry mass
Dry mass (%) Ash AIL ASL Total lignin
Glucan Xylan Arabinan Acetyl
Untreated 3.4 21.2 5.0 26.2 41 22.7 1.84 2.48
Dissolved fraction only
(DS)
Dilute NaOH
(0.2 M) 2.3 8.86 2.5 11.4 90 0.00 0.03 1.27
Acetone in water
(1:1 v/v) 1.0 10.7 2.9 13.6 74 4.30 0.14 2.06
Water 2.2 15.9 3.3 19.2 71 5.13 0.17 2.22
Lignin is reduced to 11.4 % by dilute NaOH and to 13.6 % by acetone in
water compared to the 19.2 % present in the water precipitated solids. Both dilute
NaOH and acetone in water exhibit an ability to delignify the precipitated solids
from the IL solution. The main difference between the two delignifying antisolvents
is that dilute NaOH removes the hemicellulose whereas the acetone in water
doesn’t. The results obtained for the reduction of lignin of [C4mim]Cl treated
bagasse when applying precipitation with acetone in water (from 26.2 % to 13.6 %)
are in agreement with the literature. Rogers and co-workers [101] have used the
same acetone in water mixture to precipitate oak dissolved (98.5 % mass dissolved)
in [C2mim]OAc and they reported a reduction of lignin content from 23.8 % mass in
the untreated oak to 15.5 % in treated oak. However, it must be noted that unlike
Rogers and co-workers, DMSO dilution was used in this work and this may cause
140
bias in fractionation results since DMSO / water / IL mixtures may precipitate
different biomass fractions than water / IL mixtures.
The saccharification time profile of the dissolved reprecipitated fractions is
shown in Figure 5.2.2. Note that the saccharification in this figure is expressed as %
max measured saccharification, where the extent of saccharification of the acetone
in water precipitated material at 48 h was assigned a value of 100 %. This was
necessary since it became apparent that in this experiment the moisture
determination had unacceptably high errors. However, since it is known from
previous experiments that completely dissolved and reprecipitated cellulose is
quantitatively converted to monosaccharides, the % maximum saccharification is
expected to be near equivalent to % actual saccharification. Again the differences in
saccharification profiles between antisolvents are small.
Figure 5.2.2 Enzyme saccharification of completely dissolved bagasse (DS)
precipitated from [C4mim]Cl using different antisolvents
0
10
20
30
40
50
60
70
80
90
100
0 10 20 30 40 50
glu
can
(%
ma
ss o
f m
ax
imu
m m
ea
sure
me
nt)
Time (h)
Acetone : water
dilute NaOH
water
Untreated
acetone:water
dilute NaOH
water
untreated
141
5.2.1 Summary
In all cases some lignin remained solubilised after the addition of
antisolvent. However the delignifying antisolvents (dilute NaOH and acetone in
water mixtures) removed more lignin than water. The lignin content of dilute NaOH
precipitated solids was 40 % mass lower than the water precipitated ones and the
solids precipitated with acetone in water mixtures contained 29 % mass less lignin
than water precipitated ones. Dilute NaOH delignification was accompanied with
xylan removal which was not the case with acetone in water. The delignification
efficiency was only discernible when the dissolved fraction was separated from the
undissolved material and precipitated. This indicates that the use of delignifying
solvents is only justifiable if enough bagasse has dissolved upon IL pretreatment.
Finally the antisolvents used here made little difference in the saccharification of
the recovered solids. Consequently water may be the antisolvent of preference for
these systems and indeed it is a less costly choice.
5.3 Preferential precipitation by incremental additions of water
Following the indication that water is the antisolvent of preference, the
potential of incremental additions of water to preferentially precipitate LCB
components in IL solution was investigated. Experimentation started with
determination of the pH of aqueous IL solutions of different mass ratios.
IL (5 g) was placed in a test tube and incremental additions of water were
followed by thorough agitation and a pH measurement. The resulting pH
measurements as a function of water : IL mass ratio are plotted in Figure 5.3.1 for
[C4mim]Cl and [C2mim]OAc. Aqueous solutions of [C2mim]OAc are alkaline and pH
decreases markedly with increased water : IL mass ratio while water / [C4mim]Cl
mixtures are slightly acidic and close to neutral and show a slight increase in pH.
Lignin is soluble in alkali, therefore it seemed possible to partially and fractionally
precipitate biomass dissolved in [C2mim]OAc by using small amounts of water to
precipitate cellulose while maintaining a high pH and thus keeping lignin in solution.
142
Addition of more water to lower the pH of the water / IL mixture might then cause
lignin precipitation.
As an elementary test for the validity of this hypothesis, bagasse soda lignin
and Avicel cellulose were dissolved in ILs (ca. 100 mg in 5 g of IL, 150 °C, 30 min) and
precipitated with incremental amounts of water. Solutions were centrifuged after
each water addition (10000 x g) and the water mass at first observed precipitation
was noted. The water mass where no more significant precipitate accumulated on
the centrifuge pellet was also noted. The results of these observations for lignin and
cellulose in each of the ILs are presented in Figure 5.3.2 and suggest that cellulose
and lignin may indeed precipitate at different water : IL ratios.
Figure 5.3.1: pH of [C2mim]OAc and [C4mim]Cl aqueous solutions at different
water : IL mass ratios
5
6
7
8
9
10
11
12
0 1 2 3 4
pH
water : IL mass ratio
[C2mim]OAc
[C4mim]Cl
143
Figure 5.3.2: Lignin and cellulose precipitation observed at different water : IL
mass ratios of [C2mim]OAc and [C4mim]Cl aqueous solutions
That this seems to hold true for both Ionic liquids was unexpected. Water /
[C4mim]Cl mixtures were not expected to keep lignin in solution since the pH was
not alkaline. Observations of precipitate pellet volume changes (in Figure 5.3.2)
suggest that for both water / IL mixtures, cellulose precipitated suddenly and
almost completely while the precipitation of lignin was more gradual. The likely
explanation for these observations is that cellulose is more homogeneous and less
complex than lignin. Cellulose precipitates completely before a ratio of 0.5 is
reached while most lignin precipitates between 0.5 and 1.5 in both ILs. This
outcome suggests that the preferential cellulose precipitation should be achievable
in both ILs by using 0.5 water : IL mass ratio. Moreover, increasing this ratio to 2.0
should allow for precipitation of the lignin remaining in solution for the [C2mim]OAc
system. More water and possibly some pH lowering with acid may be necessary for
recovering the lignin remaining in the water / [C4mim]Cl solution. It is surprising
that [C4mim]Cl requires more water than [C2mim]OAc to precipitate dissolved
lignin. The lower pH of [C4mim]Cl aqueous solutions does not seem to aid
precipitation. Incremental additions of water appear to have potential for fractional
precipitation of IL dissolved biomass but pH doesn’t appear to be the factor
determining lignin precipitation as originally thought. It is of note that for
[C2mim]OAc and, at least for the materials used in this experiment, it seems that
the last observable precipitation of cellulose may slightly overlap with the first
0 0.5 1 1.5 2
lignin in [C2mim]OAc
lignin in [C4mim]Cl
cellulose in [C2mim]OAc
cellulose in [C4mim]Cl
water:IL mass ratio
first precipitation
apparent completeprecipitation
144
observable precipitation of lignin. Finally it must be noted that this analysis is only
to be used as a gross indication since it is based on visual assessment of
precipitates.
5.4 Comparison of three IL pretreatment and fractionation systems
The potential of using incremental amounts of water to fractionally
precipitate IL dissolved bagasse was tested on three ILs and the mass balances of
each reaction determined (as described in Section 3.21). The schematic
representation of the fractional precipitation process designed to yield a
polysaccharide-rich and a lignin-rich fraction using two incremental additions of
water (and acidification to pH < 1.0 and a third addition of water to precipitate
remaining dissolved material) is shown in Figure 5.4.1. Preliminary studies of
fractional precipitation with water (where Avicel and soda lignin were used)
indicated that lignin precipitation was complete at a water : IL mass ratio of 2.0.
However native bagasse lignin dissolved in IL is likely to have different properties to
lignin extracted with aqueous NaOH and then dissolved in IL. Maximum lignin
recovery was ensured by acidification to a pH < 1.0 and the addition of a further 1.5
IL mass equivalents of water.
Bagasse pretreatments with [C4mim]Cl, [C2mim]Cl and [C2mim]OAc under
identical reaction conditions (35 min at 150 °C, 5 % bagasse in IL (2.5 % for
[C2mim]OAc), as described in Section 3.21) imparted partial dissolution and the
polysaccharide rich solid fractions (solid fraction 1 or SF1 in Figure 5.4.1) were
recovered using water addition (water : IL mass ratio of 0.5) as shown in Figure
5.4.1. The SF1 solids were washed and freeze-dried prior to analysis and enzyme
saccharification. Bagasse was extracted with water and ethanol prior to treatment
since better mass balance closures are obtained by removing non-structural
molecules which can interfere with characterisation of solid and liquid fractions.
145
Figure 5.4.1: Process flow chart of a fractional precipitation separation of IL
treated bagasse using incremental additions of water
biomass dissolution
precipitation in water : IL mass ratio = 0.5
LIQUID FRACTION 1 (LF1)
precipitation in water : IL mass
ratio = 2
LIQUID FRACTION 2 (LF2)
Precipitation in water : IL mass ratio = 3.5
+ acidification to pH <1.0
LIQUID FRACTION 3 (LF3)
SOLID FRACTION 3 (SF3) lignin rich
SOLID FRACTION 2 (SF2) lignin rich
SOLID FRACTION 1 (SF1)
polysaccharide rich
146
Table 5.4.1: Compositional analysis of SF1 solids from pretreatment of ethanol-extracted bagasse with three different ILs.
% dry mass ratios
Sample Mass
recovery Ash
±0.35 AIL
±0.37 ASL
±0.07 Total lignin
Glucan ±0.21
Xylan ±0.47
Arabinan ±0.08
Acetyl ±0.05
Arabinan / Xylan
Acetyl/ Xylan
Untreated
(extracted) 100 3.1 20.8 5.4 26.2 45 22.2 1.50 3.11 0.07 0.14
[C4mim]Cl 90 3.5 20.8 5.4 26.1 48 20.5 1.06 2.96 0.05 0.14 [C2mim]Cl 48 6.1 25.0 3.8 28.7 53 11.0 0.60 1.75 0.05 0.16
[C2mim]OAc 66 5.7 9.9 6.0 15.8 68 13.3 1.58 1.42 0.12 0.11
147
5.4.1 Compositional analysis
The composition of SF1 fractions from each IL pretreatment are shown in
Table 5.4.1. The dissolution extents of these reactions (at 150 °C for 35 min with 25
min temperature ramp) were not measured. However the extents of biomass
dissolution and degradation to non-recoverable material both increase with
temperature and reaction time and have been quantified. Consequently for these
experiments, the extent of dissolution can be estimated by the material balance
closures (i.e. the extent of degradation). For the [C4mim]Cl dissolution a consistent
ratio of losses to dissolution extent of 1:3 was observed (see section 4.1.2.c). This
ratio was found to be 2:5 for extracted bagasse (see Appendix II). The losses
incurred for [C4mim]Cl are ca. 10 % mass (Table 5.4.1), thus the dissolution extent
in these experiments is estimated to be around 23 % mass. The other two ILs are
known to effect different ratios of dissolution extents to losses (viz. results in
section 4.1.2.f), and the losses incurred in these ILs (35 % - 50 %) suggest that the
dissolutions are close to complete. These estimates were confirmed visually; it was
observed that [C4mim]Cl contained a very large amount of undissolved fibre,
[C2mim]OAc only a small amount and [C2mim]Cl almost none. Comparatively, Lee
et al. [68] have reported 27 % losses of maple wood flour after treatment with
[C2mim]OAc (130 °C for 90 min, 5 % biomass loading in IL).
The water : IL mass ratio used for partial precipitation of dissolved solids was
0.5. According to the observations in section 5.3, this water amount should
precipitate all cellulose and keep lignin in solution for water / [C4mim]Cl mixtures
and possibly also for water / [C2mim]OAc mixtures. Water / [C2mim]Cl solutions are
assumed to behave in a similar manner to water / [C4mim]Cl solutions.
148
Figure 5.4.2: FTIR spectra of bagasse treated with different ILs
(absorbance – common scale).
POLYSACCHARIDES
LIGNIN
Wavenumbers (cm-1)
[C2mim]Cl [C2mim]OAc [C4mim]Cl Untreated
149
For [C4mim]Cl-treated solids, the compositional changes are small with a
slight cellulose enrichment. This is primarily because only ca. 24 % of the LCB
dissolved. For [C2mim]Cl the SF1 is rich in cellulose and lignin and low in
hemicellulose saccharides. Given the fact that [C2mim]Cl dissolves biomass and
produces water soluble losses faster than [C4mim]Cl, it is not surprising that the
[C2mim]Cl solids are low in hemicellulose components. The [C2mim]OAc SF1 is rich
in cellulose and low in lignin, xylan and acetyl. Arabinose (and presumably xylose
linked to arabinosyl units) appears to survive. The [C2mim]Cl SF1 is also rich in
cellulose but also rich in lignin. Hemicellulose appears to have been substantially
removed by [C2mim]Cl treatment.
It is tempting to speculate that the acetate IL and chloride ILs cleave
different bonds. It appears that the acetate IL effects preferential delignification
and deacetylation whereas the chloride ILs effect preferential removal of arabinose
and xylan. In terms of solids composition the acetate IL appears to effect a similar
outcome to aqueous alkali treatment while the chloride ILs produce a similar
outcome to dilute acid treatment. This is a key finding. Note that the aqueous acid
and alkali treatments involve biomass dissolution and decomposition while IL
treatments involve dissolution decomposition and precipitation. Thus, the chemical
processes involved are different, and it is the gross compositional changes that
these processes effect on bagasse that are similar. The acetate may be expected to
be basic and be more reactive than chloride because acetate is a stronger base than
chloride (acetic acid pKa = 4.75 cf. HCl pKa = -7.0) and a nucleophile but these
chemical behaviours do not necessarily hold true for non-aqueous IL solutions.
5.4.2 Structural analysis by ATR-FTIR
The SF1 from each IL was also analysed using ATR-FTIR and the spectra are
shown in Figure 5.4.2. In general, these spectra reflect the compositional
characteristics discussed above. For example it is clearly discernible that the band
absorbances which are characteristic of lignin are relatively low in the spectrum of
SF1 treated with [C2mim]OAc when compared to other spectra.
150
The infrared spectra in Figure 5.4.2 were mainly used to estimate
crystallinity of each SF1. Cellulose crystallinity was estimated from absorbance band
ratios in these FTIR spectra. Infrared radiation is absorbed by molecules and causes
vibrational motion; this vibration in the cellulose molecule is influenced by inter-
and intra-molecular interactions, particularly hydrogen bonding. The molecules in
the cellulose polymer chain will vibrate differently in well-ordered crystalline phases
to less ordered amorphous phases, hence it is possible to assign absorption bands
related to crystalline and amorphous regions. In cotton, The absorbance at 1429 cm-
1 is viewed as typical of crystalline regions of cellulose and the absorption band at
893 cm-1 typical of amorphous regions; the ratio of these two bands represents a
crystallinity index (CrI) also known as lateral order index [156]. This rapid method of
determining cellulose crystallinity is less accurate than the XRD method used above.
However, it is used here due to time restrictions to determine crystallinity of
cellulose in bagasse. The corresponding absorption bands are slightly different for
bagasse (viz. 1421 cm-1 and 895 cm-1) than in cotton, due to the different molecular
interactions and morphology of the lignocellulosic matrix.
In Table 5.4.2, the ratios of the absorbance intensity of 1421 cm-1 to 895 cm-
1 at are shown as an index of cellulose crystallinity for each IL treated material.
Table 5.4.2: FTIR crystallinity indices of IL-pretreated solids
IL CrI (FTIR)
[C2mim]OAc (SF1) 0.19
[C4mim]Cl (SF1) 0.21
[C2mim]Cl (SF1) 0.37
Untreated 0.88
The estimate of the standard deviation (absolute) for this crystallinity index-
measurement is 0.02 (based on duplicate IL pretreatments, 3 df). These indices
combined with the observed shift of the 1034 cm-1 band to lower wavenumber,
represent a significant loss of crystallinity of cellulose after treatment in all three
151
ILs. Note that despite only ca. 24 % dissolution of the LCB in [C4mim]Cl the SF1 CrI is
significantly lower than starting bagasse.
In a separate experiment, extracted bagasse (0.350 g) was reacted in
[C4mim]Cl, [C2mim]Cl, [C2mim]OAc (7 g, 150 °C for 45 min). The reaction mass was
then diluted with DMSO (7 mL) and the UND and DS fractions were recovered as
described in section 3.6.2 and freeze-dried as described in Section 3.21.1. Infrared
spectra of these solids were acquired and shown in Figure 5.4.3. Some additional
information regarding preferential dissolution of components can be drawn from
these spectra. For [C4mim]Cl there seems to be no infrared evidence of preferential
dissolution since the DS and UND fractions resemble each other. For [C2mim]Cl the
DS fraction is enriched in lignin and resembles the spectrum of bagasse soda lignin.
The 1117 cm-1 band corresponding to the C-O stretching vibration of guaiacyl lignin
is particularly intense in this DS fraction. The strong lignin absorbances are possibly
a result of degradation of carbohydrate fraction leaving mostly lignin in the water
insoluble solids.
The most pronounced features in the infrared spectrum of the [C2mim]OAc
treated DS fraction are intense absorption bands characteristic of acetyl groups and
other carbonyl or ester bonds in biomass (viz. 1730 cm-1 and 1236 cm-1) in addition
to bands that are characteristic of aliphatic esters but not usually seen in biomass
spectra (viz. 1568 cm-1, 1400 cm-1). These absorbances could be indicative of
acetylation of the dissolved solids which may entail the participation of the
[C2mim]OAc anion. In fact, Kohler et al. [103] observed acetylation of cellulose
dissolved in [C2mim]OAc in the presence of acylating agents (e.g. 2-furoyl chloride).
In this case acetates formed rather than the furoyl derivatives via a furan-2-
carboxylic acetic anhydride intermediate (an acetylating agent). In addition
imidazolium ionic liquids have been shown to promote acetylation of carbohydrates
with anhydrides and acid chlorides [109]. However there are no acetylating agents
present in the bagasse [C2mim]OAc solutions (although acetic anhydride can form
from acetic acid by dehydration at very high temperatures) and compositional
analyses (by acid digestion) reported in this thesis do not support acetylation.
152
Figure 5.4.3: FTIR spectra of DS and UND bagasse treated with different ILs
(absorbance – common scale)
Untreated (extracted) Undissolved (UND) Dissolved precipitated (DS) Bagasse soda lignin
POLYSACCHARIDES
LIGNIN
[C4mim]Cl
[C2mim]Cl
[C2mim]OAc
Wavenumbers (cm-1
)
Wavenumbers (cm-1
)
Wavenumbers (cm-1
)
153
5.4.3 Enzyme saccharification
The progress of saccharification resulting from each IL treatment for
cellulose and hemicellulose (as xylan) was monitored according to Section 3.21.6
and is plotted in Figure 5.4.4 and Figure 5.4.5 respectively. Initial rates of cellulose
saccharification are very fast for all IL treatments, and this is the effect of cellulose
decrystallisation which is common to all three treatments. The extent of
saccharification is higher for the [C2mim]+ ILs than for the [C4mim]+ IL and of the
[C2mim]+ salts, the chloride anion gives a higher saccharification yield. Given that
[C2mim]OAc delignifies as well as decrystallises bagasse, it is surprising that
[C2mim]Cl yielded a higher final saccharification. This is possibly due to the different
lignin-hemicellulose bonds that survive in the solids after these two pretreatments.
The fast initial saccharification rates resulting from [C4mim]Cl treatment reflect the
presence of structural changes while the low final saccharification yields are a
consequence of a lower extent of dissolution and indicate the absence of
compositional and covalent bonding perturbation in the undissolved solids.
Figure 5.4.4: Glucan saccharification of extracted bagasse treated with 3 ILs
0
10
20
30
40
50
60
70
80
90
100
0 20 40 60 80 100
glu
can
in
pre
tre
ate
d s
oli
ds
(% m
ass
)
Time (h)
[C2mim]Cl
[C2mim]OAc
[C4mim]Cl
Untreated(extracted)
154
Figure 5.4.5: Xylan saccharification of extracted bagasse treated with 3 ILs
Interestingly, the hemicellulose saccharification of [C2mim]OAc treated
bagasse proceeds faster and closer to completion than for the other two ILs. This
shows that the delignification achieved by [C2mim]OAc results in improved
hemicellulose saccharification results, while for the chloride IL treated solids the
hemicellulose is not as accessible to enzymes due to the persistence of lignin. It is
likely that covalent linkages between lignin and hemicelluloses survive chloride IL
treatment and limit the extent of saccharification of hemicelluloses.
In the last two years there have been a number of reported studies of
biomass pretreatment using ILs. Table 5.4.3 provides a summary of the impact of a
range of ILs and treatment conditions on enzyme saccharification and
delignification of treated LCBs.
0
10
20
30
40
50
60
0 20 40 60 80 100
xy
lan
in
pre
tre
ate
d s
oli
ds
(% m
ass
)
Time (h)
[C2mim]OAc
[C4mim]Cl
[C2mim]Cl
Untreated(extracted)
155
Table 5.4.3: Mass recovery, delignification and enzyme saccharification resulting from treatment with different ILs
Entry Ionic liquid Raw material Recovery
(% mass)
Delignification
(% mass)
24 h enzyme saccharification
(% mass cellulose*)
Conditions Ref
1 [C4mim]Cl Sugarcane bagasse
(0.25 - 0.5 mm, 5 % load)
n/d 0.0 93 (3 h) 150 °C, 1.5 h
15 FPU
THIS WORK
2 [C4mim]Cl Sugarcane bagasse
(extracted, 0.25 - 0.5 mm,
5 % load)
90 0.0 56 150 °C, 0.5 h
15 FPU
Idem
3 [C2mim]Cl Idem 48 0.0 93 Idem Idem
4 [C2mim]OAc Idem 66 40.0 83 Idem Idem
5 [C2mim]OAc Wheat straw (<500 mm, 5 % load)
51.2 52.7 100 (11h) 150 °C, 1.5 h 35 U/L
Fu et al. [143]
6 [C4mim]Cl Idem 76.9 15.3 64.8 (11h) 90 °C, 24 h, 35 U/mL Idem 7 [C2mim]OAc Idem 66.0 30.3 97.6 (11h) Idem Idem 8 [C4mim]Cl Corn stover (5 % load) n/d n/d 85 150 °C, 1 h, 60 FPU Varanasi et al. [119]
9 [C2mim]OAc Idem n/d n/d 92 120 °C, 1 h, 60 FPU Idem
10 [C4mim]Cl Wheat straw (<500 mm, 4 % load)
n/d n/d 44 100 °C, 1 h 30 FPU
Li et al. [88]
11 [C2mim]OAc Idem n/d n/d 40 Idem Idem 12 [C2mim]diethy
lphosphate Idem n/d n/d 54 Idem Idem
13 Idem Idem n/d n/d 54 130 °C, 1 h, 30 FPU Idem
14 [C2mim]OAc Maple wood flour (< 0.250 mm, 5 % load)
73 63 95 130 °C, 1.5 h, 34 U/mL
Lee et al. [68]
15 Idem Southern yellow pine (< 0.125 mm, 5 % load)
59 of polysacch. 69 % of lignin
26 n/d Sun et al. [101]
16 Idem Switchgrass (<0.420 mm, 3 % load)
49 69 96 160 °C, 3 h, 5mg protein/g Li et al. [76]
17 [Allylmim]Cl Southern pine thermo-mechanical pulp (TMP)
n/d n/d 54 40 FPU Kilpellainen et al. [17]
* cellulose = cellulose recovered after pretreatment
156
Reviewing the recent studies in IL biomass pretreatment (Table 5.4.3), leads
to some interesting observations. While most of the earliest reports of biomass IL
pretreatments were at temperatures below 110 °C, many of these later studies are
at higher temperatures (and generally it is only at these higher temperatures that
high saccharification yields are achieved). [C2mim]OAc appears to impart faster
saccharification than [C4mim]Cl under all conditions and for all substrates.
Significant delignification with [C2mim]OAc is reported in all studies whereas no
delignification was observed in [C4mim]Cl treated bagasse in this study (i.e. own
data). Li et al. [88] demonstrated that [C2mim]diethylphosphate imparts faster
saccharification than most commonly used ILs at low temperatures (100 °C). This is
attributed to the low viscosity of this IL and it is argued that low viscosity ILs
dissolve more biomass at low temperatures [87, 88]. Indeed for
[C2mim]diethylphosphate pretreatment at higher temperatures (130 °C) there is no
improvement to enzymatic digestion of the cellulosic product (The cellulose
conversion at 24 h was 54 % at both 100 °C and 130 °C) [88]. The more viscous
[C2mim]OAc and [C4mim]Cl however, approach 100 % digestion at elevated
temperatures.
5.4.4 Precipitation of solid fraction 2 and 3
The second addition of water to the liquid fractions of the three IL
pretreatments to a water : IL mass ratio of 2.0 yielded lignin rich solid fractions (SF2)
which were separated from the liquid by centrifugation, washed in water, freeze-
dried and weighed. Acidification of the liquid fractions and a third water addition
yielded some additional precipitate (SF3). Acidification of the [C2mim]OAc aqueous
solution required approximately 70-fold more H2SO4 than the equivalent chloride IL
solutions to reduce pH to ≤ 1.0. This is not surprising since a buffer is formed where
acetic acid is the weak acid and acetate is the conjugate base. SF3 precipitates were
recovered by centrifugation, washed, freeze-dried, weighed. For all SF2 and SF3
samples, lignin content was measured by the acetyl bromide method (described in
Section 3.21.4) and FTIR spectra were obtained. SF2 and SF3 lignin contents and
recovered masses are reported in Table 5.4.4. The weight of all liquid fraction
precipitates and their lignin content was used to determine the total amount of
157
lignin recoverable from the liquid fraction of each IL pretreatment. The lignin
recovery from the liquid fraction of all three IL pretreatments was low (0.3 % to
11.7 % mass of starting lignin). This low lignin recovery is a striking result and it
raises the question of where the rest of the lignin goes. This will be answered in the
next Section (5.4.5), where the full mass balance results are discussed.
Table 5.4.4: Mass recovery and lignin content of solids recovered from the liquid
fraction after treatment with three ILs
recovered
mass (mg)
lignin content
(% mass)
lignin recovery
(mg)
lignin recovery
(% starting mass
of lignin)
pH
[C4mim]Cl SF2 1.2 31.1 0.4 0.3 6.1
[C4mim]Cl SF3 0.3 n/d n/d n/d 1.0
[C4mim]Cl TOTAL 1.5 0.4 0.3
[C2mim]Cl SF2 10.8 30.0 3.2 1.5 3.6
[C2mim]Cl SF3 0.3 n/d n/d n/d 0.3
[C2mim]Cl TOTAL 11.1 3.2 1.5
[C2mim]OAc SF2 58.2 23.0 13.4 9.9 7.0
[C2mim]OAc SF3 12.2 25.9 3.2 1.7 1.0
[C2mim])OAc
TOTAL
70.4 16.6 11.7
158
Figure 5.4.6: FTIR spectra of precipitate recovered after precipitation in 3.5 water : IL mass ratio (acidified to pH < 1) in three ILs
(absorbance – common scale)
Precipitate from [C2mim]Cl liquid fraction Precipitate from [C4mim]Cl liquid fraction Precipitate from [C2mim]OAc liquid fraction Untreated extracted bagasse
Wavenumbers (cm-1
)
159
The ATR-FTIR spectra for the solids precipitated from each IL pretreatment
from the 3.5 mass ratio water addition (SF3) are shown in Figure 5.4.6. Spectra of
SF2 precipitates were also obtained but were not different to those of the SF 3
precipitates. These spectra have intense absorbances at the hemicellulose
characteristic bands between 1175 cm-1 and 1000 cm -1. This indicates that the
majority of the non-lignin component of these fractions (ca. 70 % mass) is
comprised of hemicellulose. This is in agreement with previous discussion
demonstrating the preservation of lignin-hemicellulose bonding upon biomass
dissolution in ILs. The [C2mim]OAc precipitate in particular has a strong
characteristic band at 974 cm-1 indicative of arabinosyl groups. This again is in
agreement with previous discussion supporting preservation of covalent bonds to
arabinosyl moieties during [C2mim]OAc dissolution whilst they are labile in chloride
IL dissolutions.
In the preliminary study of fractional precipitation, soda lignin and Avicel
cellulose were used. Thus the interferences of hemicelluloses (and hemicelluloses-
lignin covalent bonds) were not taken into account. It is evident that a large amount
of hemicelluloses precipitates at the same water : IL ratio as lignin, most likely due
to lignin – hemicellulose bonds being preserved in the IL solvated bagasse.
Undoubtedly, a large proportion of the dissolved lignin remains in the water / IL
mixture even after addition of more water (3.5 water : IL mass ratio) and lowering
of the pH to < 1.0. In this fractional precipitation approach there are difficulties in
quantitatively recovering lignin since only a small fraction of it precipitates.
Furthermore the precipitate is far from pure lignin as it can contain up to 70 % mass
hemicellulose. While the cellulose fraction can be obtained in an enriched and
extensively decrystallised form, the poor lignin recovery and co-precipitation of
hemicelluloses argues against the use of this approach in an industrial setting.
160
Table 5.4.5: Mass balance of bulk biomass and of biomass components from three treatments with different ILs
dry mass (mg)
Bulk biomass
ash lignin
(AIL + ASL) glucan xylan arabinan acetyl HMF
(as glucan
equivalents)
furfural
(as xylan
equivalents)
[C4mim]Cl
Untreated 1461 46 383 656 324 22 46 n/a n/a
SF1 1318 47 344 627 270 14 39 n/a n/a
LF1 92 n/d 0.4* 9 47 10 7 0.6 1.2
Total mass
recovered
1411 47 344 636 317 24 46
[C2mim]Cl
Untreated 1346 42 353 605 299 20 42 n/a n/a
SF1 643 39 185 343 71 4 11 n/a n/a
LF1 446 n/d 3* 173 196 16 28 1.2 1.4
Total mass
recovered
1089 39 188 516 267 20 39
[C2mim]OAc
Untreated 699 22 183 314 155 10 22 n/a n/a
SF1 463 26 73 316 62 7 7 n/a n/a
LF1 88 n/d 17* 0 63 5 n/d 1.1 1.8
Total mass
recovered
551 26 90 316 125 12 7
* This mass does not represent all the lignin mass in the LF1 but only the recoverable lignin mass in the sum of SF2 and SF3 precipitates.
161
5.4.5 Mass recovery of bagasse components after pretreatment
Mass balances for bagasse components in the solid fraction (SF1) and liquid
fraction (LF1) of the three IL pretreatments ([C2mim]OAc, [C4mim]Cl, [C2mim]Cl)
were determined as described in Section 3.21 and are shown in Table 5.4.5. The
composition of the solid fraction and the liquid fraction (sum of monosaccharides
and oligosaccharides) were determined according to Sections 3.21.1 to 3.21.3. The
total mass accounted for by the mass balance determination protocol differs greatly
between ILs. For [C4mim]Cl the original bagasse mass accounted for is 97 % mass
(1411 mg out of 1461 mg) and this may be attributed to the low dissolution extent
achieved by this treatment. For [C2mim]Cl, the mass recovery is 81 % mass (1089
mg out of 1346 mg) and for [C2mim]OAc 79 % mass (551 mg out of 699 mg). For all
3 IL treatments, the remaining bagasse mass (not accounted for by mass balance
determinations) is mainly (more than half) lignin that was not recoverable from the
liquid fraction followed by polysaccharides (glucan followed by xylan for the
chloride ILs and xylan only for [C2mim]OAc). Note that the biomass derived acetyl
content in the liquid fraction of the [C2mim]OAc pretreatment (representing ca. 2 %
of starting bagasse mass) is not detectable since the IL counter ion is acetate.
The percent mass of each starting bagasse component in each pretreatment
fraction (viz. solid fraction, liquid fraction oligosaccharides and liquid fraction
monosaccharides) are plotted in Figure 5.4.7, Figure 5.4.8 and Figure 5.4.9 for
[C4mim]Cl, [C2mim]Cl and [C2mim]OAc, respectively. Note that the degradation
products HMF and furfural represent a small fraction of the mass in the liquid
fraction and are included in the calculations as glucan and xylan equivalents.
162
Figure 5.4.7: Mass distribution of bagasse components in [C4mim]Cl pretreatment
fractions
For [C4mim]Cl shown in Figure 5.4.7, 95 % mass of the original cellulose, 83
% of xylan and 90 % of lignin are recovered in the solid fraction. Out of the 10 %
lignin in the liquid fraction, only 0.3 % was recoverable, indicating that the vast
majority of the lignin mass remaining in the liquid fraction after addition of 0.5 IL
mass equivalents of water is in a form that cannot be recovered by further additions
of water or acidification (i.e. it is soluble and likely low molecular weight material).
Hemicellulose components are depolymerised preferentially while a big part of the
arabinose (36 %) is removed, all of which is in the monomeric form. The
arabinofuranosyl glycosidic linkages are acid labile and consequently arabinose loss
is a characteristic of acid treatments as discussed above [28]. The fact that
arabinose is found in the monomeric form indicates either that the arabinosyl
groups removed are terminal or if they are not, that the ester bonding of arabinose
to lignin is concomitantly cleaved. As the infrared analysis in Section 5.4.2 suggests
that the ester bonds are not likely to be cleaved by [C4mim]Cl, it is tempting to
speculate that only the terminal arabinosyl groups are removed by this
pretreatment.
At this point it should be noted that due to the estimated high standard
deviation of the technique measuring the small amounts of arabinose in the liquid
fraction (standard deviation = 20 % of starting bagasse mass, 3 df, see Section 3.21)
0%
20%
40%
60%
80%
100%
glucan xylan arabinan acetyl lignin
sta
rtin
g m
ass
[C4mim]Cl
LF mono
LF oligo
SF
163
, caution should be used on conclusions drawn from liquid fraction arabinose data.
Only the conclusions on whether the arabinose removed is monomeric or
oligomeric (bound to xylan) are affected by this uncertainty. The conclusions based
on removal of arabinose from the solid fraction, for all three pretreatments
investigated here, are still reliable (standard deviation for determining arabinose
content of solid fractions is low, 3 % mass of starting bagasse, 3 df).
Figure 5.4.8: Mass distribution of bagasse components in [C2mim]Cl pretreatment
fractions
Mass balance analysis for [C2mim]Cl treatment (see Figure 5.4.8) reveals the
same trends as for [C4mim]Cl but since dissolution is near complete in this IL the
trends are more pronounced. Lignin and cellulose are the predominant components
of the recovered solids reflecting the relative thermal and chemical stability of these
polymers in solution when compared to hemicelluloses. Out of 78 % mass arabinan
in the liquid fraction, 55 % mass is in monomeric form. Out of 50 % lignin expected
in the liquid fraction only 1.5 % is recoverable.
Preferential hemicellulose removal resulting in lignin and cellulose
enrichment of the treated solids is also a characteristic of dilute acid treatment. As
0%
20%
40%
60%
80%
100%
glucan xylan arabinan acetyl lignin
sta
rtin
g m
ass
[C2mim]Cl
LF mono
LF oligo
SF
164
shown in Section 4.1.4, dilute acid treatment removes all components of
hemicelluloses (xylan, arabinan, acetyl) with no apparent preference for any one
component. The chloride imidazolium ILs appear to remove arabinose
preferentially. Note that in Section 4.2.1, where the undissolved fraction of bagasse
(UND) after [C4mim]Cl dissolution was analysed, it was shown that cellulose
dissolved to a greater extent than hemicelluloses (the effect of dissolution only).
The analysis of SF1 here indicates that hemicellulose is preferentially removed (the
net effect of dissolution and reprecipitation). Cellulose is preferentially dissolved
but at a high DP and mostly recovered by precipitation with the addition of water,
whereas the little hemicellulose that dissolves, depolymerises and becomes soluble
in the water / IL mixture (i.e. does not reprecipitate).
The mass distributions of bagasse components after [C2mim]OAc
pretreatment are shown in Figure 5.4.9. While 100 % mass of the original cellulose
is recovered in the solid fraction the lignin content is reduced to 40 % mass.
Cellulose appears to get solubilised in a high DP form since it is all precipitated with
the addition of water and none of it is found in the aqueous liquid fraction. These
features indicate that [C2mim]OAc affords excellent cellulose preservation and
substantial delignification. However, out of the 60 % mass lignin extracted in the
liquid fraction, only 11.7 % was recoverable. Since arabinose is comparatively high
in the solid fraction and found in the oligomeric liquid fraction only, it can be
concluded that arabinose is preserved and that no terminal arabinosyl groups have
been removed (i.e. the arabinosyl glycosidic linkages in hemicellulose are stable in
[C2mim]OAc). Finally the acetate content of the solid fraction is reduced by 70 %
mass indicating substantial deacetylation.
The arabinan recovery is inflated (115 % mass) and this is attributed to the
large standard deviation of arabinose measurements in the liquid fraction as
discussed earlier.
165
Figure 5.4.9: Mass distribution of bagasse components in [C2mim]OAc
pretreatment fractions
Overall the [C2mim]OAc pretreatment affords distinctly different
compositional changes to the chloride IL pretreatments. These distinct differences
are delignification, deacetylation preservation of cellulose glycosidic bonds (as
deduced from the absence of cellulose mass in the liquid fraction) and preservation
of arabinosyl groups in hemicellulose. These differences are similar to the
differences between acid and alkali aqueous pretreatments.
In 2009, Sun et al. [101] reported mass balances for [C2mim]OAc treatment
of southern yellow pine (110 °C for 16 h, 5 % loading) precipitated with an acetone
in water (1:1, volume basis) antisolvent. Of note, are the total polysaccharide losses
(to the liquid fraction) measured, which amount to 41 % compared to 20 %
(comprising of xylan and arabinan) measured for [C2mim]OAc pretreatment and
reported in this thesis. Aside from different substrates and conditions, different
methods of measuring polysaccharides have been employed in the two studies and
therefore direct comparison is difficult, especially since the 13C NMR technique used
by Sun et al. does not distinguish between different polysaccharides.
0%
20%
40%
60%
80%
100%
glucan xylan arabinan acetyl lignin
sta
rtin
g m
ass
[C2mim]OAc
LF mono
LF oligo
SF
166
In 2010, Arora et al. [102] reported mass balances of [C2mim]OAc treated
switchgrass (160 °C, 3 h, 3 % loading). Their measurements account for all biomass
components, although they do not show IL mass recovery. Also the possibility of
covalent bonding of [C2mim]OAc cation or anion to biomass components (as seen
in Figure 2.4.2 for the cation and discussed in Section 5.4.2 for the anion) is not
ruled out. The recovered switchgrass solids contained 80 % of original cellulose, 35
% of lignin and 17 % of hemicelluloses while no detailed composition of
hemicelluloses was reported. Comparatively the data of this thesis shown in Figure
5.4.9 show the pretreated bagasse solids recovered contain 100 % of original
cellulose, 40 % of lignin and 40 % of xylan. The discrepancies are due to the
different substrates and conditions used. The milder conditions used in this work
are reflected in the higher recoveries of all components. Arora et al. also provide a
compositional analysis of the oligosaccharides present in the liquid effluent of a
treatment of switchgrass with [C2mim]OAc (3 h 120 °C, 3 % loading). In agreement
with Figure 5.4.9, xylan appears to be the main component of the liquid effluent.
The cellulose and hemicellulose saccharification extents achieved by each IL
at 24 h, as % mass theoretical yield on the basis of starting bagasse (prior to
pretreatment), are shown in Figure 5.4.10. Factoring in both the saccharification
extent and the loss of polysaccharide mass during pretreatment in the three ILs
provides a comparison that is more relevant to the industrial setting where avoiding
losses is crucial. Under this comparison [C2mim]OAc affords the highest cellulose
conversion due to a combination of rapid saccharification and no losses of cellulose
upon pretreatment. [C2mim]Cl and [C4mim]Cl have similar theoretical cellulose
conversions. The much higher dissolution extent in [C2mim]Cl did not benefit the
overall performance of this pretreatment since losses in [C2mim]Cl were much
higher than in [C4mim]Cl. In terms of hemicellulose conversion, [C4mim]Cl performs
best mainly due to reduced losses of xylan via hemicellulose depolymerisation.
167
Figure 5.4.10: Fraction of original bagasse polysaccharides saccharified in 24 h (15
FPU g-1
glucan) after pretreatment in three ILs
5.4.6 Mass recovery of the ionic liquid solvent after pretreatment
The recovery of the ionic liquid solvent used forms part of the mass balance
closure and it was measured using ion chromatography (described in Section 3.19.3)
of the liquid fraction of each pretreatment and the results are shown in
Table 5.4.6. The recovery of all ions is full (100 % mass) within the standard
deviation (2 %). This shows that little or no degradation of IL occurs at the reaction
conditions used. Covalent bonding of acetate ions from [C2mim]OAc to biomass (as
demonstrated in section 5.4.2) and imidazole cations with saccharides to form
imidazole glycosides (as illustrated in Figure 2.4.2) cannot be excluded by these data
since the low loading of biomass would make such ion losses small by comparison
to the 2 % standard deviation of the analysis.
0
10
20
30
40
50
60
70
80
90
[C4mim]Cl [C2mim]Cl [C2mim]OAc
% m
ass
of
the
ore
tica
l
(on
th
e b
asi
s o
f st
art
ing
bio
ma
ss)
glucan
xylan
168
Table 5.4.6: Mass recovery of ionic liquid ions after use
Recovery (% mass of
starting)
anion cation
[C2mim]OAc 100 100
[C4mim]Cl 100 103
[C2mim]Cl 98 101
5.4.7 Effect of IL anion and cation on pretreatment
In general and in agreement with the literature, for the cations, it appears
that the shorter alkyl chain of [C2mim]Cl (cf. [C4mim]Cl) imparted faster dissolution
and greater extent of saccharification. However higher dissolution rates were
accompanied by higher degradation rates. To some extent this degradation might
be reduced in an industrial setting by optimising reaction conditions or by
continuous removal of dissolved material. The anion effect is greater since it
imparts entirely distinct dissolution patterns. In the case of acetate compared to
chloride, the acetate ion appears to impart a more alkali-resembling effect while
the chloride ones a more acid-resembling effect.
5.4.8 Summary
In this section the mass balances of three IL pretreatment processes
precipitated with incremental additions of water are presented. The incremental
addition of water was successful in effecting a polysaccharide rich precipitate by
maintaining dissolved lignin in water / IL solution. Although 10 %, 50 % and 60 %
mass lignin was extracted in the liquid fractions (0.5 water : IL mass ratio) of
[C4mim]Cl, [C2mim]Cl and [C2mim]OAc pretreatments respectively, only 0.3 %, 1.5
% and 11.7 % was recovered after more water addition (3.5 water : IL mass ratio)
and acidification (pH ≤ 1). In other words the great majority of this extracted lignin
is strongly solvated in these ILs and not readily recoverable. It is also unfortunate
that the small fraction of lignin recovered from these liquid fractions contained ca.
169
70 % mass hemicellulose. It is beyond doubt that in the dissolved/extracted lignin,
covalent linkages with hemicellulose are preserved. These linkages may be different
depending on the IL used but they are certainly present in the dissolved lignin of all
three IL treatments studied here. The bagasse losses for all three IL processes are
mostly comprised of hemicellulose components. The acetate IL preferentially
removes lignin and acetyl, while it preserves arabinosyl groups. On the other hand
the chloride ILs impart an essentially opposite effect (i.e. preferential removal of
arabinosyl groups and preservation of acetyl groups and lignin). The changes
induced by the acetate IL resemble the effects of aqueous alkali pretreatments
while the changes induced by the chloride IL resemble those of aqueous acid
pretreatments. The mass of all ILs used was fully recovered although some evidence
of covalent bonding of the acetate ions from [C2mim]OAc to the LCB is provided.
These results, among others, demonstrate the role of the anion choice in ILs,
since acetate anion appears to impart an entirely different chemistry to the chloride
anion. Regarding the cation choice, it appears that the shorter alkyl chain on the
cation of [C2mim]Cl as compared to [C4mim]Cl accelerates the dissolution,
pretreatment and losses possibly via enhanced penetration of the smaller sized
cation into the tight packing of cellulose crystal structures.
The cellulose and hemicellulose saccharification rates and extents of the
precipitate recovered from the three IL pretreatments were also assessed. The 24 h
saccharification extent is combined with the mass balance data to give the percent
mass theoretical cellulose and hemicellulose saccharifications (on the basis of
starting bagasse). Using this indicator [C2mim]OAc ranks as the most suitable of the
three ILs for biomass pretreatment.
ILs directly disrupt cellulose crystallinity while they can exhibit both alkali
and acid treatment characteristics. To the best of the author’s knowledge no
conventional treatment has been demonstrated to be as versatile. The
characteristics of an ionic liquid suitable for biomass pretreatment would impart
high hydrogen bond interaction, delignification and preservation of polysaccharides.
[C2mim]OAc appears to meet these characteristics although from the perspective
170
of industrial utility it presents some technical impediments (viz. high viscosity when
in solution with bagasse, and the covalent bonding of the IL to the LCB substrate).
171
CHAPTER 6 CONCLUSIONS
The attractive characteristics of ionic liquids as a pretreatment technology
for biomass to ethanol conversion are the following:
• Thermal stability – many ILs are stable at temperatures > 170 °C and
have low or negligible vapour pressure
• Dissolution properties – Some ILs preferentially dissolve cellulose
(e.g. [C4mim]Cl) while others preferentially dissolve lignin (e.g.
[C2mim]OAc)
• Fractionation potential – wide choice of ILs that are miscible with
many solvents that can be used for preferential precipitation of
components (e.g. dilute NaOH to precipitate cellulose and keep lignin
in solution) – ILs that have the ability to be salted out and form ABSs
with aqueous salt solutions
• Decrystallisation capacity - by both precipitation of the dissolved
cellulose and by swelling of the undissolved cellulose in biomass
• Saccharification impact - by removing lignin, perturbing interpolymer
linkages and decrystallising cellulose
This thesis reports these attributes of ionic liquids as measured in selected
pretreatment processes. Out of the numerous biomass pretreatment processes that
can be envisioned with ionic liquids (see section 2.5 for examples) the following
have been studied in this research:
• Complete or partial dissolution with complete precipitation of all
water insoluble components
• Complete or partial dissolution with partial precipitation of a
cellulose-rich solid followed by a second precipitation of a lignin-rich
solid, using selected antisolvents
172
• Partial dissolution followed by the formation of an ABS with a
cellulose-rich and a lignin-rich phase
6.1 Findings
6.1.1 Chapter 4: Pretreatment
The effect of temperature and time on bagasse pretreatment with the IL
[C4mim]Cl was studied. Maximum dissolution (52 % mass dissolved) without
disproportionately increasing incurred losses (as molecules soluble in the water / IL
solution) was achieved at 150 °C and 90 min (Section 4.1). It is shown that:
• At temperatures > 150 °C and when approaching 100 % dissolution,
the dissolution rate slowed while the rate of losses continued to
rise.
• At temperatures ≤ 150 °C, dissolution extent appeared to increase
with temperature and time while the losses consistently accounted
for 1/3 of the dissolution extent.
• Up to 150 °C (90 min) no cellulose mass was lost and the losses
consisted mainly of the hemicellulose (xylan and arabinan)
fractions.
• At high temperatures (150 °C cf. 130 °C), glucose dissolved in
[C4mim]Cl is preserved from degradation (possibly by converting to
an anhydrous molecule, Section 4.1.3.c).
The effect of bagasse moisture and loading in [C4mim]Cl (150 °C) was also
studied and it is shown that:
• More than 10 % biomass moisture content could be tolerated by
the system before deceleration of dissolution was evident due to
the competition of water for hydrogen bonding (Section 4.1.2.d).
• High bagasse loading in the IL (15.3 % mass in 2 h and 20.6 % in 5 h)
was achieved by incremental additions of solids (Section 4.1.2.e).
173
Testing the effect of different ILs on bagasse dissolution (Section 4.1.2.f)
indicated that the ratio of dissolution extent to the losses was different for each IL.
The IL with short alkyl chain on its imidazolium cation and the anion with higher
hydrogen basicity (viz. acetate) seemed to impart fastest biomass dissolution.
Enzyme saccharification rates and extents along with composition of
[C4mim]Cl-treated solids from partial and complete dissolution at different
temperatures were studied in Sections 4.1.4.a and 4.1.4.b. It is shown that
pretreatment with [C4mim]Cl at 150 °C for 90 min (52 % mass dissolution) imparted
the highest saccharification rate. This rate (viz. 93 % in 3 h) was nearly as high as
that of completely solubilised bagasse (viz. 100 % in 3 h) and significantly higher
than that of the partial dissolution at 140 °C (viz. 41.5 % in 3 h). Accordingly, it was
concluded that:
• Shifting the dissolution temperature from 140 °C to 150 °C nearly
doubles the saccharification efficiency.
• Complete dissolution is not necessary in order to achieve maximum
saccharification efficiency.
These observations were attributed to structural and compositional changes
of the undissolved fraction upon dissolution. The undissolved fractions (from
dissolution reactions at different conditions) were isolated and studied separately in
Section 4.2 which showed that:
• The saccharification properties of the undissolved fraction were
enhanced with increasing severity of reaction conditions (time,
temperature) reflecting compositional and/or structural changes.
• With increasing reaction severity, the undissolved fraction was
enriched in cellulose, lignin and acetyl groups while xylan and
especially arabinose were preferentially solubilised
• A combination of high temperature phase transition of cellulose and
lignin glass transition take place in the undissolved fraction at > 140
°C.
174
• decrystallisation alone is not enough to accelerate saccharification
and the lignin-hemicellulose covalent linkages remaining in the
undissolved fraction also inhibit saccharification.
The optimised IL pretreatment ([C4mim]Cl, 150 °C, 90 min) was compared to
standard dilute acid pretreatment (160 °C, 10 min) in terms of ethanol yield and
total processing time (pretreatment + saccharification + fermentation) in Section
4.1.4.c. Ionic liquid pretreatment outperformed dilute acid both in yield (79 % cf. 52
% of theoretical on the basis of cellulose in starting biomass) and time (16.5 h cf.
36.2 h). This outcome is mainly attributed to the slow initial saccharification after
dilute acid pretreatment deriving from its inability to decrystallise cellulose.
6.1.2 Chapter 5: Fractionation
6.1.2.a Aqueous Biphasic systems
Fractionation of IL-dissolved bagasse to a polysaccharide-rich and a lignin-
rich fraction was attempted using aqueous biphasic systems and single phase
systems with preferential precipitation.
Aqueous biphasic systems were investigated (section 5.1) for their potential
to produce clean fractions of dissolved biomass while reducing the energy (cf.
distillation) needed to remove water from the IL upon solvent recycling.
Aqueous biphasic systems comprising of a [C4mim]Cl-rich top phase and a
concentrated NaOH bottom phase were assessed and the main findings are:
• Lignin reports at the top IL-rich phase rather than the alkali phase
(reverse to patent claim by Edye and Doherty [3, 134])
• Phase convergence (increasing with increasing biomass loading) and
deprotonation of the imidazolium cation in the presence of NaOH are
shown. They are both important technical difficulties.
• The use of different ILs and/or salts that are highly kosmotropic and
form less alkaline solutions were suggested as alternatives and
175
experimentation did not progress to enzyme saccharification of
recovered bagasse solids
Alternative aqueous kosmotropic salt solutions (KOH, K2CO3, Na2CO3) which
form ABSs with [C4mim]Cl were studied (excluding biomass). K2CO3 was identified
as the salt of choice while it was shown that:
• Na2CO3 presented higher phase divergence than K2CO3 but the low
water solubility of Na2CO3 limited the stability of the ABS and
increased the risk of collapse into a single phase upon migration of
water towards the IL phase.
• Deprotonation of the imidazole ring in the IL phase persisted and was
quantified at 5 % - 8 % mol depending on kosmotropic salt used.
• Metathesis reactions take place between the IL anion and the cation
of the kosmotropic salt in ABSs with K2CO3, deteriorating phase
separation.
Regardless of the numerous technical impediments, the results suggest that
a preferred composition for an ABS is attainable and these findings should be used
for guidance to selection of this composition.
6.1.2.b Single phase systems
Regarding single phase systems (section 5.2) preferential precipitation of
cellulose resulted in partially delignified pretreated solids. Completely dissolved
bagasse in [C4mim]Cl precipitated with dilute NaOH and acetone in water contained
40 % and 29 % respectively less lignin than when precipitated with water. However
these delignifications were associated with little increase of the enzyme
saccharification extent of the recovered solids. Thus, among the three antisolvents,
water was preferred since it is the most convenient and inexpensive.
Preferential precipitation of cellulose (while keeping lignin in solution) by
adding incremental amounts of water was another fractionation strategy and was
first tested on cellulose (Avicel) and lignin (bagasse soda lignin) solubilised in
176
[C4mim]Cl and [C2mim]OAc (Section 5.3). Cellulose precipitation required < 0.5
water : IL mass ratio whereas lignin precipitation was observed closer to a water : IL
mass ratio of > 1. This indicated the potential of preferential precipitation of
cellulose to lignin in a biomass IL solution simply by varying the water : IL ratio used.
This sequential precipitation was used on three partial dissolutions of bagasse in IL
(viz. [C4mim]Cl, [C2mim]Cl and [C2mim]OAc) and the mass balances of these
experiments determined.
6.1.2.c Mass balances
Mass balances of the aforementioned three IL pretreatments in [C4mim]Cl,
[C2mim]Cl and [C2mim]OAc (under identical reaction conditions, and addition of
water to reach 0.5 water : IL mass ratio) are presented in section 5.4. The results
indicate that:
• Incremental water addition is successful in extracting lignin and
producing a cellulose-rich solid in all three ILs.
• Lignin extraction: 10 %, 50 % and 60 % mass of the starting lignin
respectively, was extracted into the liquid fraction.
• Lignin recovery: Only 0.3 %, 1.5 % and 11.7 % mass of starting lignin,
respectively, was recovered after ample water addition (3.5 water :
IL mass ratio) and acidification (pH < 1).
• Lignin purity: the lignin was recovered in a solid fraction that
contained ca. 70 % mass hemicellulose in all ILs (lignin-hemicellulose
covalent linkages are preserved but are different depending on IL
used, see key finding below)
• For all three IL treatments, biomass losses to the liquid fraction
consisted mainly hemicellulose. However, the preferential
dissolution patterns differed characteristically between ILs (see key
finding below).
• The mass balance determinations accounted for 97 % of starting
bagasse mass for the [C4mim]Cl pretreatment , 81 % for [C2mim]Cl
and 79 %for [C2mim]OAc.
177
• For all three IL treatments, the remaining bagasse mass (not
accounted for by mass balance determinations) was mainly (more
than half) lignin that was not recoverable from the liquid fraction.
• After pretreatment, 100 % mass of both ions of all three ILs were
recovered in the liquid fraction.
A key finding derives from the preferential dissolution patterns identified by
the mass balance determinations. The acetate IL extracts lignin and native acetyl
groups while it preserves arabinosyl groups. The chloride ILs impart the opposite to
these trends while they remove hemicellulose preferentially to lignin. The
preferential component removal patterns in [C2mim]OAc resemble those imparted
by aqueous alkali pretreatments whilst those in [C4mim]Cl and [C2mim]Cl resemble
aqueous acid pretreatments. This pattern demonstrates the role of anion choice in
these ILs and calls for further investigation.
Aside from the anion role, some conclusions about the role of the imidazole
cation alkyl chain length are also drawn by comparing the two chloride IL
pretreatments. The shorter alkyl chain on the cation of [C2mim]Cl as compared to
[C4mim]Cl accelerates the dissolution and pretreatment possibly via enhanced
penetration of the smaller sized cation into the tightly packed cellulose crystal
structures.
Saccharification kinetics and cellulose crystallinity indices for the cellulose-
rich solids recovered from the three IL pretreatments are reported in Sections 5.4.2
and 5.4.3. FTIR analysis revealed that all three ILs caused cellulose decrystallisation.
The 24 h cellulose saccharification extents of the recovered solids from the three IL
pretreatments ranked as [C2mim]Cl>[C2mim]OAc>>[C4mim]Cl. However when the
24 h saccharification extents of these pretreatments were compared in terms of
percent mass theoretical on the basis of cellulose in the starting biomass, the order
of performance changed to [C2mim]OAc (83 %)>>[C2mim]Cl (53
%)=[C4mim]Cl(53%). This order is more practically relevant since it takes into
account cellulose loss imparted by each pretreatment. The [C2mim]Cl imparts 43 %
mass cellulose loss whereas [C2mim]OAc treatment imparts no cellulose loss
178
reflecting extensive cellulose depolymerisation and loss in the former and none in
the latter. This further confirms that [C2mim]OAc treatment acts more like an
aqueous alkali treatment since the β-glycosidic bonds are protected in alkaline
conditions.
ILs directly disrupt cellulose crystallinity while they can exhibit both alkali
and acid treatment characteristics. This combination together with the ability of ILs
to be tuned is rare among the pretreatment strategies proposed to date. ILs
dissolve biomass polymers and are compatible with an array of separation
processes including fractionation using aqueous biphasic systems or preferential
precipitation using selected antisolvents. Moreover, the choice of anion and cation
provides potential major improvements in the pretreatment performance currently
measured. Characteristics of an ionic liquid suitable for biomass pretreatment
would include low viscosity and ability for strong hydrogen bond interaction,
delignification and preservation of polysaccharides. [C2mim]OAc appears to meet
these characteristics although it presents some technical impediments such as the
high viscosity when in solution with bagasse, and the covalent bonding of the IL to
the LCB substrate, which have to be weighed against performance. The choice of IL
and its compatibility with cellulose antisolvents (e.g. water) are also going to play a
role in its utility as biomass fractionation medium.
6.2 Future work
It is indicated that the transition to the high temperature crystalline phase of
cellulose may be partly responsible for the sudden increase of saccharification rates
and extents at ca. 150 °C. This indication remains to be verified by exploring further
and more systematically the correlation of saccharification performance of IL
treated solids at different temperatures to associated crystal phase transitions as
measured by XRD analysis. The temperature at which this phase transition occurs
(in the undissolved fraction) may be associated with maximum saccharification
performance. Whether this phase transition occurs at different temperatures when
varying the IL and biomass substrate, is also a subject of further research. In this
179
regard, the techniques reported in this thesis can be used to assess more
combinations of ILs and biomass.
Alternative kosmotropic salts and/or ILs need to be tested so as to improve
the phase divergence of biphasic systems especially when biomass loadings are
elevated.
The lignin-hemicellulose covalent bonding preserved after dissolution is
possibly different for chloride and acetate ILs. This difference could be verified by
detailed structural analysis (e.g. 2D NMR) of the undissolved fractions and the
dissolved fractions of these ILs. Once the nature of the preserved bonding is
identified, studies into the possibility of separating lignin from hemicellulose by
cleaving these bonds or alternatively the possibility of using the whole lignin-
hemicellulose complex for added value by-products may be explored.
Other observations presented in this thesis that may contribute to
knowledge of ionic liquid pretreatment if investigated further are for example:
• The formation of the thermally stable 1,6-anhydro-β-D-
glucopyranose at high temperatures
• The possibility of covalent bond formation between [C2mim]OAc and
biomass which can be verified using 13C labelled [C2mim]OAc and
NMR on recovered solids
• The salt precipitation patterns in IL aqueous biphasic systems.
Overall, the possibilities of experimenting with ILs for biomass treatment are
practically infinite. Identifying the IL characteristics that impart high pretreatment
performance is essential and will prioritise research efforts towards IL systems of
high potential. One of the most alarming impediments in ionic liquid applications in
an industrial setting at the moment is their cost. Discovery of new ionic liquids with
emphasis on reduced cost of manufacturing is another urgently needed research
task.
180
Finally, the life cycle assessment of optimised ionic liquid processes needs to
be compared against conventional pretreatments and especially the ones that have
progress towards commercial level (e.g. dilute acid pretreatment). This integrated
assessment will reveal the true benefit (if any) of employing ILs in lignocellulosic
biorefineries.
181
Appendix I
Linear relationship of FTIR band heights to lignin, cellulose and glucose
concentrations in [C4mim]Cl
The FTIR spectra of glucose (2 % – 15 % mass), cellulose (1 % - 9 % mass) and
bagasse soda lignin (1 % - 20 % mass) (each dissolved in [C4mim]Cl) were obtained
and analysed. Glucose in [C4mim]Cl has a C-O-C ring stretching vibration at 1050
cm-1 that was found to be linearly related to concentration. In cellulose this C-O-C
ring stretch is shifted to 1070 cm-1 due to polymerisation. Cellulose concentration
was found to be linearly related to absorbance at 1070 cm-1. Lignin in [C4mim]Cl has
a characteristic phenolic ring vibration at 1510 cm-1 which is also linearly related to
concentration. The concentration-absorbance relationships are shown in the
accompanying figures. Absorbance data is based on peak heights with valley to
valley baselines. The FTIR software provides a method for real time monitoring of
band heights, and this method and the characteristic wavenumbers were used to
monitor biomass dissolution by ATR-FTIR.
182
R² = 0.9944
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0 5 10 15 20
Absorb
ance u
nits
mass glucose / mass [C4mim]Cl / %
Glucose at 1050 cm-1
R² = 0.9961
0
0.05
0.1
0.15
0.2
0.25
0.3
0 2 4 6 8 10
mass cellulose / mass [C4mim]Cl / %
Cellulose at 1070 cm-1
R² = 0.9988
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0 10 20 30
mass lignin / mass [C4mim]Cl / %
Lignin at 1510 cm-1
183
Appendix II
The effect of bagasse extractives on dissolution
In Section 5.4 the extent of dissolution of extracted LCB is estimated from
the measurement of loss and the mass ratio of dissolution to losses. In earlier
sections where LCB was not extracted prior to dissolution the mass ratio of
dissolution to losses was consistently 3:1. Extractives are non-structural molecules
such as sugars and waxes that are removed by prolonged exposure to water and
ethanol in a heated Sohxlet device. Extractives which represent a small fraction of
bagasse (5 % to 10 %) may have an effect on solvent-solute interactions, and
certainly have an impact on the results of characterisation. Better mass balance
closures are obtained by removing these non-structural molecules. The mass ratio
of dissolution to losses was determined for extracted bagasse under the same
conditions as non-extracted bagasse (150 °C for 90 min) and the two experiments
are compared in the accompanying figure. In the absence of extractives, bagasse
dissolution is enhanced (from 52 % dissolution to 79 %) and the losses
disproportionately increase. The dissolution to loss mass ratio changes from 3:1 to
2.3:1.
0
10
20
30
40
50
60
70
80
90
100
bagasse as is bagasse extracted with water andethanol
ba
ga
sse
(%
ma
ss)
dissolution losses
184
Appendix III
Reaction calorimetry
The same reaction as in Section 4.1.2.g was simultaneously monitored for
heat flow changes. Figure 4.1.5 shows the overtime curves for heat flow and
temperature (see Section 3.7 for details). Unfortunately, no thermal events,
characteristic of biomass material softening, were detectable at this large scale. As
demonstrated from the heat flow signal, the only significant heat flow change
detected is a large exotherm attributed to the temperature drop from 170 °C to 150
°C at 185 min.
Reaction calorimetry of bagasse dissolution in [C4mim]Cl
-150
-100
-50
0
50
100
150
200
250
300
0
20
40
60
80
100
120
140
160
180
0 50 100 150 200h
ea
t fl
ow
(W
)
T e
mp
era
ture
(°C
)
Time (min)
reaction temperature (°C )
heat flow (W)
185
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